- Email: [email protected]
Label-free immunosensor based on gold nanoparticle silver enhancement
Analytical Biochemistry 385 (2009) 128–131
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Analytical Biochemistry j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y a b i o
Label-free immunosensor based on gold nanoparticle silver enhancement Minghui Yang a, Cunchang Wang b,* a b
Center for Advanced Sensor Technology, University of Maryland, Baltimore County, MD 21250, USA Department of Chemistry, Xiangnan University, Chenzhou 423000, China
a r t i c l e
i n f o
Article history: Received 1 September 2008 Available online 01 November 2008 Keywords: CCD camera Gold nanoparticles Label-free immunosensor Silver enhancement
a b s t r a c t A label-free immunosensor for the sensitive detection of human immunoglobul in G (IgG) was prepared based on gold nanoparticle–silver enhancement detection with a simple charge-coupled device (CCD) detector. The gold nanoparticles, which were used as nuclei for the deposit of metallic silver and also for the adsorption of antibodies, were immobilized into wells of a 9-well chip. With the addition of silver enhancement buffer, metallic silver will deposit onto gold nanoparticles, causing darkness that can be optically measured by the CCD camera and quantified using ImageJ software. When antibody was immo bilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex resulted in a decrease of the darkness and the intensity of the darkness was in line with IgG concentrations from 0.05 to 10 ng/ml. The CCD detector is simple and portable, and the reported method has many desirable merits such as sensitivity and accuracy, making it a promising technique for protein detection. © 2008 Elsevier Inc. All rights reserved.
Immunosensors have been widely studied for clinical diagno sis, environmental pollutant detection, and food analysis because they are simple, sensitive, and highly selective. Typically, antigens are selectively bound to their complementary antibodies on a solid surface and then to the secondary antibody that is labeled with a probe, which is called the traditional “sandwich” assay [1–3]. Dif ferent probes have been labeled to the secondary antibody with different detection techniques such as enzymes, fluorescent dyes, and nanoparticles. Enzyme-linked immunosorbent assay (ELISA)1 is one of the oldest and most widely used laboratory methods used as a diagnostic tool in medicine as well as a quality control check in various industries [4–6]. Fluorescence immunoassays are based on antibodies labeled with fluorophores; however, the fluo rophores are easily subjected to photobleaching, which may result in reduced accuracy [7,8]. Recently, nanoparticles such as gold nanoparticles and quantum dots have been widely used as labels because they are stable and cheap [9–11]. Besides the above-mentioned traditional sandwich assay, labelfree immunosensors based on direct detection of antibody–antigen interactions have also gained growing interest. Park and coworkers immobilized antibodies on (R)-lipo-diaza-18-crown-6 self-assem bled monolayer modified gold electrodes, and the blocking effect caused by the Ab–Ag adducts formed on electrode surface resulted in an increased charge transfer resistance (Rct) of the electrode for * Corresponding author. Fax: +86 735 2653013. E-mail address: [email protected] (C. Wang). 1 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; ECL, electro chemilumin escence; CCD, charge-coupled device; SEB, staphylococcal enterotoxin B; IgG, immunoglobulin G; BSA, bovine serum albumin; RSD, relative standard deviation. 0003-2697/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.10.019
a redox probe, which provides a basis for quantification of Ag in solution [12]. Jie and coworkers immobilized CdSe nanocrystals, carbon nanotube, and chitosan composite film on electrode sur face and investigated the electrochemiluminescence (ECL) of the film. After antibody was bound to the film, the specific immunore action between antibody and antigen resulted in a decrease of ECL intensity [13]. Silver enhancement, a procedure for electroless silver deposi tion where the nanoparticles act as nuclei, has been widely used for immunoassays [14–16]. In this technique, colloidal metal par ticles act as catalysts to reduce silver ions to metallic silver, which is typically quantified by electrochemical measurement [17] or anodic stripping [18]. Examples of immunoassays using colloidal gold nanoparticles combined with silver enhancement have been demonstrated on microarrayed substrate [19], between coplanar electrodes [20], and through microfluidic channels [21]. To the best of our knowledge, no immunosensor based on silver enhancement has been reported using a charge-coupled device (CCD) camera. Previously, our lab reported a carbon nanotube-based immu nosensor for the detection of staphylococcal enterotoxin B (SEB) using a CCD camera [22]. In the current article, we report a novel method for the fabrication of label-free immunosensors using sil ver enhancement. Gold nanoparticles were immobilized in wells of 9-well chips and were used as nuclei for the deposit of metallic sil ver and also for the immobilization of antibodies. With the forma tion of antibody–antigen immunocomplex, the amount of metallic silver deposited onto the nanoparticles decreased compared with that without the immunocomplex due to the decreased contact of the gold nanoparticles with silver enhancement buffer, The dark ness of the wells in accordance with the amount of metallic silver
Label-free immunosensor / M. Yang, C. Wang / Anal. Biochem. 385 (2008) 128–131
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was optically measured by a CCD camera, and the immunosensor provided a convenient, low-cost, sensitive, and specific method for biomolecular detection. Materials and methods Materials and reagents Gold nanoparticles (G1527, » 0.75 A520 U/ml, 8.5–12.0 nm mean particle size [monodisperse], 10 nm particle size), goat antihuman immunoglobulin G (IgG) antibody, human IgG antigen, and a Silver Enhancer Kit (SE-100) were products of Sigma–Aldrich (St. Louis, MO, USA). The Silver Enhancer Kit consists of solution A (sil ver salt) and solution B (initiat or). Real human blood samples were provided by the University of Maryland Medical Center. All other reagents were of analytic al grade, and deionized water was used throughout. Apparatus For the CCD-based detection, the schematic configuration of the CCD detector is shown schematically in Fig. 1A, and Fig. 1B is a photograph of the actual detection platform. The CCD-based detec tor consists of an enclosure and an Atic-16 cooled CCD camera or SXVF-M7 (Adirondack Video Astronomy, Hudson Falls, NY, USA). Both cooled CCD cameras employ a Sony ICX-429ALL with a 752 £ 582-pixel CCD and are equipped with a 5-mm extension tube and a 12-mm Pentax f1.2 lens (Spytown, Utopia, NY, USA). The system also includes an illuminator. The 9-well sample chips used in this study were designed in CorelDraw 11 (Corel, Ontario, Canada) and were micromachined in 1/8-inch black acrylic using a computercontrolled laser cutter (Epilog Legend CO2 65W, Epilog, Golden, CO, USA). Polycarbonate film was attached to one side of the chips to hold the chemical solution (Fig. 1C). The CCD image intensities were analyzed using ImageJ software. Preparation of the immunosensor For the preparation of the immunosensor, 15 ll of gold nano particles was first dropped into the wells of the 9-well chips. When dried, the wells were washed with phosphate buffer (50 mM, pH 7.4) and 10 ll of 0.1 mg/ml antibody solution was added into the wells and incubated for 1 h. After rinsing with phosphate buffer, the chip was incubated with 10 ll of 1% bovine serum albumin (BSA) for 1 h to block nonspecific binding sites. Finally, the chip was rinsed and 10 ll of different concentrations of IgG solution were added into the wells and incubated for 45 min. Silver enhancement detection For silver enhancement detection, 10 ll of silver enhancement buffer (mixture of the two solutions from the Silver Enhancer Kit in a 1:1 volume ratio prepared just prior to use) was added into the wells of the chip, the chip was incubated in the dark for 10 min, and then picture was taken by the CCD detector immediately.
Fig. 1. The CCD detector: (A) schematic configuration of the detector; (B) photo graph of the actual detection platform; (C) actual 9-well sample chip. 1, plastic enclosure; 2, Atic-16 CCD camera [2]. A 5-mm extension tube (3) was attached to the 12-mm Pentax f1.2 lens (4). A black acrylic shelf box (5) was designed to hold the sample chip (6) and fluorescence illumination module (7).
(silver ions and initiator) and incubation in the dark for 10 min, the picture was taken using the CCD camera. Under illumination, well A appears to be totally white because there was no gold nanopar ticles and so no metallic silver deposited and the light from the illuminator was collected by the CCD. On the contrary, in well B, when gold nanoparticles were introduced, the gold nanoparticles served as nucleation sites to catalyze the reduction of silver ions to metallic silver and were enlarged in the process by up to five orders of magnitude [16]. As a result, the light from the illuminator was partially blocked, displaying a decrease in light intensity. In well C, after immobilization of the antibody and then antigen, the light intensity increased. The reason was that the formed immuno complex after specific immunoreaction greatly inhibited the con tact of gold nanoparticles with silver buffer, so that the amount of metallic silver deposited onto gold surface decreased and the light intensity was proportional to the concentration of antigen. The CCD data were quantified using ImageJ software. A circle of 20 pixels in radius (1250-pixel area) was used as a uniform region of interest, which covers roughly 75% of the surface of each well of the chip. The value for the individual wells was calculated as the average of the intensity values of the respective pixels. After proving the possibility of using this method to detect the concentration of IgG, we investigated the effect of the silver deposit time. In the wells of the chip, after gold nanoparticles were immobilized and silver enhancement buffer was added, we tested different incubation times. From Fig. 3A, we can see that with an increase of the incubation time from 1 to 5 min, the light inten sity of the wells decreased greatly, meaning that the amount of metallic silver deposited increased and so the light intensity of
Results and discussion In this article, we have developed a simple method for the prep aration of label-free immunosensor based on silver enhancement through detection with a CCD camera. First, the possibility of such a method for the detection of antigens was investigated. As shown in Fig. 2 for 3 wells of the 9-well chip, well A was blank, well B was immobilized with only gold nanoparticles, and well C was immobilized with gold nanoparticles and then antibody–antigen immunocomplex. After the addition of silver enhancement buffer
Fig. 2. Immunosensor based on silver enhancement detected by CCD camera. (A) Blank well. (B) Well immobilized with only gold nanoparticles. (C) Well immobilized with gold nanoparticles and antibody–antigen immunocomplex. Exposure: 1 s.
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the antibody loading, and then more antigens will be captured to form a more “compact” film on the gold surface to inhibit silver reduction and, as a result, the sensitivity will increase. In further increasing the antibody concentration from 0.1 to 0.3 mg/ml, the sensitivity did not change too much, and from 0.3 mg/ml onward increasing the antibody concentration resulted in a sensitivity decrease. The reason for the decreased sensitivity may be that at a high antibody concentration the adsorption of antibody onto gold surface already formed a compact film, and so at this time the con tact of the gold nanoparticles with silver enhancement buffer was already greatly inhibited and further capture of antigen will not help too much of the film to inhibit the contact. An antibody con centration of 0.1 mg/ml was used in this experiment. Different concentrations of IgG were tested using the 9-well chip to try to draw the calibration curve. The light intensity of the well in the presence of IgG was higher than that in the absence of IgG, and it increased gradually with the increasing concentrations of IgG. The reason was that at higher concentrations of IgG, more IgG molec ules were captured by the antibody, leading to the form ing of a more compact immunocomplex film. The more compact the immunocomplex film, the less contact between the gold nano particles and the silver enhancement buffer, and as a result less sil ver was deposited. So, we can see that the IgG concentration could be determined by this CCD–silver enhancement measurement. The standard calibration curve for the IgG detection is shown in Fig. 3C, and a linear range from 0.05 to 10 ng/ml was obtained. The reproducibility of the measurement was estimated. A series of five measurements were prepared for the detection of 0.1 and 1 ng/ml IgG. The relative standard deviations (RSDs) of measure ment for the 0.1 and 1 ng/ml IgG were 6.1 and 5.7%, respectively, suggesting the reliability of the measurement. The possibility of using the method for clinical applications was investigated by detecting real samples. Human blood samples were diluted to different concentrations using phosphate buffer and were measured using the CCD camera following the above methods. The results were compared with those of the ELISA method and are shown in Table 1. It can be seen that there is no significant differ ence between the results obtained by the CCD and ELISA methods, indicating that the developed immunoassay method could be used for the determination of IgG levels in human serum.
Concentration of IgG (ng/ml) Fig. 3. (A) Effect of gold nanoparticles and silver enhancement buffer (10 ll) incu bation time on the amount of metallic silver deposited. (B) Effect of antibody con centration (10 ll) on the sensitivity of the immunosensor to 1 ng/ml human IgG. (C) Calibration curve of the immunosensor to different concentrations of human IgG. The error bars show standard deviat ions (n = 3).
the well decreased. With a further increase of the time to 10 min, the change in the light intensity leveled off and after 10 min not too much change was observed. From these results, we can draw the conclusion that after 10 min not too much silver will continue being deposited onto the gold nanoparticles, and so the incubation time of 10 min was selected for further experiments. During the experiments, we found that the concentration of antibody solution affected the sensitivity of the detection. As shown in Fig. 3B, for the detection of 1 ng/ml IgG, when the con centration of antibody solution was increased from 0.01 to 0.1 mg/ ml, the sensitivity increased correspondingly. The reason may be that increasing the concentration of antibody solution will increase
Table 1 Comparison of IgG detections on human serum samples by CCD detector and ELISA. Serum samples
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Our method (ng/ml) ELISA (ng/ml)
0.11 0.14
0.53 0.47
0.98 1.08
2.35 2.54
5.68 5.36
Conclusions In this article, we have reported a novel strategy for the fabri cation of label-free immunosensors using a simple and portable CCD detector. The detection scheme is based on gold nanoparticleinduced silver enhancement. With gold nanoparticles immobilized into the wells of a 9-well chip, metallic silver can be deposited onto the gold, causing darkness. Pictures were taken by the CCD camera, and data were quantified using ImageJ software. When antibody was immobilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex inhibited the contact of the gold nanoparticles with the silver enhancement buffer, decreas ing the amount of metal silver deposited, and the light intensity change is in line with IgG concentrations from 0.05 to 10 ng/ml. By combining the high sensitivity of CCD–silver enhancement mea surement with the specificity of immunoreaction, the developed immunosensor was tested for the detection of IgG in human serum sample with satisfactory results. This CCD-based detection is sim ple, easy, and sensitive, and it could find wide application in ana lytical systems and biosensors. Acknowledgment We greatly appreciate the support of the Hunan Provincial Nat ural Science Foundation of China (07JJ3017).
Label-free immunosensor / M. Yang, C. Wang / Anal. Biochem. 385 (2008) 128–131
References [1] S. Viswanathan, L.C. Wu, M.R. Huang, J.A. Ho, Electrochemical immunosen sor for cholera toxin using liposomes and poly(3, 4-ethylenedioxythiophene)coated carbon nanotubes, Anal. Chem. 78 (2006) 1115–1121. [2] C.L. Morgan, D.J. Newman, C.P. Price, Immunosensors: Technology and oppor tunities in laboratory medicine, Clin. Chem. 42 (1996) 193–209. [3] D.P. Tang, R. Yuan, Y.Q. Chai, Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer, Anal. Chem. 80 (2008) 1582– 1588. [4] N. Takamune, H. Hamada, H. Sugawara, S. Misumi, S. Shouji, Development of an enzyme-linked immunosorbent assay for measurement of activity of myri stoyl–coenzyme A:protein N-myristoyltransferase, Anal. Biochem. 309 (2002) 137–142. [5] V. Kourilov, M. Steinitz, Magnetic-bead enzyme-linked immunosorbent assay verifies adsorption of ligand and epitope accessibility, Anal. Biochem. 311 (2002) 166–170. [6] M. Steinitz, Quantitation of the blocking effect of Tween 20 and bovine serum albumin in ELISA microwells, Anal. Biochem. 282 (2000) 232–238. [7] G. Macbeath, S.L. Schreiber, Printing proteins as microarrays for high-through put function determination, Science 289 (2000) 1760–1763. [8] D.J. Stephens, V.J. Allan, Light microscopy techniques for live cell imaging, Sci ence 300 (2003) 82–86. [9] A.P. Fan, C.W. Lau, J.Z. Lu, Magnetic bead-based chemiluminescent metal immunoassay with a colloidal gold label, Anal. Chem. 77 (2005) 3238– 3243. [10] J. Wang, D. Xu, A. Kawde, R. Polsky, Metal nanoparticle-based electrochemi cal stripping potentiometric detection of DNA hybridization, Anal. Chem. 73 (2001) 5576–5581. [11] M. Dequaire, C. Degrand, B. Limoges, An electrochemical metalloimmunoassay based on a colloidal gold label, Anal. Chem. 72 (2000) 5521–5528.
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[12] J.Y. Park, Y.S. Lee, B.H. Kim, S.M. Park, Label-free detection of antibody–antigen interactions on (R)-lipo-diaza-18-crown-6 self-assembled monolayer modi fied gold electrodes, Anal. Chem. 80 (2008) 4986–4993. [13] G.F. Jie, J.J. Zhang, D.C. Wang, C. Cheng, H.Y. Chen, J.J. Zhu, Electrochemilum inescence immunosensor based on CdSe nanocomposites, Anal. Chem. 80 (2008) 4033–4039. [14] Z.P. Chen, Z.F. Peng, Y. Luo, B. Qu, J.H. Jiang, X.B. Zhang, G.L. Shen, R.Q. Yu, Suc cessively amplified electrochemical immunoassay based on biocatalytic depo sition of silver nanoparticles and silver enhancement, Biosens. Bioelectron. 23 (2007) 485–491. [15] Q. Pan, R.Y. Zhang, Y.F. Bai, N.Y. He, Z.H. Lu, An electrochemic al approach for detection of specific DNA-binding protein by gold nanoparticle-catalyzed sil ver enhancement, Anal. Biochem 375 (2008) 179–186. [16] S. Gupta, S. Huda, P.K. Kilpatrick, O.D. Velev, Characterization and optimiza tion of gold nanoparticle-based silver-enhanced immunoassays, Anal. Chem. 79 (2007) 3810–3820. [17] H. Cai, Y. Wang, P. He, Y. Fang, Electrochemical detection of DNA hybridization based on silver-enhanced gold nanoparticle label, Anal. Chim. Acta 469 (2002) 165–172. [18] X. Chu, X. Fu, K, Chen, G. L. Shen, R. Q. Yu, An electrochemical stripping metal loimmunoassay based on silver-enhanced gold nanoparticle label, Biosens. Bioelectron. 20 (2005) 1805–1812. [19] R.Q. Liang, C.Y. Tan, K.C. Ruan, Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement, J. Immunol. Methods 285 (2004) 157–163. [20] O.D. Velev, E.W. Kaler, In situ assembly of colloidal particles into miniaturized biosensors, Langmuir 15 (1999) 3693–3698. [21] S.K. Sia, V. Linder, B.A. Parviz, A. Siegel, G.M. Whitesides, An integrated approach to a portable and low-cost immunoassay for resource-poor settings, Angew. Chem. Intl. Ed. 43 (2004) 498–502. [22] M. H. Yang, Y. Kostov, H. A. Bruck, A. Rasooly, Carbon nanotubes with enhanced chemiluminescence immunoassay for CCD-based detection of staphylococcal enterotoxin B in food, Anal. Chem. in press, doi:10.1021/ac801418n.
Contents lists available at ScienceDirect
Analytical Biochemistry j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y a b i o
Label-free immunosensor based on gold nanoparticle silver enhancement Minghui Yang a, Cunchang Wang b,* a b
Center for Advanced Sensor Technology, University of Maryland, Baltimore County, MD 21250, USA Department of Chemistry, Xiangnan University, Chenzhou 423000, China
a r t i c l e
i n f o
Article history: Received 1 September 2008 Available online 01 November 2008 Keywords: CCD camera Gold nanoparticles Label-free immunosensor Silver enhancement
a b s t r a c t A label-free immunosensor for the sensitive detection of human immunoglobul in G (IgG) was prepared based on gold nanoparticle–silver enhancement detection with a simple charge-coupled device (CCD) detector. The gold nanoparticles, which were used as nuclei for the deposit of metallic silver and also for the adsorption of antibodies, were immobilized into wells of a 9-well chip. With the addition of silver enhancement buffer, metallic silver will deposit onto gold nanoparticles, causing darkness that can be optically measured by the CCD camera and quantified using ImageJ software. When antibody was immo bilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex resulted in a decrease of the darkness and the intensity of the darkness was in line with IgG concentrations from 0.05 to 10 ng/ml. The CCD detector is simple and portable, and the reported method has many desirable merits such as sensitivity and accuracy, making it a promising technique for protein detection. © 2008 Elsevier Inc. All rights reserved.
Immunosensors have been widely studied for clinical diagno sis, environmental pollutant detection, and food analysis because they are simple, sensitive, and highly selective. Typically, antigens are selectively bound to their complementary antibodies on a solid surface and then to the secondary antibody that is labeled with a probe, which is called the traditional “sandwich” assay [1–3]. Dif ferent probes have been labeled to the secondary antibody with different detection techniques such as enzymes, fluorescent dyes, and nanoparticles. Enzyme-linked immunosorbent assay (ELISA)1 is one of the oldest and most widely used laboratory methods used as a diagnostic tool in medicine as well as a quality control check in various industries [4–6]. Fluorescence immunoassays are based on antibodies labeled with fluorophores; however, the fluo rophores are easily subjected to photobleaching, which may result in reduced accuracy [7,8]. Recently, nanoparticles such as gold nanoparticles and quantum dots have been widely used as labels because they are stable and cheap [9–11]. Besides the above-mentioned traditional sandwich assay, labelfree immunosensors based on direct detection of antibody–antigen interactions have also gained growing interest. Park and coworkers immobilized antibodies on (R)-lipo-diaza-18-crown-6 self-assem bled monolayer modified gold electrodes, and the blocking effect caused by the Ab–Ag adducts formed on electrode surface resulted in an increased charge transfer resistance (Rct) of the electrode for * Corresponding author. Fax: +86 735 2653013. E-mail address: [email protected] (C. Wang). 1 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; ECL, electro chemilumin escence; CCD, charge-coupled device; SEB, staphylococcal enterotoxin B; IgG, immunoglobulin G; BSA, bovine serum albumin; RSD, relative standard deviation. 0003-2697/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.10.019
a redox probe, which provides a basis for quantification of Ag in solution [12]. Jie and coworkers immobilized CdSe nanocrystals, carbon nanotube, and chitosan composite film on electrode sur face and investigated the electrochemiluminescence (ECL) of the film. After antibody was bound to the film, the specific immunore action between antibody and antigen resulted in a decrease of ECL intensity [13]. Silver enhancement, a procedure for electroless silver deposi tion where the nanoparticles act as nuclei, has been widely used for immunoassays [14–16]. In this technique, colloidal metal par ticles act as catalysts to reduce silver ions to metallic silver, which is typically quantified by electrochemical measurement [17] or anodic stripping [18]. Examples of immunoassays using colloidal gold nanoparticles combined with silver enhancement have been demonstrated on microarrayed substrate [19], between coplanar electrodes [20], and through microfluidic channels [21]. To the best of our knowledge, no immunosensor based on silver enhancement has been reported using a charge-coupled device (CCD) camera. Previously, our lab reported a carbon nanotube-based immu nosensor for the detection of staphylococcal enterotoxin B (SEB) using a CCD camera [22]. In the current article, we report a novel method for the fabrication of label-free immunosensors using sil ver enhancement. Gold nanoparticles were immobilized in wells of 9-well chips and were used as nuclei for the deposit of metallic sil ver and also for the immobilization of antibodies. With the forma tion of antibody–antigen immunocomplex, the amount of metallic silver deposited onto the nanoparticles decreased compared with that without the immunocomplex due to the decreased contact of the gold nanoparticles with silver enhancement buffer, The dark ness of the wells in accordance with the amount of metallic silver
Label-free immunosensor / M. Yang, C. Wang / Anal. Biochem. 385 (2008) 128–131
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was optically measured by a CCD camera, and the immunosensor provided a convenient, low-cost, sensitive, and specific method for biomolecular detection. Materials and methods Materials and reagents Gold nanoparticles (G1527, » 0.75 A520 U/ml, 8.5–12.0 nm mean particle size [monodisperse], 10 nm particle size), goat antihuman immunoglobulin G (IgG) antibody, human IgG antigen, and a Silver Enhancer Kit (SE-100) were products of Sigma–Aldrich (St. Louis, MO, USA). The Silver Enhancer Kit consists of solution A (sil ver salt) and solution B (initiat or). Real human blood samples were provided by the University of Maryland Medical Center. All other reagents were of analytic al grade, and deionized water was used throughout. Apparatus For the CCD-based detection, the schematic configuration of the CCD detector is shown schematically in Fig. 1A, and Fig. 1B is a photograph of the actual detection platform. The CCD-based detec tor consists of an enclosure and an Atic-16 cooled CCD camera or SXVF-M7 (Adirondack Video Astronomy, Hudson Falls, NY, USA). Both cooled CCD cameras employ a Sony ICX-429ALL with a 752 £ 582-pixel CCD and are equipped with a 5-mm extension tube and a 12-mm Pentax f1.2 lens (Spytown, Utopia, NY, USA). The system also includes an illuminator. The 9-well sample chips used in this study were designed in CorelDraw 11 (Corel, Ontario, Canada) and were micromachined in 1/8-inch black acrylic using a computercontrolled laser cutter (Epilog Legend CO2 65W, Epilog, Golden, CO, USA). Polycarbonate film was attached to one side of the chips to hold the chemical solution (Fig. 1C). The CCD image intensities were analyzed using ImageJ software. Preparation of the immunosensor For the preparation of the immunosensor, 15 ll of gold nano particles was first dropped into the wells of the 9-well chips. When dried, the wells were washed with phosphate buffer (50 mM, pH 7.4) and 10 ll of 0.1 mg/ml antibody solution was added into the wells and incubated for 1 h. After rinsing with phosphate buffer, the chip was incubated with 10 ll of 1% bovine serum albumin (BSA) for 1 h to block nonspecific binding sites. Finally, the chip was rinsed and 10 ll of different concentrations of IgG solution were added into the wells and incubated for 45 min. Silver enhancement detection For silver enhancement detection, 10 ll of silver enhancement buffer (mixture of the two solutions from the Silver Enhancer Kit in a 1:1 volume ratio prepared just prior to use) was added into the wells of the chip, the chip was incubated in the dark for 10 min, and then picture was taken by the CCD detector immediately.
Fig. 1. The CCD detector: (A) schematic configuration of the detector; (B) photo graph of the actual detection platform; (C) actual 9-well sample chip. 1, plastic enclosure; 2, Atic-16 CCD camera [2]. A 5-mm extension tube (3) was attached to the 12-mm Pentax f1.2 lens (4). A black acrylic shelf box (5) was designed to hold the sample chip (6) and fluorescence illumination module (7).
(silver ions and initiator) and incubation in the dark for 10 min, the picture was taken using the CCD camera. Under illumination, well A appears to be totally white because there was no gold nanopar ticles and so no metallic silver deposited and the light from the illuminator was collected by the CCD. On the contrary, in well B, when gold nanoparticles were introduced, the gold nanoparticles served as nucleation sites to catalyze the reduction of silver ions to metallic silver and were enlarged in the process by up to five orders of magnitude [16]. As a result, the light from the illuminator was partially blocked, displaying a decrease in light intensity. In well C, after immobilization of the antibody and then antigen, the light intensity increased. The reason was that the formed immuno complex after specific immunoreaction greatly inhibited the con tact of gold nanoparticles with silver buffer, so that the amount of metallic silver deposited onto gold surface decreased and the light intensity was proportional to the concentration of antigen. The CCD data were quantified using ImageJ software. A circle of 20 pixels in radius (1250-pixel area) was used as a uniform region of interest, which covers roughly 75% of the surface of each well of the chip. The value for the individual wells was calculated as the average of the intensity values of the respective pixels. After proving the possibility of using this method to detect the concentration of IgG, we investigated the effect of the silver deposit time. In the wells of the chip, after gold nanoparticles were immobilized and silver enhancement buffer was added, we tested different incubation times. From Fig. 3A, we can see that with an increase of the incubation time from 1 to 5 min, the light inten sity of the wells decreased greatly, meaning that the amount of metallic silver deposited increased and so the light intensity of
Results and discussion In this article, we have developed a simple method for the prep aration of label-free immunosensor based on silver enhancement through detection with a CCD camera. First, the possibility of such a method for the detection of antigens was investigated. As shown in Fig. 2 for 3 wells of the 9-well chip, well A was blank, well B was immobilized with only gold nanoparticles, and well C was immobilized with gold nanoparticles and then antibody–antigen immunocomplex. After the addition of silver enhancement buffer
Fig. 2. Immunosensor based on silver enhancement detected by CCD camera. (A) Blank well. (B) Well immobilized with only gold nanoparticles. (C) Well immobilized with gold nanoparticles and antibody–antigen immunocomplex. Exposure: 1 s.
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the antibody loading, and then more antigens will be captured to form a more “compact” film on the gold surface to inhibit silver reduction and, as a result, the sensitivity will increase. In further increasing the antibody concentration from 0.1 to 0.3 mg/ml, the sensitivity did not change too much, and from 0.3 mg/ml onward increasing the antibody concentration resulted in a sensitivity decrease. The reason for the decreased sensitivity may be that at a high antibody concentration the adsorption of antibody onto gold surface already formed a compact film, and so at this time the con tact of the gold nanoparticles with silver enhancement buffer was already greatly inhibited and further capture of antigen will not help too much of the film to inhibit the contact. An antibody con centration of 0.1 mg/ml was used in this experiment. Different concentrations of IgG were tested using the 9-well chip to try to draw the calibration curve. The light intensity of the well in the presence of IgG was higher than that in the absence of IgG, and it increased gradually with the increasing concentrations of IgG. The reason was that at higher concentrations of IgG, more IgG molec ules were captured by the antibody, leading to the form ing of a more compact immunocomplex film. The more compact the immunocomplex film, the less contact between the gold nano particles and the silver enhancement buffer, and as a result less sil ver was deposited. So, we can see that the IgG concentration could be determined by this CCD–silver enhancement measurement. The standard calibration curve for the IgG detection is shown in Fig. 3C, and a linear range from 0.05 to 10 ng/ml was obtained. The reproducibility of the measurement was estimated. A series of five measurements were prepared for the detection of 0.1 and 1 ng/ml IgG. The relative standard deviations (RSDs) of measure ment for the 0.1 and 1 ng/ml IgG were 6.1 and 5.7%, respectively, suggesting the reliability of the measurement. The possibility of using the method for clinical applications was investigated by detecting real samples. Human blood samples were diluted to different concentrations using phosphate buffer and were measured using the CCD camera following the above methods. The results were compared with those of the ELISA method and are shown in Table 1. It can be seen that there is no significant differ ence between the results obtained by the CCD and ELISA methods, indicating that the developed immunoassay method could be used for the determination of IgG levels in human serum.
Concentration of IgG (ng/ml) Fig. 3. (A) Effect of gold nanoparticles and silver enhancement buffer (10 ll) incu bation time on the amount of metallic silver deposited. (B) Effect of antibody con centration (10 ll) on the sensitivity of the immunosensor to 1 ng/ml human IgG. (C) Calibration curve of the immunosensor to different concentrations of human IgG. The error bars show standard deviat ions (n = 3).
the well decreased. With a further increase of the time to 10 min, the change in the light intensity leveled off and after 10 min not too much change was observed. From these results, we can draw the conclusion that after 10 min not too much silver will continue being deposited onto the gold nanoparticles, and so the incubation time of 10 min was selected for further experiments. During the experiments, we found that the concentration of antibody solution affected the sensitivity of the detection. As shown in Fig. 3B, for the detection of 1 ng/ml IgG, when the con centration of antibody solution was increased from 0.01 to 0.1 mg/ ml, the sensitivity increased correspondingly. The reason may be that increasing the concentration of antibody solution will increase
Table 1 Comparison of IgG detections on human serum samples by CCD detector and ELISA. Serum samples
1
2
3
4
5
Our method (ng/ml) ELISA (ng/ml)
0.11 0.14
0.53 0.47
0.98 1.08
2.35 2.54
5.68 5.36
Conclusions In this article, we have reported a novel strategy for the fabri cation of label-free immunosensors using a simple and portable CCD detector. The detection scheme is based on gold nanoparticleinduced silver enhancement. With gold nanoparticles immobilized into the wells of a 9-well chip, metallic silver can be deposited onto the gold, causing darkness. Pictures were taken by the CCD camera, and data were quantified using ImageJ software. When antibody was immobilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex inhibited the contact of the gold nanoparticles with the silver enhancement buffer, decreas ing the amount of metal silver deposited, and the light intensity change is in line with IgG concentrations from 0.05 to 10 ng/ml. By combining the high sensitivity of CCD–silver enhancement mea surement with the specificity of immunoreaction, the developed immunosensor was tested for the detection of IgG in human serum sample with satisfactory results. This CCD-based detection is sim ple, easy, and sensitive, and it could find wide application in ana lytical systems and biosensors. Acknowledgment We greatly appreciate the support of the Hunan Provincial Nat ural Science Foundation of China (07JJ3017).
Label-free immunosensor / M. Yang, C. Wang / Anal. Biochem. 385 (2008) 128–131
References [1] S. Viswanathan, L.C. Wu, M.R. Huang, J.A. Ho, Electrochemical immunosen sor for cholera toxin using liposomes and poly(3, 4-ethylenedioxythiophene)coated carbon nanotubes, Anal. Chem. 78 (2006) 1115–1121. [2] C.L. Morgan, D.J. Newman, C.P. Price, Immunosensors: Technology and oppor tunities in laboratory medicine, Clin. Chem. 42 (1996) 193–209. [3] D.P. Tang, R. Yuan, Y.Q. Chai, Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer, Anal. Chem. 80 (2008) 1582– 1588. [4] N. Takamune, H. Hamada, H. Sugawara, S. Misumi, S. Shouji, Development of an enzyme-linked immunosorbent assay for measurement of activity of myri stoyl–coenzyme A:protein N-myristoyltransferase, Anal. Biochem. 309 (2002) 137–142. [5] V. Kourilov, M. Steinitz, Magnetic-bead enzyme-linked immunosorbent assay verifies adsorption of ligand and epitope accessibility, Anal. Biochem. 311 (2002) 166–170. [6] M. Steinitz, Quantitation of the blocking effect of Tween 20 and bovine serum albumin in ELISA microwells, Anal. Biochem. 282 (2000) 232–238. [7] G. Macbeath, S.L. Schreiber, Printing proteins as microarrays for high-through put function determination, Science 289 (2000) 1760–1763. [8] D.J. Stephens, V.J. Allan, Light microscopy techniques for live cell imaging, Sci ence 300 (2003) 82–86. [9] A.P. Fan, C.W. Lau, J.Z. Lu, Magnetic bead-based chemiluminescent metal immunoassay with a colloidal gold label, Anal. Chem. 77 (2005) 3238– 3243. [10] J. Wang, D. Xu, A. Kawde, R. Polsky, Metal nanoparticle-based electrochemi cal stripping potentiometric detection of DNA hybridization, Anal. Chem. 73 (2001) 5576–5581. [11] M. Dequaire, C. Degrand, B. Limoges, An electrochemical metalloimmunoassay based on a colloidal gold label, Anal. Chem. 72 (2000) 5521–5528.
131
[12] J.Y. Park, Y.S. Lee, B.H. Kim, S.M. Park, Label-free detection of antibody–antigen interactions on (R)-lipo-diaza-18-crown-6 self-assembled monolayer modi fied gold electrodes, Anal. Chem. 80 (2008) 4986–4993. [13] G.F. Jie, J.J. Zhang, D.C. Wang, C. Cheng, H.Y. Chen, J.J. Zhu, Electrochemilum inescence immunosensor based on CdSe nanocomposites, Anal. Chem. 80 (2008) 4033–4039. [14] Z.P. Chen, Z.F. Peng, Y. Luo, B. Qu, J.H. Jiang, X.B. Zhang, G.L. Shen, R.Q. Yu, Suc cessively amplified electrochemical immunoassay based on biocatalytic depo sition of silver nanoparticles and silver enhancement, Biosens. Bioelectron. 23 (2007) 485–491. [15] Q. Pan, R.Y. Zhang, Y.F. Bai, N.Y. He, Z.H. Lu, An electrochemic al approach for detection of specific DNA-binding protein by gold nanoparticle-catalyzed sil ver enhancement, Anal. Biochem 375 (2008) 179–186. [16] S. Gupta, S. Huda, P.K. Kilpatrick, O.D. Velev, Characterization and optimiza tion of gold nanoparticle-based silver-enhanced immunoassays, Anal. Chem. 79 (2007) 3810–3820. [17] H. Cai, Y. Wang, P. He, Y. Fang, Electrochemical detection of DNA hybridization based on silver-enhanced gold nanoparticle label, Anal. Chim. Acta 469 (2002) 165–172. [18] X. Chu, X. Fu, K, Chen, G. L. Shen, R. Q. Yu, An electrochemical stripping metal loimmunoassay based on silver-enhanced gold nanoparticle label, Biosens. Bioelectron. 20 (2005) 1805–1812. [19] R.Q. Liang, C.Y. Tan, K.C. Ruan, Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement, J. Immunol. Methods 285 (2004) 157–163. [20] O.D. Velev, E.W. Kaler, In situ assembly of colloidal particles into miniaturized biosensors, Langmuir 15 (1999) 3693–3698. [21] S.K. Sia, V. Linder, B.A. Parviz, A. Siegel, G.M. Whitesides, An integrated approach to a portable and low-cost immunoassay for resource-poor settings, Angew. Chem. Intl. Ed. 43 (2004) 498–502. [22] M. H. Yang, Y. Kostov, H. A. Bruck, A. Rasooly, Carbon nanotubes with enhanced chemiluminescence immunoassay for CCD-based detection of staphylococcal enterotoxin B in food, Anal. Chem. in press, doi:10.1021/ac801418n.