Protein-Functionalized Gold Nanoparticles for ... - JST

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Introduction

Nanotechnology promotes the development of unique strategies for the detection and regulation of diverse biomedical processes at the nanometre scale, which have been employed for bioanalytical and biomedical applications. Nanoparticles (NPs), especially gold nanoparticles (AuNPs), are ideal substrates for biosensing assays and biomedical applications because of their unique properties such as high dispersibility in water, facile synthesis, tunability of morphology and size, and chemical modulation of surface functionalities.1,2 AuNPs can be synthesized and modified with various chemical functional groups that facilitate the conjugation of low-molecular weight ligands, polymers, peptides, proteins, and DNAs with the NPs.3–7 This conjugation ability imparts stability, functionality, and biocompatibility to the functionalized AuNPs, thus widening their application scope in bioanalysis and biotechnology.

AuNPs can be synthesized by various physical and chemical methods such as the Turkevich method, which is based on the reduction of Au salt by citrate.8 Another method is the Brust–Schiffrin method, in which Au clusters are formed by the reduction of Au ions using borohydride ions in a single-phase or two-phase aqueous–organic media.9 However, these methods

generally require harsh reaction conditions such as high temperature, elevated pressure, and organic solvents. Thus, an alternative approach for the synthesis of AuNPs by using enzymes under mild conditions has been proposed; AuNPs are produced by the bio-reduction of Au ions by NADH with NAD+-dependent enzyme.10,11 However, this method also requires the addition of chemical reagents such as NADH.

To overcome the limitations of the above-mentioned methods, the synthesis of AuNP by using peptides and proteins has been reported, wherein the reducing ability of amino acid residues such as cysteine and tyrosine is utilized.12,13 For example, AuNPs with a size of 10 – 15 nm were synthesized by an A3 peptide (AYSSGAPPMPPF) using tyrosine residues as the reducing agents.14,15 Slocik et al. also reported AuNP synthesis using YYY peptide.16 Various proteins such as bovine serum albumin (BSA) and lysozyme having such amino acids with reduction ability can also be used for AuNP synthesis.13

Herein, AuNPs were synthesized in a one-pot synthetic reaction using reduced BSA (rBSA) as the reducing agent. The free thiol group in rBSA can conjugate with the surface of AuNPs via the Au-Sulfur interaction, thereby forming functionalized AuNPs (rBSA-AuNPs). The prepared rBSA-AuNPs were used for the detection of antibodies that can bind to the protein on the AuNP surface. It is presumed that rBSA-AuNPs were recognized by the anti-BSA antibody (anti-BSA), and the interaction between anti-BSA and rBSA on the AuNP surface led to the formation of large aggregates, which enabled the detection of anti-BSA (Fig. 1).

The formation of the anti-BSA-induced rBSA-AuNP aggregates was analyzed by darkfield microscopy (DFM), which

2021 © The Japan Society for Analytical Chemistry

† To whom correspondence should be addressed.E-mail: [email protected], Current address: Department of Marine, Faculty of Fisheries and Marine, Universitas Airlangga, Campus C Mulyorejo, Surabaya 60115, Indonesia.

Protein-Functionalized Gold Nanoparticles for Antibody Detection Using the Darkfield Microscopic Observation of Nanoparticle Aggregation

Ken YOSHIMURA,* PATMAWATI,** Mizuo MAEDA,*** Noriho KAMIYA,** and Tamotsu ZAKO*†

* Department of Chemistry and Biology, Graduate School of Science and Engineering, Ehime University, 2-5 Bunkyo, Matsuyama, Ehime 790–8577, Japan

** Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819–0395, Japan

*** Bioengineering Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama 351–0198, Japan

Gold nanoparticles (AuNPs) are commonly used in biosensing applications. In this study, AuNPs were synthesized by using reduced bovine serum albumin (rBSA) as the reducing agent. The rBSA conjugated with AuNPs via Au-Sulfur interactions to form rBSA-functionalized AuNPs (rBSA-AuNPs). The interaction of the rBSA moieties on the rBSA-AuNP surface with an anti-BSA antibody (anti-BSA) led to AuNP aggregation, which enabled the successful detection of anti-BSA at a concentration as low as 20 nM through darkfield microscopy (DFM). This study demonstrates the potential applications of protein-functionalized AuNPs in the bioanalysis of substances through DFM.

Keywords Gold nanoparticles, synthesis, aggregation, antibody detection, darkfield microscopy

(Received October 29, 2020; Accepted December 2, 2020; Advance Publication Released Online by J-STAGE December 11, 2020)

Anal. Sci., 2021, 37, 507–511DOI:10.2116/analsci.20SCP12

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detects scattered light from individual nanostructures.17,18 We have previously demonstrated the application of DFM for the facile and sensitive detection of target molecules such as DNA, thrombin, and protein amyloids by analysing the AuNP aggregates formed by each of the target molecules.19–21 In this study, DFM results revealed the formation of the rBSA-AuNP aggregates upon the addition of 20 nM anti-BSA. Because AuNPs can be synthesized using various proteins,13 this study indicates that the DFM-based analysis of AuNP aggregation is an effective approach for antibody detection, enabling the potential biomedical applications of protein-modified AuNPs, such as disease diagnosis.

Materials and Methods

Preparation of rBSAThe rBSA was prepared according to a previously reported

method.22,23 Briefly, 300 μM BSA (Wako, Osaka, Japan) was incubated with 60 mM Tris(2-carboxyethyl)phosphine (TCEP, Wako) in a buffer consisting of 25 mM Tris-HCl, 8 M urea and 0.1 mM EDTA at 25°C for 1 h. The resulting solution was dialyzed against 500 mL of 1 mM HCl for 3 days at 4°C using a 12 – 14 kDa molecular weight cut off dialysis membrane (Spectra/Por, Waltham, MA). The medium was changed 4 – 6 times to remove excess urea and TCEP. The concentration of the rBSA was measured using the Bradford assay, and the solution was stored at 4°C for further analysis.

Preparation of rBSA-functionalized AuNPsAuNPs were synthesized in the presence of rBSA or BSA

according to the method described for AuNP synthesis using the A3 peptide.15 A solution consisting of AuCl4

– (0.25 mM), rBSA or BSA (2 μM), and HEPES buffer (10 mM, pH 8) was incubated overnight at 27°C. Presumably, this method produced relatively large AuNPs with a size suitable for DFM observation.15 To purify the AuNPs, the solution was centrifuged at 10000 × g for 15 min at 4°C to remove unbound rBSA or BSA, and the AuNPs were re-dispersed in a 10-times diluted PBS buffer and stored at 4°C. The formation of AuNPs was verified by dynamic light scattering (DLS) analysis (Zetasizer Nano-ZS, Malvern, Worcestershire, UK).

Evaluation of interaction between rBSA/BSA and anti-BSA using a dot blot assay

The binding of anti-BSA (rabbit, product number A11133, Molecular Probes, OR, USA) to the rBSA or BSA was evaluated by a dot blot assay. Six microliters (2 μL × 3) of rBSA or BSA (100 μg/mL) was spotted onto a nitrocellulose membrane. After blocking with 5% skim milk in TBS containing 0.01% Tween

20 (0.01% TBST) for 1 h at room temperature, the membrane was incubated with anti-BSA (1:5000) for 1 h at room temperature, followed by a secondary HRP-conjugated goat anti-rabbit IgG antibody (1:5000, product number HAF008, R&D Systems, Minneapolis, MN) for 1 h at room temperature. Proteins on the spots were visualized using the Clarity Western ECL Substrate (Bio-Rad, Hercules, CA). Luminescence was detected with a LAS 4000 mini luminescent Image Analyzer (Fujifilm, Tokyo, Japan), using the Image Reader LAS4000 software. The ImageJ software was used to determine the averaged intensity of each dot area.

Anti-BSA detection by analyzing rBSA-modified AuNP aggregationAnti-BSA was detected by analyzing the aggregation of

rBSA-AuNPs. Various concentrations of anti-BSA (0 – 40 nM) were added to the rBSA-AuNP solutions (OD630 = 0.75), and the resulting samples were incubated at room temperature for 1 h. As a control experiment, 40 nM BSA was added in one of the samples to inhibit the interaction between the anti-BSA and rBSA-AuNPs. The anti-BSA-induced aggregation of rBSA-AuNPs was evaluated through UV-VIS analysis (Shimadzu, Kyoto, Japan) and DFM, as described below.

DFM observationThe AuNP samples were spotted on a 3-Aminopropyl-

triethoxysilane (APTES)-treated slide glass (APTES-glass) for DFM imaging. The APTES-treated glass was prepared by following a previously described method.20 Briefly, the glass slide was immersed in a 1% APTES solution (Tokyo Chemical Industry, Tokyo, Japan) for 30 min at room temperature, rinsed with ultrapure water, and sonicated for 10 min. The glass was dried and stored at room temperature. The AuNP solutions (3 μL) were dropped on the APTES-glass, and then covered with a cover glass. DFM images were captured using a BX53 microscope (Olympus, Tokyo, Japan) equipped with a UDCW darkfield condenser, UPlanFLN 60× objective lens, and DP73 CCD camera (Olympus). The CellSens Standard software Ver. 1.6 (Olympus) was used. The intensity of each DFM spot was evaluated using the ImageJ software.19,24 To obtain the aggregation ratio of AuNPs, bright spots having an intensity higher than 100 were defined as aggregates since 90% of the negative control samples (samples without anti-BSA) had an intensity lower than 100.

Results and Discussion

Preparation of rBSA-functionalized AuNPsAuNPs were successfully prepared from a chloroauric acid

(HAuCl4) solution using BSA or rBSA as reducing agents. The

Fig. 1　Schematic illustration of the anti-BSA-induced aggregation of rBSA-AuNPs.

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DLS analysis revealed that the average size of the synthesized AuNPs was approximately 60 nm (Fig. 2A). In addition, the size distributions of the AuNPs synthesized using rBSA or BSA were similar, suggesting that there were sufficient amino acid residues with good reducing abilities, such as cysteine and tyrosine for AuNP synthesis (35 cysteine and 21 tyrosine residues in BSA). This is supported by the previous report showing that one or three tyrosine is enough for AuNP synthesis using peptide.15,16 The bluish color of the sample solutions (Fig. 2B) was similar to that reported for the AuNP solutions synthesized using the A3 peptide at pH 8.15 The TEM images of

the AuNPs synthesized using A3 revealed the formation of sea urchin-shaped AuNPs.15 Thus, it is presumed that sea urchin-shaped AuNPs were also synthesized using BSA or rBSA.

Evaluation of binding of anti-BSA to rBSA/BSA using a dot blot assay

The binding of anti-BSA to rBSA or BSA was examined

Fig. 3　Affinity of anti-BSA toward BSA/rBSA.A. Dot blot assay to estimate the binding of anti-BSA to BSA or rBSA. Three spots from the same amout of BSA or rBSA (6 μL of 100 μg/mL rBSA or BSA) are shown.B. Intensity of the spots. The average and standard deviation values of the intensities from the three spots in Fig. 3A are indicated.

Fig. 2　Synthesis of AuNPs using rBSA or BSA.A. Size distribution of the synthesized NPs.B. Color of the synthesized AuNP solutions.

Fig. 4　Anti-BSA-induced aggregation of rBSA-AuNPs.A. UV-VIS spectra of rBSA-AuNP samples incubated with anti-BSA. BSA was added as a control (rBSA-AuNP+BSA+anti-BSA).B. Color of the rBSA-AuNP solutions (1: rBSA-AuNP, 2: rBSA-AuNP+anti-BSA, and 3: rBSA-AuNP+BSA+anti-BSA).

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using a dot blot assay. The same amounts of rBSA or BSA were spotted on the membrane, and the amount of bound anti-BSA was evaluated (Figs. 3A and 3B). The affinity of anti-BSA toward rBSA was approximately 1.7-fold higher than that toward BSA. It is noted that no signal was detected for the rBSA sample without anti-BSA but with the secondary antibody (data not shown), supporting that non-specific interaction of the secondary antibody to rBSA was negligible. Presumably, the epitopes recognized by an antibody are exposed in rBSA, i.e. when BSA is reduced and denatured, which improves its

reactivity toward antibodies. Thus, rBSA-AuNPs were used for further analysis.

Anti-BSA detection by rBSA-AuNP aggregationThe aggregation of rBSA-AuNPs upon the addition of anti-

BSA was preliminarily examined via the UV-VIS analysis and color change of the solution (Fig. 4). As shown in Fig. 4A, the absorption intensity decreased and the peak red-shifted when anti-BSA was added, indicating the formation of aggregates. These findings are consistent with a recent report by Zheng

Fig. 5　Detection of anti-BSA using DFM.A. DFM images of the rBSA-AuNP samples incubated with different anti-BSA concentrations ranging from 0 to 40 nM. BSA (40 nM) was added as a negative control (40 nM anti-BSA+40 nM BSA).B. Intensity histogram of AuNP aggregates observed via DFM.C. Ratio of aggregated AuNPs at various anti-BSA concentrations. BSA (40 nM) was added as a negative control (40 nM anti-BSA+40 nM BSA). The ratio of the aggregated AuNPs was obtained using the intensity histogram from each image, and the average and standard deviation values from three different images are shown.

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et al.25 The aggregation of rBSA-AuNPs was also verified by visually observing the color change of the solution (Fig. 4B). Importantly, the color and UV-VIS spectrum of the solution remained largely unchanged in the control experiment, i.e. when BSA was added to the rBSA-AuNP solution prior to the addition of anti-BSA in an aim to inhibit interaction between anti-BSA and rBSA on the AuNP surface (Fig. 4). This indicates that the aggregation of rBSA-AuNPs occurs because of the interaction between anti-BSA and rBSA on the AuNP surface.

Anti-BSA detection by DFM observationThe formation of the anti-BSA-induced rBSA-AuNP aggregates

was investigated at different anti-BSA concentrations by using DFM (Fig. 5). DFM images revealed the formation of large AuNP aggregates upon the addition of 20 nM anti-BSA solution (Fig. 5A). Moreover, the intensity histogram indicates that the brightness of the spots increased with an increase in the concentration of anti-BSA, thus verifying that the aggregation of rBSA-AuNPs is dependent on anti-BSA (Fig. 5B). In addition, the aggregation of rBSA-AuNPs did not occur in the presence of free BSA even at high anti-BSA concentrations. The data from the intensity histograms were used to calculate the ratio of aggregated rBSA-AuNPs at different anti-BSA concentrations (Fig. 5C). The aggregate ratio was also dependent on the anti-BSA concentration. There was a significant difference in the aggregation ratio between the control sample (without anti-BSA) and the sample with 20 nM of anti-BSA, indicating that the proposed approach can be used to detect anti-BSA with a concentration as low as 20 nM. In addition, a previous report has demonstrated the application of DFM to detect AuNP aggregation in colored solutions, such as blood, wherein the color change of the solution due to AuNP aggregation cannot be observed.20 These findings demonstrate the efficiency of DFM for the detection of anti-BSA via AuNP aggregate formation.

Conclusions

In this study, AuNPs were synthesized using rBSA as the reducing agent. In addition, rBSA conjugated with AuNPs via Au-Sulfur interactions form rBSA-AuNPs. The prepared rBSA-AuNPs were used for the detection of anti-BSA as a model by observing the AuNP aggregation induced by the interaction of anti-BSA with rBSA. Anti-BSA was successfully detected at a concentration as low as 20 nM by using the proposed approach. Since protein-modified AuNPs can be synthesised using various proteins that possess cysteine and other amino acid residues with reducing ability such as tyrosine, this study demonstrates the potential applications of such protein-modified AuNPs in the field of bioanalysis. Furthermore, the DFM-based analysis of the protein-modified AuNP aggregation is confirmed to be effective for antibody detection. Therefore, the proposed novel approach could be useful in biomedical applications such as disease diagnosis.

Acknowledgements

This work was supported mainly by JSPS KAKENHI grant

number JP19H02527 (T. Z.) and Ehime University (Research Unit for Advanced Nano-Bioanalysis), and in part by JSPS KAKENHI grant number JP19H00841 (N. K.).

References

1. P. Baptista, E. Pereira, P. Eaton, G. Doria, A. Miranda, I. Gomes, P. Quaresma, and R. Franco, Anal. Bioanal. Chem., 2008, 391, 943.

2. T. Zako, Anal. Sci., 2020, 36, 509. 3. J. Chen, I. R. Corbin, H. Li, W. Cao, J. D. Glickson, and G.

Zheng, J. Am. Chem. Soc., 2007, 129, 5798. 4. Q. Mu, F. M. Kievit, R. J. Kant, G. Lin, M. Jeon, and M.

Zhang, Nanoscale, 2015, 7, 1. 5. M. J. Meziani and Y. P. Sun, J. Am. Chem. Soc., 2003, 125,

8015. 6. A. Csáki, P. Kaplanek, R. Möller, and W. Fritzsche,

Nanotechnology, 2003, 14, 1262. 7. N. Sakono, K. Nakamura, T. Ohshima, R. Hayakawa, and

M. Sakono, Anal. Sci., 2019, 35, 79. 8. J. Turkevich, P. C. Stevenson, and J. Hillier, Discuss.

Faraday Soc., 1951, 11, 55. 9. M. Brust, M. Walker, D. Bethell, D. J. Schiffrin, and R.

Whyman, J. Chem. Soc. Chem. Commun., 1994, 801. 10. Y. Xiao, V. Pavlov, S. Levine, T. Niazov, G. Markovitch,

and I. Willner, Angew. Chem., Int. Ed., 2004, 43, 4519. 11. Y. Djohan, T. Azukizawa, Patmawati, K. Sakai, Y. Yano, F.

Sato, R. Takahashi, M. Yohda, M. Maeda, N. Kamiya, and T. Zako, Biomater. Sci., 2019, 7, 1.

12. C. L. Chen and N. L. Rosi, Angew. Chem., Int. Ed., 2010, 49, 1924.

13. C. Hart, N. Abuladel, M. Bee, M. C. Kreider, A. C. Cvitan, M. M. Esson, A. Farag, T. Ibeh, E. N. Kalivas, D. M. Larco, A. Walker Long, L. Lymperopoulos, Z. Mendel, N. Miles, C. M. Zareba, J. C. Schwabacher, H. Slucher, J. Vinals, J. M. Heddleston, W. Li, D. M. Fox, and M. R. Hartings, Dalton Trans., 2017, 46, 16465.

14. J. M. Slocik, M. O. Stone, and R. R. Naik, Small, 2005, 1, 1048.

15. K. Shimojo, T. Niide, T. Taguchi, H. Naganawa, N. Kamiya, and M. Goto, Analyst, 2012, 137, 2300.

16. J. M. Slocik, R. R. Naik, M. O. Stone, and D. W. Wright, J. Mater. Chem., 2005, 15, 749.

17. X. Huang, P. K. Jain, I. H. El-Sayed, and M. A. El-Sayed, Nanomedicine, 2007, 2, 681.

18. H. Jans, X. Liu, L. Austin, G. Maes, and Q. Huo, Anal. Chem., 2009, 81, 9425.

19. T. Bu, T. Zako, M. Fujita, and M. Maeda, Chem. Commun., 2013, 49, 7531.

20. T. Bu, T. Zako, and M. Maeda, Anal. Sci., 2016, 32, 307. 21. Y. Yano, M. Nisougi, Y. Yano-Ozawa, T. Ohguni, A. Ogawa,

M. Maeda, T. Asahi, and T. Zako, Anal. Sci., 2019, 35, 685. 22. Y. Sun and Y. Huang, J. Mater. Chem. B, 2016, 4, 2768. 23. M. A. Razi, R. Wakabayashi, M. Goto, and N. Kamiya,

Langmuir, 2019, 35, 2610. 24. C. A. Schneider, W. S. Rasband, and K. W. Eliceiri, Nat.

Methods, 2012, 9, 671. 25. B. Zheng, J. Li, Z. Zheng, C. Zhang, C. Huang, J. Hong, Y.

Li, and J. Wang, Opt. Laser Technol., 2021, 133, 106522.