In this article, a facile and effective technique is described to prepare a complex of gold nanoparticles (GNPs)/protein A (PrA) for biosensors in virus detection. GNPs were synthesized by the reduction of tetrachloroauric (III) acid trihydrate using sodium ascorbate, and then coated with PrA via ultracentrifugation. The complex of GNPs/PrA was characterized using UV-vis spectroscopy and transmission electron microscopy. The immunogold labeling method of scanning electron microscopy was also used to verify the capacity for the detection and binding of GNPs/PrA to H$_{1}$N$_{1}$ influenza A virus particles. The results showed that GNPs were spherical, uniform shape, and approximately 10 nm in size. Noticeably, the complex of GNPs/PrA could detect and bind effectively to H$_{1}$N$_{1}$ influenza A virus particles by a large number of GNPs surrounded. The advantage of the complex of GNPs/PrA showed a highly potential application in biosensors with the improvement of the sensitivity and transducing signal for virus detection

Huy, Tran Quang

Tuan, Mai Anh

Vietnam Academy of Science and Technology: Journals Online

Communications in Physics, Vol. 21, No. 4 (2011), pp. 333-339
SYNTHESIS OF GOLD NANOPARTICLES CONJUGATED
WITH PROTEIN A: TOWARDS THE APPLICATION
IN BIOSENSORS FOR VIRUS DETECTION
TRAN QUANG HUY
National Institute of Hygiene and Epidemiology
MAI ANH TUAN
International Training Institute for Materials Science,
Hanoi University of Science and Technology
Abstract. In this article, a facile and effective technique is described to prepare a complex of gold
nanoparticles (GNPs)/protein A (PrA) for biosensors in virus detection. GNPs were synthesized
by the reduction of tetrachloroauric (III) acid trihydrate using sodium ascorbate, and then coated
with PrA via ultracentrifugation. The complex of GNPs/PrA was characterized using UV-vis
spectroscopy and transmission electron microscopy. The immunogold labeling method of scanning
electron microscopy was also used to verify the capacity for the detection and binding of GNPs/PrA
to H1N1 influenza A virus particles. The results showed that GNPs were spherical, uniform shape,
and approximately 10 nm in size. Noticeably, the complex of GNPs/PrA could detect and bind
effectively to H1N1 influenza A virus particles by a large number of GNPs surrounded. The
advantage of the complex of GNPs/PrA showed a highly potential application in biosensors with
the improvement of the sensitivity and transducing signal for virus detection.
I. INTRODUCTION
Gold nanoparticles (GNPs) have been known to have unique and superior proper-
ties due to high surface-to-volume ratio, high surface energy, high biocompatibility and
ability to facilitate electron transfer between redox proteins and electrode surfaces [5, 9].
GNPs and their products have been widely used in many fields of life sciences such as
immuno-cytochemistry, pathogen detection and biosensors [7, 10, 13, 15, 18]. It has been
shown that the optical and electrical properties of GNPs strongly depend on their size,
shape and nanostructure, therefore the controllable synthesis of GNPs with well-defined
geometry and properties is a key challenge to promote their applications [11]. Recent
development of nanotechnology showed a number of methods to synthesize GNPs, called
“top-down” and “bottom-up” resulted in desired nanostructure, size, and shape [5, 9, 14].
In these methods, the reduction of gold salts has more interest thanks to ease, low cost
and uniform shape of GNPs formed, it is also easy to protect these nanoparticles from
aggregation when using some stabilizing agents such as surfactants, polymers, micelles
and biomolecules [5]. In biomedical appplications, GNPs usually use bio-molecules for
protection and functionalization [6] such as protein A, because this molecule has a high
affinity to GNPs. Protein A also bind and orientate well some antibodies immobilized on
the solid surfaces [2, 8].
334 TRAN QUANG HUY AND MAI ANH TUAN
Recently, there has been an increasing demand for the development of biosensors
in rapid and label free detection of pathogens [16]. It revealed that the reliability of anti-
body based-biosensors strongly depends on both the number of antibody immobilized on
the sensor surface and the design structure of transducers. Several methods were devel-
oped to improve the sensitive and selective performance of biosensors such as minimized
electrodes with micro- or nano sizes; and/or the optimal immobilization of antibody on
the sensor surface; and/or the modification of sensor surface with nanomaterials (GNPs,
silver nanoparticles, carbon nanotubes, etc) [3,11,13,20,21]. Despite all these efforts, the
improvement of the sensitivity and selectivity of biosensors has not been comparable to
the requirement of the market yet. In fact, a few or large number of viruses depends
strongly on different clinical samples, this makes the difficulty of most biosensors in virus
detection.
In the present work, we describe a facile synthesis of the complex of GNPs/PrA for
the effective detection of virus particles as well as improvement of signal between virus
particles and electrodes of biosensors.
II. EXPERIMENTAL
Reagents
Tetrachloroauric (III) acid trihydrate 99.5% (HAuCl4*3H2O), postasium carbon-
ate (K2CO3), protein A (PrA), bovine serum albumin (BSA)were purchased from Merck.
Sodium ascorbate (C6H7NaO6) and polyethylene glycol (PEG) were purchased from Sigma.
Distilled water used was purified through a Millipore system. All other chemicals were of
analytical grade.
The samples of inactivated H1N1 influenza A virus and human serum containing
antibodies to H1N1 influenza A virus were provided by the National Institute of Hygiene
and Epidemiology (NIHE) of Vietnam.
Synthesis of GNPs
0.5 ml of l% HAuCl4 and 0.5 ml of 0.1M K2CO3 were mixed in 12 ml of distilled
water at 4˚ C. Then, added quickly 1 ml of 0.7% C6H7NaO6 into the above solution during
stirring. The color would become immediately purple-red. Distilled water was then added
to the solution till 50 ml of volume and boiled until the color became red. The morphology
and size distribution of GNPs were immediately checked by using the transmission electron
microscopy (JEM 1010, JEOL) operating at 80 kV
Preparation of GNPs/PrA complex
1 mg/ml of PrA was mixed with 15 ml of the GNPs solution. After 5 min, 0.15 ml of
0.02 % PEG was added to stabilize the complex. GNPs/PrA solution was ultracentrifuged
(Airfuge, Beckman Coulter) for 45 min at 70,000 rpm. The supernatant was removed
carefully. The pellet was dissolved in the remained solution and layered over a 50% glycerol
gradient containing 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.4, and 0.02% sodium
azide. The gradient was centrifuged for 20 min at 20,000 rpm. A solution of GNPs/PrA
complex was obtained by collecting the red zone of the gradient. The final product of
GNPs/PrA was investigated using the UV-vis spectrometry (HP 8453 Spectrophotometer)
SYNTHESIS OF GOLD NANOPARTICLES CONJUGATED WITH PROTEIN A ... 335
in the wavelength region 300 – 900 nm and transmission electron microscopy (JEM 1010,
JEOL) operating at 80kV.
Immuno-gold labeling test
Firstly, H1N1 influenza A virus was inactivated and fixed in 2.5% glutaraldehyde
for 1 hr at 4˚ C. After that, dehydration by series of alcohol concentration and a droplet
of virus sample was place on the aluminum surface, treated with 2 % BSA/PBS for 20
min to block non-specific antigenic sites. The sample was then incubated with the diluted
human serum in 0.5% BSA/PBS for 45 min. After careful washes with 0.2 % BSA/PBS,
the sample was subsequently incubated with the GNPs/PrA complex (diluted 1:60 in 0.5%
BSA/PBS) for 30 min, washed with PBS and air-dried. The sample was coated by a thin
platinum (Pt) layer before observation with a high resolution scanning electron microscopy
(FESEM- S4800, Hitachi).
III. RESULTS AND DISCUSSION
UV–vis spectroscopy analysis
The GNPs were obtained by the reduction from a gold salt using sodium ascor-
bate. This process occurred through the electrons transfer from the hydroxyl groups of
the sodium ascorbate to the Au3+ ions leading to the formation of Au0. The obtained
solution was checked by UV-vis spectrometry. Fig. 1 shows UV–vis absorption spectra
of GNPs/PrA complex. The surface plasmon resonance (SPR) peak of GNPs was found
at the wavelength of 524 nm, indicated the formation of the GNPs. The absorbance is
extremely sensitive to the morphology and nature of GNPs formed, their inter-particle
distances and the surrounding media [12]. According to the Mie’s theory, the averaged
GNPs diameter could be estimated from the maximum absorption band, the diameter of
GNPs increased when the maximum absorption band shifts to longer wavelength [4]. It
is important to note that the use of PrA as a stabilizer resulting in the absorption peak
of GNPs/PrA complex nearly unchanged even after 6 months in storage at – 20˚ C. These
results indicated that PrA could also stabilize GNPs as well as protect them from ag-
gregation and precipitation over the time. Moreover, PrA could retain well its biological
activity at this temperature [1].
Stability of GNPs size distribution
The morphology and size distribution of GNPs in the colloidal solution were investi-
gated using transmission electron microscopy. Fig. 2A shows GNPs before treatment with
PrA, these GNPs are mainly spherical, but some of GNPs clustered and void spaces made
which could lead to the aggregation of GNPs. Fig. 2B shows the distribution histogram of
GNPs reduced in which the average size is about 10 nm, this result is also highly agreed
with the UV-vis investigation. Furthermore, the image revealed GNPs formed with a
complete reduction due to sodium ascorbate, this reductant was also preferred to form
GNPs in several studies [19,22].
After treatment with PrA, a mono-dispersed and non-clustered distribution of GNPs
was found (Fig. 3A). This can be understood that the use of ultracentrifugation and
glycerol gradient resulted in the adhesion of PrA molecules around GNPs, and prevented
336 TRAN QUANG HUY AND MAI ANH TUAN
Fig. 1. UV-vis absorption spectra of the complex of GNPs/PrA synthesized,
and after six months in storage at −20˚ C. The inset is the colloidal solution of
GNPs/PrA synthesized.
Fig. 2. TEM image of GNPs before treatment with PrA (A), and sizes distribu-
tion histogram of 386 GNPs in the colloidal solution (B)
SYNTHESIS OF GOLD NANOPARTICLES CONJUGATED WITH PROTEIN A ... 337
GNPs from aggregation. The result showed that PrA revealed as a good stabilizer in
comparison with other stabilizers such as polymers and micelles [5,9]. GNPs also remained
spherically and homogeneously over the time. The complex of GNPs/PrA stabilized for
more than six months in storage at -20˚ C [Fig. 3B]
Fig. 3. GNPs after treatment with PrA. (A) The distribution of GNPs and the
left corner inset of GNPs of approximately 10 nm in size. (B) the stability of
GNPs/PrA distribution after 6 months in storage at -20˚ C
Immunogold labeling reaction with H1N1 A influenza virus particles
In order to estimate the biological activity of the complex of GNPs/PrA synthe-
sized, this product was used for immunogold labeling methods in the detection of H1N1
A influenza virus particles by scanning electron microscopy. Fig. 4a shows the image of
many H1N1 A influenza virus particles (arrows) before the immunogold labeling reaction,
this image revealed the morphology of H1N1 A influenza virus particles with spherical-
and rod-like shapes and withour the attachment of GNPs. Whereas after the reaction
with GNPs/PrA associated with human serum containing antibodies to H1N1 influenza
A virus, the results of the Fig. 4b revealed these virus particles localized by many GNPs
(head arrows). A large number of GNPs surrounded around H1N1 influenza A virus par-
ticles demonstrated that many viruses could be detected by the localization of GNPs.
In this method, the Fc fragment of the anti-viral IgG antibodies would bind strongly to
the PrA, and the Fab fragments bind specifically to the viral antigen sites [1]. It also
proved that many anti - viral antibodies could be attached on the surface of GNPs via
PrA molecules [10,15].
Towards the application of GNPs/PrA complex in biosensors for virus detec-
tion
GNPs have excellently biocompatible and potential carriers, especially their ability
to facilitate the direct transfer of electrons between redox proteins and micro-electrodes
leading to the performance of the electrochemical sense even without electron transfer
mediators [13]. In a recent review, Yuanyuang et al. showed the extensive application of
GNPs in construction of electrochemical biosensors in which GNPs play crucial roles both
338 TRAN QUANG HUY AND MAI ANH TUAN
Fig. 4. SEM photograph of H1N1 influenza A virus particles: (a) without the
use of GNPs/PrA complex, and (b) with the use of GNPs/PrA complex (arrows:
H1N1 influenza A virus particles; arrowheads: GNPs).
in the enhancement of the electrochemical signal biosensing process and their mechanism
for improving analytical performance, such as: electron transfer “electron wires”, immo-
bilization platform and electrocatalyst [11]. Moreover, PrA is very heat-stable and retains
the native conformation with some physical and chemical factors, it binds specifically to
the Fc region of the immunoglobulin molecules without disturbing the binding of antigen
[1]. Chih et al. reported that using PrA is a good way of immobilizing antibodies onto
the silica based surface [2]. Another paper also reported that 30%–70% higher antibod-
ies could be bound on silica-tagged PrA than on uncoated silica surface [8]. In a recent
study, we have demonstrated that the use of prA is an advantageous way to immobilize
serum antibodies for electrochemical biosensors [20]. In addition, our results of this work
also show the comlex of GNPs/PrA could detect and bind well to H1N1 A influenza virus
particles. Hence, the combination of GNPs and PrA is expected to be a good idea for
improvement of the sensitivity and transducing signal in biosensors for virus detection.
IV. CONCLUSION
In this paper, we represented the facile synthesis of GNPs/PrA complex for biosensor
applications in virus detection. The results showed that this technique provided GNPs
spherically, homogeneously and approximately 10 nm in size. The use of PrA as the
stabilizer resulted in a mono-dispersed and non-clustered distribution of GNPs in solution
over the time. Furthermore, the complex of GNPs/PrA formed could detect and bind
well to H1N1 influenza A virus particles, and it showed the potential applications in the
improvement of the sensitivity and transducing signal in biosensors for virus detection.
ACKNOWLEDGEMENTS
The authors would like to thank MSc. Nguyen Thanh Thuy, National Institute
of Hygiene and Epidemiology for the kind support of SEM immunogold labelling tests;
SYNTHESIS OF GOLD NANOPARTICLES CONJUGATED WITH PROTEIN A ... 339
Dr. Le Anh Tuan, Advanced Intitute for Science and Technology, Hanoi University of
Science and Technology for helpful comments. This work was financially supported by
Vietnam’s National Foundation for Science and Technology Development (NAFOSTED),
project code: 106.16.181.09.
REFERENCES
[1] A. Surolia, D. Pain and M.I. Kahn, Trend. Biochem. Sci. 7 (1982) 74
[2] C. S. Chih, Z.W. Tzong, K.C. Li, H.Y Hui and F.T Dar, Anal. Chim. Acta 479 (2003) 117
[3] D.L. Alfredo, M. Escosura, P. Claudio and M. Arben, Materialstoday 13 (2010) 24
[4] G. Mie. Ann. Phys. (Leipzig) 25 (1908) 377
[5] G. Shaojun and W. Erkang. Anal. Chim. Acta 598 (2007) 181
[6] H. Abdelghani , A. Marya, L. Shiyong and N. Ravin. J. Phys. Chem. C112 (2008) 12282
[7] H.M.E. Azzazy and M.H.M. Mansour, Clin. Chim. Acta 403 (2009) 1
[8] H.Y. Takeshi, N. Ken-ichi, I. Yoshiaki, T. Keigo, A. Satoka, H. Ryuichi, and K. Akio, Anal. Biochem.
385 (2009) 132
[9] J. Zhou, J. Ralston, R. Sedev and D.A. Beattie. J. Colloid Interface Sci. 331 (2009) 251
[10] J.W. Slot, and H.J. Geuze, J. Cell Biol. 90 (1981) 533
[11] L. Yuanyuang, J.S. Hermann and X. Shunquing, Gold Bulletin 10 (2010) 29
[12] M. Audrey and G. Fr\‘ed\‘e ric, New J. Chem. 30 (2006) 1121
[13] M.P. Jose, Y. Paloma, and G.C. Araceli, Electrochimica Acta 53 (2008) 5848
[14] N.T. H. Lien, L.T. Huyen, V.X. Hoa, C.V. Ha, N.T. Hai, L.Q. Huan, F. Emmanuel, D.Q. Hoa, and
T.H. Nhung, Adv. Nat. Sci.: Nanosci. Nanotechnol. 1 (2010) 025009
[15] N.V. Man et al., C. R. Acad. Sci. Paris. Life Sciences 324 (2001) 815
[16] P. Bobby, D.M. Roland, and P. Gordon, Analyst 131 (2006) 1979
[17] P. Setua, A. Chakraborty, D. Seth, M.U. Bhatta, P.V. Satyam and N. Sarkar. J. Phys. Chem. C111
(2007) 3901
[18] S.A. Sarit, R. Subinoy, H.P. Myoung, K.K. Chae, C.Y. Chang and M.R. Vincent, Adv. Drug Deliv.
Reviews 62 (2010) 316
[19] S.H. Ding, W.P Qian, Y. Tan, and Y. Wang, Langmuir 22 (2006) 7105
[20] T. Q. Huy, N. T. H. Hanh, P. V. Chung, D. D. Anh, P. T. Nga and M. A. Tuan, Appl. Sci. Surf.
257 (2011) 7090
[21] V. V Jithesh and Y Kaiming, Biotech. Progress 23 (2007) 517
[22] Y. Tan, W. P. Qian, S. H. Ding, and Y. Wang, Chem. Mater. 18 (2006) 3385
Received 5 June 2011.


Synthesis of Gold Nanoparticles Conjugated with Protein A: Towards  the Application in Biosensors for Virus Detection

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