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A label-free visual immunoassay on solid support with silver nanoparticles as plasmon resonance scattering indicator
Analytical Biochemistry 383 (2008) 168–173
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A label-free visual immunoassay on solid support with silver nanoparticles as plasmon resonance scattering indicator Jian Ling, Yuan Fang Li, Cheng Zhi Huang * College of Chemistry and Chemical Engineering and College of Pharmaceutical Sciences, MOE Key Laboratory on Luminescence and Real-Time Analysis, Southwest University, 400715 Chongqing, People’s Republic of China
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Article history: Received 30 April 2008 Available online 26 August 2008 Keywords: Immunoassay Silver nanoparticles Plasmon resonance scattering (PRS)
a b s t r a c t By taking silver nanoparticles (Ag-NPs) as plasmon resonance scattering (PRS) indicator considering that Ag-NPs have strong plasmon resonance light scattering signals corresponding to their plasmon resonance absorption (PRA), we propose a label-free visual immunoassay on the solid support of glass slides. Our investigations showed that Ag-NPs could be adsorbed on the surface of glass slides where immunoreactions between a previously immobilized antigen and its antibody have occurred if the glass slides were immersed in an Ag-NP suspension whose pH value has been carefully adjusted. The optimal pH of the AgNP suspension depends on the nature of previously immobilized antigen and its antibody. It was found that the adsorption of negative-charged Ag-NPs on the surface of glass slides depends only on the content of antibody under optimal conditions. With a common spectrofluorometer to measure the PRS signals of the Ag-NPs adsorbed on the surface, we could detect antibody in the range of 10 to 160 ng ml 1. If a white light-emitting diode (LED) torch is employed to illuminate the glass slides, we can make visual detection of the antibody by the naked eye. Ó 2008 Elsevier Inc. All rights reserved.
Immunoassays have been now widely applied in clinical tests [1–3], environmental monitoring [4], and trials for construction of early warning systems for the detection of chemical and biological warfare agents [5]. Owing to time-consuming sample purification or separation, incubation, and rinsing steps prior to detection, conventional immunoassays such as the enzyme-linked immunosorbent assay (ELISA)1 are not feasible for building up a warning system. Label-free immunoassays, including surface plasmon resonance (SPR) [6–11], SPR imaging [12–14], localized SPR [15–17], plasmon resonance absorption (PRA) [18,19], piezoelectrical [20,21], electrochemical [22,23], and atomic force microscopy [24], although avoiding labeling such as radioisotopes [25] and fluorophores [26–29], also suffer from drawbacks such as lower sensitivity and expensive apparatus. Therefore, developing simple, low-cost, and sensitive label-free immunoassays is attractive for researchers. Owing to the excitation of the collective oscillations of conducting electrons known as plasmon resonances or surface plasmons [30], nanoparticles such as gold and silver nanoparticles exhibit * Corresponding author. Fax: +86 23 68866796. E-mail address: [email protected] (C.Z. Huang). 1 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance; PRA, plasmon resonance absorption; PRS, plasmon resonance scattering; Ag-NP, silver nanoparticle; LED, light-emitting diode; SEM, scanning electron microscope; H-IgG, human immunoglobulin G; APTES, 3-aminopropyltriethoxysilane; BSA, bovine serum albumin; GA, glutaraldehyde; PBS, phosphatebuffered saline. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.08.019
unique optical properties in the visible spectral range. The plasmon resonance scattering (PRS) features of gold nanoparticles, nanorods, and nanocages, for instance, have found wide applications in bioaffinity sensing [31,32] and cellular imaging [33–35]. It has been known that silver nanoparticles (Ag-NPs) have size- and shape-dependent PRA and PRS properties in the spectral range from 400 to 600 nm [36,37] and can exhibit strong blue PRS signals that can be seen by the naked eye under the illumination of white light [38]. In this article, we apply Ag-NPs as a PRS indicator to identify the interaction of antigen and antibody on glass slides and further propose a direct visual immunoassay. Different from the common schemes of labeling immunoassays and sandwich structure-based assays, which need to employ a primary antibody and a labeled secondary antibody for the recognition of the antigen [39–41], our current contribution involves the adsorption of the Ag-NPs on the surface of glass slides where immunoreactions have occurred between the immobilized antigen and free antibody. It was found that the negative-charged Ag-NPs capped with citrate ion can be adsorbed in the area on the surface of glass slides where the immunoreactions have occurred only under optimal adsorption conditions and that the adsorption increases only with the antibody content. Due to the strong PRS property of adsorbed Ag-NPs, the blue scattering light signals from the Ag-NPs on glass slides can be characterized and measured with a common spectrofluorometer under the synchronous mode of kem = kex and also can be seen clearly by the naked eye under white
Label-free visual immunoassay on solid support / J. Ling et al. / Anal. Biochem. 383 (2008) 168–173
light illumination using a common light-emitting diode (LED) torch. Materials and methods Apparatus The PRS measurements were made with an F-4600 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) by simultaneously scanning the excitation and emission monochromators of the spectrofluorometer with kem = kex. For the detection of light scattering signals of Ag-NPs on glass slides, a very easily prepared homemade holder [42] for glass slides was mounted in the sample chamber of the spectrofluorometer (see supplementary material for the design and principles). The size of prepared Ag-NPs was imaged with a Hitachi S-4800 scanning electron microscope (SEM), and their PRAs on glass slides were measured on a Hitachi U-3010 spectrophotometer. The PRS imaging of Ag-NPs on the glass slides was made with an Olympus BX51 microscope coupled with a highly numerical dark field condenser (U-DCW, 1.2–1.4), which delivers a very narrow beam of white light from a tungsten lamp from the bottom of the sample, and a 100 1.3 oil Iris objective (UPlanFLN), which was employed to collect the scattered light from the samples. The dark field light scattering imaging pictures of single Ag-NPs and photographs of glass slides were captured with a Nikon 4500 digital camera. Reagents Antigen, human immunoglobulin G (H-IgG), antibody, goat anti-human IgG antibody (Fc specific) and 3-aminopropyltriethoxysilane (APTES) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA) was purchased from Shanghai Biology Products Institute (Shanghai, China). Other commercial reagents, including glutaraldehyde (GA), silver nitrate, and trisodium citrate, were analytical reagent grade and used without further purification. In addition, 0.01 mol L 1 phosphate-buffered saline (PBS, pH 7.4), prepared by dissolving 0.296 g NaH2PO42H2O, 2.90 g Na2HPO412H2O, 8.0 g NaCl, and 1.0 g KCl in 1 L of ultrapure water (18.2 MX), was used as the buffer for immunoreactions. Also, 0.1 mol L 1 citric acid/Na2HPO4 buffer solution (pH 6.8) and Britton–Robinson buffer solution (pH 5.02–9.15) was used to adjust the pH of Ag-NP suspension for light scattering indication. All water used was ultrapurified with an LD-50G-E Ultra-Pure Water System (Lidi Modern Water Equipment, Chongqing, China).
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and boiling for 30 min, the mixture gradually changed and became yellow after approximately 10 min and then turned to brown yellow. After that, the colloidal Ag-NP solution was stirred continuously until it cooled to room temperature. It should be noted that controlling the reaction time is very important to make AgNPs of identical diameter for PRS detection (see supplementary material). The size of Ag-NPs as prepared was measured with an SEM, and the concentration of colloidal Ag-NPs was calculated according to the absorbance of Ag-NP solution [38] (e = 4.5 1010 for 46 nm Ag-NPs). Procedures Following traditional solid support immunoassay methods, we employed glass slides (12.7 76.2 1 mm, Shitai Experimental Instrument, Haimen, Jiangsu, China) as the solid support for immunoassays, and they were pretreated similarly to common procedures widely accepted for the cleaning and immobilization of antigen [42,45]. Shortly thereafter, the glass slides were ultrasonically washed with the detergent and soaked in a piranha solution (H2SO4/H2O2, 3:1 [v/v]) for 12 h. After washing thoroughly by ultrapure water of 18.2 MX and drying with nitrogen, the glass slides were soaked in acetone for approximately 2 min and then dried with a nitrogen gas flow. The cleaned and dried glass slides were immersed in 2% (v/v) APTES acetone solution for 3 min, washed three times with ultrapure water, and dried completely in an oven for 30 min at 110 °C. To immobilize antigen onto the surface of amino-silanized glass slides, the GA covalent coupling strategy was applied to modify the glass slides. Amino-silanized glass slides were at first immersed in 2.5% (v/v) GA solution for 1 h at 37 °C, washed thoroughly with ultrapure water, and then immersed in a plastic cell containing 1 ml of 0.1 to 1 lg ml 1 H-IgG in PBS (pH 7.4) solution to bind the antigen. After being left for 9 h at 4 °C, the glass slides were soaked in 1 mg ml 1 BSA for 1 h at room temperature to block the active site of unreacted GA. After the above widely employed procedures for immobilizing the antigen on the solid support of glass slides, the H-IgG immobilized glass slides were transferred for immunoreactions into the plastic cell containing goat anti-human IgG antibody in PBS solution for 1 h at 37 °C. After washing with PBS solution and ultrapure water three times, the glass slides were immersed in the plastic cells containing 1.0 ml of carefully pH-adjusted Ag-NP suspension for several minutes. Then the glass slides were washed and nitrogen-dried before being transferred to PRS measurements. Results and discussion
Preparation of Ag-NPs Spectral features of Ag-NPs adsorbed on glass slides Ag-NPs were prepared according to the modified Lee–Meisel method [43] that was similar to that described by Jarvis and Goodacre [44]. All facilities were cleaned thoroughly with detergent and aqua regia. Then 50.0 ml of solution containing 1.0 mmol L 1 of AgNO3 was boiled in a conical flask and 2.0 ml of 1% (w/v) trisodium citrate was introduced to the flask. Under continuous stirring
Scheme 1 shows our protocol of label-free immunoassay for the detection of antibody with Ag-NPs as PRS indicator. After first chemically immobilizing antigen on the surface of glass slides and making the immunoreactions occur with antibody, we transferred the glass slides into an Ag-NP suspension so that Ag-NPs
Scheme 1. Label-free immunoassay based on the PRS signals of adsorbed Ag-NPs on the surface of glass slides.
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Selective adsorption of Ag-NPs on the solid supports
Fig. 1. PRS spectra of Ag-NPs adsorbed on glass slides. Conditions: antibody (from 1 to 4), 0, 50, 120, and 250 ng ml 1, respectively; antigen immobilized, 0.25 lg ml 1; Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; adsorption time, 10 min. The inset shows the PRA spectra of glass slides under the same conditions.
could be adsorbed in the area where the immunoreactions have occurred. Fig. 1 shows the light scattering spectra of glass slides and those on which Ag-NPs have been adsorbed. It can be seen that the PRS spectra of adsorbed Ag-NPs have a characteristic PRS peak located at 405 nm and increase with increasing antibody. The characteristic PRS features are strongly related to the PRA [37]. As the inserted PRA spectra in Fig. 1 show, Ag-NPs display PRA features characterized near 400 nm similar to those reported extensively in aqueous medium [46,47], and the increase of absorbance also confirms the increasing adsorption of Ag-NPs on antibody bound on the surface of glass slides. The microstructure and optical property of single Ag-NPs, which adsorbed on the glass slides with a beautiful blue emission of scattered light, was studied intensively with an SEM and a dark field microscope. The SEM image (Fig. 2A) shows that Ag-NPs as prepared in this study have an average diameter of 46 nm and are dispersed on the glass slides. However, the shapes of Ag-NPs are not only spheres but also a few rods. This might be attributed to the multiple distribution of our prepared Ag-NPs in size and shape. It is known that the PRA and PRS properties of Ag-NPs are size and shape dependent in the spectral range from 400 to 700 nm [37]; therefore, the light scattering image of single Ag-NPs on glass slides with the dark field microscope shows that Ag-NPs scatter mainly blue light besides some green or red light (Fig. 2B). The green and red dots in Fig. 2B are the results of other shapes and sizes of the prepared nanoparticles owing to the uneven distribution.
Because the protein is positive or negative charged depending on the pH of the aqueous medium and the isoelectric points (pI0), there is a possibility that antigen or antibody will be positive and could have electrostatic attraction with Ag-NPs. So, if the pI0 values of immobilized antigen (H-IgG) and the antibody (goat anti-human IgG) are different, the selective adsorption of Ag-NPs on the solid supports could be realized by adjusting the pH values of the Ag-NP suspension in which the antibody bound on the surface of immunocomplex glass slides is positively charged, whereas the antigen immobilized glass slides is not. In such cases, controlling the pH value of Ag-NP suspension in the absorption step is crucial to make the adsorption of Ag-NPs be dependent only on the content of antibody and not on that of antigen. Although human IgG has a structure similar to that of goat IgG, the antigen covalently immobilized on glass slides should have different features, particularly the different pI0 values, given the acylamidate group of the antigen resulting from the immobilizing reaction with GA. Therefore, in a certain medium of pH lower than its pI0 value, the antigen immobilized on the surface of glass slides might not be positively charged for the acylamidation of the amido group that has stereo-obstruction to the adsorption of the negative-charged Ag-NPs, whereas the antibody bound on the glass slides could be positive so that the adsorption of the negative-charged Ag-NPs is dependent only on the content of antibody. Fig. 3 shows the dependence of the adsorption of Ag-NPs on the pH values of the Ag-NP suspension after the immunoreactions between a given content of antigen immobilized on the surface of the glass slides and a given content of antibody. From the intensity of
Fig. 3. pH dependence of colloidal silver on the adsorption process. Conditions: antigen immobilized, 0.25 lg ml 1; antibody, 0.25 lg ml 1; Ag-NPs, 1.1 10 10 M; adsorption time, 10 min; k(IPRS) = 405 nm. Ab, antibody.
Fig. 2. SEM image (A) and dark field light scattering microscopic image (B) of Ag-NPs on glass slides. Conditions: antigen immobilized, 0.25 lg ml Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; adsorption time, 10 min.
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PRS signals of Ag-NPs adsorbed on the surface of glass slides, we found that Ag-NPs could be adsorbed only on antibody-bound glass slides. With decreasing pH, the antigen immobilized on glass slides is also positive charged (as is the bound antibody), resulting in the adsorption of Ag-NPs on the glass slides without bound antibody. With increasing pH values higher than 8.5, the isoelectric point of antibody, the adsorption of Ag-NPs decreases dramatically with the disappearance of electrostatic interaction between the antibody and the Ag-NPs. However, the adsorption of Ag-NPs on antibody-bound glass slides is also present at pH values higher than the isoelectric point of antibody, indicating that the interactions between the Ag-NPs and proteins is not only electrostatic attraction but also chemisorptions of proteins on the Ag-NP surface. Experiments showed that the detection of antibody concentration was dependent on the amount of antigen immobilized on glass slides. Theoretically, the more antigens immobilized on glass slides, the more antibodies could bind to the glass slides. Immobilizing much more antigen on glass slides, however, would result in the chemisorptions of Ag-NPs on the antigen immobilized glass slides even if no antibody is present. By optimizing antigen concentration during the process of immobilization, we found that the maximum enhanced PRS signals (DIPRS) of glass slides could be obtained if 0.25 to 0.30 lg ml 1 antigen was used during the immobilization (Fig. 4). Adsorption time is another factor that affects the adsorption of Ag-NPs only on antibody-bound glass slides. Fig. 5 shows that longer adsorption time (ta) could result in chemisorptions of AgNPs on the antigen immobilized glass slides even if antibody is not present. Consequently, the adsorption of Ag-NPs on only the antibodybound glass slides depends on the pH of Ag-NP solution, the amount of antigen immobilized on glass sides, and the Ag-NP adsorption time. Controlling these experimental conditions carefully could result in the selective adsorption of Ag-NPs on the glass slides bound with antibody.
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Fig. 5. Dependence of adsorption time on Ag-NPs. Conditions: antigen immobilized, 0.25 lg ml 1; antibody, 0.25 lg ml 1, Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; adsorption time, 10 min; k(IPRS), 405 nm. Ab, antibody.
Fig. 6. Competition experiment in the presence of free antigen. Conditions: antigen immobilized, 0.25 lg ml 1; antibody, 0.25 lg ml 1; Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; k(IPRS), 405 nm. Ab, antibody.
Competitive assay Analytical detection of antibody To demonstrate the reliability of this immunoassay, a competitive experiment was performed under optimal conditions. In the competitive assay, the free antigen in the solution competes with the antigen immobilized on the glass to react with the antibody. Fig. 6 shows that the PRS signal of Ag-NPs can be effectively reduced in the presence of free antigen.
Under optimal conditions, it was found that the intensity of PRS signals of Ag-NPs adsorbed selectively on antibody-bound glass slides is linearly dependent on the content of antibody. Fig. 7 shows that a good linear relationship, DIPRS = 12.83c 10.31, between the PRS intensity (DIPRS) at kmax = 405 nm and antibody
Fig. 4. Effect of antigen concentration on the immobilization process. Conditions: antibody concentration, 1 lg ml 1; Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; adsorption time, 10 min; k(IPRS), 405 nm. Ab, antibody.
Fig. 7. Relationship between the scattering intensity and the antibody concentration. Conditions: antigen immobilized, 0.25 lg ml 1; Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; adsorption time, 10 min, k(IPRS), 405 nm.
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Fig. 8. Visual detection of antibody on glass slides under the illumination of an LED torch. Conditions: antigen immobilized, 0.25 lg ml pH, 6.8; adsorption time, 10 min; antibody (from A to G), 10, 30, 60, 100, 150, 200, and 250 ng ml 1, respectively.
concentration (cAb) is found in the range between 10 and 160 ng ml 1 with R2 = 0.9836 and a detection limit of 5.6 ng/ml. Due to the strong PRS emission of Ag-NPs, the attractive signals from the PRS of Ag-NPs could be clearly seen by the naked eye under a common white LED light excitation. Then a visual label-free immunoassay based on the PRS of Ag-NPs was established to detect antibody on common glass slides with simplicity and low cost. Fig. 8 shows a series of glass slides bound with different amounts of antibody under the illumination of an LED torch in a dark room. Because the amount of Ag-NPs adsorbed depends on the amount of antibody bound on the glass slides, clear blue PRS light of Ag-NPs seen from the glass slides can be used to visually estimate the concentration of antibody. Because they are adsorbed on the surface of glass slides only where the antibody is bound, the Ag-NPs could be used as an indicator to localize the immunoreactions between antigen and antibody on the glass slides. With the PRS light of Ag-NPs from the immune–glass slides, we can see the location where immunoreactions happened and get more information about the immunoreactions on the glass slides. According to the PRS image of immune–glass slides (Fig. 8), the immunoreactions happened over all of the antigen immobilized surface of the glass slides where abundant antibody (more than the antigen immobilized) is present (panels F and G), but they happened only at certain areas of the glass slides randomly where there is a lack of antibody (panels A–E). Therefore, the PRS-based light scattering method with AgNPs selectively adsorbed on the antibody where immunoreactions happened is suitable to investigate the reaction between antigen and antibody on solid support. Conclusion In this work, a novel, label-free, visual immunoassay method, using the PRS signals of the Ag-NP electrostatic adsorbed on glass slides where antibody is bound, has been established to distinguish the immunoreactions on glass slides with a common LED torch. We discussed the mechanism of this method and investigated the effect of experimental conditions with the scattering signals of AgNPs measured on a common spectrofluorometer. Under optimal conditions, antibody over the range between 10 and 160 ng ml 1 could be detected quantitatively with spectrofluorometer or just an LED torch and the naked eye. The method based on the adsorption of Ag-NPs as an indicator to distinguish the different electrostatic properties between the immobilized molecule and the specific bound molecule on glass slides could find more applications in the label-free study of protein–protein, DNA–protein, and saccharide–protein interactions. Acknowledgment The authors greatly appreciate the financial support from the National Natural Science Foundation of China (20425517).
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A label-free visual immunoassay on solid support with silver nanoparticles as plasmon resonance scattering indicator Jian Ling, Yuan Fang Li, Cheng Zhi Huang * College of Chemistry and Chemical Engineering and College of Pharmaceutical Sciences, MOE Key Laboratory on Luminescence and Real-Time Analysis, Southwest University, 400715 Chongqing, People’s Republic of China
a r t i c l e
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Article history: Received 30 April 2008 Available online 26 August 2008 Keywords: Immunoassay Silver nanoparticles Plasmon resonance scattering (PRS)
a b s t r a c t By taking silver nanoparticles (Ag-NPs) as plasmon resonance scattering (PRS) indicator considering that Ag-NPs have strong plasmon resonance light scattering signals corresponding to their plasmon resonance absorption (PRA), we propose a label-free visual immunoassay on the solid support of glass slides. Our investigations showed that Ag-NPs could be adsorbed on the surface of glass slides where immunoreactions between a previously immobilized antigen and its antibody have occurred if the glass slides were immersed in an Ag-NP suspension whose pH value has been carefully adjusted. The optimal pH of the AgNP suspension depends on the nature of previously immobilized antigen and its antibody. It was found that the adsorption of negative-charged Ag-NPs on the surface of glass slides depends only on the content of antibody under optimal conditions. With a common spectrofluorometer to measure the PRS signals of the Ag-NPs adsorbed on the surface, we could detect antibody in the range of 10 to 160 ng ml 1. If a white light-emitting diode (LED) torch is employed to illuminate the glass slides, we can make visual detection of the antibody by the naked eye. Ó 2008 Elsevier Inc. All rights reserved.
Immunoassays have been now widely applied in clinical tests [1–3], environmental monitoring [4], and trials for construction of early warning systems for the detection of chemical and biological warfare agents [5]. Owing to time-consuming sample purification or separation, incubation, and rinsing steps prior to detection, conventional immunoassays such as the enzyme-linked immunosorbent assay (ELISA)1 are not feasible for building up a warning system. Label-free immunoassays, including surface plasmon resonance (SPR) [6–11], SPR imaging [12–14], localized SPR [15–17], plasmon resonance absorption (PRA) [18,19], piezoelectrical [20,21], electrochemical [22,23], and atomic force microscopy [24], although avoiding labeling such as radioisotopes [25] and fluorophores [26–29], also suffer from drawbacks such as lower sensitivity and expensive apparatus. Therefore, developing simple, low-cost, and sensitive label-free immunoassays is attractive for researchers. Owing to the excitation of the collective oscillations of conducting electrons known as plasmon resonances or surface plasmons [30], nanoparticles such as gold and silver nanoparticles exhibit * Corresponding author. Fax: +86 23 68866796. E-mail address: [email protected] (C.Z. Huang). 1 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance; PRA, plasmon resonance absorption; PRS, plasmon resonance scattering; Ag-NP, silver nanoparticle; LED, light-emitting diode; SEM, scanning electron microscope; H-IgG, human immunoglobulin G; APTES, 3-aminopropyltriethoxysilane; BSA, bovine serum albumin; GA, glutaraldehyde; PBS, phosphatebuffered saline. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.08.019
unique optical properties in the visible spectral range. The plasmon resonance scattering (PRS) features of gold nanoparticles, nanorods, and nanocages, for instance, have found wide applications in bioaffinity sensing [31,32] and cellular imaging [33–35]. It has been known that silver nanoparticles (Ag-NPs) have size- and shape-dependent PRA and PRS properties in the spectral range from 400 to 600 nm [36,37] and can exhibit strong blue PRS signals that can be seen by the naked eye under the illumination of white light [38]. In this article, we apply Ag-NPs as a PRS indicator to identify the interaction of antigen and antibody on glass slides and further propose a direct visual immunoassay. Different from the common schemes of labeling immunoassays and sandwich structure-based assays, which need to employ a primary antibody and a labeled secondary antibody for the recognition of the antigen [39–41], our current contribution involves the adsorption of the Ag-NPs on the surface of glass slides where immunoreactions have occurred between the immobilized antigen and free antibody. It was found that the negative-charged Ag-NPs capped with citrate ion can be adsorbed in the area on the surface of glass slides where the immunoreactions have occurred only under optimal adsorption conditions and that the adsorption increases only with the antibody content. Due to the strong PRS property of adsorbed Ag-NPs, the blue scattering light signals from the Ag-NPs on glass slides can be characterized and measured with a common spectrofluorometer under the synchronous mode of kem = kex and also can be seen clearly by the naked eye under white
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light illumination using a common light-emitting diode (LED) torch. Materials and methods Apparatus The PRS measurements were made with an F-4600 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) by simultaneously scanning the excitation and emission monochromators of the spectrofluorometer with kem = kex. For the detection of light scattering signals of Ag-NPs on glass slides, a very easily prepared homemade holder [42] for glass slides was mounted in the sample chamber of the spectrofluorometer (see supplementary material for the design and principles). The size of prepared Ag-NPs was imaged with a Hitachi S-4800 scanning electron microscope (SEM), and their PRAs on glass slides were measured on a Hitachi U-3010 spectrophotometer. The PRS imaging of Ag-NPs on the glass slides was made with an Olympus BX51 microscope coupled with a highly numerical dark field condenser (U-DCW, 1.2–1.4), which delivers a very narrow beam of white light from a tungsten lamp from the bottom of the sample, and a 100 1.3 oil Iris objective (UPlanFLN), which was employed to collect the scattered light from the samples. The dark field light scattering imaging pictures of single Ag-NPs and photographs of glass slides were captured with a Nikon 4500 digital camera. Reagents Antigen, human immunoglobulin G (H-IgG), antibody, goat anti-human IgG antibody (Fc specific) and 3-aminopropyltriethoxysilane (APTES) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA) was purchased from Shanghai Biology Products Institute (Shanghai, China). Other commercial reagents, including glutaraldehyde (GA), silver nitrate, and trisodium citrate, were analytical reagent grade and used without further purification. In addition, 0.01 mol L 1 phosphate-buffered saline (PBS, pH 7.4), prepared by dissolving 0.296 g NaH2PO42H2O, 2.90 g Na2HPO412H2O, 8.0 g NaCl, and 1.0 g KCl in 1 L of ultrapure water (18.2 MX), was used as the buffer for immunoreactions. Also, 0.1 mol L 1 citric acid/Na2HPO4 buffer solution (pH 6.8) and Britton–Robinson buffer solution (pH 5.02–9.15) was used to adjust the pH of Ag-NP suspension for light scattering indication. All water used was ultrapurified with an LD-50G-E Ultra-Pure Water System (Lidi Modern Water Equipment, Chongqing, China).
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and boiling for 30 min, the mixture gradually changed and became yellow after approximately 10 min and then turned to brown yellow. After that, the colloidal Ag-NP solution was stirred continuously until it cooled to room temperature. It should be noted that controlling the reaction time is very important to make AgNPs of identical diameter for PRS detection (see supplementary material). The size of Ag-NPs as prepared was measured with an SEM, and the concentration of colloidal Ag-NPs was calculated according to the absorbance of Ag-NP solution [38] (e = 4.5 1010 for 46 nm Ag-NPs). Procedures Following traditional solid support immunoassay methods, we employed glass slides (12.7 76.2 1 mm, Shitai Experimental Instrument, Haimen, Jiangsu, China) as the solid support for immunoassays, and they were pretreated similarly to common procedures widely accepted for the cleaning and immobilization of antigen [42,45]. Shortly thereafter, the glass slides were ultrasonically washed with the detergent and soaked in a piranha solution (H2SO4/H2O2, 3:1 [v/v]) for 12 h. After washing thoroughly by ultrapure water of 18.2 MX and drying with nitrogen, the glass slides were soaked in acetone for approximately 2 min and then dried with a nitrogen gas flow. The cleaned and dried glass slides were immersed in 2% (v/v) APTES acetone solution for 3 min, washed three times with ultrapure water, and dried completely in an oven for 30 min at 110 °C. To immobilize antigen onto the surface of amino-silanized glass slides, the GA covalent coupling strategy was applied to modify the glass slides. Amino-silanized glass slides were at first immersed in 2.5% (v/v) GA solution for 1 h at 37 °C, washed thoroughly with ultrapure water, and then immersed in a plastic cell containing 1 ml of 0.1 to 1 lg ml 1 H-IgG in PBS (pH 7.4) solution to bind the antigen. After being left for 9 h at 4 °C, the glass slides were soaked in 1 mg ml 1 BSA for 1 h at room temperature to block the active site of unreacted GA. After the above widely employed procedures for immobilizing the antigen on the solid support of glass slides, the H-IgG immobilized glass slides were transferred for immunoreactions into the plastic cell containing goat anti-human IgG antibody in PBS solution for 1 h at 37 °C. After washing with PBS solution and ultrapure water three times, the glass slides were immersed in the plastic cells containing 1.0 ml of carefully pH-adjusted Ag-NP suspension for several minutes. Then the glass slides were washed and nitrogen-dried before being transferred to PRS measurements. Results and discussion
Preparation of Ag-NPs Spectral features of Ag-NPs adsorbed on glass slides Ag-NPs were prepared according to the modified Lee–Meisel method [43] that was similar to that described by Jarvis and Goodacre [44]. All facilities were cleaned thoroughly with detergent and aqua regia. Then 50.0 ml of solution containing 1.0 mmol L 1 of AgNO3 was boiled in a conical flask and 2.0 ml of 1% (w/v) trisodium citrate was introduced to the flask. Under continuous stirring
Scheme 1 shows our protocol of label-free immunoassay for the detection of antibody with Ag-NPs as PRS indicator. After first chemically immobilizing antigen on the surface of glass slides and making the immunoreactions occur with antibody, we transferred the glass slides into an Ag-NP suspension so that Ag-NPs
Scheme 1. Label-free immunoassay based on the PRS signals of adsorbed Ag-NPs on the surface of glass slides.
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Selective adsorption of Ag-NPs on the solid supports
Fig. 1. PRS spectra of Ag-NPs adsorbed on glass slides. Conditions: antibody (from 1 to 4), 0, 50, 120, and 250 ng ml 1, respectively; antigen immobilized, 0.25 lg ml 1; Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; adsorption time, 10 min. The inset shows the PRA spectra of glass slides under the same conditions.
could be adsorbed in the area where the immunoreactions have occurred. Fig. 1 shows the light scattering spectra of glass slides and those on which Ag-NPs have been adsorbed. It can be seen that the PRS spectra of adsorbed Ag-NPs have a characteristic PRS peak located at 405 nm and increase with increasing antibody. The characteristic PRS features are strongly related to the PRA [37]. As the inserted PRA spectra in Fig. 1 show, Ag-NPs display PRA features characterized near 400 nm similar to those reported extensively in aqueous medium [46,47], and the increase of absorbance also confirms the increasing adsorption of Ag-NPs on antibody bound on the surface of glass slides. The microstructure and optical property of single Ag-NPs, which adsorbed on the glass slides with a beautiful blue emission of scattered light, was studied intensively with an SEM and a dark field microscope. The SEM image (Fig. 2A) shows that Ag-NPs as prepared in this study have an average diameter of 46 nm and are dispersed on the glass slides. However, the shapes of Ag-NPs are not only spheres but also a few rods. This might be attributed to the multiple distribution of our prepared Ag-NPs in size and shape. It is known that the PRA and PRS properties of Ag-NPs are size and shape dependent in the spectral range from 400 to 700 nm [37]; therefore, the light scattering image of single Ag-NPs on glass slides with the dark field microscope shows that Ag-NPs scatter mainly blue light besides some green or red light (Fig. 2B). The green and red dots in Fig. 2B are the results of other shapes and sizes of the prepared nanoparticles owing to the uneven distribution.
Because the protein is positive or negative charged depending on the pH of the aqueous medium and the isoelectric points (pI0), there is a possibility that antigen or antibody will be positive and could have electrostatic attraction with Ag-NPs. So, if the pI0 values of immobilized antigen (H-IgG) and the antibody (goat anti-human IgG) are different, the selective adsorption of Ag-NPs on the solid supports could be realized by adjusting the pH values of the Ag-NP suspension in which the antibody bound on the surface of immunocomplex glass slides is positively charged, whereas the antigen immobilized glass slides is not. In such cases, controlling the pH value of Ag-NP suspension in the absorption step is crucial to make the adsorption of Ag-NPs be dependent only on the content of antibody and not on that of antigen. Although human IgG has a structure similar to that of goat IgG, the antigen covalently immobilized on glass slides should have different features, particularly the different pI0 values, given the acylamidate group of the antigen resulting from the immobilizing reaction with GA. Therefore, in a certain medium of pH lower than its pI0 value, the antigen immobilized on the surface of glass slides might not be positively charged for the acylamidation of the amido group that has stereo-obstruction to the adsorption of the negative-charged Ag-NPs, whereas the antibody bound on the glass slides could be positive so that the adsorption of the negative-charged Ag-NPs is dependent only on the content of antibody. Fig. 3 shows the dependence of the adsorption of Ag-NPs on the pH values of the Ag-NP suspension after the immunoreactions between a given content of antigen immobilized on the surface of the glass slides and a given content of antibody. From the intensity of
Fig. 3. pH dependence of colloidal silver on the adsorption process. Conditions: antigen immobilized, 0.25 lg ml 1; antibody, 0.25 lg ml 1; Ag-NPs, 1.1 10 10 M; adsorption time, 10 min; k(IPRS) = 405 nm. Ab, antibody.
Fig. 2. SEM image (A) and dark field light scattering microscopic image (B) of Ag-NPs on glass slides. Conditions: antigen immobilized, 0.25 lg ml Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; adsorption time, 10 min.
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PRS signals of Ag-NPs adsorbed on the surface of glass slides, we found that Ag-NPs could be adsorbed only on antibody-bound glass slides. With decreasing pH, the antigen immobilized on glass slides is also positive charged (as is the bound antibody), resulting in the adsorption of Ag-NPs on the glass slides without bound antibody. With increasing pH values higher than 8.5, the isoelectric point of antibody, the adsorption of Ag-NPs decreases dramatically with the disappearance of electrostatic interaction between the antibody and the Ag-NPs. However, the adsorption of Ag-NPs on antibody-bound glass slides is also present at pH values higher than the isoelectric point of antibody, indicating that the interactions between the Ag-NPs and proteins is not only electrostatic attraction but also chemisorptions of proteins on the Ag-NP surface. Experiments showed that the detection of antibody concentration was dependent on the amount of antigen immobilized on glass slides. Theoretically, the more antigens immobilized on glass slides, the more antibodies could bind to the glass slides. Immobilizing much more antigen on glass slides, however, would result in the chemisorptions of Ag-NPs on the antigen immobilized glass slides even if no antibody is present. By optimizing antigen concentration during the process of immobilization, we found that the maximum enhanced PRS signals (DIPRS) of glass slides could be obtained if 0.25 to 0.30 lg ml 1 antigen was used during the immobilization (Fig. 4). Adsorption time is another factor that affects the adsorption of Ag-NPs only on antibody-bound glass slides. Fig. 5 shows that longer adsorption time (ta) could result in chemisorptions of AgNPs on the antigen immobilized glass slides even if antibody is not present. Consequently, the adsorption of Ag-NPs on only the antibodybound glass slides depends on the pH of Ag-NP solution, the amount of antigen immobilized on glass sides, and the Ag-NP adsorption time. Controlling these experimental conditions carefully could result in the selective adsorption of Ag-NPs on the glass slides bound with antibody.
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Fig. 5. Dependence of adsorption time on Ag-NPs. Conditions: antigen immobilized, 0.25 lg ml 1; antibody, 0.25 lg ml 1, Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; adsorption time, 10 min; k(IPRS), 405 nm. Ab, antibody.
Fig. 6. Competition experiment in the presence of free antigen. Conditions: antigen immobilized, 0.25 lg ml 1; antibody, 0.25 lg ml 1; Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; k(IPRS), 405 nm. Ab, antibody.
Competitive assay Analytical detection of antibody To demonstrate the reliability of this immunoassay, a competitive experiment was performed under optimal conditions. In the competitive assay, the free antigen in the solution competes with the antigen immobilized on the glass to react with the antibody. Fig. 6 shows that the PRS signal of Ag-NPs can be effectively reduced in the presence of free antigen.
Under optimal conditions, it was found that the intensity of PRS signals of Ag-NPs adsorbed selectively on antibody-bound glass slides is linearly dependent on the content of antibody. Fig. 7 shows that a good linear relationship, DIPRS = 12.83c 10.31, between the PRS intensity (DIPRS) at kmax = 405 nm and antibody
Fig. 4. Effect of antigen concentration on the immobilization process. Conditions: antibody concentration, 1 lg ml 1; Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; adsorption time, 10 min; k(IPRS), 405 nm. Ab, antibody.
Fig. 7. Relationship between the scattering intensity and the antibody concentration. Conditions: antigen immobilized, 0.25 lg ml 1; Ag-NPs, 1.1 10 10 M; adsorption pH, 6.8; adsorption time, 10 min, k(IPRS), 405 nm.
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Fig. 8. Visual detection of antibody on glass slides under the illumination of an LED torch. Conditions: antigen immobilized, 0.25 lg ml pH, 6.8; adsorption time, 10 min; antibody (from A to G), 10, 30, 60, 100, 150, 200, and 250 ng ml 1, respectively.
concentration (cAb) is found in the range between 10 and 160 ng ml 1 with R2 = 0.9836 and a detection limit of 5.6 ng/ml. Due to the strong PRS emission of Ag-NPs, the attractive signals from the PRS of Ag-NPs could be clearly seen by the naked eye under a common white LED light excitation. Then a visual label-free immunoassay based on the PRS of Ag-NPs was established to detect antibody on common glass slides with simplicity and low cost. Fig. 8 shows a series of glass slides bound with different amounts of antibody under the illumination of an LED torch in a dark room. Because the amount of Ag-NPs adsorbed depends on the amount of antibody bound on the glass slides, clear blue PRS light of Ag-NPs seen from the glass slides can be used to visually estimate the concentration of antibody. Because they are adsorbed on the surface of glass slides only where the antibody is bound, the Ag-NPs could be used as an indicator to localize the immunoreactions between antigen and antibody on the glass slides. With the PRS light of Ag-NPs from the immune–glass slides, we can see the location where immunoreactions happened and get more information about the immunoreactions on the glass slides. According to the PRS image of immune–glass slides (Fig. 8), the immunoreactions happened over all of the antigen immobilized surface of the glass slides where abundant antibody (more than the antigen immobilized) is present (panels F and G), but they happened only at certain areas of the glass slides randomly where there is a lack of antibody (panels A–E). Therefore, the PRS-based light scattering method with AgNPs selectively adsorbed on the antibody where immunoreactions happened is suitable to investigate the reaction between antigen and antibody on solid support. Conclusion In this work, a novel, label-free, visual immunoassay method, using the PRS signals of the Ag-NP electrostatic adsorbed on glass slides where antibody is bound, has been established to distinguish the immunoreactions on glass slides with a common LED torch. We discussed the mechanism of this method and investigated the effect of experimental conditions with the scattering signals of AgNPs measured on a common spectrofluorometer. Under optimal conditions, antibody over the range between 10 and 160 ng ml 1 could be detected quantitatively with spectrofluorometer or just an LED torch and the naked eye. The method based on the adsorption of Ag-NPs as an indicator to distinguish the different electrostatic properties between the immobilized molecule and the specific bound molecule on glass slides could find more applications in the label-free study of protein–protein, DNA–protein, and saccharide–protein interactions. Acknowledgment The authors greatly appreciate the financial support from the National Natural Science Foundation of China (20425517).
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M; adsorption
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