Asymmetrically Functionalized AntibodyâGold Nanoparticle

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Asymmetrically Functionalized Antibody-Gold Nanoparticle Conjugates to Form Stable Antigen-Assembled Dimers Alexandra Mandl, Seth L. Filbrun, and Jeremy D. Driskell Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00459 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on October 1, 2016

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Bioconjugate Chemistry

Asymmetrically Functionalized Antibody-Gold Nanoparticle Conjugates to Form Stable Antigen-Assembled Dimers Alexandra Mandl, Seth L. Filbrun, and Jeremy D. Driskell* Department of Chemistry, Illinois State University, Normal, IL 61790 *corresponding author ABSTRACT Biomolecular assays based on the aggregation of modified gold nanoparticles (AuNPs) have been developed to provide low detection limits and rapid results with a simple one-step, wash-free procedure. However, a relatively narrow dynamic range, low sensitivity, and poor precision due to time-sensitive readout limit the application of these assay platforms. In this work we synthesized asymmetrically functionalized antibody-AuNP conjugates that are rationally designed to overcome the limitations of aggregation-based immunoassays. Solid-phase synthesis was used to chemically passivate the majority of the AuNP surface and restrict antibody immobilization into a small area of the AuNP surface. These asymmetric conjugates assembled into dimers with the addition of antigen and were stable for over 24 hours. In contrast, conventional antibody-AuNP conjugates which are symmetrically modified with antibody assembled into large aggregates that continuously increased in size with the addition of target antigen. These results suggest that asymmetric antibody-AuNP conjugates have the potential to significantly improve the analytical performance of aggregation-based immunoassays.

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In recent years, gold nanoparticles (AuNPs) modified with biomolecules (proteins, DNA, receptors, etc.) have received much attention due to their potential ability to revolutionize the medical field through the development of new detection methods1, 2 and drug delivery systems.3 Central to the success of these novel applications is the surface modification of the AuNP. Consequently, the focus of this communication is on the synthesis of rationally designed modified AuNPs to enhance the analytical performance of nanoparticle-enabled detection technologies. Numerous AuNP aggregation based assays have been developed in which antigenmediated aggregation is rapidly and easily detected by a color change,4, 5 spectrophotometry,6, 7 and more recently DLS.8-12 This assay platform affords a single-step, wash-free protocol with assay times on the order of 30 minutes to 2 hours.5, 11, 13 This is in contrast to more conventional immunoassay formats, such as ELISA, which require many labor-intensive and time-consuming steps with total assay times on the order of 6-24 hours. Commonly, these aggregation based detection strategies employ AuNPs that are fully modified with a molecular recognition element such as an antibody. Often antibody immobilization onto the AuNP is achieved via direct adsorption at an appropriate pH or NHS-coupling chemistry to produce fully and symmetrically modified AuNP.14-16 However, with these approaches to modification, proteins orient randomly on the AuNP surface to reduce antibody binding activity and ultimately assay reproducibility and sensitivity.17 One approach to improve AuNP aggregation based assays is to employ an alternative modification method, many of which focus on directed orientation to improve binding. Popular mediation techniques noted in literature include Protein A or G,18 carbohydrate coupling,19 and click chemistry.20 While these immobilization strategies have provided increased particle proficiency, they have several associated limitations such as complex procedures,


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inability to be universally applied, and the potential requirement of protein alteration before AuNP modification. Moreover, for aggregation-based assays, regardless of random or controlled orientation, fully and symmetrically modified AuNPs assemble into large and heterogeneously sized aggregates upon introduction of the antigen that continuously increase in size with time. This uncontrolled aggregation and aggregate instability limits the potential application of nanoparticle-based aggregation assays. For example, heterogeneously sized aggregates limit the inter-assay precision and negatively impact quantitative results. Time-dependent, continuous aggregation necessitates precise endpoint detection, another factor that introduces variability to negatively affect quantitative analysis. In light of these limitations, we hypothesize that functionalized AuNPs rationally designed to controllably assemble into uniformly sized aggregates with the introduction of antigen, that are stable over time, can lead to enhanced analytical performance for quantitative aggregation-based assays as well as other nanoparticleenabled assays.13, 21, 22 In this communication, we demonstrate the synthesis of asymmetrically functionalized antibody-AuNP conjugates and controlled antigen-mediated assembly of the conjugates into dimers. A solid-phase synthetic approach was used to asymmetrically modify AuNP in which antibody is regiospecifically localized into a small area of the particle while the remaining area is passivated. While synthetic approaches to asymmetrically functionalize AuNP have been demonstrated using bifunctional small molecule linkers23, 24 and DNA21 as a means to controllably assemble AuNPs, asymmetric functionalization of AuNP with antibody has not been reported. Asymmetric functionalization of particles with protein has been limited to much larger micron-sized silica spheres.25 Herein, we establish that antigen-assembly of asymmetrically functionalized antibody-AuNP conjugates form uniform dimers that remain stable for an



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extended period of time, whereas AuNPs fully modified with antibody form large, heterogeneous aggregates that settle out of suspension within a few hours and continue to grow in size with time. Asymmetrically functionalized 60 nm AuNPs were synthesized using a modified solid phase synthesis protocol illustrated in Scheme 1.23 Scheme 1. Synthesis of asymmetrically modified antibody-AuNP conjugates.

Glass substrates were cleaned in piranha solution and functionalized with (3-aminopropyl) triethoxysilane (APTES) to present a terminal primary amine. AuNPs were then physisorbed onto the surface of the glass to form a densely packed, single layer (Figure S1). The immobilized AuNPs were immersed in a solution of bovine serum albumin (BSA), resulting in the partial modification of the AuNP via direct adsorption of BSA to the exposed regions of the AuNPs. BSA was selected as a passivating molecule to modify the majority of the AuNP surface because


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it is a common stabilizing agent for AuNPs and blocking agent to minimize non-specific proteinprotein interactions in assays.26, 27 The partially modified AuNPs were then removed from the glass slide via sonication in the presence of goat anti-mouse IgG polyclonal antibody. Upon release from the glass, the antibody directly adsorbed onto the unmodified region of the AuNP that was previously in contact with the glass surface to form the final asymmetrically modified AuNP. Conventional fully modified antibody-AuNP conjugates were also prepared and investigated in parallel with the asymmetric modified conjugates. These symmetrically modified conjugates were a necessary control in order to confirm successful asymmetric modification and to evaluate the potential benefits for aggregation-based detection platforms. To this end, antibody was directly adsorbed onto unmodified AuNP suspended in pH 8.5 buffer (Scheme 2). It has been well-established that antibody adsorbs onto AuNP through non-specific hydrophobic and electrostatic interactions when the pH is adjusted to be slightly basic of the antibody isoelectric point. It is important to note that polyclonal antibody was used to prepare both asymmetric and symmetric conjugates in order to allow for multiple conjugates to bind a single antigen. Aggregation is also possible for conjugates synthesized with monoclonal antibodies; however, in this case, the conjugates would require synthesis with a mix of two different monoclonal antibodies targeting unique epitopes, i.e., match pairs, to enable antigen-induce aggregation. DLS, UV/vis spectrophotometry, and TEM were used to analyze the asymmetric and symmetric antibody-AuNP conjugates before and after antigen-mediated assembly.



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Scheme 2. Synthesis of symmetrically modified antibody-AuNP conjugates.

Prepared asymmetric and symmetric conjugate suspensions were centrifuged to remove excess antibody and resuspended in 0.1% BSA solution containing 1% sodium chloride. Both conjugates remained red in color suggesting that they were stable and therefore protected by a protein adlayer. UV-visible extinction spectra of unmodified AuNP and both conjugates suspended in the saline solutions are provided as Supporting Information (Figure S2). The characteristic plasmon resonance peak shifted 4 nm for both conjugates and arises from the change in local refractive index at the AuNP surface after modification with proteins.28 Importantly, no extinction bands at longer wavelengths resulting from aggregates were observed indicating that the conjugates were stable in the saline environment. Additionally, DLS was conducted to confirm protein adsorption and that the conjugates were stable in the high ionic strength solutions. The mean hydrodynamic diameter (DH) of the asymmetric and symmetric AuNP conjugates measured 80.0 ± 2.0 nm and 83.6 ± 4.0 nm, respectively, via DLS for three independent preparations. These size increases relative to unconjugated 60 nm AuNP (DH = 64.0 nm) are consistent with an adlayer of protein given the size of IgG and BSA.10, 11, 29 Additionally, the absence of larger sized aggregates demonstrates that both protocols, asymmetric and symmetric, modified the AuNPs in a manner that produced stable particles under biologically relevant ionic strength. Further, the symmetrically modified conjugates had a DH of 83.6 nm and the DH of the asymmetric conjugates measured slightly smaller at 80.0 nm. While not


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confirmatory, these data support that a significant portion of the asymmetric conjugate was modified with BSA, a smaller molecule than IgG antibody.30, 31 The asymmetric spatial distribution of the bioactive antibody and BSA of the asymmetric conjugate was confirmed via reaction with antigen and DLS analysis of the assembled product. The concentrations of the symmetric and asymmetric conjugates were adjusted to be equivalent. A sample of the target antigen (10 µL of 5 ng/mL mouse IgG) was added to each of the conjugates (90 µL) and allowed to react for 2 hours. As a negative control, PBS, e.g., 0 ng/mL, was added to the conjugates. Following incubation, particle sizes were measured using DLS. No increase in the size of either conjugate was observed for the 0 ng/mL negative control while the 5 ng/mL antigen sample produced a measureable increase in the mean DH for both conjugates. Importantly, these data illustrate aggregate formation was initiated by the introduction of the antigen and not conjugate instability. The DLS histogram reveals that the addition of the 5 ng/mL antigen sample to the symmetric AuNP conjugates resulted in the formation of large aggregates with a wide distribution of sizes and a mean DH of 204 nm (Figure 1). This size suggests conjugates formed clusters consisting of more than two AuNPs. The DLS histogram shows that the aggregates formed by the introduction of 5 ng/mL antigen to the asymmetric AuNP conjugates were predominately uniform with a mean DH of 136 nm (Figure 1). Comparison of the DLS histograms for the antigen-assembled AuNP conjugates suggests asymmetric AuNP conjugates experienced controlled aggregation to form dimers, whereas, the symmetric AuNP conjugates underwent uncontrolled aggregation to produce large heterogeneous aggregates. Unlike asymmetric AuNP conjugates where the Ab is localized to a select region of the AuNP surface to facilitate binding a single antigen, symmetric AuNP conjugates are fully modified with Ab to generate multiple points of interaction between the



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particle and multiple antigen molecules; hence, the production of dimers and large aggregates of variable sizes, respectively (Figures 2A and 2B).

Figure 1. DLS histograms of asymmetric and symmetric AuNP conjugates. Conjugates were mixed with PBS (0 ng/mL) as a negative control or 5 ng/mL antigen as a positive control and allowed to react for 2 hours.

To further exemplify the differences in aggregation for the asymmetric and symmetric conjugates, TEM images of the AuNP conjugates incubated for 2 hours with the 5 ng/mL antigen sample were acquired. As anticipated, asymmetric AuNP conjugates were present as predominately dimers and single unreacted particles (Figure 2C; Figure S3). In contrast, symmetric AuNP conjugates formed large aggregates consisting of many conjugates which vary in the number of particles (Figure 2D; Figure S3). Histograms based on TEM analysis demonstrate that 71% of aggregates formed by asymmetric conjugates are dimers while only 26% of aggregates formed by symmetric aggregates are dimers (Figure S3). Few aggregates were observed in the TEM images of the 0 ng/mL negative control samples (Figure S4). These images conclusively demonstrate spatially confined immobilization of the antibody into a small region of the asymmetric conjugate that minimizes the ability of each conjugate to bind antigen


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and sterically limits the size of aggregates that can effectively form.

Figure 2. Illustration of antigen-mediated aggregation of asymmetric conjugates (A) and symmetric conjugates (B). Representative TEM images of the aggregate product formed after the addition of 5 ng/mL mouse IgG to asymmetric conjugates (C) and symmetric conjugates (D). Scale bar is 500 nm.

A control study was performed to establish that the antibody adsorbed onto the unmodified gold upon removal from the glass surface and did not displace the adsorbed BSA. To this end, gold nanoparticles were symmetrically modified with BSA. The BSA conjugates were then incubated with 30 µg/mL of antibody for 2 hours to allow for the potential displacement of BSA by antibody. Excess antibody was then removed from the suspension and the conjugate was mixed with 0 ng/mL, 5 ng/mL, and 500 ng/mL antigen. The conjugate size did not change with the addition of antigen even at the highest concentration indicating that the conjugates did not



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bind with the antigen. These results confirm that antibody can not displace BSA once adsorbed onto the gold nanoparticle and that the antibody immobilized onto the asymmetric conjugates takes place at a region of unmodified gold. The dimer-sized aggregates formed by asymmetric conjugates upon introduction of antigen suggests that the majority of the conjugate was passivated by BSA and a minor portion of the AuNP conjugate was modified with antibody. To qualitatively assess relative surface coverage of BSA and antibody, asymmetric conjugates were prepared in which the locations of BSA and antibody were switched. It was anticipated that these asymmetric conjugates first modified with antibody while immobilized on glass followed by BSA modification after sonication would lead to functionalization of the major portion of the AuNP surface with antibody and BSA would be localized into a smaller region of the AuNP. Thus, these particles were hypothesized to aggregate to a greater extent, approaching the behavior of symmetric conjugates, due to more bioactive surface area. As an alternative possibility, similar antigenmediated aggregation regardless of the order of BSA/antibody functionalization would suggest the two proteins cover equivalent surface area of the AuNP. To test this hypothesis, two types of asymmetric conjugates were synthesized in which the BSA and antibody were switched; both conjugates were mixed with antigen and allowed to assemble into aggregates for 2 hours prior to DLS analysis. The asymmetric conjugates first modified with antibody formed significantly larger aggregates than those illustrated in Scheme 1 (Figure S5). This control study qualitatively supports the claim that the AuNP is asymmetrically modified, and the majority of the surface is modified while the AuNP is tethered to the glass surface and a minority of the surface is modified after removal from the glass surface.


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Further analysis to quantify the relative surface coverage of the BSA and antibody on the asymmetric conjugates was based on the catalytic activity of unmodified, partially modified, and fully modified conjugates (Supporting Information; Figure S6). While this analysis lacked the sensitivity to quantify the surface area of the AuNP occupied by the antibody, these results support that BSA covers much of the AuNP surface and a relatively small region remains unmodified for localization of the antibody in a subsequent step. In addition to improving precision with controlled, uniform conjugate assembly, aggregation-based assays would benefit from the formation of stable aggregates. Typically, signal transduction in aggregation-based assays are time-sensitive; thus, readout is performed at a defined endpoint typically ranging from 30 minutes to 2 hours.5, 11, 13 Large aggregates formed by symmetric conjugates sediment within a few hours and large aggregates can continue to couple together over time provided that one aggregate contains a captured antigen that is accessible on the surface of the conglomerate. We hypothesize that the antigen-assembled asymmetric conjugate dimers are stable over a long period of time because the BSAfunctionalized portion of the asymmetric conjugate dimer is exposed, thereby resisting interactions with neighboring dimers. To study aggregate stability, conjugates mixed with 0 ng/mL and 5 ng/mL antigen where also analyzed after 24 hours of incubation with DLS. The asymmetric dimers did not significantly change size between 2 and 24 hours of incubation with antigen, whereas, the symmetric aggregates increased from 204 nm to 721 nm during this incubation time (Figure 3). Both conjugates were stable and no size change was detected for the negative control samples incubated for 24 hours. These results support our hypothesis that once dimerized via antigen, asymmetric dimers are rendered unreactive to yield stable aggregates.



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Moreover, these data further validate that the regioselective asymmetric modification was realized as proposed.

Figure 3. Mean hydrodynamic diameter of asymmetric and symmetric AuNP conjugates mixed with PBS (0 ng/mL) as a negative control or 5 ng/mL antigen as a positive control. The sizes were measured after 2 hours and 24 hours of incubation with sample to assess the stability of the conjugates and antigenassembled aggregates.

CONCLUSIONS In summary, our work demonstrates the first use of a novel solid-phase synthesis protocol for the asymmetric protein modification of AuNPs. This protocol allows for the regiospecific immobilization of antibody that spatially limits the region of the AuNP surface that exhibits binding reactivity. Like traditional symmetric AuNP conjugates, asymmetric AuNP conjugates were stable under biologically relevant conditions. However, unlike symmetric AuNP conjugates, asymmetric AuNP conjugates undergo controlled antigen-mediated aggregation and afford aggregate stability over time. These qualities have the potential to improve analytical


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performance of nanoparticle-enabled aggregation assays as detection methods with respect to precision, sensitivity, and stability.

ASSOCIATED CONTENT Supporting Information The supporting Information is available free of charge on the ACS Publications website at DOI: 00.0000/acs.bioconjchem.XXXXXXX Experimental procedures, Results and Discussion, SEM, UV-visible extinction spectra, TEM histograms, TEM, DLS

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the Defense Threat Reduction Agency, Basic Research Award # HDTRA1-13-1-0028. Additional funding was provided by Illinois State University’s Department of Chemistry and College of Arts and Sciences.

REFERENCES (1)

Jans, H., and Huo, Q. (2012) Gold nanoparticle-enabled biological and chemical detection and analysis. Chem. Soc. Rev. 41, 2849-2866.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(2) (3) (4)

(5)

(6) (7) (8)

(9) (10)

(11)

(12)

(13)

(14) (15)

(16) (17) (18) (19) (20)

Zhou, W., Gao, X., Liu, D. B., and Chen, X. Y. (2015) Gold Nanoparticles for In Vitro Diagnostics. Chem. Rev. 115, 10575-10636. Ghosh, P., Han, G., De, M., Kim, C. K., and Rotello, V. M. (2008) Gold nanoparticles in delivery applications. Adv. Drug Deliver. Rev. 60, 1307-1315. Kang, J.-H., Asami, Y., Murata, M., Kitazaki, H., Sadanaga, N., Tokunaga, E., Shiotani, S., Okada, S., Maehara, Y., and Niidome, T. (2010) Gold nanoparticle-based colorimetric assay for cancer diagnosis. Biosens. Bioelectron. 25, 1869-1874. Medley, C. D., Smith, J. E., Tang, Z., Wu, Y., Bamrungsap, S., and Tan, W. (2008) Gold nanoparticle-based colorimetric assay for the direct detection of cancerous cells. Anal. Chem. 80, 1067-1072. Rosi, N. L., and Mirkin, C. A. (2005) Nanostructures in biodiagnostics. Chem. Rev. 105, 15471562. Zhao, W., Brook, M. A., and Li, Y. (2008) Design of gold nanoparticle-based colorimetric biosensing assays. ChemBioChem 9, 2363-2371. Chun, C., Joo, J., Kwon, D., Kim, C. S., Cha, H. J., Chung, M.-S., and Jeon, S. (2011) A facile and sensitive immunoassay for the detection of alpha-fetoprotein using gold-coated magnetic nanoparticle clusters and dynamic light scattering. Chem. Commum. 47, 11047-11049. Driskell, J. D., Jones, C. A., Tompkins, S. M., and Tripp, R. A. (2011) One-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles. Analyst 136, 3083-3090. James, A. E., and Driskell, J. D. (2013) Monitoring gold nanoparticle conjugation and analysis of biomolecular binding with nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). Analyst 138, 1212-1218. Jans, H., Liu, X., Austin, L., Maes, G., and Huo, Q. (2009) Dynamic light scattering as a powerful tool for gold nanoparticle bioconjugation and biomolecular binding studies. Anal. Chem. 81, 9425-9432. Liu, X., and Huo, Q. (2009) A washing-free and amplification-free one-step homogeneous assay for protein detection using gold nanoparticle probes and dynamic light scattering. J. Immunol. Methods 349, 38-44. Lopez, A., Lovato, F., Oh, S. H., Lai, Y. H., Filbrun, S., Driskell, E. A., and Driskell, J. D. (2016) SERS immunoassay based on the capture and concentration of antigen-assembled gold nanoparticles. Talanta 146, 388-393. Geoghegan, W. D., Ambegaonkar, S., and Calvanico, N. J. (1980) Passive gold agglutination. An alternative to passive hemagglutination. J. Immunol. Methods 34, 11-21. Wagner, P., Hegner, M., Kernen, P., Zaugg, F., and Semenza, G. (1996) Covalent immobilization of native biomolecules onto Au (111) via N-hydroxysuccinimide ester functionalized selfassembled monolayers for scanning probe microscopy. Biophys. J. 70, 2052. Hermanson, G. T. (2013) Chapter 6 - Heterobifunctional Crosslinkers, in Bioconjugate Techniques (Third edition) pp 299-339, Academic Press, Boston. Trilling, A. K., Beekwilder, J., and Zuilhof, H. (2013) Antibody orientation on biosensor surfaces: a minireview. Analyst 138, 1619-1627. Bae, Y. M., Oh, B. K., Lee, W., Lee, W. H., and Choi, J. W. (2005) Study on orientation of immunoglobulin G on protein G layer. Biosens. Bioelectron. 21, 103-10. Duval, F., van Beek, T. A., and Zuilhof, H. (2015) Key steps towards the oriented immobilization of antibodies using boronic acids. Analyst 140, 6467-6472. Finetti, C., Sola, L., Pezzullo, M., Prosperi, D., Colombo, M., Riva, B., Avvakumova, S., Morasso, C., Picciolini, S., and Chiari, M. (2016) Click Chemistry Immobilization of Antibodies on Polymer Coated Gold Nanoparticles. Langmuir 32, 7435-7441.


Page 14 of 16

Page 15 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(21)

(22)

(23)

(24) (25)

(26) (27)

(28) (29)

(30) (31)

Guo, L. H., Xu, Y., Ferhan, A. R., Chen, G. N., and Kim, D. H. (2013) Oriented Gold Nanoparticle Aggregation for Colorimetric Sensors with Surprisingly High Analytical Figures of Merit. J. Am. Chem. Soc. 135, 12338-12345. Yan, N., Zhu, Z. L., Jin, L. L., Guo, W., Gan, Y. Q., and Hu, S. H. (2015) Quantitative Characterization of Gold Nanoparticles by Coupling Thin Layer Chromatography with Laser Ablation Inductively Coupled Plasma Mass Spectrometry. Anal. Chem. 87, 6079-6087. Sardar, R., Heap, T. B., and Shumaker-Parry, J. S. (2007) Versatile solid phase synthesis of gold nanoparticle dimers using an asymmetric functionalization approach. J. Am. Chem. Soc. 129, 5356-+. Sardar, R., and Shumaker-Parry, J. S. (2008) Asymmetrically functionalized gold nanoparticles organized in one-dimensional chains. Nano Lett. 8, 731-736. Chen, B., Jia, Y., Gao, Y., Sanchez, L., Anthony, S. M., and Yu, Y. (2014) Janus Particles as Artificial Antigen-Presenting Cells for T Cell Activation. ACS applied materials & interfaces 6, 1843518439. Brewer, S. H., Glomm, W. R., Johnson, M. C., Knag, M. K., and Franzen, S. (2005) Probing BSA binding to citrate-coated gold nanoparticles and surfaces. Langmuir 21, 9303-9307. Singh, A. V., Bandgar, B. M., Kasture, M., Prasad, B., and Sastry, M. (2005) Synthesis of gold, silver and their alloy nanoparticles using bovine serum albumin as foaming and stabilizing agent. J. Mater. Chem. 15, 5115-5121. Anker, J. N., Hall, W. P., Lyandres, O., Shah, N. C., Zhao, J., and Van Duyne, R. P. (2008) Biosensing with plasmonic nanosensors. Nat. Mater. 7, 442-453. Tsai, D.-H., DelRio, F. W., Keene, A. M., Tyner, K. M., MacCuspie, R. I., Cho, T. J., Zachariah, M. R., and Hackley, V. A. (2011) Adsorption and conformation of serum albumin protein on gold nanoparticles investigated using dimensional measurements and in situ spectroscopic methods. Langmuir 27, 2464-2477. Bohidar, H. (1989) Light scattering study of solution properties of bovine serum albumin, insulin, and polystyrene under moderate pressure. Colloid Polym. Sci. 267, 292-300. Jøssang, T., Feder, J., and Rosenqvist, E. (1988) Photon correlation spectroscopy of human IgG. J. Protein Chem. 7, 165-171.



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Asymmetrically Functionalized AntibodyâGold Nanoparticle

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Asymmetrically Functionalized AntibodyâGold Nanoparticle