We present a novel one-step flow process to synthesize biocompatible gold nanorods with tunable absorption and biocompatible surface ligands. Photothermal optical coherence tomography (OCT) of human breast tissue is successfully demonstrated using tailored gold nanorods designed to have strong absorption in the near-infrared range.National Science Foundation (U.S.) (grant CHE-0714189)National Institutes of Health (U.S.) (R01-CA75289-15)National Institutes of Health (U.S.) (K99-EB010071-01A1)United States. Air Force Office of Scientific Research (FA9550-10-1-0063)Deutsche Forschungsgemeinschaft (DFG-GSC80-SAOT)Spain. Ministerio de Educación y Ciencia (MEC)US-UK Fulbright Commissio

Bar-Ilan

Chao Zhou

Connor

Halder

Huang

James G. Fujimoto

Jana

Jiang

Klavs F. Jensen

Koeppl

Leonov

Marre

Martin F. Kraus

Murphy

Niidome

Orendorff

Seung-Kon Lee

Shukla

Víctor Sebastián

Wang

Zhong

Zhou

Chemical Communications

PubMed

Sebastián, Víctor

Lee, Seung-Kon

Zhou, Chao

Kraus, Martin Franz Georg

Fujimoto, James G.

Jensen, Klavs F.

Sebastian, Victor

DSpace@MIT

One-step Continuous Synthesis of Biocompatible GoldNanorods for Optical Coherence Tomography‡Víctor Sebastián#,&, Seung-Kon Lee#, Chao Zhou†, Martin F. Kraus†,+, James G. Fujimoto†,and Klavs F. Jensen#James G. Fujimoto: jgfuji@mit.edu; Klavs F. Jensen: kfjensen@mit.edu#Department of Chemical Engineering, Massachusetts Institute of Technology, 77 MassachusettsAvenue, Cambridge, MA 02139, USA†Department of Electrical Engineering and Computer Science and Research Laboratory ofElectronics, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA02139, USAAbstractWe present a novel one-step flow process to synthesize biocompatible gold nanorods with tunableabsorption and biocompatible surface ligands. Photothermal optical coherence tomography (OCT)of human breast tissue is successfully demonstrated using tailored gold nanorods designed to havestrong absorption in near-infrared range.Gold nanorods (GNRs) have received much attention because of their potential in a widevariety of applications such as, biological imaging, sensors, surface enhanced Ramanspectroscopy, drug delivery and photothermal therapy of cancer cells1,2 Seed mediatedgrowth3 in the presence of cetyltrimethyl ammonium bromide (CTABr) is the most widelyused synthesis method to produce GNRs, comprising of at least two steps: synthesis of goldseeds, and then addition of the gold seeds to a CTABr/Au solution for growth of nanorods.CTABr is the structure-directing agent used to control nanorod shape, but CTABr-cappedGNRs have been reported to be cytotoxic.4 CTABr resulting from incomplete purification ofthe GNRs or desorption from the bound bilayer is toxic to cells as a detergent that adverselyaffect cell membranes.5 Hence, proper purification of the GNRs is a key step for any in vivowork. Various approaches have been employed to retard CTABr desorption and to eliminatefree CTABr molecules in nanoparticle solutions.6 For example, the use of silver ions toassist in the growth of GNR is commonly used in seed-mediated approach. Silver ions arereduced to form monolayer or submonolayer of silver.7 Fast silver deposition followed bystrong CTABr binding inhibits gold growth on the sides of the rods leading to preferentialanisotropic growth of gold at the ends.8 Unfortunately, the presence of silver exacerbates thetoxicity problem.9 The seeded growth methods have also been reported to suffer fromlimited reproducibility perhaps attributable to the number of the steps involved.10,11Therefore, development of an alternative approach is necessary for the synthesis ofbiocompatible GNRs by means of a simple and versatile process.‡Electronic Supplementary Information (ESI) available© The Royal Society of ChemistryCorrespondence to: James G. Fujimoto, jgfuji@mit.edu; Klavs F. Jensen, kfjensen@mit.edu.&Present Addresses: Universidad de Zaragoza, Departamento de Ingenieria Quimica, Instituto de Nanociencia de Aragon, C/PedroCerbuna 12, 50009 Zaragoza (Spain)+Pattern Recognition Lab, Graduate School in Advanced Optical Technologies (SAOT), University Erlangen-Nuremberg, 91054Erlangen, GermanyNIH Public AccessAuthor ManuscriptChem Commun (Camb). Author manuscript; available in PMC 2012 July 07.Published in final edited form as:Chem Commun (Camb). 2012 July 7; 48(53): 6654–6656. doi:10.1039/c2cc32969g.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptIn order to circumvent the toxicity and multistep issues associated with typical GNRssynthesis, we report a single-step process with lysine, a biocompatible amino acid, as thecapping agent and without the presence of silver/CTABr.12 Moreover, we have adapted thesingle-pot synthesis carried out in a batch reactor to a continuous flow process for improvedsynthesis control and reproducibility. Specifically, a solution of Au(OH)4- at pH 10.3 isreduced in flow by sodium borohydride in presence of several amino acids: lysine,adenosine and alanine. The results demonstrate that rod-shape gold nanoparticles withstrong absorption in the near infrared wavelength range (700∼900 nm) can be prepared bytuning the lysine concentration. Selection of the optimal gold precursor species, pH andligands are critical to the successful growth of nanorods.Figure 1 shows the absorption spectra of the growth products made with various molar ratiosof lysine/gold (Lys/Au). Gold products synthesized with a Lys/Au molar ratio below 10exhibit only transverse plasmon band (TPB). Upon further increase in the Lys/Au molarratio, the TPB decreases in intensity and stays at 520 nm, becoming a shoulder of thelongitudinal plasmon band (LPB), suggesting the conversion of nanospheres into nanorods.Finally, the LPB redshifts to the near infrared wavelength range with increases in width ofthe absorption. Transmission electron microscopy (TEM) images are in agreement with theoptical data and shows that a there is a transition from spherical-shape nanoparticles tonanorods as the Lys/Au ratio was increased ultimately resulting in nanorods with an aspectratio of 4.5-5 at 180 Lys/Au (Fig. 2 and Fig. S1). Synthesis of GNRs was not feasible usingother aminoacids such as adenosine or alanine (Fig. S2a). In addition to the carboxylic acidfunctionality, lysine possesses two chemically distinct amino groups allowing both cappingand bridging roles13. Lysine is thought to interact with the gold surface via the α-aminegroup, whereas the terminal amino group forms hydrogen bonds with the carboxylic acidgroup of surrounding lysine molecules, which promotes the gold nanoparticle linearassembly. The bridging role of lysine is pH dependent and it is only effective at pH higherthan its pK2 (8.5) (Fig.S2b). Above this pH, the amino terminal group is deprotonated,leading to capped gold nanoparticles with negative charged surfaces.13 The unidirectionalgrowth best minimizes the electrostatic repulsion forces, so as the concentration of lysineincreases, the dual role of lysine capping/bridging promotes the growth of anisotropic goldnanostructures (Fig 1, Fig. 2a and b). However, the directionality created by the lysineligand is not perfect and some gold nanoparticles assembly in L or Y shapes, which wouldbroaden the LPB. Moreover, poor mixing efficiency in the batch reactor tend to acerbate thebroadening for fast reaction systems like the present one.Slow and inhomogeneous mixing in batch systems leads to an uneven concentrationdistribution as well as a limitation in mass transfer.14 Therefore, we adapted the batchsynthesis to a continuous microfluidic flow process for a better control in the synthesis ofGNR (Fig S3). Moreover, being able to manipulate separate streams of the gold precursorand reducing agent enabled fast screening and optimization of the synthesis conditions. Withapplication of the continuous reactor, optical properties of the resulting GNRs weresignificantly improved. Moreover, a a high throughput was obtained as a result of thereduced characteristic time of the transport processes at micrometer length scale (Fig. S4).UV-VIS spectra show a better definition of the TPB and a narrower LPB absorption (Fig.2c). From the statistical characterization, the aspect ratio of the resulting nanorods was about3.5, with uniform diameter of 15 nm (Fig. 2d and e).As shown in Fig. 3a and b, the lattice planes in the facets of the GNR continuously extendedto form single crystals. These characterizations indicate a strong cross-linking interactionbetween the fused gold nanoparticles with the oriented attachment mechanism, where‡Electronic Supplementary Information (ESI) availableSebastián et al. Page 2Chem Commun (Camb). Author manuscript; available in PMC 2012 July 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptadjacent particles self-organize so that they share a common crystallographic orientation.15TEM images show that {111} plane is corresponding to the longitudinal axis of the GNRs.XRD data in Fig. 3c also support the TEM observations. Compared with the XRD spectra ofthe reference bulk gold, {111} orientations are significantly extended as much as 202 %. Itis known that the crystal orientations of short GNRs are dominated by {100} and {110}while long GNRs are dominated by {111} and {110}, when they are grown with CTABr.16However, for our short GNRs grown with lysine, {111} plane was significantly dominantover {100} and {110}. Crystallite size characterized by X-ray diffraction (XRD) are 14.1,11.2, 13.4 nm for {111}, {100}, {110} plane, respectively.Lysine is reported to promote the formation of gold linear chains at basic pH values,13 butno LPB has been reported for these nanostructures. To demonstrate that not only the pH butalso the gold species are important to obtain lysine capped GNRs, a synthesis of GNRs wascarried out using fresh AuCl4- solution at pH value of 10.3, where hydrolysis of Au-Cl doesnot occur completely.17 Figure S5 shows that the incomplete hydrolysis of Au-Cl bondsspecies promotes the formation of linear gold chains with a high degree of cross-linking andonly TPB absorption, whereas the overnight aged AuCl4- solution with Au(OH)4-speciesfavors the formation of nanorods with both TPB and LPB absorption. Au-Cl species can bereduced faster than Au(OH)4- 17, which promotes the fast addition of gold colloids to thelinear chains and high crosslinking. In contrast, the slow reduction of Au(OH)4- promoteskinetic control of gold addition and rod shape formation.Optical coherence tomography (OCT) is an emerging biomedical imaging tool for assessingtissue architectural morphology in three-dimensions with resolutions approaching that ofstandard histopathology.18 Contrast in conventional OCT is derived from endogenousoptical scattering changes within the tissue. Recently, there has been an increased interest inusing exogenous contrast agents, such as gold nanoparticles, for OCT applications.19 Fig. 4shows cross-sectional OCT images of the human breast tissue in gray scale with thephotothermal OCT signal superimposed as pseudocolor (see S.I. for further details). In thecontrol experiment, where the 830 nm photothermal modulation beam was turned off, anincreased scattering signal was observed at the nanorod injection site (yellow arrow). Theincreased scattering signal due to the nanorods, however, is indistinguishable from intrinsicscattering variations from stromal tissues in the breast (red arrow). In addition, nophotothermal signal was observed (Fig. 4a). When a 20 kHz, 11 mW photothermal beam isapplied to the tissue, localized photothermal signal was clearly observed at the nanorodsinjection site (Fig. 4b). Here, a SNR threshold of 5 was selected to highlight the regions ofinterest where nanorods were injected. A maximum SNR of 29 was achieved with the 20kHz photothermal modulation.ConclusionsThese results demonstrate the feasibility of using gold nanorods synthesized in continuousflow with lysine, as the contrast agent for photothermal OCT. This work not only advancesthe synthesis approaches to obtain biocompatible nanomaterials, but also provides apotential application of using them to register 3D morphological and pathological state oftissues with micro scale resolution. Since the lysine molecules have amine and acid groupssimultaneously, they would have potential to specifically bind to DNAs, proteins and otherbiomolecules.AcknowledgmentsAuthors acknowledge US NSF grant (CHE-0714189, K.F.J.), NIH grants (R01-CA75289-15, J.G.F. and K99-EB010071-01A1, C.Z.) and Air Force Office of Scientific Research contract (FA9550-10-1-0063, J.G.F.) forSebastián et al. Page 3Chem Commun (Camb). Author manuscript; available in PMC 2012 July 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptfunding this research. V.S. acknowledges the support of the Fulbright Commission and the Ministry of Education inSpain and M.K. acknowledges the support from Deutsche Forschungsgesellschaft (DFG-GSC80-SAOT)Notes and references1. Huang XH, El-Sayed IH, Qian W, El-Sayed MA. Journal of the American Chemical Society. 2006;128:2115–2120. [PubMed: 16464114]2. Murphy CJ, Gole AM, Hunyadi SE, Stone JW, Sisco PN, Alkilany A, Kinard BE, Hankins P.Chemical Communications. 2008:544–557. [PubMed: 18209787]3. Jana NR, Gearheart L, Murphy CJ. Journal of Physical Chemistry B. 2001; 105:4065–4067.4. Niidome T, Yamagata M, Okamoto Y, Akiyama Y, Takahashi H, Kawano T, Katayama Y, NiidomeY. Journal of Controlled Release. 2006; 114:343–347. [PubMed: 16876898]5. Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD. Small. 2005; 1:325–327. [PubMed:17193451]6. Leonov AP, Zheng JW, Clogston JD, Stern ST, Patri AK, Wei A. Acs Nano. 2008; 2:2481–2488.[PubMed: 19206282]7. Liu MZ, Guyot-Sionnest P. Journal of Physical Chemistry B. 2005; 109:22192–22200.8. Orendorff CJ, Murphy CJ. Journal of Physical Chemistry B. 2006; 110:3990–3994.9. Bar-Ilan O, Albrecht RM, Fako VE, Furgeson DY. Small. 2009; 5:1897–1910. [PubMed: 19437466]10. Koeppl S, Solenthaler C, Caseri W, Spolenak R. Journal of Nanomaterials. 201111. Jiang XC, Pileni MP. Colloids and Surfaces a-Physicochemical and Engineering Aspects. 2007;295:228–232.12. Shukla R, Bansal V, Chaudhary M, Basu A, Bhonde RR, Sastry M. Langmuir. 2005; 21:10644–10654. [PubMed: 16262332]13. Zhong ZY, Luo JZ, Ang TP, Highfield J, Lin JY, Gedanken A. Journal of Physical Chemistry B.2004; 108:18119–18123.14. Marre S, Jensen KF. Chemical Society Reviews. 2010; 39:1183–1202. [PubMed: 20179831]15. Halder A, Ravishankar N. Advanced Materials. 2007; 19:1854.16. Wang ZL, Mohamed MB, Link S, El-Sayed MA. Surface Science. 1999; 440:L809–L814.17. Kah JCY, Phonthammachai N, Wan RCY, Song J, White T, Mhaisalkar S, Ahmad I, Sheppard C,Olivo M. Gold Bulletin. 2008; 41:23–36.18. Huang D, Swanson EA, Lin CP, Schuman JS, Stinson WG, Chang W, Hee MR, Flotte T, GregoryK, Puliafito CA, Fujimoto JG. Science. 1991; 254:1178–1181. [PubMed: 1957169]19. Zhou C, Tsai TH, Adler DC, Lee HC, Cohen DW, Mondelblatt A, Wang YH, Connolly JL,Fujimoto JG. Optics Letters. 2010; 35:700–702. [PubMed: 20195324]Sebastián et al. Page 4Chem Commun (Camb). Author manuscript; available in PMC 2012 July 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 1.Absorption spectra and TEM images of the gold nanorods (GNRs) grown with varyingamounts of the lysine/gold precursor (Lys/Au) ratio. Various colors of the GNR solutionscorrespond to the absorption spectra of different Lys/Au ratios (top). Corresponding TEMimages showing structural evolution (middle, also see Fig. S1). Scale bar is 30 nm.Sebastián et al. Page 5Chem Commun (Camb). Author manuscript; available in PMC 2012 July 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 2.TEM images and absorbance spectra of the Au nanostructures. (a) TEM image of Aunanostructures obtained at Lys/Au molar ratio of 2, in a batch reactor. (b) At molar ratio of180, in a continuous flow reactor. Insets of (a) and (b) Optical images from gold colloidsobtained at different Lys/Au ratios. (c) Comparison of absorbance spectra from GNRsobtained in a batch reactor and continuous flow reactor (oxidant flow rate of 40 μL/min andreductant flow rate of 10 μL/min) with same Au/Lys molar ratio of 180. (d), (e) Statisticalanalysis of produced GNRs obtained in continuos flow: (d) aspect ratio and (e) diameter.Sebastián et al. Page 6Chem Commun (Camb). Author manuscript; available in PMC 2012 July 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 3.HR-TEM and XRD analysis of the gold nanorods. (a), (b) HRTEM image with latticeorientations; {111}, {100} and {110}. (b) HRTEM images correspond to the colored boxesA, B and C in (a) presented in the left. Right images show Fourier transform of A, B and C,respectively. (c) XRD spectrum from dried bulk GNRs (Black). Spectra from reference bulkgold (Red). Small sub-peak at 33° is attributed to crystalline L-lysine from the residue.Sebastián et al. Page 7Chem Commun (Camb). Author manuscript; available in PMC 2012 July 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 4.OCT images with (a) or without (b) photothermal modulation on freshly excised humanbreast tissue using injected nanorods as contrast agents. Photothermal OCT signal (inarbitrary units) with a SNR>5 is superimposed as pseudocolor on the gray scale OCTstructural image. The yellow arrow indicates the injection site of the nanorods, while the redarrow indicates fibrostromal regions with strong backscattering.Sebastián et al. Page 8Chem Commun (Camb). Author manuscript; available in PMC 2012 July 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

One-step continuous synthesis of biocompatible gold nanorods for optical coherence tomography

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One-step continuous synthesis of biocompatible gold nanorods for optical coherence tomography

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