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US 20160176988A1 as) United States cz) Patent Application Publication (10) Pub. No.: US 2016/0176988 Al Zhang et al. (43) Pub. Date: Jun. 23, 2016 (54) DNA-NANOPARTICLE CONJUGATES Publication Classification (71) Applicant: Brookhaven Science Associates, LLC, (51) Int. Cl. Upton, NY (US) CO7K 17/14 (2006.01) C30B 29/68 (2006.01) (72) Inventors: Yugang Zhang, Middle Island, NY C30B 29/38 (2006.01) (US); Fang Lu, Middle Island, NY (US); GOIN 33/58 (2006.01) Daniel van der Lelie, Chapel Hill, NC CO7K 14/36 (2006.01) (US); Oleg Gang, Setauket, NY (US) (52) ssssesssseeee CO7K 17/14 (2013.01); GOIN 33/588 (2013.01); CO7K 14/36 (2013.01); C30B 29/58 (21) Appl. No.: 14/876,899 (2013.01); C30B 29/68 (2013.01) (57) ABSTRACT (22) Filed: Oct. 7, 2015 The bio-programmable crystallization of multi-component functional nanoparticle systems is described, as well as meth- ods for such bio-programmable crystallization, and the prod- Related U.S. Application Data ucts resultant from such methods. Specifically, the systems (62) Division of application No. 14/111,732, filed on Feb, “closed and taught herein are directed to improved strate- ae gies for the DNA-mediated self-assembly of multi-compo- 25, 2014, now abandoned, filed as application No. rn . . : PCT/US12/33380 on Apr. 12, 2012. nent functionalized nanoparticles into three-dimensional ° order superlattices, wherein the functionalization of the nano- (60) Provisional application No. 61/475,172, filed on Apr. _ particles with DNA is independent of either the composition 13, 2011. of the material, or the shape of the nanoparticles.
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Patent Application Publication Jun. 23,2016 Sheet 1 of 78 US 2016/0176988 Al FIG. 1A FIG. 18 FIG, iC
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Patent Application Publication Jun. 23,2016 Sheet 2 of 78 US 2016/0176988 Al EES NO FIG, 2A FIG. 2B FIG. 2D FIG, 2C
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Patent Application Publication Jun. 23,2016 Sheet 7 of 78 US 2016/0176988 Al aie T + t ? t GOO 002 ODE ONG RG G4e O42 16 Q FIG. 8G omen Intensity from Sys-TAu, 36SAu,, 35 intensity from melting Sys-TAu, 365Au,, 38 5800 4 40004 300B 4 2600 +f FIG, 5H
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Patent Application Publication Jun. 23,2016 Sheet 9 of 78 US 2016/0176988 Al FIG, 6A
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Patent Application Publication Jun. 23,2016 Sheet 10o0f78 US 2016/0176988 Al Pande FIG. 6B
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Patent Application Publication Jun. 23,2016 Sheet 22 0f78 US 2016/0176988 Al * t r t ¥ OR G07 OO 086 ot Oi Ri ote 3 HOC ON (GO OK GOR UNO Os he FIG. 9B
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Patent Application Publication Jun. 23,2016 Sheet 23 of 78 US 2016/0176988 Al FIG. 9C FIG. 9D aod} a4 t oon t OnE
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Patent Application Publication Jun. 23,2016 Sheet 240f78 US 2016/0176988 Al 2544 Gos 0M 002 004 O06 O68 Ot ote O18 FIG. 9E i ss es 3 i r r r C00 G02 O04 EGG 60R O30 OTR Oe Q FIG, OF
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Patent Application Publication Jun. 23,2016 Sheet 250f78 US 2016/0176988 Al t59 | 48 26 004. 008 0.12 FIG. 10
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Patent Application Publication Jun. 23,2016 Sheet 260f78 US 2016/0176988 Al ie Da Ob v t 3% aan Oe - OX O02 O08 G06 ~ONe 01 OT O44 a FIG. 11A QD:AB-DNA&TH 1120) FIG, 11B
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Patent Application Publication Jun. 23,2016 Sheet 270f78 US 2016/0176988 Al 2B 264 384 w® 304 OS $ messager $ v r t v C00 C82 684 806 (O08 Ot 812 BAF a FIG. 11¢ ey ado 902 6M 006 908 GIG Ofte. One FIG. 11D
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Patent Application Publication Jun. 23,2016 Sheet 28 0f78 US 2016/0176988 Al Boy = 10:1:20, 184 a4 ho4 t T t T T t r ss GMO 0.02 0.04 0.08 0B MR Q FIG. 11E
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Patent Application Publication Jun. 23,2016 Sheet 29 0f78 US 2016/0176988 Al FIG. 11F | idati thu i rirlil L S | ° ih | im di ih duals Li | 1 Lu Lalit ithe 0.00 6.02 0.04 606 008 O10 0.12 0.14 FIG. 11G
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Patent Application Publication Jun. 23,2016 Sheet 30 of 78 US 2016/0176988 Al t T T T T + t G0) 802 OFA 9OR BEE OW UR oe a FIG. 124 , ay t t r t r POS O8e GN GOR ONE Gin hye 38 & FIG, 22B
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Patent Application Publication Jun, 23,2016 Sheet 31 of 78 US 2016/0176988 A1 24 0.0: r t t t T T 5 £00 802 004 O08 008 079 G52 dae Q FIG. 12€ 284 C64 Gad 24 a0 tT r T t t BM 052 $04 005 O88 816 GR tld FIG. 12D
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Patent Application Publication Jun. 23,2016 Sheet 32 of 78 US 2016/0176988 Al FIG. 12E t t 7 r t T T T oOo 062 8.08 O06 O88 Of O42 Oe FIG. 12F
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Patent Application Publication Jun, 23,2016 Sheet 33 of 78 US 2016/0176988 Al 2a~ mn 404 t 8 t a T T t r OR 864 GOH O88 G30 Bie OM FIG. 12G OM OO 004 AOR NOB G1 Oe ote Q FIG. 12H
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Patent Application Publication Jun. 23,2016 Sheet 34 0f78 US 2016/0176988 Al 0 Sys-Q7A18gg 26 t t z ss t T 0G 202 8.0% 906 O48 Of G42 B44 Q FIG. 13A Vas Sys-Q7A16y9 53 6 144 : 134 0.94 BB+ t t T t t t BOO DUZ O04 Ons O88 GIG GN2 D4 FIG. 13B
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Patent Application Publication Jun. 23,2016 Sheet 350f78 US 2016/0176988 Al Particle diameter [Ay 100 FIG. 13C
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Patent Application Publication Jun. 23,2016 Sheet 36 0f78 US 2016/0176988 Al me Sys QTA_Conirot ¥ We date ‘ae4 enone SYE-QIA TS ond omomae Fits seven ByS-OTARE i . amine SSCITASO | an baamnetiy 054 QE=1.14*exp(-Dv15.3) e » wey 2 Ss | “ B aad « ee aa = 4 ae ® : om ee 702 780 ako 4 Cae aie ee ee er ee Wavelength (nen) Surtace-tonirtsns 1D (nt) Fig. 144 Fig. 14B Fig. 154
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Patent Application Publication Jun. 23,2016 Sheet 370f78 US 2016/0176988 Al Particle diameter [A 400 v9 intensity eS Es & t icf uorBannG oSeniea = T oO & Sa Residuais fawrsadrignd | femoris oy t 7 senna Sees an Wee tae ET z 3 a fb & F Be 3 8 eo 4 Ra UARSapdtatiate PGI ION FSP te8 2, Moe DONE et OG “Tas, Doe 14 ete, ase mR PLL
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Patent Application Publication Jun. 23,2016 Sheet 38 of 78 US 2016/0176988 Al o 14 a r 3 + T 8.98 Hed one O32 Q FIG. 16A . . t . 7 7 t $00. OR 106 GOS. 8.08. 040 faz ote FIG. 16B
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Patent Application Publication Jun. 23,2016 Sheet 39 of 78 US 2016/0176988 Al FIG. 16C t t T r T T O00 C82 B08 008 OR 810 B42 Oe FIG. 16D
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Patent Application Publication Jun. 23,2016 Sheet 40 of 78 US 2016/0176988 Al FIG. 16E FIG. 16F
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Patent Application Publication Jun. 23,2016 Sheet 41 0f78 US 2016/0176988 Al FIG. 17A
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Patent Application Publication Jun. 23,2016 Sheet 42 0f 78 US 2016/0176988 Al
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Patent Application Publication Jun. 23,2016 Sheet 43 of 78 US 2016/0176988 Al B48 i r t ss t T Hob 602 804 O08 BOF OI ete Mw Q FIG. 184 FIG. 18B
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Patent Application Publication Jun. 23,2016 Sheet 44 of 78 US 2016/0176988 Al FIG. 18C 204 +54 41044 t Tr Tv t t r : ooh 0.02 O88 666 OB O18 GAR B14 FIG. 18D
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Patent Application Publication Jun. 23,2016 Sheet 45o0f78 US 2016/0176988 Al i : . “45b spacer, 15-bp linker» & eee he eS ineb spacer FIG. 19
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Patent Application Publication Jun. 23,2016 Sheet 46 0f78 US 2016/0176988 Al : . * araseoneeapanseneneys 3 Bue OOF Mk 8G OS ute te oad isi FIG. 20A Qa Gy FIG. 208
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Patent Application Publication Jun. 23,2016 Sheet 470f78 US 2016/0176988 Al acl 4 FIG, 20C FIG. 20D
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Patent Application Publication Jun. 23,2016 Sheet 48 of 78 US 2016/0176988 Al FIG. 21A
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Patent Application Publication Jun. 23,2016 Sheet 49 of 78 US 2016/0176988 Al FIG. 21B
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US 2016/0176988 A1 Jun. 23, 2016 Sheet 50 of 78 Patent Application Publication feocccenvoveemnoccnnnooreneoc IT? ‘Sd st : : y / OTE Oe BAZ serwommennitay pees RecA
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Patent Application Publication Jun. 23,2016 Sheet 51o0f78 US 2016/0176988 Al FIG. 21D
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Patent Application Publication Jun. 23,2016 Sheet 53 0f78 US 2016/0176988 Al au FIG. 21F 2a
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Patent Application Publication Jun. 23,2016 Sheet 570f78 US 2016/0176988 Al
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Patent Application Publication Jun. 23,2016 Sheet 58 o0f78 US 2016/0176988 Al ain 204 Sys Shig a Gm Uo4 z T r O00 902 806 Gus bob O10 B42 04d Q s oa a scotty t ¢ r OOo ORE OG4 GOR 6.08 a FIG. 23B
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Patent Application Publication Jun. 23,2016 Sheet 59 of78 US 2016/0176988 Al FIG, 23C FIG. 23D
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Patent Application Publication Jun. 23,2016 Sheet 60 of 78 US 2016/0176988 Al e 3s " peep OH ORS G04 ROE OS OH Gt ote FIG. 244 FIG. 24B
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Patent Application Publication Jun. 23,2016 Sheet 61 0f78 US 2016/0176988 Al FIG, 24C FIG. 24D
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Patent Application Publication Jun. 23,2016 Sheet 62 0f78 US 2016/0176988 Al FIG. 24F
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Patent Application Publication Jun. 23,2016 Sheet 63 of 78 US 2016/0176988 Al a * [ee toyie = & oh z = 5 «0 ‘4 8 i 20. 0 18 20 Wavelength (ern) Surface-to-Surface Distance (nm) FIG. 25A FIG. 25B mR RR R= trosllsas } 40. intonsity (3.u } & Enhancement-to-quenching factor (%) 8 — ol a P a an 0 70 6 2 ‘Wavelength ‘nen ‘Surface-to-Surface Distance (nm) FIG. 25C FIG. 25D
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Patent Application Publication Jun. 23,2016 Sheet 64 0f78 US 2016/0176988 Al FIG. 26 8.04 0.08 O48 FIG, 27 ‘rome Syvs-QFO Control 1804 sonseone Sy5-GPDIB eee SyS-QPUED Bo a0 Intensity 80 BS
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US 2016/0176988 A1 Jun. 23, 2016 Sheet 65 of 78 Patent Application Publication 82 Sid SSHN/Jda — HOOD 2QoudospAH
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Patent Application Publication Jun. 23,2016 Sheet 66 0f78 US 2016/0176988 Al Catalytic Pd Magnetic Fe,0; f=15~25 f=3~8 2 's0nm_, ee 009000? 60000 0 60000 H (Oe) FIG. 29 FIG. 30
Page 68
Patent Application Publication Fluorescent QD f=20~40 CdSe/ZnS, CdTe/ZnS intensity (a.u.) Jun. 23, 2016 Sheet 67 of 78 Plasmonic Au f=45~60 ic, eS 450 «600750 Wavelength (nm) FIG. 31 400 600 Wavelength (nm) FIG. 32 US 2016/0176988 A1
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Patent Application Publication Jun. 23,2016 Sheet 68 of 78 US 2016/0176988 Al FIG. 33A FIG. 33B
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Patent Application Publication FIG. 33C FIG. 33D Jun, 23, 2016 Sheet 69 of 78 US 2016/0176988 Al “"" 400 200 300 & (nm) @ SAXS Data 39 | DC-WLC Model 204 40
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Patent Application Publication Jun. 23,2016 Sheet 70 of 78 US 2016/0176988 Al FIG. 34A Ww fy ee Sys_FeO ° FeO_Augs 65 S(q) ' peed HO t : itera serccammaneang v FeO_Au, 515 T (3) 8 egy 3 % H a » ANY ' 4 o STV_Augs 45 + STV_Auy 45 0.03 0.06 0.09 0.4
Page 72
Patent Application Publication FIG. 34B Jun, 23, 2016 Sheet 71 of 78 FIG, 34C US 2016/0176988 A1 @ Feo NP '® Au@Feo NP FeO_Auss 45 —- rw FeO_Ausg 35 ~@- \ _*s “se ‘ Phase_Fo ‘Ny. —m “@- ~@! 4 + 40 40
Page 73
Patent Application Publication Jun. 23,2016 Sheet 72 0f78 US 2016/0176988 Al
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Patent Application Publication Jun. 23,2016 Sheet 73 of 78 US 2016/0176988 Al 0.03 0.06 0.09 0.12 q FIG, 34E
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Patent Application Publication Jun. 23,2016 Sheet 74 0f78 US 2016/0176988 Al FIG. 35A FIG. 35B & om QD : x CdSe ran ye fs 4 oud * : Q5_Aug 4 5 CdSe S(q) Ss@
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Patent Application Publication Jun. 23,2016 Sheet 750f78 US 2016/0176988 Al FIG. 35C 180 150; 120 7 — @aunp 708 604 - drt tf @Q7 a GAunPia7 |, 4 40 80 420 160 € (nm) 30
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Patent Application Publication 35 304 Dy, (nm) 20; 15- Jun. 23, 2016 Sheet 76 of 78 ie 45 oy 145 dine ‘DC Model k 4 Q7_Au Q6_Au Q5_Au FIG. 35D US 2016/0176988 A1
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Patent Application Publication Jun. 23, 2016 Sheet 77 of 78 US 2016/0176988 A1 150 Ae 8100 Q7 eS Assembly —d 2 g 50 g Me, Ebene —= 600 6680 700 750 Free QD Superlattice — Wavelength (nm) porn 70) ~~ = === gan +. | Acceptor 4 H qo0 FSi Ro © | Donor i=526nm i hl] || aglee ence re e--- $-) 10 2 1] Setar 40 8 0 time (ns) Free QD Superlattice FIG. 35E FIG, 35F
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Patent Application Publication Jun. 23,2016 Sheet 78 of 78 US 2016/0176988 Al Component A . A FIG.36A ge sata QD gt 50) om r —_ “Wl FCCHike Crystals 40; |@ * Bcc a A Cluster A 30] ty oe ™ § 4 Weakly ordered E 20 q Q 8 10 A A \ 0 10 20 30 40 50 FIG. 36B 95] rc moons 99 | mm RB ~ 25 § 2 9 a 45 104 54 o4 Au_Q5 Au_Q7? AuPC Au PD Q7_Q5 Q7_PD Q7_PC PD_PC
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US 2016/0176988 Al DNA-NANOPARTICLE CONJUGATES CROSS-REFERENCE TO A RELATED APPLICATION [0001] This application isa divisional application related to US. application Ser. No. 14/111,732, filed on Feb. 25, 2014, which is the U.S. National Phase of International Patent Application Serial No. PCT/US2012/033380, filed Apr. 12, 2012, which claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 61/475,172 filed on Apr. 13, 2011, the contents of which are incorporated herein in their entireties. STATEMENT OF GOVERNMENT LICENSE RIGHTS [0002] This invention was made with Government support under contract number DE-AC02-98CH10886, awarded by the U.S. Department of Energy. The Government has certain rights in the invention. 1. FIELD OF THE INVENTION [0003] The inventions disclosed and taught herein relate generally to the field of DNA-mediated particle assembly, and, more specifically, to DNA-mediated self-assembly of multicomponent functionalized nanoparticles into three-di- mensional (3D) ordered superlattices. 2. BACKGROUND [0004] The ability to control and regulate the kinetic behav- ior of DNA-based nanosystems is required for emerging nanoparticle applications in sensing, nano-device assembly, and gene delivery, among other applications. DNA-based methodology takes advantage of the tunable and program- mable hybridization between DNA-capped nanomaterials. This approach has allowed for the development of sensitive detection systems based on the optical and physical proper- ties of assembled nanoparticles, as well as detection based on their novel melting/disassembly properties. [0005] In 1996, the Mirkin and Alivisatos groups showed that thiolated deoxyribonucleic acid (DNA) oligonucleotides can be attached onto gold nanoparticle surfaces to direct the formation of larger aggregations (Mirkin, C.A., etal., Nature, 1996. 382(6592): p. 607-609; Alivisatos, A. P., et al., Nature, 1996. 382(6592): p. 609-611, each of which is incorporated by reference in its entirety). Since then, there have been many efforts to use the lock-and-key property of DNA to achieve ordered arrangements of gold nanoparticles. Only very recently, several groups independently demonstrated the suc- cessful DNA-guided three-dimensional crystallization of gold nanoparticles (Nykypanchuk, D., et al., Nature, 2008. 451(7178): p. 549-552; Park, S. Y., et al., Nature, 2008. 451 (7178); p. 553-556; Xiong, H. M., D. van der Lelie, and O. Gang, Physical Review Letters, 2009. 102(1): p. 015504-(1- 4); and Macfarlane, R. J., et al., Angewandte Chemie-Inter- national Edition, 2010. 49(27): p. 4589-4592, each of which is incorporated herein by reference in its entirely). In these studies, it was found that either face-centered cubic (FCC) or body-centered cubic (BCC) structures with tunable lattice parameters can be formed by controlling the type, number and length of the DNA sequences. DNA length, rigidity, and number were proven to be the key parameters for gold nano- particle crystallization. Jun. 23, 2016 [0006] During the last decade, functional nanomaterials have become a hot research topic due to their importance and wide-spread application potential, ranging from magnetic recording media, catalysts, solar cells, biomedicine, and so on. The ability to assemble multi-component nanoparticles into three-dimensional ordered superstructures is of particu- lar interest for building advanced metamaterials with novel magnetic, plasmonic, photonic, and catalytic properties. Among many assembly techniques, DNA-mediated nanopar- ticle assembly has emerged as a powerful and versatile strat- egy that has many advantages due to the synthetically pro- grammable length and recognition properties of DNA. [0007] However, up to now, assembly in organized struc- tures of DNA-functionalized objects has mainly been limited to gold nanoparticles. The main reason being that gold nano- particles can be easily coated with a dense DNA shell by simply replacing the weak surfactants, e.g., citrate, cetyltri- methylammonium bromide (CTAB) etc., used during the syn- thesis process, by thiolated DNA. For synthesis of nanopar- ticles with different composition other than gold or gold materials with more complex morphologies, the functional- ization is very difficult because surfactants that are routinely used with these nanoparticles bind tightly to the surface, making their removal very difficult. For instance for Au nano- particles with complex shapes, such as Au rhombic dodeca- hedra and octahedra with cetylpyridinium chloride (CPC) as surfactant, directly replacing CPC with thiolated DNA will result in Au particle aggregation due to the low DNA-CPC exchange efficiency. In this case, fortunately, since CPC is not a very strong surfactant, one can first use a high CTAB con- centration to partially exchange CPC, and then replace CTAB by thiolated DNA (Jones, M. R., et al., Nature Materials, 2010. 9(11): p. 913-917, which is incorporated herein by reference in its entirety). [0008] However, for Au polyhedrons synthesized with much stronger ligands or long polymers as surfactants, like poly-diallyl-dimethylammonium chloride (PDDA) and poly- vinyl-pyrrolidone (PVP), the surfactants are very difficult to replace and consequently, to date, there are no reports on their functionalization with DNA and use in programmable assem- blies. For materials other than gold, such as palladium nano- particles synthesized with PVP, direct thiolated DNA fune- tionalization is impossible due to difficulty to DNA penetration and the much weaker thiol-palladium affinity. As a result of these functionalization problems, the components for DNA directed ordered nanoparticle assembly and crystal- lization have been limited to gold. Additionally, although there have been some recent reports on extending the particle component to other inorganic materials, suchas silver (Lee, J. S., et al., Nano Letters, 2007. 7(7): p. 2112-2115; Pal, S., et al., Chemical Communications, 2009(40): p. 6059-6061, each of which is incorporated herein by reference in its entirety), quantum dots (Maye, M. M., et al., Chemical Com- munications, 2010. 46(33): p. 6111-6113, which is incorpo- rated herein by reference in its entirety), silica (Hilliard, L.R., etal., Analytica Chimica Acta, 2002. 470(1): p. 51-56, which is incorporated herein by reference in its entirety) and iron oxides (Cutler, J. I., et al., Nano Letters, 2010. 10(4): p. 1477-1480; Lee, C. W., et al., Journal of Magnetism and Magnetic Materials, 2006. 304(1): p. B412-E414, each of which is incorporated herein by reference in its entirety), there are still no reports on incorporating such materials into three-dimensional (3D) ordered structures using the concept
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US 2016/0176988 Al of programmable assembly offered by functionalization with biological compounds, including nucleic acids, preferably DNA, and proteins. [0009] For most types of particles used for catalysis and other advanced applications, surface capping with high affin- ity ligands or long polymers is inevitable during their synthe- sis process. This makes it hard for DNA to replace or pen- etrate the ligand shell, and thus functionalization becomes a challenge. Furthermore, the application of strong ligands is not only limited to nano particles with composition different from gold, for instance quantum dots (QD) (Murray, C. B., et al., Journal of the American Chemical Society, 1993. 115(19): p. 8706-8715; Dabbousi, B. O., et al., Journal of Physical Chemistry B, 1997. 101(46): p. 9463-9475, each of which is incorporated herein by reference in its entirety) or palladium (Lim, B.., et al., Advanced Functional Materials, 2009. 19(2): p. 189-200, which is incorporated herein by reference in its entirety), but also to synthesize and preserve the shapes of non-spherical particles, even for Au (Sun, Y. G. and Y. N. Xia, Science, 2002. 298(5601): p. 2176-2179, which is incorpo- rated herein by reference in its entirety). [0010] Insum, several challenges remain for the full exploi- tation of DNA-mediated assembly of heterogeneous nanopar- ticle assembly. DNA-functionalized nano objects are mainly limited to gold nanoparticles. Materials coated with high affinity ligands or polymers, such as palladium nanoparticles coaled with PVP, or gold nanoparticles coated with PVP or PDDA, fail to be further functionalizable with biological molecules with the current state of the art. The range of nano objects successfully used for DNA-directed crystallization has been limited to gold nanoparticles. Although there are a few, limited reports of DNA functionalized nanoparticle other than gold, such as silver, quantum dots, silicon and iron oxides, these nanoparticles have never been exploited as nanoparticle building blocks that were subsequently used for the programmable assembly of 3D artificial materials. The structures of DNA-guided nanoparticle-nanoparticle assem- blies have so far been limited to body-centered cubic (BCC) and face-centered cubic (FCC) structures, which compro- mises novel structure-related properties and their advanced applications. Additionally, the cost of using thiolated DNA for gold nanoparticle functionalization is very high compared to using biotinylated DNA. SUMMARY [0011] The present disclosure describes a general strategy for DNA-mediated self-assembly of multicomponent func- tionalized nanoparticles into three-dimensional (3D) ordered superlattices. The generally applicable strategy either allows for removal of the high affinity ligands that bind to the nano- particle surface and their replacement with other ligands that do allow for subsequent functionalization with biological groups (mostly for hydrophilic nanoparticles), or provision of an additional ligand layer that allows for further functional- ization with biological groups (mostly for hydrophobic nano- particles), which can prevent irreversible and uncontrolled aggregation of nanoparticles while preserving their unique structures and physical properties. Such nanomaterials can then be applied in various programmable assembly strategies. [0012] The disclosure also demonstrates a generally appli- cable strategy of how to functionalize nanoparticles with DNA, independent of the composition of the material or the shape of the nanoparticles. The generally applicable strategy includes three steps, namely, carboxylic group grafting, Jun. 23, 2016 streptavidin (STV)-conjugation, and biotinylated-DNA attachment. In the first step, the ligands having a carboxylic group are adopted for the nanoparticles by replacing the origi- nal high affinity ligands or providing additional ligands with the carboxylic acid functional groups. In particular, short mercapto acid ligands, such as mercaptoundecanoic acid, and amphiphilic polymers, such as lipid-PEG carboxylic acid, may be used. [0013] The subsequent two steps rely on | -ethyl-3-[3-dim- ethylaminopropyl]-carbodiimide hydrochloride (EDC)-as- sisted chemistry and high specific and strong STV-biotin binding. This DNA functionalization strategy is very versatile and can be applied to a broad range of functional nanopar- ticles. In the EDC-assisted streptavidin (STV)-conjugation. the conjugate streptavidin can be covalently bound to the particle surface by a reaction between the carboxyl (COOH) group of the ligand and the amine (NH,) groups abundant on the streptavidin (STV) surface. Finally, biotinylated-DNA is coupled with STV on the particle surface due to the specific binding between biotin and STV. This strategy has been suc- cessfully demonstrated to assemble organized superstruc- tures with magnetic (Fe,O5), plasmonic (Au), photonic (quantum dot), and catalytic (Pd) materials, and protein (such as STV), as well as combinations thereof. Also demonstrated is that these ordered structures possess rich phases that until now could not be obtained using the current state of the art in nanomaterial assembly approaches. BRIEF DESCRIPTION OF THE DRAWINGS [0014] The following Figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these Figures in combination with the detailed description of specific embodi- ments presented herein. [0015] FIG. 1A shows SEM and TEM images for Pd nano- octahedra (NO). [0016] FIG. 1B shows SEM and TEM images for Pd nanocubes (NC). [0017] FIG. 1C shows SEM and TEM images for Pd nan- ododecahedra (ND). [0018] FIG.2A shows TEM images of Pd NCs with an edge size of 640.5 nm. [0019] | FIG.2BisaTEM image of Pd NCs with an edge size of 100.8 nm. [0020] FIG.2C isa TEM image of Pd NCs with an edge size of 1220.9 nm. [0021] FIG. 2D shows TEM images of Pd NCs with an edge size of 232.6 nm. [0022] FIG. 2E shows TEM images for Pd NOs with an edge size of 15+1.3 nm. [0023] FIG. 3 is a schematic illustration of the assembly system for direct hybridization of binary nanoparticles or nanoparticles and protein entities. [0024] FIG. 4A is a TEM image of thiol-DNA capped Au nanoparticles with a diameter of 6.2+1 nm. [0025] FIG. 4B is a TEM image of thiol-DNA capped Au nanoparticles with a diameter of 8.81.7 nm. [0026] FIG. 4C is a TEM image of thiol-DNA capped Au nanoparticles with a diameter of 12.5+1.8 nm. [0027] FIG. 4D is a TEM image of thiol-DNA capped Au nanoparticles with a diameter of 14.72 nm. [0028] FIG. 4E is a TEM image of streptavidin (STV)- capped Au nanoparticles with diameter of 16.6+1.5 nm.
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US 2016/0176988 Al [0029] FIG. 5A illustrates a 2D SAXS pattern and its cor- responding S(q) of the Sys-AA9, s. [0030] FIG. 5B illustrates a 2D SAXS pattern and its cor- responding S(q) of the Sys-AA9,,. [0031] FIG. 5C illustrates a 2D SAXS pattern and its cor- responding S(q) of the Sys-AA9<o. [0032] FIG. 5D illustrates a 2D SAXS pattern and its cor- responding S(q) of the Sys-AA9go. [0033] FIG. 5E illustrates a 2D SAXS pattern and its cor- responding S(q) of the Sys-AA6.o. [0034] FIG. 5F illustrates a 2D SAXS pattern and its cor- responding S(q) of the Sys-AA125,. [0035] FIG. 5G illustrates a 2D SAXS pattern and its cor- responding S(q) of the Sys-AA1555. [0036] FIG. 5H illustrates a 2D SAXS pattern of the melt- ing Sys-AA9,,. at 710° C., with the gray and black 1D curves corresponding to the scattering intensity of melting and assembled Sys-AA9<o, respectively. [0037] FIG. 51 illustrates fitting of the melting curve of Sys AAD so. [0038] FIG. SJillustrates a 2D SAXS pattern of the melting Sys-AA15,, and its fitting. [0039] FIG. 6A illustrates an exemplary schematic of the Cu,Au structure (bottom) and the calculated S(q) for this structure using Powder Cell in a two-atom system with an atom number ratio (AR) of 17. [0040] FIG. 6Billustrates the calculated S(q) for the Cu, Au structure with an AR of 5. [0041] FIG. 6C illustrates a schematic of the NaT] structure (bottom) and the calculated S(q) for this structure with an AR. of 17. [0042] FIG. 6D illustrates the calculated S(q) for the NaT1 structure with an AR of 5. [0043] FIG. 6E illustrates the calculated S(q) for the NaT1 structure with an AR of 2. [0044] FIG. 6F illustrates the calculated S(q) for the NaT1 structure with an AR of 1.5. [0045] FIG. 7A illustrates the 2D SAXS pattern and corre- sponding S(q) for the Sys-POA. [0046] FIG. 7B illustrates the 2D SAXS pattern and corre- sponding S(q) for the Sys-PCA. [0047] FIG. 7C illustrates the 2D SAXS pattern and corre- sponding S(q) for the Sys-PDA,, [0048] FIG. 7D illustrates the 2D SAXS pattern and corre- sponding S(q) for the Sys-PDA,,. [0049] FIG. 7E illustrates the 2D SAXS pattern and corre- sponding S(q) for the Sys-PDAso. [0050] FIG. 7F illustrates the 2D SAXS pattern and corre- sponding S(q) for the Sys-PDAgg. [0051] FIG. 7G illustrates the 2D SAXS pattern and corre- sponding Ip(q) for a system of Pd NDs and Au without a linker. [0052] FIG. 7H illustrates the 2D SAXS pattern and corre- sponding S(q) for Sys-PDA,, at 710° C. (black curve), and after cooling down (gray curve). [0053] FIG. 8A shows a TEM image of Q705, where the QD has elongated shape, and the size distribution histograms of long axis length and short axis length of the QDs in the image. [0054] FIG. 8B illustrates Ip(q), the fitting, and size distri- bution for Q705. [0055] FIG. 8C illustrates Ip(q), the fitting, and size distri- bution for Q605. Jun. 23, 2016 [0056] FIG. 8D illustrates Ip(q), the fitting, and size distri- bution for Q525. [0057] FIG. 9A illustrates the 2D SAXS pattern and corre- sponding S(q) for Sys-Q7A,, for n=15. [0058] FIG. 9B illustrates the 2D SAXS pattern and corre- sponding S(q) for Sys-Q7A,, for n=18. [0059] FIG. 9C illustrates the 2D SAXS pattern and corre- sponding S(q) for Sys-Q7A,, for n=30. [0060] FIG. 9D illustrates the 2D SAXS pattern and corre- sponding S(q) for Sys-Q7A,, for n=50. [0061] FIG. 9E illustrates the 2D SAXS pattern and corre- sponding S(q) for Sys-Q7A,, for n=80. [0062] FIG. 9F illustrates the 2D SAXS pattern and corre- sponding Ip(q) for a system of Q705 and Au without a linker. [0063] FIG. 10 illustrates the temperature-dependent phase behavior for Sys-Q7A,, without pre-annealing. [0064] FIG. 11A illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-Q7A,, with a mole ratio of QD:Au: Biotin DNA::1:1:10. [0065] FIG. 11B illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-Q7A3, with a mole ratio of QD: Au: Biotin-DNA::1:1:120. [0066] FIG. 11C illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-Q7A3o With a mole ratio of QD: Au: Biotin-DNA::1:2:80. [0067] FIG. 11D illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-Q7A3, with a mole ratio of QD: Au: Biotin-DNA::2:1:40. [0068] FIG. 11E illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-Q7A,, with a mole ratio of QD: Au: Biotin-DNA::10:1:20. [0069] FIG. 11F illustrates the calculated S(q) for a La,O, structure using Powder Cell in a two-atom system with atom number ratio (AR) labeled in the figure. [0070] FIG. 11G illustrates a schematic of the La,O, struc- ture. [0071] FIG. 12A illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-QA6,, with n=15. [0072] FIG. 12B illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-QA6,, with n=30. [0073] FIG. 12C illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-QA6,, with n=50. [0074] FIG. 12D illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-QA6,, with n=80. [0075] FIG. 12E illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-QA5 with n=15. [0076] FIG. 12F illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-QA5,, with n=30. [0077] FIG. 12G illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-QAS5,, with n=50. [0078] FIG. 12H illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-QAS,, with n=80. [0079] FIG. 13< illustrates the 2D SAXS pattern and cor- responding S(q) Sys-Q7A16,, at 260° C. [0080] FIG. 13B illustrates the 2D SAXS pattern and cor- responding S(q) Sys-Q7A16,9 at 530° C. [0081] FIG. 13C illustrates the 2D SAXS pattern, its Ip(q), and the fitting at 710° C. for the melting system. [0082] FIG. 14A illustrates the photoluminescence of Sys- QUA. [0083] FIG. 14B illustrates a plot of the quenching effi- ciency of Sys-Q7A against the surface-to-surface distance between the QD and Au obtained by SAXS. The solid line is a fitting using an exponential decay model.
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US 2016/0176988 Al [0084] FIG. 15A illustrates a TEM image of iron oxide Fe,0, (also refered to as IO or FeO) nanoparticles. [0085] FIG. 15B illustrates the SAXS Ip(q) and the fitting for the 1O nanoparticles, which indicate that they have spheri- cal shapes with diameters of 10.20.7 nm. [0086] FIG. 16A illustrates the 2D SAXS pattern and cor- responding 5(q) for Sys-IA,, with n=15. [0087] FIG. 16B illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-1A,, with n=30. [0088] FIG. 16C illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-1A,, with n=50. [0089] FIG. 16D illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-1A,, with n=80. [0090] FIG. 16F illustrates the 2D SAXS pattern and cor- responding S(q) for the mixture of STV-IO and Au particles without Biotin-DNA. [0091] FIG. 16F illustrates the 2D SAXS pattern for the mixture of STV-IO and Biotin-DNA without Au particles. [0092] FIG. 17A illustrates S(q) as a function of tempera- ture for Sys-IAjo. [0093] FIG. 17B illustrates S(q) as a function of tempera- ture for Sys-IA,,. [0094] FIG. 18A illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-IA3, with the mole ratio IO:Au: Biotin-DNA::1:1:7. [0095] FIG. 18B illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-IA,, with the mole ratio IO:Au: Biotin-DNA::1:1:60. [0096] FIG. 18C illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-IA,, with the mole ratio IO:Au: Biotin-DNA::1:5:75. [0097] FIG. 18D illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-IA3, with the mole ratio 1O:Au: Biotin-DNA::5:1:75. [0098] FIG. 19 is a schematic illustration of the assembly system for linker assisted hybridization of binary nanopar- ticles or nanoparticles and protein entities. [0099] FIG. 20A illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-IAL,,, for n=O. [0100] FIG. 20B illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-IAL,,, for n=30. [0101] FIG. 20C illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-IAL,,, for n=70. [0102] FIG. 20D illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-IAL,,, for n=170. [0103] FIG. 214A illustrates a schematic of and the calcu- lated S(q) for CsCl using Powder Cell in a two-atom system with an atom number ratio (AR) of 2.6. [0104] FIG. 21B illustrates a schematic of and the calcu- lated S(q) for a-ReO, using Powder Cell in a two-atom sys- tem with an atom number ratio (AR) of 2.6. [0105] FIG. 21C illustrates a schematic of and the calcu- lated S(q) for AuCu, using Powder Cell in a two-atom system with an atom number ratio (AR) of 2.6. [0106] FIG. 21D illustrates a schematic of and the calcu- lated S(q) for La,O, using Powder Cell in a two-atom system with an atom number ratio (AR) of 2.6. [0107] FIG. 21E illustrates a schematic of and the calcu- lated S(q) for NaT1 using Powder Cell in a two-atom system with an atom number ratio (AR) of 2.6. [0108] FIG. 21F illustrates a schematic of and the calcu- lated S(q) for NaCl using Powder Cell in a two-atom system with an atom number ratio (AR) of 2.6. Jun. 23, 2016 [0109] FIG. 21G illustrates a schematic of and the calcu- lated S(q) for ZnS using Powder Cell in a two-atom system with an atom number ratio (AR) of 2.6. [0110] FIG. 21H illustrates a schematic of and the calcu- lated S(q) for CaF, using Powder Cell in a two-atom system with an atom number ratio (AR) of 2.6. [0111] FIG. 22A illustrates the magnetic field-dependent 2D SAXS pattern and corresponding S(q) for Sys-IA3o. [0112] FIG. 22B illustrates the magnetic field-dependent 2D SAXS pattern and corresponding S(q) for Sys-IAL, 30, [0113] FIG. 23< illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-SA,,, for n=15. [0114] FIG. 23B illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-SA,,, for n=18. [0115] FIG. 23C illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-SA,,, for n=30. [0116] FIG. 23D illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-SA,, for n=50. [0117] FIG. 24< illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-Q77, for n=3 [0118] FIG. 24B illustrates the 2D SAXS pattern for Sys- Q77,, for n=15. [0119] FIG. 24C illustrates the 2D SAXS pattern for Sys- Q77, for n=30. [0120] FIG. 24D illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-Q75,,, for n=3. [0121] FIG. 24E illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-Q75,,, for n=30. [0122] FIG. 24F illustrates the 2D SAXS pattern and cor- responding S(q) for Sys-Q75,,, for n=50. [0123] FIG. 25A depicts the photoluminescence of Sys- Q77,, including the control system (a mixture of Q7 and Q7 without biotin-DNA), for the n=18, 30 and 50 systems. [0124] FIG. 25B illustrates the enhancement factor (EF) of Sys-Q77 against the surface-to-surface distance between Q7 and Q7. [0125] FIG. 25C illustrates the photoluminescence of Sys- Q75,, including the control system (a mixture of Q7 and Q5 without biotin-DNA), for the n=18, 30 and 50 systems. [0126] FIG. 25D illustrates the enhancement-to-quenching factor (EQF) of Sys-Q75 against the surface-to-surface dis- tance between Q7 and Q5. [0127] FIG. 26 illustrates the 2D SAXS patterns and cor- responding S(q) for Sys-QPD,, at different temperatures. [0128] FIG. 27 illustrates the photoluminescence of Sys- QPD. [0129] FIG. 28 is a schematic illustration of the three-step strategy for DNA functionalization of hydrophilic and hydro- phobic nanoparticles (f—denotes the number of grafting DNA on the nanoparticles). [0130] FIG. 29 are schematics and SEM images for bioti- nylated DNA-tethered palladium nano-cube (NC), octahe- dron (NO), and dodecahedron (ND) that were coated with PVP. [0131] FIG. 30 is a schematic, TEM image (inset is HRTEM), and hysteresis loop for biotinylated DNA-grafted JO nanoparticles originally capped by oleic acid. [0132] FIG. 31 is a schematic, TEM image (inset for HRTEM), and photoluminescence spectra for biotinylated DNA-attached CdSe/ZnS QDs (QD525, denoted by Q5, and QD605, denoted by Q6) and CdTe/ZnS QDs (Q705, denoted by Q7). TEM image is for Q7.
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US 2016/0176988 Al [0133] FIG. 32 is a schematic, TEM (for 10 nm Au nano- particles), and UV-Vis spectra for thiolated DN-functional- ized Au nanoparticles, including 10, 15, 20 nm, originally capped by citrate. [0134] FIG. 33< illustrates plots of shape-dependent struc- ture factors (S(q)) extracted from SAXS patterns of direct hybridization (DH) systems with short DNA. [0135] FIG. 33B illustrates in the top portion the Au nano- particle size-dependent S(q) evolution of ND-Au DH sys- tems, including PD hybridized with 15 nm and 20 nm Au. [0136] FIG. 33C illustrates the effects of nanoparticle shape on the correlation length (€) of binary systems assembled by shaped and spherical NPs. [0137] FIG. 33D is a plot showing the nearest neighbor particle surface-to-surface distance (D,,), as illustrated by inset, for ND-10 nm Au systems. [0138] FIG. 34A illustrates plots of shape-dependent struc- ture factors (S(q)) extracted from SAXS patterns of DH sys- tems for Fe,O, (denoted as FeO in figures) and Au nanopar- ticles. (1): S(q) for non-specific interaction induced Fe,0, aggregates. (2): A DNA base number (N)-dependent evolu- tion of S(q) from the single component Phase-F to a DNA- directed Au-IO binary superlattice upon introducing Au nanoparticles, tethering DNA direct complementary to that on IO surface, into Sys_FeO; (3) S(q) for a DH system assembled by STV and Au nanoparticles with longer and shorter DNA. [0139] FIG. 34B is a 3D schematic illustration for structure switch between Phase_F and Phase_D via introducing Au nanoparticles or elevating temperature. [0140] FIG. 34C shows the assembly kinetics for Phase-F and Phase-D. The inset is a 2D schematic for phase-D. [0141] FIG. 34D isa plot ofthe D,, for IO-Au direct hybrid- ization systems and the a calculated from geometrical con- sideration based on the D,, values as a function of N. Inset illustrates the definition of D,, and @ in the Au-IO supperlat- tice. [0142] FIG. 34E shows the experimental configuration for SXAS measurement in a magnetic field (top) and the S(q) magnetic response (bottom) of the IO-Au direct hybridization systems. [0143] FIG. 354A is a plot showing component-dependent S(q) evolution of DH systems for QD-Au nanoparticles. [0144] FIG. 35B shows the DNA-spacer length dependent S(q) evolution of Q7-Au systems (top) and S(q) of a well ordered Q7-Au system, which involves both flexible and rigid DNA regions (bottom). [0145] FIG. 35C is a plot showing the change of composi- tional order parameter (ny) and correlation length (§) with DNA base number (N) for DH Q7-Au systems. The inset sketches the compositional order-to-disorder transition with 1 from | to 0 ina CsCl lattice formed in the binary Au and QD systems. [0146] FIG. 35D isa plot of Dss for QD-Au DH systems. [0147] FIG. 35E is a plot of steady-state and time-resolved PL spectra collected from Q7-Au direct hybridization sys- tems. [0148] FIG. 35F illustrates a sketch ofa CsC1 lattice formed by Q7 and QS directed by DNA. FIG. 35F also shows a plot of the lifetime (t) for donor (Q5) and acceptor (Q7) in the free-dispersed states and superlattice Q7_Q5, 5. [0149] FIG. 36A is a phase diagram for the heterogeneous binary ~10 nm nanoparticle systems. Jun. 23, 2016 [0150] FIG. 36B is a diagram showing an example (N=30, DH systems) for the predictable interparticle center-to-center distances (D...) for heterogeneous binary systems. DETAILED DESCRIPTION [0151] In accordance with aspects of the present invention, applicants have developed a general strategy for multi-com- ponent DNA-guided three-dimensional (3D) assembly of functional nanoparticles. The disclosure demonstrates a gen- erally applicable strategy of how to functionalize nanopar- ticles with DNA, independent of the composition of the mate- rial or the shape of the nanoparticles. The disclosure further demonstrates a programmable assembly of the DNA-func- tionalized nanoparticles into predefined multi-dimensional and multi-component, such as, but not limited to, magnetic (Fe,O, and other magnetic materials), plasmonic (Au and other metals), photonic (quantum dot, QD), and catalytic (Pd, Pt, and others) materials, and protein (such as STV) struc- tures. Described herein is a general strategy for DNA-medi- ated self-assembly of multicomponent functionalized nano- particles into three-dimensional (3D) ordered superlattices. Also described are exemplary embodiments of DNA-medi- ated heterogeneous assemblies of nanoparticles including new phases of known nanoparticle assemblies. (A) DNA Functionalization [0152] The generally applicable strategy either allows for removal of the high affinity ligands that bind to the nanopar- ticle surface and their replacement with other ligands that do allow for subsequent functionalization with biological groups (mostly for hydrophilic nanoparticles), or provision of an additional ligand layer that allows for further functionaliza- tion with biological groups (mostly for hydrophobic nanopar- ticles), which can prevent irreversible and uncontrolled aggregation of nanoparticles while preserving their unique structures and physical properties. Such nanomaterials can then be applied in various programmable assembly strategies. [0153] The general strategy for multi-component DNA- guided 3D assembly of functional nanoparticles is described herein. First, a generally applicable strategy of how to func- tionalize nanoparticles with DNA, independent of the com- position of the material or the shape of the nanoparticles, will be described. For DNA functionalization, there is provided a facile method for the synthesis of non-commercially avail- able nanoparticles with uniform size and shape. In a second step, either the original high affinity ligands are replaced by or additional ligands are provided with carboxylic acid func- tional groups. In a third step, 1-ethyl-3-[3-dimethylamino- propyl]-carbodiimide hydrochloride (EDC)-assisted chemis- try is adapted to covalently conjugate streptavidin onto the particle surface due to the reaction between the carboxylic (COOH) groups of the ligands and the primary amine (NH,) groups that are abundant on the STV surface. Finally, bioti- nylated-DNA is coupled with STV on the particle surface due to the strong and specific affinity of biotin to STV. This functionalization strategy is very versatile and robust. Certain examples demonstrate how to assemble organized super- structures with iron oxide (IO; such as magnetic Fe,0;), plasmonic (Au), photonic (QD), and catalytic (Pd) materials, and protein (STV), as well as combinations thereof. Also demonstrated is that these ordered structures possess rich phases that until now could not be obtained using the current state of the art in nanomaterial assembly approaches.
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US 2016/0176988 Al [0154] The methods of the present disclosure provide vari- ous examples to illustrate the general process of the invention for nanoparticle synthesis and subsequently DNA function- alization. Depending on the capping agent used for their synthesis, the nanoparticles can be divided into two classes, namely, hydrophilic and hydrophobic. For hydrophilic nano- particles, the initial step is to first replace the original ligand by mercapto acid (MA), e.g., mercaptoundecanoic acid, and thereafter to conjugate it with STV, and then finally couple it with biotinylated-DNA. For hydrophobic nanoparticles, the initial step is to either replace the original ligands or provide additional ligands. In one embodiment, the initial step is to treat the nanoparticles with one or more amphiphilic poly- mers, such as lipid-PEG carboxylic acid, followed br a con- jugation with STV and coupling with biotinylated-DNA. The general procedure is shown in FIG. 28. [0155] To demonstrate the universal applicability of this strategy with respect to the hydrophilic nanoparticles, palla- dium nanoparticles with different shapes are used as examples. Palladium nanoparticles are important for hydro- genation catalysis. To demonstrate the universal applicability of this strategy with respect to the hydrophobic nanoparticles, the iron oxide (IO) capped with oleic acid (OA), and quantum dots (QD) capped with trioctylphosphine (TOPO) nanopar- ticles are used as examples. Iron oxide is a typical magnetic material and QD can be used as highly efficient luminescent nanocrystals. (B) Assembly of 3D Ordered Structure by Multi-Component Functional Nanoparticles [0156] Once nanoparticles are successfully encoded with DNA, it is possible to either hybridize DNA-encoded nano- particles or nanoparticles and proteins, independent of the particle’s component, size, or shape, into 3D aggregations due to the specific interaction of DNA. The 3D ordered phases can be obtained by carefully controlling the interplay of inter- particle attraction and repulsion energies, which can be experimentally achieved in a variety of ways, such as by controlling DNA sequence length, number and structure of DNA molecules, and DNA structure hybridization tempera- ture, [0157] FIGS. 3 and 19 show a schematic illustration of an assembly system for direct hybridization (DH) and linker hybridization (LH) of binary nanoparticles, or nanoparticles and proteins, respectively. In a DII assembly system, nano- particles can be functionalized with DNA that has two func- tional parts. One is non-complementary and forms the inter- nal spacer part, which is designed to tune the repulsive interaction between particles, and the other is complemen- tary, forming the outer recognition sequence part, and which provides the attraction interaction for nanoparticle assembly. The spacer part on particle A (B) can be designed as X, (X;) poly T bases and is denoted X,-b (X,-b) spacer in FIG. 3. The total base number (N) is defined as X.,+X-b. Alterna- tively, in a LH assembly system, nanoparticles can be func- tionalized with DNA that has two functional parts, but neither one complementary to provide the attraction interaction for nanoparticle assembly. However, while the outer spacer regions are non-complementary to each other, they are complementary to the respective base ends ofa ssDNA linker, which has a central flexible part (base number denoted by L,,-b) separating the two ends. N is defined as X ,+X,+L,,-b in LH systems. Generally, DH systems reveal quicker assem- bly kinetics in comparison with LH systems involving similar Jun. 23, 2016 DNA length. While the LH strategy proves more flexible for system design, for example, regulation of the interparticle distance can be achieved by simple tuning linker base number without changing grafting DNA types. [0158] The following examples are included to demon- strate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor(s) to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the scope of the invention. EXAMPLES Example 1 Synthesis of Palladium Nanoparticles with Controlled Shapes (Octahedral, Cubic, and Dodecahedral) and Size [0159] Palladium (Pd) nanoparticles were synthesized in an aqueous solution by a modifying the procedure described in Lim et al. (2009). In the original reported procedure, only Pd nanoparticles with cubic shape were obtained. Here, two new shapes (octahedral and dodecahedral) were obtained by either changing the KBr concentration or by using potassium iodide (KI), which was an important modification of the reported procedure. [0160] Water soluble inorganic Pd salts, such as Na,PdCL, or K,PdCl,, were used as a palladium source. Poly-vinyl- pyrrolidone (PVP) (having a typical molecule weight (M.W.) ranging from ~30,000 to 100,000) was used both as reluctant and surfactant. Alkali metal bromides or iodides, such as NaBr, KBr, Nal, and KI, were used as shape-controlling agents. Bromides were used for the synthesis of nano-octa- hedrons (NOs), nanocubes (NCs), and nanododecahedrons (NDs), while iodides were used for the synthesis of dodeca- hedrons. Ina typical synthesis procedure, a mixture of Pd salt and alkali metal halide was first heated to about 80-100° C. with a standard reflux system and kept at that temperature for about 30 minutes. Then a pre-heated PVP solution was injected into the mixture solution. The reaction was allowed to continue for about 3-5 hours. For the synthesis of Pd NOs, the mole ratio between Pd salt, bromide, and PVP was approximately 1: (3-30):3-8) for temperatures around 80-90° C. and approximately 1:(3-15):(3-8) for temperatures around. 90-100° C. For the synthesis of Pd NCs, the mole ratio between Pd salt, bromide, and PVP was about 1:(15-30):(3-8) for temperatures around 90-100° C. For the synthesis of Pd NDs, if bromide was used in the reaction, the mole ratio between Pd salt, bromide, and PVP was about 1:(30-60):(3-8) for temperatures around 80-100° C. The Pd NDs may also be obtained by introducing trace iodide to the reaction. [0161] The mole ratio of Pd salt to bromide, iodide, and PVP can be around 1:(3-60):(0.01-0.1):(3-8) and reaction temperatures can be around 80-100° C. For the above three synthesis reactions, Pd salt concentration typically ranges between about 10 mmol/l to about 30 mmol/l. After the reac- tion, the nanoparticle products were collected by centrifuga- tion, and then purified by washing once with acetone and subsequently three times with ethanol or water. The as-ob-
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US 2016/0176988 Al tained nanoparticles can be well dispersed in ethanol or water. The Pd nanoparticles obtained by such methods are uniform in shape with no more than 15% unexpected shape, and also have a narrow size distribution (<10%). The yield of nano- particle for NOs, NCs, and NDs are about 70%, 50%, and 40%, respectively, calculated from the transformation of Pd from salt form to nanoparticle form. [0162] By regulating the synthesis parameters, such as reactant ratio and concentration, temperature, and reaction time, one can control the edge size of NOs, NCs, and NDs ranging from about 6 to 13 nm. Generally, higher tempera- ture, lower halide concentration, and longer reaction duration will produce bigger nanoparticles. FIGS. 1A, 1B, and 1C show scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images for the prepared Pd NOs, NCs, and NDs, respectively. The edge size of Pd NOs, NCs, and NDs are 8.6+0.8 nm, 10+] nm, and 9+0.9 nm, respec- tively. The synthesis parameters for Pd nanoparticles shown in FIG. 1 are as follows: for NOs, [Na,PdC1,]-58 mM, mole ratio Na,PdCl,:KBr:PVP (M.W. ~50,000)=1:20:5, tempera- ture ~80° C., and the reaction time is about 3 h; for NCs, [Na,PdCL,]=58 mM, mole ratio Na,PdCL:KBr:PVP (M.W. ~50,000)=1:20:5, temperature ~100° C., and the reaction time is about 3 h; for NDs, [Na,PdCl,]=58 mM, mole ratio Na,PdCl,:KBr:PVP (M.W. ~50,000)=1:50:5, temperature =100° C., and the reaction time is about 3 h. [0163] To grow larger palladium (Pd) nanoparticles, a seed- mediated method was developed wherein small sized nano- particles are used as seeds and Pd (0) is reduced and deposited onto the surface of the seeds. Generally, using such a method one can predictably produce Pd nanoparticles with good con- trol of shape as well as with precise size control, even at the nm level. The nanoparticle shape mainly depends on the ratio of Pd salt to bromide or iodide, and such ratios for synthesis of larger Pd NOs, NCs, and NDs are roughly the same as that described above for the synthesis of the corresponding nano- particles. The nanoparticle sizes depend on the ratio between seeds and Pd salt, and a higher ratio of Pd salt will produce bigger nanoparticles. For instance, one can use small sized NCs as seeds to grow big sized NOs, NCs, and NDs. FIG. 2A shows Pd NCs with an edge size of 6+0.5 nm, for which the synthesis parameters are set as [Na,PdCl,]=58 mM, mole ratio Na,PdCl,:KBr:PVP (M.W. ~50,000)=1:20:5, tempera- ture=100° C. Using such NCs as seeds, bigger NCs with edge sizes of 10+0.8 mm (FIG. 2B), 12+0.9 nm (FIG. 2C), 2342.6 nm (FIG. 2D), and bigger NOs with an edge size of 15+1.3 nm (FIG. 2E) can be obtained. [0164] The synthesis parameters for Pd nanoparticles shown in FIGS. 2A through 2E are as follows: for NCs, the growth solution is that [Na,PdCL.J=l1 mM, mole ratio Na,PdCl,:KBr:PVP_ (M.W. ~50,000)=1:20:5, tempera- ture=100° C., the reaction time is 3 h, and the mole ratio between 6 nm NCs seeds and Na,PdCl, is 10° for 10nm NCs, 2:10° for 10 nm NCs, and. 2:10° for 23 nm NCs; for NOs, the growth solution is that [Na,PdCL,J=1 mM, mole ratio Na,PdCl,:;KBr:PVP (M.W. ~50,000)=1:10:5, tempera- ture=80° C., the reaction time is 3 h, and the mole ratio between 6 nm NCs seeds and Na,PdC1, is 1.5°10° for 15 nm NOs. Example 2 Functionalization of Pd Nanoparticles with Mercapto Acid by a Ligand-Exchange Process [0165] The PVP cap on the surface of Pd nanoparticles, including NOs, NCs, and NDs, can be replaced with mercapto Jun. 23, 2016 acid by a ligand-exchange process. The carbon number of alkane can range between about 2 to 18, but a longer carbon chain length may be better for stabilizing the nanoparticles. The thiol group number in MA can be one, two, or more. The typical ligand-exchange process can be described in three steps. First, the pH value of the freshly prepared PVP-capped Pd nanoparticles in aqueous solution was adjusted to about 6-9 by buffer, which contains about 0.01% to 1% (by volume) surfactant. The buffer can be phosphate buffer, borate buffer, etc., and the pH value can range between about 6-9. The surfactant can be Tween (such as Tween 20), Triton (such as Triton 100), sodium dodecyl sulfate (SDS) and so on. Mer- capto acid (MA) in ethanol, for instance 11-mercaptounde- canoic acid (MUA), is mixed with the above solution. [0166] The mole ratio of mereapto acid can be about 10° to 10’ times to that of nanoparticles depending on the surface area of nanoparticle, e.g., for Pd nanocubes with an edge size of 10 nm the ratio can be about 2:10°. In the second step, the above mixture was incubated at about 50-90° C, for about 3 to 12 hours after brief sonication for about 20 minutes to 1 hour. Finally, the as-functionalized nanoparticles were purified by a centrifugation-wash cycle procedure, where the particles are washed two times with ethanol and three times with the above buffer with surfactant. Such a functionalization proce- dure produces MA-capped Pd nanoparticles which are well dispersed in buffer or aqueous solution. This functionaliza- tion method is robust and can also be applied for hydrophilic materials other than Pd and other surfactants than PVP. The materials can be gold, silver, platinum, and so on. The original surfactant can be very broad and their charge can be varied from negative charge, such as citrate, positive charge, such as cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), poly-diallyl-dimethylammonium chloride (PDDA), to neutral charge, such as Pluronic P-123, Car- boxymethy! Cellulose Sodium (CMC). Example 3 Conjugation of Pd Nanoparticles with STV [0167] The as-prepared MA-capped Pd nanoparticles (or other component nanoparticles) can be conjugated with STV by formation of an amide bond between carboxylic groups on the nanoparticles, provided by the ligand, and primary amine groups of STV through 1-ethyl-3-[3-dimethylaminopropy]]- carbodiimide hydrochloride (EDC)-assisted chemistry. Typi- cally, MA-capped Pd nanoparticles in buffer with pH about 6-8 are first mixed with freshly prepared EDC (about 0.1 mg/ml to 1 mg/ml), N-hydroxysulfosuccinimide (NHS, about 0.1 mg/ml to 1 mg/ml) and STV. The quantity of STV can be about 10 to 100 times that of the Pd nanoparticles. The mix- ture is allowed to incubate either at room temperature for about 1 to 4 hours or at 4° C. for about 6 to 12 hours. Finally, the nanoparticles are collected by a centrifugation-wash cycle procedure, where the particles can be washed three times by water or the above motioned surfactant-containing buffer. After purification, the nanoparticles are dispersed in surfac- tant-containing buffer. Example 4 Functionalization of Pd Nanoparticles with Biotinylated-DNA [0168] The as-prepared STV-capped Pd nanoparticles (or other component nanoparticles) were coupled with biotiny-
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US 2016/0176988 Al lated-DNA because of the strong and specific affinity of biotin to STV. The DNA sequence from 5' to 3' of the recognition part on A has a sequence TAC TTC CAA TCC AAT [SEQ 1] and is complementary to the sequences on B, which is ATT GGA TTG GAA GTA [SEQ 2] from 5' to 3'. The system was denominated as Sys-Ajp,,,.,,X4B 5,2. where the subscript D, and D, or E, and E, denote the diameter or emission wavelength (for QD) of particle A and B, respectively. [0169] The STV-capped nanoparticles were mixed with biotinylated-DNA, which amounts can be used to control DNA number on the particle surface, and the mixture was allowed to incubate for several hours at room temperature. Finally, the nanoparticles were collected by a centrifugation- wash cycle procedure, where the particles were washed three times by water or the above-mentioned surfactant-containing buffer. After purification, the nanoparticles were dispersed in surfactant-containing buffer. [0170] The three kinds of Pd nanoparticles had uniform shape and size and displayed the similar volume correspond- ing to about 11 nm spherical particles as illustrated in FIG. 29. The attached DNA number (f) was typically 15-25. Example 5 Synthesis of IO and QD Particles [0171] The synthesis of iron oxide (IO; e.g., Fe,O,) nano- particles with sizes from about 4 to about 16 nm followed procedures published by Hyeon, T., et al. Vournal of the American Chemical Society, 2001. 123(51): p. 12798-12801; incorporated herein by reference). Synthesis of quantum dots (QDs) with emission wavelengths of 400 nm to 780 nm fol- lowed procedures published in Dabbousi (1997) and Med- intz, I. L., et al., (Nature Materials, 2005. 4(6): p. 435-446, incorporated herein by reference). Example 6 Functionalization of IO and QD with Carboxylic Acid Groups [0172] For the first method, iron oxide (Fe,O3) nanopar- ticles or quantum dots (QDs) dispersed in an organic solvent, such as toluene or chloroform, were first mixed with MA (usually 3-mercaptopropionic acid (MPA)) in ethanol or methanol solvent. Then the mixture was heated at about 50° C. to 70° C, for about 4 to 12 hours after brief sonication for about 5 to 30 minutes. Finally, the nanoparticles were col- lected by a centrifugation-wash cycle procedure, where the particles can be washed three times by water or the above mentioned surfactant-containing buffer. After purification, the nanoparticles were dispersed in surfactant-containing buffer. This is similar to the procedure for QD published by Kang, S. H., et al., (Applied Physics Letters, 2008. 93(19): p. 191116-1 to -3, which is incorporated herein by reference in its entirety). [0173] Forthe second method, Fe,O or QD dispersed in an organic solvent, such as toluene or chloroform, were first mixed with amphiphilic polymers, such as poly(maleic anhy- dride alt-1-tetradecene), lipid-PEG carboxylic acid, which have hydrophobic chains interacting with ligands on the nanoparticles and carboxylic acid groups for further function- alization. Then the mixture was incubated for about 2 to 4 hours at room temperature. After complete evaporation of the organic solvent, the residual solid was purified by a centrifu- gation-wash cycle procedure, where the particles are washed Jun. 23, 2016 three times by water or buffer with pH about 7 to 9, such as borate, TBE. After purification, the nanoparticles were dis- persed in water or buffer. A similar procedure has been reported by Pellegrino, T., et al. (Nano Letters, 2004. 4(4): p. 703-707; incorporated herein by reference in its entirety). Example 7 Conjugation with STV and Biotinylated-DNA. [0174] These two steps are nearly the same as those described above for Pd nanoparticles. Although STV has been used to functionalize Fe,O,, (Lee 2006 and Shen, T. T., et al., Bioconjugate Chemistry, 1996. 7(3): p. 311-316, which is incorporated herein by reference in its entirety) and QD (Glazer, A. N., Bioconjugate techniques—Hermanson, G T. Nature, 1996. 381(6580): p. 290-290, which is incorporated herein by reference in its entirety), the resultant nanoparticles have not undergone 3D crystallization. [0175] Using the above procedure, uniform ~10 nm (diam- eter) spherical nanoparticles of Fe.0, capped with oleic acid (OA) having superparamagnetic properties were synthesized as illustrated in FIG. 30. Hydrophobic commercially-avail- able QD capped with trioctylphosphine oxide (TOPO) of three different emission peaks (Am) centered at 525 (core- shell CdSe/ZnS), 605 (core-shell CdSe/ZnS), and 705 nm (core-shell CdTe/ZnS) were also synthesized using the above procedure as illustrated in FIG. 31. All the particles showed a slightly elongated shape and the hard-core particle size was about 2~3, 4~6, and 6~7 nm. Citrate-capped Au nanoparticles of three different size (~10, 15, 20 nm) were also synthesized with dense thiol-DNA as shown in FIG. 32. The attached DNA number (f) on 16.8-nm Au, QD, and 10-nm IO nano- particles is about 20 (45~60 for 10 nm Au), about 20-40, and 3-8, respectively. Example 8 Particle Assembly [0176] For particle assembly, a defined ratio of particles A and B was mixed in 10 mM phosphate buffer with 0.14 M NaCl, pH=7.1 at room temperature. The particles were allowed to assemble into aggregates for from several minutes to days, depending on the particle concentration. Subse- quently the precipitates were split into two parts, one for melting temperature measurements and the other, after trans- ferring into a capillary, for structure measurement. The melt- ing temperature was determined using UV-Vis spectroscopy, monitoring the change in absorbance at the nanoparticles’ predominant absorption peak. The structure of the assembly was analyzed by synchrotron-based small-angle X-ray scat- tering (SAXS), which was performed at the National Syn- chrotron Light Source X-9A beam line. If not specifically mentioned, the samples in the capillary were annealed at a temperature several (about 1 to 5) degrees below their melting temperature for ten minutes to several hours and then slowly cooled down to room temperature for several hours before SAXS measurements. [0177] For SAXS data analysis, the scattering data were collected witha MAR CCD area detector and converted to 1D scattering intensity vs. wave vector transfer, q=(42v/.) sin(6/ 2), where . and 6 are the wavelength of incident X-ray and the scattering angle, respectively. The structure factor S(q) was calculated as I,(q)/1,(q), where 1,(q) and I, (q) are background corrected 1D scattering intensities extracted by angular aver-
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US 2016/0176988 Al aging of CCD images for assembled systems and un-aggre- gated particles, respectively. The peak positions in S(q) are determined by fitting to the Lorentzian equation. [0178] To analyze the structure of the assembly, the peak position ratio (Qx/Q1) from the structure factor as well as the relative peak intensity are initially used to propose possible structure models, and then such proposed models are com- pared with first peak positions (q1) to calculate the nearest neighbor particle center-to-center distances (D,.M) in the assembly, and finally the most probable model is obtained by comparing the D..M and the distances (D.C) calculated in real space from the designed system configuration. Example 9 Au and Au with Different Sizes [0179] Thiolated DNA-capped Au nanoparticles (TA) (functionalization methods can be found in the reports of Nykypanchuk 2008 and Park 2008) and biotinylated DNA- capped Au nanoparticles (SA) (the functionalization method is similar to that of Pd nanoparticles) are used as particle models to illustrate the phase behavior for the hybrid system composed of Au nanoparticles with different sizes and sur- face chemistries. FIGS. 4A, 4B, 4C, and 4D show TEM images for the four kinds of TA with corresponding diameters of 6.2+1 nm, 8.8+1.7 nm, 12.5+1.8 nm, and 14.7+2 nm, each of which was used to hybridize with DNA-biotin-STV capped Au (SA) with diameter of 16.6+1.5 nm (FIG. 4E). For the hybrid system 9-nm TAu with 16.8-nm SAu, fourkinds of internal spacer sets (Xa-Xb), namely, 15-3, 15-15, 35-35, and 65-65, were used, and the systems were denominated as Sys- AAQ,, and n=18, 30, 50, and 80, respectively. For the hybrid systems 6-nm, 12.5-nm, and 15-nm TA with 16.8 nm SA, all the internal spacer sets (Xa-Xb) were designated as 35-35, and the systems were accordingly nominated as Sys-AA650, Sys-AA12,o, and Sys-AA15,,. [0180] FIGS. 5A, 5B, SC, 5D, SE. SF, and 5G show the 2D SAXS pattern and corresponding S(q) of the Sys-AA9,s, Sys-AA939, Sys-AA9<5, Sys-AA9go, Sys-AA65o, Sys- AAI2s0, and Sys-AA15so, respectively. Here, S(q)=1,(q)/1, (q); the melting system was used for I,(q). Interestingly, the first peak in Sys-AA9 and Sys-AA6, which contain particles of big size difference, has weaker intensity compared with the second one; while for Sys-AA12 and Sys-AA15 the first peaks are always the strongest ones, which is the same as the reported results of single component systems (Nykypanchuk 2008 and Park 2008). By fitting the 1D scattering curves from the melting system, the size distribution of the particles in the system can be obtained. [0181] FIG. 5H shows the 2D SAXS pattern of the melting Sys-AA9,, and the gray and black 1D curves correspond to the integrated scattering intensity of melting and assembled Sys-AA9,., respectively. The fitting of the melting curve (FIG. 51) using the Irena 2 macros package gives two particle size distributions with diameters of about 9 nm and about 16 nm, which confirms that the system was assembled by two different sizes of nanoparticles. FIG. 5J shows the 2D SAXS pattern of the melting Sys-AA15., and the fitting of the melt- ing curve, which indicates the single size distribution due to the similar size of the two particles in this system. [0182] To analyze the assembled structure, first consider Sys-AA12 and Sys-AA15, where Q,/Q,#1:V3:V7 and such ratios correspond to a body-centered cubic (BCC) structure. The BCC structure is expected for the hybrid system with two Jun. 23, 2016 types of Au nanoparticles of similar size, and the result is in coincidence with the reports of Nykypanchuk 2008 and Park 2008. For Sys-AA9,, and Sys-AA6<o, all the systems have similar structures and Sys-AA9.,. was used to analyze their structure. In Sys-AA9<o, Q/Q, -1:1.71:3.0:4.1:4.95:6.0; interestingly, Q./Q.=1:1.75:2.19:2.89:3.5. Considering the two ratios, the structure is similar to a type of face-centered cubic (FCC) with Q,/Q, as 1:1.63:2.31:2.83:3.41 from dif- fraction planes (111), (220), (400), (422), (531), while the first extinction peak of (100) also appears. [0183] Two possible structure models, similar to the crys- talline organizations of either Cu,Au or NaTI, are proposed for the system. The Cu,Au phase corresponds to the Pm 3 m space group with group number 221, and Cu sits in 3c sites, and Au sits in the 1a site. (See the schematic in FIG. 6A.) The NaTI phase corresponds to the Fd3 m space group with group number 227, and Na sits in 8a sites, and TI sits in 8b sites. (See the schematic in FIG. 6C.) The scattering ability (Is) of the Au particles used is then calculated. The Is can be roughly esti- mated as Ban rdlS ge pl Gen edger Va tal [Gotu Fer outer) "Va, eo) and o,=p*Z/M,,, where o, is the electron density of the par- ticles, p is the material density, Z is the material atomic or molecular electrons, and M,, is the material atomic or molecular weight. The IS ,,..).6/IS.4,..6 and IS 4,,.6/IS4,,.9 Were calculated as 295 and 31, respectively. [0184] We then use an atomic system to calculate the S(q) using software PowderCell, where Cu;Au and NaTI struc- tures containing two atoms with an atom number ratio of 17 (~7295, resembling Sys-AA6) and 5 (~V31, resembling Sys- AA9) were used. The same lattice constant was used to cal- culate the S(q). The calculated S(q) for Cu,Au structure are shown in FIGS. 6A and 6B, which correspond to atom num- ber ratios (AR) of 17 and 5 (heavy atom at Ja site), respec- tively. FIGS. 6C and 6D accordingly correspond the calcu- lated S(q) for NaTI structure with AR of 17 and 5 (heavy atom at 8a site). NaTI fits well with the experimental results. When the particles on the Na and TI sites are the same, the NaT1 structure degenerates to a BCC structure. FIGS. 6E and 6F show the calculated S(q) with AR of 2 and 1.5, corresponding, to Sys-AA12 and Sys-AA15, respectively. The calculated results show that S(q) changes into a BCC structure with the decrease of the particle size difference. Therefore, the results suggest that all these systems, no matter the size difference, are actually in a NaTI structure. This structure has very recently been reported for the assembly system of Au nano- particles and protein particles (QB phage capsid), where the two particles have the same size (Cigler, P., et al., Nature Materials, 2010. 9(11): p. 918-922, which is incorporated herein by reference in its entirety). [0185] With the proposed NaT1 structure, D,.M in the assembly can be calculated using Q,. For Sys-AA9,5, Sys- AA9 59, Sys-AAI 50, SyS-AAY9 59, S¥S-AAG5, Sys-AA12, and Sys-AA15, Q, are correspondingly 0.0177, 0.0168, 0.0144, 0.0114, 0.0141, 0.0216, and 0.0204 A’. For Sys-AA12 and Sys-AA15, D..M=v6*2/Q, since the first peak comes from (220) in NaTI structure, and the values are 35.6 and 37.7 nm, respectively. For Sys-AA6 and Sys-AA9, D,,M=1.5*2/Q, since the first peak comes from (111) in NaT] structure, and the values are 26.6, 28.1, 32.7, 41.3, and 33.4 nm for Sys- AAD. s, Sys-AA939, Sys-AA9so, Sys-AAIg,, and Sys- AA6s50, respectively.
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US 2016/0176988 Al [0186] To validate the proposed model, D,,, was estimated using the following methods. For the configuration shown in FIG. 3, DCR HD cast Dep tp AX Ap Mig No where R,, and R, correspond to the radius of particles A and. B, T,.4.5 and T,z., correspond to the characteristic length of X,- and X,-base ssDNA tethered on particles, and A is the DNA shrinkage length due to hybridization (roughly related to the X tethered base), X,,, is the hybridized base, and N, the DNA coverage on the particles. Here, R,=4.5 nm and R,=12.9 nm (considering STV has a diameter of 4.5 nm). Then T was estimated by the Daoud-Cotton blob model and the parameters used are: persistent length (1,) as 1 nm; salt concentration (C,) as 0.14 M; and the DNA number (N,,) on. 6, 9, 12, 15, and 16.8 nm Au are 30, 65, 70, 100, and 20, respectively. A for different X ,-X, sets was obtained from a known BCC structure assembled by all 9-nm Au nanopar- ticles, and we obtained A=3.8, 6.9, and 7.3 for 15-15, 35-35, and 65-65 X ,-X,, sets, respectively. Using the above model, the calculated D..C=25.8, 27.4, 31.1, 39.6, 30.6, 35.1, and 36.8 for Sys-AA9,., Sys-AA9,, Sys-AA9.,, Sys-AA9 9, Sys-AA6;5, Sys-AA12 and Sys-AAIS5, respectively. The consistent with D,.M, which confirms the proposed NaT1 structure. [0187] These results indicate that all the Au—Au systems where at least one of the two particles is coated with STV, no matter the size difference, actually do have a NaTI structure. This is the first time that this kind of structure has been reported for bioassembled inorganic materials. This finding is unexpected, as previous assemblies of Au particles with thi- olated DNA were always reported to form a CsCl structure (Nykypanchuk 2008 and Park 2008), which however, cannot be distinguished from the NaTl structure when using Au particles with same sizes for the assembly. Example 10 Au and Catalytic Pd [0188] Thiolated DNA-capped Au nanoparticles and bioti- nylated DNA-capped Pd nanoparticles of different shapes were used as particle models to illustrate phase behavior for the hybrid system of Au and Pd nanoparticles. Each type of Pd nanoparticle, including NOs, NCs, and NDs shown in FIGS. 1A through 1C, was used to hybridize with 9-nm Au nano- particles to form Sys-POA, Sys-PCA, and Sys-PDA, respec- tively. For both Sys-POA and Sys-PCA, the X_,-X, sets were designed as 3-15. For Sys-PDA, the X,-X, sets were designed as 3-15, 15-15, 35-35, and 65-65, and the systems were nominated as Sys-PDAs, with n=18, 30, 50, and 80, respectively. [0189] FIGS. 7A through 7F show the 2D SAXS pattern and corresponding S(q) of Sys-POA, Sys-PCA, Sys-PDA,,, Sys-PDA4o, Sys-PDAso, and Sys-PDAgo, respectively. Here, S(q)=1.(@/1,(q). 1, (q) was obtained from the control system, which is the mixture of STV-capped Pd nanoparticles and thiol-DNA-capped Au nanoparticles without biotinylated- DNA. FIG. 7G gives the 2D SAXS pattern and corresponding 1,(q) for an example control system, Pd NDs and Au (PDA- C). The control system does not show any diffraction patterns, which indicates that the STV-Pd nanoparticles are stable and verify the DNA-mediation role for the assembly as well. Such Pd—Au systems show different phase behaviors with tem- perature from the reported Au—Au systems and the above- Jun. 23, 2016 described Au—Au with different size systems, where the assembly becomes dissociated at the melting temperature (M7) and show no diffraction peaks for SAXS. However, the Pd—aAu systems still show several peaks even at tens of degrees higher than M; determined by UV measurements. For example, FIG. 7H gives the 2D SAXS pattern and corre- sponding S(q) (black curve) for Sys-PDA,, at 71° C., which shows two peaks centered at 0.0387 and 0.0795 Av. Inter- estingly, when the system was cooled down, these two peaks would disappear and S(q) be restored back to the original state, as evidenced by the gray curve in FIG. 7H. [0190] The structures of the Pd—Au system can be deter- mined by using similar structure analysis methods as described for the Au—Au system. All the Pd—Au systems could have similar structures due to their similar structure factors as shown in FIGS. 7A through 7F. Taking Sys-PDA,, for example, Q/Q)=1:1.8:2.64:3.49, and such values resemble the peak position ratios, which are 1:1.73:2.65:3.46, of diffraction planes (110), (211), (321), and (422) to (110) of a BCC structure. The Is of the Pd and Au particles is then calculated. B41 pa UGosu-Cermper)*V aul (CePa-Sebugo)* Val and the Is,,,/Isp, was calculated as 0.63. According to their similar scattering ability, the BCC structure can either be CsCl or NaTI, but these two structures are impossible to distinguish as stated for Sys-AA. [0191] Using the proposed CsCl or NaTI structure, D,.M for the Pd—Au system are calculated using Q,. For systems shown in FIGS. 7A through 7F, the Q,’s are correspondingly 0.0340, 0.0330, 0.0330, 0.0303, 0.0248, and 0.0198 A~!, and using D,.M=v6*z/Q,, the D,.M are correspondingly 22.6, 23.3, 23.3, 25.4, 30.9, and 38.9 nm. For the calculation of D.C, the following parameters were used: R.,=4.5 nm for Au nanoparticle and R,=10, 11, 11 nm (including STV) for NO, NC, ND, respectively, DNA number on Pd nanoparticle=20; and the other parameters are the same as that used for Au—Au system. For the above systems, the calculated D,,C=23.2, 24.0, 24.0, 25.8, 29.6, and 38.3 nm, which agrees with the corresponding D,.M. Not wishing to be bound by the theory, it appears that although CsCl and NaTI structures can’t be distinguished in the present systems, a NaTI structure is more reasonable for such STV-capped Pd and thiol-Au system considering the NaTI structure for STV-capped Au and thiol- Au system. [0192] For the melted Sys-PDA,, system (FIG. 7II), D,.M is 16.2 nm using D,.M=2*a/Q,, since this system resembles an amorphous structure. This value approaches that of the distance (18.5 nm) between two DNA-capped PD particles. Therefore, the structure of the melted system actually comes from the aggregation of PD particles. Not wishing to be bound by the theory, it may happen that with the dissociation of the hybrid DNA at the melting temperature the Au particles release from the assembly due to the high repulsive interac- tion related to high DNA coverage, while the Pd particles in the assembly collapse together possible reasons including depletion interaction from DNA-Au particle, weak magnetic interaction from Pd particles themselves, weak repulsive interaction related to low DNA coverage, or high local con- centration of Pd particles. However, this kind of Pd particle aggregation is reversible and the Pd particles can re-hybridize with Au particles to form the CsCl or NaTI structure. [0193] The CsCl (or NaTI) structure is expected for the system assembled from two types of spherical nanoparticle
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US 2016/0176988 Al with similar size, as evident by reports of Nykypanchuk 2008 and Park 2008 and the above-described Au—Au system. However, herein, three kinds of Pd polyhedrons are used to hybridize with spherical Au, so other structures than BCC, such as simple cubic (SC) for NC-Au and FCC for ND-Au, are expected due to the anisotropic shape effect. The only observed BCC phase possibly resulted from the actual loss of the anisotropic property of Pd polyhedrons because of their thicker capping soft molecular layers (typically 7 to 16 nm) in comparison with their hard core size (typically 4 to 6 nm). Such Pd—Au systems may find important applications in the catalysis area because of the good catalytic properties of Pd nanoparticles, unique plasmonic-related properties of Au nanoparticles and the quite open framework of the assembled structure. [0194] FIG.33A (top) shows the structure factor S(q) (sym- bols) extracted from SAXS patterns for three DH systems, accordingly corresponding to 10 nm Au hybridized with NC for NC Au,_,;, with NO for NO Au, |, and with ND for ND Aug 15. The three systems show similar structures, including similar first peak positions (q,) accordingly centered at 0.0339, 0.0333, and 0.0345 A~’; however, their correlation length (&) depends on the particle shape and increases for shape being more spherical-like, as shown in FIG. 33C, For example, § increases from 37 nm to 46 and 52 nm when Pd nanoparticles change from NC to NO and ND. To verify the universality of this shape effect, a spherical Au—Au system was built, Au_Au,; ,; with similar DNA design. The S(q) is given in FIG. 33A (bottom), which indeed shows a larger € of 60 nm as well as a similar structure as Pd—Au systems. The nature of the driving force for the Pd—Au systems was exam- ined. A control system was created by a mixture of Au and Pd. nanoparticles but lacking the recognition of DNA sequences. No aggregates formed in this system, which indicates that the STV-Pd nanoparticles are stable and also verifies the DNA- mediation role for the assembly. [0195] DNA flexibility is necessary for the crystallization of DNA-Au Nanoparticles. The design of DNA witha certain length was found to really facilitate the ordering of shaped Pd—Au and spherical Au—Au, although the spherical sys- tems can attain more profound ordered states than shaped systems. Take dodecahedron Pd and Au system for example with N from 45 to 145 in direct hybridization and N from 60 to 130 in linker hybridization systems. It was found that q, shifted to small values with increasing the N indicating the increase of the interparticle distances. At the same time, § increased from 56 nm to 124 nm and then decreased to 91 nm with N increased from 45 to 130 (ND_Au,,,, with S(q) displayed in the middle panel of FIG. 33B) and 145. In con- trast, the spherical Au—Au reach a well ordered state for N as 90 with § of 310 nm, and this system is denoted by Au_Au,, 39, which S(q) is displayed in the bottom panel of FIG. 33B. [0196] Using the CsC1 lattice, the experimental S(q) can be fitted well, especially for Au_Au,3o, as shown by black solid lines in FIGS. 33A and 33B. Due to the similar form factors (AP(q)) for 10 nm Au and 11 nm Pd nanoparticles, such binary CsCl lattices actually show BCC patterns with first peak from (110), which is the same as single component Au systems. One way to confirm this type of lattice is to adjust the AP(q), and thus a SC pattern with (100) as first peak will display. Other systems comprising components with distinct form factors were also constructed. Au size was increased from 10 nm to 15 nm and 20 nm while keeping ND nanopar- ticle size unchanged. FIG. 33B (top) shows S(q) for two Jun. 23, 2016 representative systems for ND with 15 nm Au nanoparticles and ND with 20 nm Au nanoparticles. In comparison with systems for ND and 10 nm Au, a weak peak with q centered at 1/21/72 of the original first peak gradually emerges with increase Au nanoparticle size. This peak was assigned as (100) peak from a SC structure. Therefore, above all, the Pd and Au Nanoparticles formed a CsC] superlattice, as sche- matically shown in FIG. 33C (insert). Distinct from the expected NaCl or FCC-like phase, the only observed CsCl lattice could be resulted from the effective shape transforma- tion from anisotropic to isotropic shape due to the thick cap- ping soft molecular layers. The spherical-like particle favors such shape transformation, and thus favors the CsCl lattices because they are the stable structures for spherical binary DNA nanoparticles. [0197] Based on the CsCl structure, the nearest neighbor particle surface-to-surface distance (D,,) for ND-Au systems was calculated. FIG. 33D summarizes the D,, for the ND and 10nm Au systems, which displays a range from ~12 to 30 nm. While the interparticle distances can be regulated by the change of ionic strength, it can also be achieved by varying the DNA length. The established DNA structure medel allows us to predict the D,,. A Daoud-Cotton (DC) blob model anda worm-like chain (WLC) model were used to calculate the tethered DNA thickness and linker length, respectively. FIG. 33D shows that the model distances agree well with the experimental data, especially for systems with shorter length DNA, and the accuracy is limited in ~12%. Such Pd—Au systems could be attractive for optical and catalytic-related studies due to the intrinsic merits of Pd and Au nanoparticles, possible energy transfer between them, and the quite open framework of the assembled structure. Example 11 Au and Fluorescent QD [0198] First take 9.0-nm thiolated DNA-capped Au nano- particles and biotinylated DNA-capped QD with an emission wavelength centered at 705 nm (denoted Q7) as an example to illustrate the phase behavior for the hybrid system of Au and QD nanoparticles. The systems were obtained by mixing DNA-Au with biotin-DNA first and then with STV-QD, and the mole ratio of QD to Au and biotin-DNA is 1:1:40. The size and shape of the QD was characterized by TEM and SAXS. FIG. 8A shows the TEM images of Q7, where the QD has an elongated shape. The size distribution histogram of Q7 gives the long axis length and short axis length as 14+2.5 nm and 621.5 nm, respectively. FIG. 8B shows the 1,(q), the fitting, and size distribution for Q7. The fitting gives two size distri- butions, 1321.5 nm and 6.1™1.1 nm, which accordingly corresponds to the long and short axis of Q7. For this hybrid system, the X_,-X, sets were designated as 0-15, 3-15, 15-15, 35-35, and 65-65, and the systems were denominated as Sys- Q7A,, with n=15, 18, 30, 50, and 80, respectively. [0199] FIGS. 9A through 9E give the 2D SAXS pattern and corresponding S(q) for Sys-Q7A,,, and the images 9A through 9E corresponds to n=15, 18, 30, 50, and 80, respectively. Here, S(q)-1,(@)/,(q), 1,(q) was obtained from either the control system or the melting system. The S(q) shows that the first peak intensity is weaker than the second one for all the system, and becomes much weaker for longer DNA spacer. The control system is the mixture of STV-capped QD and Thiol-DNA capped Au nanoparticles without biotinylated- DNA. FIG. 9F gives the 2D SAXS pattern and its correspond-
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US 2016/0176988 Al ing L(@) for a control system of Q7 and Au (Q7A-C), which does not show any diffraction patterns. [0200] To investigate the temperature-dependent phase behavior, Sys-Q7A3, without pre-annealing was selected as an instance and the results are shown in FIG. 10. The as- assembled system without annealing does not show a long- range ordered structure, as demonstrated by the broad rings in 2D pattern and the broad and few diffraction peaks of the structure factor. This Sys-Q7A,, can be crystallized by annealing at 48° C. (about 10° C. below the M,) for about 20 min. Upon further increasing temperature to 59° C., no struc- ture was found, which means Q7 and Au nanoparticles can be re-dispersed in the solution after DNA de-hybridization. To investigate the thermal stability of STV on the QD surface, the system was further heated to 75° C. and kept at tempera- ture for | hour. After cooling down to 26° C., the system again showed crystallization, which indicates the high thermal sta- bility of capped STV. Such phase behavior is similar to the reported Au—Au systems and the above-described systems of Au—Au with different particle sizes. [0201] The effects of biotin-DNA number (N) and particle ratio on the assembly phase behavior were also investigated. First the biotin-DNA number was changed while the mole ratio of QD to Au was maintained at 1:1. FIGS. 11A and 11B show the 2D SAXS pattern and corresponding S(q) for Sys- Q7Agp With the mole ratio of biotin-DNA to Q7 (Au) as N=10 and 120, respectively. Compared with the structure factors of systems for N=10, 40 (FIG. 9C), and 120, one can conclude that a certain amount of biotin-DNA, at least ten times the Au particle amount, is required for good crystallization, but excess biotin-DNA seems not to frustrate the crystallization of the QD-Au system. Then the particle ratio was altered and the ratio of QD:Au:biotin-DNA was set at 1:2:80, 2:1:40, 10:1:20. After assembly, there were no visible particles in the supernatant for any of the above systems except the system with QD: Au:biotin-DNA as 10:1:20, which contains QD as easily observed by a UV lamp. The 2D SAXS patterns and S(q) corresponding to the above three systems are shown in FIGS. 11C and 11D. It was found that all the systems (pre- annealed) show similar structure factors and the particle ratios in this studied range don’t have important effects on the structure. Therefore, as long as the QD and Au systems crys- tallize after annealing, they actually show the same structure, which may be in a global energy minimum state and thus independent of the initial state and assembly pathway. [0202] To analyze the assembly structure, the peak position ratios were calculated. Since all the systems show the same structure, Sys-Q7A,, was taken as the example. For this sys- tem, Q./Q,=1:1.36:2:2.63:3.19:3.9:4.8, and such values resemble the peak position ratios, which are 1:1.41:2:2.65:3. 19:3.87:4.79, of diffraction planes (110), (200), (220), (321), (420), (521), (611) to (110) of a BCC structure. The relative scattering ability of Au to Q7 (Is,,/Isg;) was calculated as ~18. Supposing the assembly has a CsCI structure, the calcu- lated results by the method used for Pd—Au system show that this system displays its intrinsic SC diffraction pattern. The relative peak intensity of (100) to (110) is about 0.4, and so the first diffraction peak should be (100) and the peaks with Q/Q, at 2.63, 3.9, and 4.8 should never appear, which con- tradicted the observed results. Therefore, either QD or Au should pack in a BCC structure, and the other one sit on some sites of this BCC frame. [0203] Considering the relative intensity of (110) and (200) (utoyT(200)), a BCC with a sub-SC structure is proposed, Jun. 23, 2016 where one kind of particle sits on BCC sites and combines with another type of particle to form an SC subunit. Such structure is a cubic La,O,-like structure (the high temperature X-phase described in Aldebert, P., et al., (Journal De Phy- sique, 1979, 40(10): p. 1005-1012, which is incorporated herein by reference in its entirety), which corresponds to the Im 3 m space group with group number 229. [0204] In this prototype structure, La sits in 2a sites, and O is randomly distributed over the 6b sites with a 50% probabil- ity that any one site is occupied. For an A-B particle system with this structure, and suppose particle A sits on 2a sites and B on 6b sites, the Is ,/Isy-dependent diffraction behavior is calculated by PowderCell and the results are shown in FIG. 11F, where the two numbers correspond to A and B atom number and the square of their ratio is roughly equal to Is ,/Is. When Is,,>>Isg, this structure shows a BCC diffrac- tion pattern and the first peak is from (110) and has the strongest intensity. The 1,,;oyIjooq) decreases with the decrease of Is ,/Isz; when Is ,=Isz, this structure shows a SC diffraction pattern and I,, 19) becomes 0 while 299) becomes strongest. With the further decrease of Is ,/Is;, I(, 19) increases and T.10)/T,200) becomes ~0.4 when Is <<Isz. According to the calculated results and Is ,,/Isg;, the Sys-Q7A corresponds to the case 80:20 in FIG. 11F, and Au and QD particles are on 2a and 6b sites, respectively. The Iq 10)/T2o0) of Sys-Q7A for short DNA spacers agrees with the calculated results, while this value decreases with spacer length and deviates from the calculated results. This spacer length-dependent intensity change may be related to the decreased correlation length with the increase of spacer length, which leads to diffraction (200) from subunit lattice stronger but (110) from unit lattice weaker. The proposed structure is also shown in FIG. 11F, where the QD was proposed to link two Au particles through its short axis direction since it can maximize the hybrid DNA number in this way. [0205] Using the proposed La,O, structure, D,.M for Sys- Q7Aare calculated using Q,. For systems shown in FIGS. 9A. through 9F, Q, are correspondingly 0.0230, 0.0223, 0.0201, 0.0152, and 0.0116 A~', and using D,.M=¥2*7/Q, , the D,.M are correspondingly 19.3, 19.9, 22.1, 29.2, and 38.3 nm. For the calculation of D.C, the following parameters were used: R,,=4.5 nm, Ro75=7.5 nm (including STV), DNA number on Q7-20, and the other parameters are the same as that used for the Au—Au system. The calculated D,.C for the above sys- tems is accordingly as 20.5, 21.1, 23.2, 27.5, and 36.3 nm, which agrees with the D..M. [0206] Two other kinds of QD with emission wavelengths centered at 605 nm (Q6) and 525 nm (Q5) were used to hybridize with Au nanoparticles. FIGS. 8C and 8D show the 1,(q), the fitting, and the size distribution for Q6 and QS, respectively. The fitting results show that 06 and Q5 also have elongated shape, and the long and short axis length are 1] nm and 5 nm for Q6, and 7 nm and 3 nm for QS. Q6 and Q5 were accordingly hybridized with 9-nm Au nanoparticles to form Sys-QA6 and Sys-QAS, and the X_,-Xz, sets were designated as 0-15, 15-15, 35-35, and 65-65, and the systems were denominated as Sys-QA6,, and Sys-QAS,, and n=15, 30, 50, and 80, respectively. FIGS. 12A through 12H give the 2D SAXS pattern and corresponding S(q) for Sys-QA6, and Sys- QAS,, and the images in FIGS. 12A through 12D and 12E through 12H correspond ton=15, 30, 50, and 80, respectively. All the systems were proposed to be La,O; structure due to
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US 2016/0176988 Al their similar S(q) to Sys-Q7A. With the decrease of QD size for QD-Au system, the Iq 1oy/I(200) increases, which agrees with he calculated S(q). [0207] The Sys-QA6 and Sys-QA5 also have spacer Jength-dependent intensity change behavior. Q, was used to calculate the DM. For Sys-QA6, Q, are 0.0238, 0.0209, 0.0182, and 0.01490 A~!, corresponding to n=15, 30, 50, and 80, respectively, and D,,.M are correspondingly 18.6, 21.2, 24.3, and 29.6 nm. For Sys-QA5, Q, are 0.0245, 0.0205, 0.0187, and 0.0138 A~', corresponding to n=15, 30, 50, and 80, respectively, and D..M are correspondingly 18.1, 21.6, 23.7, and 32.1 nm. For the calculation of D.C, the following parameters were used: R ,,=4.5 nm; Ry,s=6.5 nm (including STV); Rgss=5.5 nm (including STV); DNA number on Q6 or Q5 is 20; and the other parameters are the same as that used for the Au—Au system. The calculated D.C for Sys-QA6,, are 19,8, 22.5, 26.6, and 35.6 nm for n=15, 30, 50 and 80, and for Sys-QA5 are 19.1, 21.9, 26.2, and 35.3 nm for n=15, 30, 50 and 80, respectively. D.C agrees with the D..M, espe- cially for the short DNA spacer case. [0208] The Au size effect on the Au-QD assembly structure was also investigated. Here, a system was constructed by assembling 16.6-nm STV-Au and Q7 and the X,-X,, sets were designed as 15-15 and the system was denoted as Sys- Q7A16;,. FIGS. 134 and 13B show the 2D SAXS pattern and corresponding S(q) at 26° C. and 53° C., and FIG. 13C shows the 2D SAXS pattern, its 1,(q), and the fitting at 71°C. for the melting system. The Q/Q, for this system at 53°C. is 1:1.73:2.38:3.2:3.92, and can be assigned to a BCC structure, which corresponds the case ~80:1 in FIG. 11F. So this system also has a cubic La,O,-like structure. [0209] Therefore, these results show that all the hybrid systems of QD and Au have cubic La,O,-like structures, and this is the first time that this kind of structure has been reported for bioassembled materials. Without wishing to be bound by the theory, it is considered that the elongated shape of QD is important for the formation of this novel structure, and we predict that other nanoparticles with similar shapes might result in the creation of similar assemblies. [0210] The photoluminescence properties of the Au-QD systems were also measured. Take Sys-Q7A for the example. FIG. 14A shows the photoluminescence of Sys-Q7A,, including the control system, with n=15, 30, 50, and 80 sys- tems. It can be seen that the Au-QD systems show a distance- dependent fluorescence quenching behavior. The quenching efficiency (QE) of Sys-Q7A against the surface-to-surface distance between the QD and Au is given in FIG. 14B. The decay curve can be fitted by an exponential decay model where QE=QE, ‘exp (-d/d,) (see, e.g., Zheng, W. M. and L. He, Journal of Physical Chemistry C, 2010. 114(41): p. 17829-17835, which is incorporated herein by reference in its entirety), where QE, is the quenching efficiency when QD were directly attached to the Au surface, dy is the distance constant within which fluorescence quenching occurs, and d the separating distance between the QD and Au surfaces. The fitting yielded an equation of QE=1.14-exp (-d/15.3), and the fitted do agrees with the experimental values obtained by Zheng (2010). Example 12 Au and Magnetic Iron Oxide Particles [0211] About 9 nm thiolated DNA-capped Au nanopar- ticles and biotinylated DNA-capped IO nanoparticles were Jun. 23, 2016 used as particle models to illustrate phase behavior for the hybrid system of Au and IO nanoparticles. The size and shape of the JO were characterized by TEM and SAXS. FIG. 15A. shows the TEM image, FIG. 15B the SAXS 1,(q) and FIG. 15C the fitting of 1, which indicate that the IO have spherical shapes with diameters of 10.2+0.7 nm. For the Au and IO hybrid system, the ratio of IO to Au and biotin-DNA was set as 1:1:15, and the X,,-X,, sets were designed as 0-15, 15-15, 35-35, and 65-65, and the systems were nominated as Sys- JA,,, with n=15, 30, 50, and 80, respectively. [0212] FIGS. 16A through 16D give the 2D SAXS pattern and corresponding S(q) for Sys-IA,,, and the images in FIGS. 16A through 16D correspond to n=15, 30, 50, and 80, respec- tively. Here, S(q)=1,(qV/I,(q), 1,(q) was obtained from the melting system of Sys-1A3). Two control systems (ICA-I and TAC-II) were designed, and IAC-I is the mixture of STV-IO and Au particles without biotin-DNA, and IAC-II is the mix- ture of STV-IO and biotin-DNA without Au particles. FIGS. 16E and 16F give the 2D SAXS pattern and corresponding S(q) for IAC-I and IAC-II, respectively. Both the control systems have aggregations and show similar S(q) and three peaks near about 0.033, 0.059, 0.102 A~!, which indicates that this structure actually comes from JO-IO aggregation. Such aggregation may be induced by the depletion attraction by biotin-DNA or DNA-Au particles because STV-IO doesn’t form aggregates in solution as shown in FIG. 15A. Interestingly. these three peaks from IO disappear for short DNA spacers, such as n=15 and 30, but not for long DNA spacers like n=50 and 80. This result indicates that the DNA with shorter spacer has higher hybrid energy and thus can break the 1O-IO aggregation to form IO-Au aggregation. Therefore, the Sys-IA consists of only IO-Au aggregation for short DNA spacers (n=15, 30), but IO-IO and IO-Au aggre- gation for long DNA spacers (n=50, 80). [0213] The temperature-dependent phase behavior of Sys- IO was investigated. We found that Sys-IO showed different behavior, which depended on the spacer length. FIGS. 174 and 17B show the S(q) as a function of temperature for Sys-1A,. and Sys-IAso, respectively. For Sys-IA39, when this system is heated to 55° C., all the diffraction peaks disappear, which means IO and Au nanoparticles in this system can be re-dispersed in the solution after DNA de-hybridization. After cooling down the system, IO-Au hybrid structures form again, but it will take a long time (days) to form better struc- tures. It should be noted that if the system is kept in the melting state (55° C.) for hours, the IO-IO aggregation will form and the aggregation has a structure similar to that shown in FIGS. 16E and 16F. But such IO-IO aggregation will even- tually convert back into a 1O-Au structure when the system cooled down. However, for Sys-IA,,, when this system is melted, only its first original peak disappears and the other peaks with Q at ~0.033, 0.059, 0.102 A-', which are the same peaks in 1O-IO aggregation, still exist. This result again shows that Sys-IA;, actually consists of two kinds of aggre- gations, namely, [0-IO and Au-IO, and also indicates that the kinds of 10-10 interaction are not related to the specific DNA hybridization. [0214] The effects of biotin-DNA number (N) and particle ratio on the assembly phase behavior were investigated. First the biotin-DNA number was changed while the mole ratio of IO to Au was kept at 1:1. FIGS. 184 and 18B show the 2D SAXS pattern and corresponding S(q) for Sys-IA3, with the mole ratio of biotin-DNA to IO (Au) as N=7 and 60, respec- tively. Compared with the structure factors of systems for
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US 2016/0176988 Al N=15 (FIG. 16A) and the control system, one can conclude that an appropriate amount of biotin-DNA is required to break down the IO-IO aggregation and to form the IO and Au assembly. In the case of too little biotin-DNA it doesn’t pro- vide enough driving force for 1O and Au assembly; while for too much biotin-DNA it may introduce excess depletion attraction that makes IO-IO aggregation uneasily broken as well. Then the particle ratio was changed and IO:Au:biotin- DNA was set to 1:5:75 and 5:1:75. The 2D SAXS patterns and S(q) corresponding to the two systems are given in FIGS. 17C and 17D, and the results indicate that less IO than Au is not favorable for IO-Au assembly. This may be caused by the blocking of the IO surface DNA by excess Au particles due to the lower DNA coverage on IO (~10 DNA on 10-nm IO). [0215] A linker DNA was used to assemble STV-IO capped with biotin-DNA and Au particle capped with thiol-DNA. A linker system was designed as illustrated in FIG. 19, and the L,, spacer was designed as different number of poly T and the system was nominated as Sys-[AL,,. FIGS. 20A through 20D. show the 2D SAXS pattern and corresponding S(q) for Sys- TAL,,, and the images in FIGS. 20A through 20D correspond to n=0, 30, 70, and 170, respectively. Compared with the 10-10 structure, the Sys-IAL shows peaks all coming from JO-Au aggregation for short DNA linkers (n-0), and shows peaks both coming from IO-IO and JO-Au for long DNA linkers (n=30, 70, 170). The linker system also shows similar temperature-dependent phase behavior to the direct hybrid system, namely, peaks from IO-Au rather than 1O-IO aggre- gation disappear above melting temperature. [0216] To analyze the assembly structure, the peak position ratios were calculated. Only systems displaying Au-IO peaks, such as Sys-IA,;, Sys-1A3o, and Sys-LALo, were used to calculate Q,/Q,, and the ratio obtained was 1:1.7~1.8:2.3~2. 5. A similar ratio was also obtained from other systems if the peaks from IO-IO were subtracted. According to the Q,/Q,, the structure may be SC with Q/Q, as 1:1.73:2.45 from diffraction planes (100), (111), (211), or BCC with Q/Q, as 1:1.73:2.45 from diffraction planes (110), (211), (222), or FCC with Q/Q, as 1:1.63:2.31:2.52 from diffraction planes (111), (220), (400), (331). For a binary SC system, the struc- ture model can be CsCl, a-ReO3, or AuCu; fora binary BCC system, the structure model can be La,O,; for a binary FCC system, the structure model can be NaTl, NaCl, ZnS (zincblende), or CaF,. The Is4,/Is7o is calculated as 2.6 for Sys-Au-IO, and the proposed structure for CsCl, a-ReO,, AuCu;, La,O;, NaTl, NaCl, ZnS and CaF, with the corre- sponding calculated S(q) is accordingly shown in FIGS. 21A. through 21H, respectively. The models, including CsCl with Q, from (110), AuCu, with Q, from (111), NaTI withQ, from (220), and ZnS with Q, from (111), seem possible in com- parison of their relative peak intensity with experimental results. The D,.M can be calculated as V6*/Q,, V6*/Q,, V6*W/Q,, and 1.5*2/Q,, for CsCl, AuCu,, NaTl, and ZnS, respectively. For Sys-IA,,, Q, are 0.0246, 0.023, 0.018, and 0.0138 Av, corresponding to n=15, 30, 50, and 80, respec- tively, and D..M are correspondingly 31.3, 33.5, 42.8, and $5.8 nm for the CsCl, AuCu,, and NaT] models, and 19.2, 20.5, 26.2, and 34.1 nm for the ZnS model. For Sys-IAL,,, Q; are 0.0213, 0.0177, 0.0156, and 0.015 A~!, corresponding to n=O, 30, 70, and 170, respectively, and D.,.M are correspond- ingly 36.1, 43.5, 49.3, and 51.3 nm for the CsCl, AuCus, and NaTI models, and 22.1, 26.6, 30.2, and 31.4 nm for the ZnS model. Jun. 23, 2016 [0217] To calculate the D.C for Sys-IA,, the following parameters were used: R.,,, 4.5 nm; R,9=9.5 nm (including STV); DNA number on JO=10; and the other parameters are the same as that used for the Au—Au system. The calculated D.C for the Sys-LA,, is 21.7, 23.7, 27.2, and 35.4 nm, corre- sponding to n=15, 30, 50, and 80, respectively. D.C agrees with the D,.M of ZnS model. Therefore, our results show that all the hybrid systems of IO and Au have zincblende struc- tures, and this is the first time that this kind of structure has been reported for bioassembled materials. [0218] The magnetic field (B) effects on the phase behavior of the IO-Au hybrid systems were measured. Take Sys-IA39 (a system having only IO and Au aggregation) and sys- 1AL130 (a system containing a mixture of IO and Au aggre- gation and IO and 10 aggregation system) for examples to illustrate such B effects. FIGS. 22A and 22B accordingly show the magnetic field-dependent 2D SAXS pattern and corresponding S(q) for Sys-IA,, and Sys-IAL, 3. For Sys- TA3o with the increase of B, the third diffraction peak (331) of ZnS structure first disappears, and then the second peak (220) disappears, and finally only the first peak (111) survives at the highest B in this study. Interestingly, both the second and third peaks appear again after the removal of B, which indicates the B-dependent phase behavior is reversible. The sys-[AL, 3 also shows a reversible B-dependent phase behavior. With the increase of B, the first peak (111) from IO-Au aggregation disappears, while the other peaks from IO-IO aggregation remain nearly constant. Such a change law of S(q) with B is similar to that of S(q) with T. For the above two systems, the first peak position nearly remains constant but its width becomes much broader with the increase of B, which indi- cates that the mean particle distance remains unchanged but the particles’ position fluctuation increases. Such position fluctuation with B is related to the DNA stiffness. In compari- son with Sys-IA,o, Sys-IAL,39 is easier to subject to this fluctuation due to its longer and more flexible DNA linker, and this leads to the loss of the first peak of Sys IAL, 3, while not of Sys-IA3, at the same B. Example 13 Au and Magnetic Iron Oxide Particles [0219] In these systems, besides the DNA specific interac- tions between IO and Au, there are remarkable non-specific interactions, such as weak magnetic attraction and van der Waals interaction related to the limited DNA number, between IO nanoparticles. The assembly rules should be dif- ferent from the Au—Au and Au—Pd systems. Moreover, a route with controllable interplay between the specific and non-specific interactions is promising for switchable struc- tures. It was found that DNA-capped IO were ready to form aggregates. The S(q) is given in FIG. 34A (1), which corre- sponds to asystem, denoted by Sys-FeO, containing IO nano- particles capped with one type of 30-base biotinylated-DNA. The spectrum, denoted by Phase-F, shows two broad peaks centered at 0.033 and 0.059 A~", respectively. This phase was assigned as a weak-ordered FCC structure, as indicated by the fit shown as black line in FIG. 34A (1). The Phase-F was triggered by the non-specific interactions, as evidenced by the temperature-dependent study, which displayed an absence of thermal dissociation process for such aggregates. [0220] Interestingly, these non-specific interaction induced aggregates can switch into a binary component superlattice directed by DNA hybridization. FIG. 34A (2) shows a DNA
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US 2016/0176988 Al length-dependent structure evolution of Phase-F by introduc- ing direct complementary Au nanoparticles. In these direct hybridization systems, with N decrease from 145 to 85, 45, and 30, a new phase emerges accompanied by the consump- tion of the initial Phase-F. However, distinct from Phase-F, this new phase revealed a thermally reversible dissociation- association behavior, indicating that it was indeed a DNA- driven assembly by IO and Au nanoparticles. This new phase was denoted by Phase-D. The longer spacer systems for N=145 (FeO_Augs ¢5) and 70 (FeO_Au,; 35) show a mix- ture phase-F and D, while shorter ones for N=45 (FeO_Au,, 15)and30(FeO_Au, ,;) show a pure phase-D as well as with improved structure order. This behavior is distinct from Pd—Au systems and Au—Au systems, where longer DNA favors better structure. This DNA-length dependent structure evolution seems universal for IO-Au systems and is also observed in the linker hybridization systems, which demon- strates that the longer linkers (N=90, 130, 190, and 230) produce a mixture phase of JO and IA, and shorter linker (N=60) gives the pure phase-D. Moreover, this binary IO and Au phase can transform back into IO phase by decreasing the DNA attraction forces. For example, upon keeping the Phase-D at T,, (typically ~55° C.) for hours rather than cool- ing directly to room temperature, the Phase-F will form although this phase can eventually transformed into pure Phase-D if cooling down this system. Therefore, a switchable phase transition between different-component phases can be realized with the regulation of interplay between non-specific and specific interactions. [0221] To further elaborate the DNA length effects on the phase switch behavior, the assembly kinetics of the two phases were analyzed upon introducing complementary Au nanoparticles into Phase-F. Two representative systems were investigated, including FeO_Au,, ,; for assembly of pure Phase-FA and FeO_Au,; 3; for assembly of mixed Phase-F and -D. Based on the time-dependent development of SAXS patterns, the § of the two phases was derived and plotted in FIG. 34C. In comparison with longer spacer system, the shorter one demonstrates a short time-scale for the develop- ment of Phase-D and a complete elapse of Phase-F at ~40 h. The slower kinetics and smaller € for longer spacer system might be caused by the lower penetration capability into the Phase-F due to the higher entropic penalty, their lower effec- tive DNA hybridization concentration, and the higher posi- tional fluctuation due to more soft repulsive potentials. [0222] Structural analysis suggested Phase-D with an Au nanoparticles-based FCC structure, where only Au nanopar- ticles show positional order. Compared with other types of possible lattices, such as CsCl, such FCC structure gives the best fit, as given by black solid line in FIG. 34A (2) for FeO_Auy ,;- Due to the limited number of STV on the IO nanoparticles surface, the investigation of systems compris- ing Au and STV is helpful to understand the Phase-D struc- ture. Two systems, namely STV_Au,; ,; and STV_Auy 5 were constructed, and their S(q) are given in FIG. 34A (3). Interestingly, the STV_Au,; ;; shows similar S(q) as phase- D, and meanwhile the similar fit quality was exhibited using the Au nanoparticles-based FCC structure. Different from FeO_Auy ,5, the STV_Au, ,; could achieve a highly ordered state, which can be well fitted by such FCC structure. Above all, it’s reasonable to such structure as a FCC for Au nano- particles, which are surrounded by STV for STV-Au or IO for JO-Au as linker shells. The 3D and 2D schematics are illus- trated FIGS. 34B and 34C, respectively. Jun. 23, 2016 [0223] Based on this FCC structure, the D,, for IO-Au DH systems was calculated and plotted as black spherical sym- bols in FIG. 34D. As the DC-model is applicable for the Au—Au and Pd—Au systems, it was also adopted to calcu- late D,,. Due to the linker roles for IO connecting two Au Nanoparticles, there could be variable angle (a), which formed between two adjacent Au-IO connections, as shown in FIG. 34D. Interestingly, it was found that D,, data fell in the calculations with a between 180° (upper straight line in FIG. 34D) and 109° (lower straight line in FIG. 34D). We then calculated a and gave the values in FIG. 3¢, which showed that a changes from ~170° to 109° with the increase of N from 45 to 145. This result implies that the IO shifts its position from a two Au Nanoparticles center to a three Au Nanopar- ticles (triangle) center or to a four Au Nanoparticles (tetrahe- dron) center with the increase of spacer number. [0224] The magnetic response for JO-Au systems was also investigated. By changing the sample-magnet distance as shown in FIG. 34H, the magnetic field (B)-dependent response of two representative IO-Au systems were mea- sured, namely, FeO_Au,, ,; (Phase-D) and FeO_Au, 39 (mixed Phase-F and -D). For FeQ_Au,; ,s, the diffraction peaks became broader and even disappeared with B. The q; disappears for B at 0.11 T, and further increase of B to 0.16 T leads to the diminished q, and the residue of q,. FeO Au; 39 shows a more profound B-response for Phase-FA and an inert response for Phase-F. That is q, from Phase-D disappears for B at 0.16 T and other peaks from Phase-F display subtle changes. The S(q) can convert back to the initial states for the both systems, indicating a reversible B response. The softer potential from longer DNA and lower hybridization effi- ciency might be responsible for the more responsive behavior of FeO_Au, 30 system. This result suggested that through rational DNA design, one can fabricate systems with B-re- sponse switchable superlattice of different states, which could be interesting for smart responsive materials. Example 14 Au and Protein (Streptavidin) [0225] Nine-nm thiolated DNA-capped Au nanoparticles were used to hybridize with streptavidin (STV). The ratio of STV to Au and biotin-DNA was set as 1:20:100, and the X,-Xz sets were designed as 0-15, 3-15, 15-15, and 35-35, and the systems were nominated as Sys-SA,,, with n=15, 18, 30, and 50, respectively. [0226] FIGS. 23A through 23D give the 2D SAXS pattern and corresponding S(q) for Sys-SA,,, and the images in FIGS. 23A through 23D correspond to n=15, 18, 30, and 50, respec- tively. Here, S(q)=1,(qy/1,(q), 1,(q) was obtained from the melting system. The Q./Q, were calculated as 1:(1.69~1.78): (2.3~2.5):(2.7~2.8):(3.1~3.2). According to the Q,/Q,, the structure can be SC with Q/Q, as 1:1.73:2.45:2.83:3.16 from diffraction planes (100), (111), (211), (220), and (310) or BCC with Q/Q, as 1:1.73:2.45:2.83:3.16 from diffraction planes (110), (211), (222), (400), and (420) or FCC with Q,/Q; as 1:1.63:2.31:2.52:2.83:3.26 from diffraction planes (111), (220), (400), (331), (422), and (440). For a binary SC system, the structure model can be CsCl, a-ReOs, or AuCu; for a binary BCC system, the structure model can be La,O,; for a binary FCC system, the structure model can be NaTI, NaCl, ZnS (zincblende), or CaF. The Isy,/Issyy is roughly considered as 0, and according to the calculated results the possible models are La,O, NaCl, ZnS, and CaF,. The D,.M
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US 2016/0176988 Al can be calculated as V2*1/Q,, V3*/Q,, V1.5*7/Q,, and 1.5*1/Q, for La,O, NaCl, ZnS, and CaF, respectively. For Sys-SA,,, Q; are 0.0288, 0.0273, 0.0246, and 0.0225 Av}, corresponding to n=15, 18, 30, and 50, respectively, and D,.M are correspondingly 15.4, 16.3, 18.1, and 21.2 nm for the La,O, models, 18.9, 19.9, 22.1, and 25.9 nm for the NaCl model, and 16.4, 17.3, 19.2, and 22.4 nm for the ZnS and CaF, models. [0227] To calculate the D_.C for Sys-SA,, the following parameters were used: R,,=4.5 nm, Rg-,=3 nm (including STV), DNA number on STV=4, and the other parameters are the same as that used for the Au—Au system. The calculated C for Sys-IA,, is 15.9, 16.4, 18.1, and 21.6 nm, corre- sponding to n=15, 18, 30, and SO, respectively. D,.C agrees with the D,.M of the La,0;, ZnS and CaF, models. Since STV doesn’t give sufficient scattering, the precise location of the organic compound of the assembly can’t be predicted, and the predicted La,O,, ZnS and CaF, structures are based on the positions of the Au particles. However, considering STV has four binding sites for biotin, the most likely model is the CaF,-like crystalline organization. Example 15 QD and QD [0228] STV-Q7 was used to hybridize with STV-Q7 and STV-Q5 to form Sys-Q77 and Sys-Q75 systems, respectively. The ratio of QD to QD and biotin-DNA was set as 1:1:40, and the X,-X,, sets were designed as 3-3, 0-15, and 15-15 for Sys-Q77, and the systems were denoted Sys-Q77,, with n=3, 15, and 30, respectively. The X,-X, sets were designed as 3-3, 15-15, and 35-35 for Sys-Q75, and the systems were denoted Sys-Q75,,, with n=3, 30, and 50, respectively. [0229] FIGS. 24A through 24F give the 2D SAXS pattern and corresponding S(q) for Sys-Q77,, and Sys-Q75,, and the images in FIGS. 24A through 24C correspond to n=3, 15, and 30 for Sys-Q77,,, and the images in FIGS. 24D through 24F correspond to n=3, 30, and 50 for Sys-Q75,,, respectively. Here, S(q)=1,(qV1,(q), 1,(q) was obtained from the corre- sponding melting system. The Q,/Q, were calculated as 1:(1. 76~1.85):(2.65~2.73):(~3.4). According to the Q/Q,, and the above analysis for Au—Au and Pd—Au systems, the structure can be either the CsCl or the NaTI structure. The D,.M for these two structure can be calculated as V6*2/Q,. For Sys-Q77,,, Q; are 0.0288, 0.0282, and 0.0258 A-’, cor- responding to n=3, 15, and 30, respectively, and D..M are correspondingly 26.7, 27.2, and 29.8 nm. For Sys-Q75,,, Q; are 0.0312, 0.0258, and 0.0234 A7!, corresponding to n=3, 30, and 50, respectively, and D,,.M are correspondingly 24.7, 29.8, and 32.9 nm. [0230] To calculate the D,.C for Sys-QD,,, the following parameters were used: Rg7=12 nm (long axis size including STV), Rgs=10 nm (long axis size including STV), DNA number on QD=20, and the other parameters are the same as that used for the Au—Au system. The calculated D.C for the Sys-Q75, is 25.1, 29.1, and 32.1 forn=3, 15, and 30, respec- tively, and for the Sys-Q77, is 26.8, 28.2, and 30.6 forn=3, 30, and 50, respectively. D.C agrees with the D.,. M of both the CsCl and NaTI models, so the possible structures for STV and. Au system are CsCl and NaTI. [0231] The photoluminescence (PL) properties of the QD- QD systems, including Sys-Q77 and Sys-Q75, were mea- sured. FIG. 25A shows the photoluminescence of Sys-Q77,,, including the control system (a mixture of Q7 and Q7 without Jun. 23, 2016 biotin-DNA), and n=18, 30, and 50 systems. Different from the Au-QD systems, the Q7-Q7 systems show a distance- dependent fluorescence-enhancing behavior. The enhance- ment factor (EF) of Sys-Q77 against the surface-to-surface distance between Q7 and Q7 is given in FIG. 25B. EF=(I,,- 1,)/I,, where I,, and I, correspond to the PL intensity of Sys- Q77,,and the control system, respectively. The EF is inversely proportional to the surface-to-surface distance. FIG. 25C shows the photoluminescence of Sys-Q75,, including the control system (a mixture of Q7 and Q5 without biotin- DNA), and n=18, 30, and SO systems. Such Q7-Q5 systems show a distance-dependent fluorescence quenching of Q5 and enhancing of Q7 behavior. The enhancement-to-quenching factor (EQF) of Sys-Q75 against the surface-to-surface dis- tance between Q7 and QS is given in FIG. 25D. EQF=(R,,- R,VR,, where R=I,,5/I;,5, and the subscript n and ¢ denotes respectively Sys-Q75, and the control system, and I,,; and 1,55 correspond to the PL intensity at 705 nm and 525 nm of Sys-Q75, respectively. It can be seen that the EQF is propor- tional to the surface-to-surface distance. Example 16 QD and Pd [0232] STV-Q7 was used to hybridize with STV-Pd NDs to form the Sys-QPD system. The ratio of QD to Pd and biotin- DNA was set as 1:1:40, and the X,-X, sets were designed 3-3, 15-15, and 35-35, and the systems were nominated as Sys-QPD,,, with n=3, 30, and 50. FIG. 26 gives the 2D SAXS pattern and corresponding S(q) for Sys-QPD5, at different temperatures. This system show a similar temperature-depen- dent phase behavior as system IO-Au with long DNA spacers, and the Pd particles can’t be re-dispersed into solution. This system doesn’t have long-range order since it actually only shows one peak. [0233] The photoluminescence properties of the Pd-QD systems were also investigated. FIG. 27 shows the photolu- minescence of Sys-QPD,,, including the control system (a mixture of STV-PD and Q7 without biotin-DNA) andn=3 and 50 systems. Similar to the Au-QD systems, the Pd-QD sys- tems also show a distance-dependent fluorescence quenching behavior. To compare the QE of these two systems, we selected Sys-QPD;, and Sys-Q7Aso, and found that the QE of Sys-QPD,, and Sys-Q7A;, was 0.26 and 0.14, respectively. Additionally, the particle surface-to-surface distance in Sys- QPD5p is bigger than that in Sys-Q7A5, due to an additional STV on PD surface. Therefore, in comparison to the Au-QD systems, the Pd-QD systems show a more profound distance- dependent fluorescence quenching behavior. Example 17 IO and 10 [0234] STV-IO was used to hybridize with STV-IO to form the Sys-II system. The ratio of IO to IO and biotin-DNA was set as 1:1:15, and the X_,-X, sets were designed as 15-15, and the systems were nominated as Sys-II,,. FIG. 24 gives the 2D SAXS pattern and corresponding S(q) for Sys-II3. This sys- tem shows very broad peaks and doesn’t have long-range order. Example 18 QD and Au [0235] These systems comprised QD and Au nanoparticles. Specifically, the 3D assembly of Au nanoparticles with three
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US 2016/0176988 Al
types of QD, namely QD705 (Q7), QD605 (Q6), and QD525
(QS). Due to the comparable DNA grafting number (f) and
hydrodynamic radius between QD and Au, a CsCl superlat-
tice similar to Au—Au system would be expected. However,
additional detailed structure information, such as composi-
tional disorder, can be studied in the QD-Au systems thanks
to the remarkable AP(q) but similar effective size for Au and
component/size-tunable QD.
[0236] Similar to Pd nanoparticles, the DNA-grafted QDs
are well-dispersed and stable in an aqueous solution. The S(q)
of three DH systems with N=30, namely, Q7_Aup ,;,
Q6_Aup 15, and Q5_Aup ;5, are given in FIG. 354. Their
S(q) displayed similar peak ratios and were assigned as SC
patterns for the binary CsCl structures. Similar to the Au
size-dependent S(q) evolution behavior in PdA—Au systems,
as the QD changes from ~2 nm CdSe/ZnS to ~6 nm CdTe/
Zn8, the intensity ratio of (110) to (100) increases caused by
the decrease of AP(q) between QD and Au NPs, which fea-
tures the binary CsCl lattice. A fit for Q7_Aup 15 using CsCl
lattice is given as black line in FIG. 35A.
[0237] The interparticle distances in these QD and Au
binary systems can also be facilely tuned by regulating DNA
length. The N was varied from 30 to 33, 45, 85, and 145 forall
these three types of binary superlattices. FIG. 35B (top) gives
the S(q) of three representative Au-Q7 systems, including
Q7_Auy js. Q7_Au,; 35, and Q7_Augs 65. The D,, of these
QD-Au systems was calculated from the SAXS data and
summarized the results as symbols in FIG. 35D, which exhib-
ited that the D,, can be tuned from ~12 to 31 nm. For short
DNA length systems, superlattices comprising larger size QD
display smaller D,., which consists with the DC-model pre-
dicted tendencies (line in FIG. 35D). However, similar D,,
were observed for all types of QD-Au superlattices with long
DNA, and this might be related to compositional disorder.
[0238] Similar to Au—Pd systems, the structural order was
improved with N and the § increased from 82 nm to 168 nm
with the increase N from 30 to 145. Besides the increase of £,
the first two peak intensity ratio (I,/I,) also increases with N
regardless of types of QD. Such intensity modulation does not
result from different nanoparticles size in systems for Pd
hybridized with Au of different sizes and Au with QD of
different types. This S(q) evolution results from the compo-
sitional order-to-disorder (OTD) transition in the binary CsCl
structures. Such OTD transition has been extensively studied
in atomic systems, such as ZnCu alloys, and recently was
demonstrated in a computational work on DNA-assembled
Au systems, which show a ODT transition with elevated
temperature approach T,,,. The ODT process can be described
by means of a long-range order parameter 1, defined as
ne(.-F ,V0-F_ma(t,-F ,/C-EF.,), where ris fraction of A
sites occupied by the “right” particles, i.e. A particles, and F ,
is fraction of A particles in the lattice. The value of r,=1, q=1
and r,=F,, 10 respectively correspond to compositional
ordered and disordered lattice.
[0239] In the case of a CsCl lattice, such a transition is
schematically illustrated in FIG. 35C, which implies that a
diffraction pattern evolution from SC to BCC will emerge as
1 decreases from | to 0. With increasing DNA length in our
QD-Au systems, the gradual increase of I,/I, values, a feature
for SC to BCC pattern evolution, indicates a smaller y for
longer DNA systems. The increase of softness of interparticle
repulsive potential with DNA length might be responsible for
this ODT transition. It’s also reasonable that the D,, becomes
similar for longer DNA Au-QD systems regardless of QD
Jun. 23, 2016
kinds because Au and QD are more like one type of “average”
nanoparticle in the superlattice for smaller Obviously, such
OTD transition can be only observed in heterogeneous sys-
tems, comprising components with quite different P(q),
which could explain the absence of such ODT transition in
our Pd (~11 nm)-Au (~10 nm) systems. The dark lines in FIG.
35B give the fit using CsCl lattice with and without consid-
eration of compositional order. By the fit, the N-dependent
for Q7-Au systems was obtained and plotted in FIG. 35C,
which shows that y decreases from ~0.98 to ~0.54 with N
increase from 30 to 145.
[0240] The N-dependent compositional and_ structural
order behaviors hints to a certain balance between DNA flex-
ibility and rigidity that is crucial for ordering. According to
this guidance, a modified-linker hybridization system was
designed, denoted by Q7_Au;3oc24, where the central
24-base segments of the 30-base linker part in Q7_Au,3. are
hybridized into a rigid duplex. FIG. 35C (Bottom) gives the
S(q) and the CsCl lattice fit for this system. Indeed,
Q7_AU, 30¢24 shows large § (>700 nm) and highly improved
crystalline quality.
[0241] The photoluminescence (PL) properties of the QD-
Au systems were also examined. FIG. 4E gives a set of
steady-state and time-resolved PL spectra collected from
Q7_Au DH systems, including N change from 145 to 85, 45
33 and 30, anda free dispersed biotinylated DNA-capped Q7
solution. A progressive PL quenching of QD is clearly
observed as decreasing N, especially for N in the range from
45 to 30. In comparison with free QD, the PL intensity of
superlattice decreases by about 8%, 20%, and 60% for
N=145, 45, and 30, respectively. The lifetime (t) also pro-
gressive decreases ftom 62.1 ns for free QD to 59.2 ns, 44.5 ns
and 16.6 ns for superlattice accordingly corresponding to
N=130, 30, and 15. The quenching efficiency, E=1-(t/t),
where 1, and t, are accordingly the lifetime of QD in the
superlattice and free-states, reached ~0.74 for the system with
N-=30, which the D,, is ~12 nm.
Example 19
Binary Systems without Au
[0242] The arbitrary binary combination of different types
of QD (Q7, Q6, and Q5), different shape of Pd (PD, PC, PO),
and JO were investigated. It was found that the grafting DNA
number on the nanoparticles (f) plays a crucial role for assem-
bly behavior. For example, QD of each three types (with f
20~40) and Pd of each three shapes (with f<15~25) can
hybridized with other into a superlattice, but the systems
containing IO nanoparticles (with {~3~8) only form non-
specific induced clusters with the size typically less than 100
nm. All these systems display thermally reversible dissocia-
tion-association behaviors, implying the DNA-directed
assembly. Structural analysis indicates that all these superlat-
tices can be assigned with CsC] lattices but of quite different
degree of structure order.
[0243] The PL behaviors of QD-based binary systems was
also investigated. The lifetime is summarized in FIG. 35F.
The superlattice shows an energy transfer process, where
involves ~20% decrease in donor lifetime and ~12% increase
in acceptor lifetime in comparison with free particles. The
current studies on fluorescence behavior of QD near metal
NPs and QD most focused on clusters, the present QD-Auand
QD-QD superlattice provide a platform to study the collectivePage 97
US 2016/0176988 Al optical properties in 3D lattice due to their well-controlled structural ordering and lattice parameter. Example 20 Summary of DNA-Mediated Assembly [0244] The phase diagram for the assembled systems is summarized in FIG. 36A. Based on all the systems investi- gated, several important factors of the phase behavior of heterogeneous binary ~10 nm NP-A and B systems are derived. i) Two threshold values, f;, and f;,, are accordingly required for the assembly of particle into micro-scale (or to form participate in solution) and into well ordered crystals. As plotted in FIG. 36A, if the two components have similar grafting DNA number, f,, is about 20 for 10 nm NPs; and n,. could be relaxed to ~3-8 if the other component possesses a high DNA number, i.e. f,,~60 for 10 nm NPs in the experi- mental limit. f,, is about 30. ii) For systems involving NP-A (for example IO) with considerable non-specific interactions, besides the required f,, for NP-B short length DNA is neces- sary to break and transform the non-specific interactions- induced aggregates into DNA-driven dominant assemblies. iii) For anisotropic shaped NPs involved systems, more spherical-like NPs (e.g Pd-dodecahedrons) generate better structure orders of superlattices. v) For systems involving one type NPs with high f (e.g. Au) and the other with low f (e.g. QD), shorter (rigid) DNA benefits the compositional order and longer (flexible) DNA favors better structure order. A deliberate balance between DNA rigidity and flexibility is crucial for improving the ordering degree. This behavior is different for systems with high f for both types NPs, and in those systems DNA flexibility is the most important factors for ordering. If both components possess low f, a composi- tional disorder is favorable even for short DNA, as indicated in QD and Pd systems that the first peak diffracted from (110) planes. [0245] The quantitative structural analysis demonstrated the interparticle center-to-center distances (D.,..) of any binary systems can be predicted from their corresponding single systems. Taking DH systems with N=30 for example, the effective R, (=D,,/2) of five components were calculated, including Au, Q7, Q5, PD, and PC NPs based on the SXSA. data from single component systems. The R, of each compo- nent is represented by black and gray bars in FIG. 55. These data agree well with the DC models. The values simply by the sum of two R, agree well with the D,.. (represented by solid narrow bars in FIG. 36B) obtained from the corresponding binary systems. Such consistency might help one to distin- guish the lattice types because of the dependence of D_,, on lattice types with known SAXS data, and also benefit the understanding of DNA configurations on NPs surfaces as well as between nanoparticles. [0246] Insummary for the assembly part described in these examples, several examples for DNA-mediated assembly of binary systems have been presented, which show rich phases, such as Au and Au with both same and different size forming a NaTI structure, Au and Pd most likely forming a NaTl structure, Au and QD forming a La,O, structure, Au and IO forming a zincblende structure, Au and STV most likely forming a CaF, structure, QD and QD with same/different size forming a NaTI or CsCl structure, and the system com- prised of Pd and QD or IO and IO that most likely having an amorphous phase. Although the examples shown are for Jun. 23, 2016 binary systems, the use of DNA to assemble three or more multi-component systems is also possible. [0247] Data on the fluorescence properties of metal (Au, Pd) and fluorescent particle (QD) systems, QD and QD sys- tems, and the magnetic field effects on the phase behavior of a metal (Au) and magnetic particle (IO; Fe,O,) system were also obtained. All the measured metal-fluorescent systems showed a distance-dependent fluorescence quenching behav- ior. Incomparison to the Au-QD systems, the Pd-QD systems showed a more profound quenching effect. For QD and QD systems, the system with same types of QD showed a dis- tance-dependent fluorescence enhancement behavior, and the system comprised of different types of QD showed a fluores- cence quenching for small QD and enhancement for big QD. Metal-magnetic particle systems show a reversible magnetic field intensity-modulation phase behavior. [0248] Other and further embodiments utilizing one or more aspects of the inventions described above can be devised without departing from the spirit of Applicant’s invention. For example, specific embodiments have been described using gold and palladium nanoparticles of an approximate diameter of about 10 nm, but particles of other materials (metallic, semi-conductive, magnetic, dielectric, etc.) of various dimensions may be substituted and still be within the confines of this disclosure. In addition, although the examples have, for purposes of concreteness, been described with reference to DNA functionalization, micro- and nano-objects can be functionalized similarly in accor- dance with the methods of the present disclosure using RNA or PNA, as both RNA and PNA have the same addressable properties as does DNA, and similar melting temperatures and structure. PNA is artificial and is therefore more resistant to degraedation than is DNA, allowing it to be used under conditions inimical to DNA, including but not limited to non-aqueous solvents. Further, DNA and RNA may be used in concert, as appropriate. Further, the various methods and embodiments of the functionalization of DNA as described herein can be included in combination with each other to produce variations of the disclosed methods and embodi- ments. Discussion of singular elements can include plural elements and vice-versa. [0249] The order of steps can occur in a variety of sequences unless otherwise specifically limited. The various steps described herein can be combined with other steps, interlineated with the stated steps, and/or split into multiple steps. Similarly, elements have been described functionally and can be embodied as separate components or can be com- bined into components having multiple functions. [0250] The inventions have been described in the context of preferred and other embodiments and not every embodiment of the invention has been described. Obvious modifications and alterations to the described embodiments are available to those of ordinary skill in the art. The disclosed and undis- closed embodiments are not intended to limit or restrict the scope or applicability of the invention conceived of by the Applicants, but rather, in conformity with the patent laws, Applicants intend to fully protect all such modifications and improvements that come within the scope or range of equiva- lent of the following claims. INCORPORATION OF SEQUENCE LISTING [0251] Incorporated herein by reference in its entirety is the Sequence Listing forthe application. The Sequence Listing is disclosed on a computer-readable ASCII text file titled, “sequence_listing.txt”, created on Dec. 22, 2015. The sequence_listing. txt file is 1 kb in size.
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US 2016/0176988 Al 19 Jun. 23, 2016 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 2 <210> <211> <212> <213> <220> <223> SEQ ID NO 1 LENGTH: 15 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic sequence <400> SEQUENCE: 1 tacttccaat ccaat <210> <211l> <212> <213> <220> <223> SEQ ID NO 2 LENGTH: 15 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic sequence <400> SEQUENCE: 2 attggattgg aagta 15 15 1.-19. (canceled) 20. A three-dimensional (3D) ordered superlattice com- prising a plurality of DNA-nanoparticle conjugates assembled into one or more superlattices by a direct or linker- mediated hybridization, wherein the DNA-nanoparticle con- jugate, comprises: a functionalized nanoparticle; a protein covalently bound to the functionalized nanoparticle; and biotinylated DNA molecules coupled with the protein. 21. The three-dimensional (3D) ordered superlattice of claim 20, wherein the number of the hybridized bases between complementary DNA is 15. 22. (canceled) 23. The three-dimensional (3D) ordered superlattice of claim 20, wherein the protein is streptavidin. 24. The three-dimensional (3D) ordered superlattice of claim 20, wherein the covalent bond between streptavidin and the functionalized nanoparticle is an amide bond. 25. The three-dimensional (3D) ordered superlattice of claim 20, wherein the functionalized nanoparticle is function- alized with a mercapto acid ligand. 26. The three-dimensional (3D) ordered superlattice of claim 20, wherein the functionalized nanoparticle is function- alized with a mercapto acid ligand that is a mercaptounde- canoic acid. 27. The three-dimensional (3D) ordered superlattice of claim 20, wherein the functionalized nanoparticle is function- alized with an amphiphilic polymer. 28. The three-dimensional (3D) ordered superlattice of claim 20, wherein the functionalized nanoparticle is function- alized with an amphiphilic polymer that is a lipid-PEG car- boxylic acid. 29. The three-dimensional (3D) ordered superlattice of claim 20, wherein the functionalized nanoparticle comprises a magnetic material, a plasmonic material, a photonic mate- rial, a catalytic material, or a biological material, and has a shape of an octahedron, a cube or a dodecahedron. 30. The three-dimensional (3D) ordered superlattice of claim 20, wherein the functionalized nanoparticle further comprises a magnetic material of Fe,O,. a photonic material that is a quantum dot selected from CdSe/ZnS or CdTe/ZnS, acatalytic material that is selected from Pdor Pt, a plasmonic material of Au, and a biological material that is a protein. 31. (canceled) 32. The three-dimensional (3D) ordered superlattice of claim 20, wherein at least one functionalized nanoparticle within the superlattice is made from a material different than at least one other functionalized nanoparticle within the same superlattice. 33. The three-dimensional (3D) ordered superlattice of claim 29, wherein at least one functionalized nanoparticle within the superlattice is palladium (Pd) and at least one other functionalized nanoparticle within the superlattice is gold (Au), wherein at least one functionalized nanoparticle within the superlattice is iron oxide (Fe,O,) and at least one other functionalized nanoparticle within the superlattice is gold (Au), or wherein at least one functionalized nanoparticle within the superlattice is a CdSe/ZnS or CdTe/ZnS quantum dot and at least one other functionalized nanoparticle within the superlattice is gold (Au). 34,-35. (canceled) 36. The three-dimensional (3D) ordered superlattice of claim 20, wherein the DNA-nanoparticle conjugate further comprises DNA molecules attached to the functionalized nanoparticle, a number of DNA molecules attached to the functionalized nanoparticle ranges between 3 and 60 and a length of DNA attached to the functionalized nanoparticle ranges between 30 and 180 nucleotide bases. 37. (canceled) 38. (canceled) 39.-40. (canceled) 41.-42. (canceled)
Source notes & attribution
- https://rexresearch.com/GangDNASilica/US2016176988A1.pdf