Anal. Chem. 2002, 74, 96-99
Application of the Biological Conjugate between Antibody and Colloid Au Nanoparticles as Analyte to Inductively Coupled Plasma Mass Spectrometry Chao Zhang,† Zhenyu Zhang,† Binbing Yu,‡ Jinjun Shi,† and Xinrong Zhang*,†
Department of Chemistry and Department of Biological Sciences and Biotechnology, Tsinghua University, 100084, Beijing, P.R. China
This paper describes the study of atomization of nanoparticles by inductively coupled plasma mass spectrometry (ICPMS) and developes a novel nonisotopic immunoassay by coupling sandwich-type immunoreaction to ICPMS. The goat-anti-rabbit immunoglobulin G (IgG) labeled with colloidal gold nanoparticles served as an analyte in ICPMS for the indirect measurement of rabbitanti-human IgG. Matrix effect studies showed the gold signal was not sensitive to the organic matrix. A relatively good correlation (r2 ) 0.9528) between the proposed method and enzyme-linked immunosorbent assay has been obtained. The method may have significant potential as an important ICPMS-based nonisotopic immnoassay method for the simultaneous determination of biologic analytes of interest by labeling different kinds of inorganic nanoparticles. Since its introduction in the early 1960s, radioimmunoassay (RIA) has revolutionized the biological measurement due to its extremely high sensitivity and specificity.1 Even today, radioimmunoassay still plays a major role in clinic diagnoses, medicine and related areas.2 The special disposal of radioactive waste after use and the potential radioactive hazards, however, has been the big problem. The sensitively nonisotopic methods may overcome the drawbacks of radioactive detection and have great potential in many research fields such as DNA sequencing, clinical diagnostics, and biological assays.3-6 Therefore, a plethora of nonisotopic labels has been employed, such as chemiluminescent, fluorescent labels, metal atoms, as the alternatives of RIA method.7-9 * Corresponding author: (tel) +86-10-62787678; (fax) +86-10-62770327; (email)
[email protected]. † Department of Chemistry. ‡ Department of Biological Sciences and Biotechnology. (1) Chard, T. An introduction to radioimmunoassay and related techniques, 2nd ed.; Elsevier Biomedical Press: Amsterdam, 1982. (2) Development in Radioimmunoassay and Related Procedures; IAEA: Vienna, 1991 (Proc. Symp. Vienna, 1991). (3) Kricka, L., Ed. Nonisotopic probing, blotting, and sequencing; Academic Press: NewYork, 1995, (4) Knemeyer, J. P.; Marme, N.; Sauer, M. Anal. Chem. 2000, 72, 3717-3724. (5) Gelmini, S.; Caldini, A.; Becherini, L.; Capaccioli, S.; Pazzagli, M.; Orlando, C. Clin. Chem. 1998, 44, 2133-2138. (6) Rossler, A. Clin. Chim. Acta 1998, 270, 101-114. (7) Martin, C.; Bresnick, L.; Juo, R. R.; Voyta, J. C.; Bronstein, I. Biotechniques 1991, 11, 110-113. (8) Gite, S.; Mamaev, S.; Olejnik, J.; Rothschild, K. Anal. Biochem. 2000, 279, 218-225.
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The expanding availability of a variety of nanostructures with unique properties at nanoscale dimensions has attracted widespread attention in their use in biotechnological systems. Besides, since nanoparticles are similar in size range to many common biomolecules, they are more suitable for integration with biological systems.10-13 For instance, the use of highly luminescence semiconductor nanocrystals (quantum dots) as fluorescent biological labels has been used in ultra-sensitive biological detection.14 Inductively coupled plasma mass spectrometry (ICPMS), as an outstanding method for trace element determination, has gained a very wide acceptance due to its extremely high sensitivity and element specificity. However, the excellent performances of ICPMS mainly focus on the field of inorganic element analysis. It is well-known that apart from high sensitivity, ICPMS can offer powerful ability for simultaneous determination of inorganic elements in a few minutes. In principle, by selecting proper nanoparticles of elements and labeling them to the biological molecules, ICPMS-based nonisotopic immunoassay may open new possibility to simultaneously detect biological analytes of interest. At the present level of development, colloidal Au nanoparticlelabeled antibody has been chosen as the model protein and the possibility of a sandwich-type immunoreaction coupled to ICPMS was explored. Colloidal Au nanoparticles are ideal markers in biotechnological systems for several reasons: first, they can be readily prepared in a wide range of sizes, from about 2 nm to above 100 nm; second, the specific activities of micromolecules can be retained when coupling micromolecules to colloidal Au nanoparticles; third, the gold particles can be easily visualized as dense strictures within biological entities in the transmission electron microscopy.15 As an excellent biological tag, colloidal Au nanoparticles have been extensively employed to label a broad range of biological receptors, such as protein A, immunoglobulin G (IgG), and glucose oxidase, and applied to surface-enhanced (9) Li. M.; Selvin, P. R. Bioconjugate Chem. 1997, 8, 127-132. (10) Reichert, J.; Csaki, A.; Kohler, J. M,; Fritzsche, W. Anal. Chem. 2000, 72, 6025-6029. (11) Taton, T. A.; Mirkin, C. A.; Letsinger, R. Science 2000, 289, 1757-1760. (12) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 1214212150. (13) Sondi, I.; Siiman, O.; Koester, S.; Matijevic, E. Langmuir 2000, 16, 31073118. (14) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018. (15) Verkleij, A. J.; Leunissen, J. L. M. Immuno-gold-labeling in cell biology; CRC Press: Boca Raton, FL, 1989. 10.1021/ac0103468 CCC: $22.00
© 2002 American Chemical Society Published on Web 11/30/2001
Table 1. Operating Conditions of Elan 6000 ICPMS rf power coolant argon flow auxiliary argon flow nebulizer argon flow operating frequency sample uptake rate detector mode scanning mode dwell time sweeps per reading readings per replicate replicates internal standard used
1100 W 15 L min-1 1.2 L min-1 1 L min-1 40 MHz 1.4 mL min-1 pulse mode peak hopping 50 ms 3 60 1 In
Raman scattering (SERS), surface plasmon resonance (SPR), and immunoblotting.16-18 In the proposed paper, the atomization of different kinds of nanosized materials including TiO2, La2CuO4, Y2O3, and colloidal Au nanoparticles in the ICP torch has been investigated. Also, a sandwich-type immunoreaction using colloidal Au nanoparticlelabeled antibody coupled to ICPMS was demonstrated. Human IgG was first immobilized to the solid phase. Afterward, the bound antigen was allowed to capture rabbit-anti-human IgG antibody specifically followed by detection of goat-anti-rabbit second antibody labeled with colloidal Au nanoparticles with ICPMS. Results indicated that the ICPMS-based nonisotopic immunoassay may have potential for the determination of biological analytes of interest. EXPERIMENTAL SECTION Instrumentation. A Elan 6000 ICPMS (PE- Sciex, Concord, Canada) was used for this experiment. The instrumental optimal operating parameters are summarized in Table 1. Prior to analysis, the X, Y positions of the torch, rf power, nebulizer gas flow, and lens were optimized using 10 µg/L Mg, Rh, and Pb in 2% nitric acid. A multielement solution containing 10 µg/L Be, Co, In, and Pb, respectively, was used to calibrate the lens autosettings and to establish a linear relationship between lens voltage and mass. The enzyme-linked immunosorbent assay (ELISA) results were obtained by measuring the absorbance at 490 nm with the BioRad model 550 microplate reader. Reagents and Immunoreaction Buffers. Deionized water (18 MΩ cm) was used in all the experiment (Beijing ShaungFeng purity water equipment factory). Polystyrene 96-well microtiter plates (Nanc) were used to perform the immunoreaction. Human IgG, goat IgG, rabbit-anti-human IgG antibody, and bovine serum albumin (BSA) were purchased from Beijing Xin Jing Ke biotechnology Co. Ltd. (Beijing, China). TiO2, La2CuO4, Y2O3, colloidal Au nanoparticles, and goat-anti-rabbit colloidal Au conjugate were synthesized in our laboratory. The buffers used were as follows: (A) coating buffer, 0.05 M carbonate/bicarbonate buffer solution, pH 9.6; (B) assay buffer, 0.01 M sodium phosphate-buffered saline (PBS) containing 1% BSA, pH 7.4; (C) washing buffer, buffer B with 0.05% Tween 20 (16) Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71, 4903-4908. (17) Lyon, L. A.; Musick, M. D.; Natan, M. J. Anal. Chem. 1998, 70, 51775183. (18) Chevallet, M.; Procaccio, V.; Rabilloud, T. Anal. Biochem. 1997, 251, 6972.
Figure 1. TEM photograph of colloidal Au.
Preparation of Colloidal Gold Nanoparticles. Colloidal Au was prepared according to the literature with slight modification.15,19 Briefly, after boiling 0.01% HAuCl4 with 0.05% trisodium citrate in aqueous solution for 15-30 min, the resulting colloidal suspension was cooled and filtered through a 0.45-µm Millipore membrane. The diameter of particle was ∼15 nm (Figure 1), as confirmed by Hitachi H-800 transmission electron microscopy. Preparation of a Colloidal Gold-Antibody Conjugate. Antibody-colloidal conjugates were prepared according to the modification in the literature.15-17 The goat-anti-rabbit antibody (10% more than the minimum amount, which was determined using a flocculation test) was added to 1 mL of pH-adjusted colloidal Au suspension followed by incubation at room temperature for 1 h. The conjugate was centrifuged at 45000g for 30-60 min, and the soft sediment was resuspended in 0.01mol L-1 Trisbuffered saline. Addition of glycerol to a final concentration of 50% allows storage of the colloidal Au goat-anti-rabbit conjugate at -20 °C for several months. Immunoassay Protocol. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction (Figure 2). Initially, a polystyrene 96-well microtiter plate was coated using 200 µL of human IgG (diluted to 10 µg/well with bicarbonate buffer, pH9.6) and incubated at 4 °C overnight. The unbound antigen was washed away with PBS containing 0.05% Tween-20 (PBS-T). After washing, the wells were incubated with PBS containing 1% BSA for 1 h at 37 °C. Afterward, the plate was washed three times with PBS-T; Series dilutions of rabbit-antihuman IgG antibodies with assay buffer were pipetted into the wells and incubated for 2 h at 37 °C. Plates were washed six times with PBS-T followed by addition of colloidal Au-labeled goat-anti(19) Frens, G. Nat. Phys. Sci. 1973, 241, 20-22.
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Figure 2. Scheme of sandwich-type immunoreaction. Table 2. Comparison of Atomization of Nanoparticle Suspensions and Their Solutions relative intensitya
TiO2 Y2O3 La2CuO4 Au
a
in solution
nanoparticle suspension
100 ( 3.3 100 ( 2.9 100 ( 2.1 100 ( 1.8
98.6 ( 3.2 100.5 ( 1.7 101.2 ( 3.4 100.8 ( 3.7 (colloidal Au) 99.1 ( 2.5 (colloidal Auantibody conjugate)
Five replicates; concentrations, 10 ng/mL
rabbit antibody (1:200 dilution with PBS containing 1% BSA) to each well and incubation for 4 h at 37 °C followed by washing six times with PBS-T. External calibration was used for the quantitative determination of rabbit-anti-human IgG. In was used as internal standard element to correct the fluctuation of the instrument. A 200-µL aliquot of 1% HNO3 solution containing 1 ng/mL In was added to each well. Samples to be analyzed were placed at room temperature for 3 min and introduced to the ICPMS by peristaltic pump. RESULTS AND DISCUSSION Atomization of Nanosized Meterials in ICP. For most atomic spectroscopic methods, it is necessary to solubilize the sample in a suitable solvent before it can be introduced into the instrument. The atomization, therefore, is an important factor that affects the sensitivity of the analyte of interest. In this paper, a comparison of atomization of TiO2, La2CuO4, Y2O3, and Au nanoparticle suspensions as well as their solutions was conducted in ICP. La2CuO4 and Y2O3 nanoparticle suspensions were divided into two groups (nanoparticle suspensions and their corresponding solutions dissolved by 1% nitric acid) and introduced into the ICP system. The same concentrations of Ti and Au standard solutions were prepared in order to compare the results with that obtained from TiO2, colloidal Au nanoparticles in ICP. The results in this experiment shown in Table 2 suggested that the nanoparticles in ICP have the same atomization efficency as that of ion solutions, indicating the excellent atomic efficiency in the ICP torch. Study of the Matrix Effect of Colloidal Au NanoparticleLabeled Antibody. Sample composition and interaction in plasma commonly affect the result in ICPMS determinations when a nebulizer chamber system is used. These interferences are generally the result of either sample matrix effects that influence 98 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
Table 3. Nonspecific Binding and Specificity for the Determination of Rabbit-Anti-Human IgG capture antibody
analyte
Relative Au intensity (n)4)
human IgG human IgG human IgG none goat IgG
blank goat-anti-human IgG rabbit-anti-human IgG rabbit-anti-human IgG rabbit-anti-human IgG
3.5 ( 0.17 5.9 ( 0.31 100 ( 6.5 4.8 ( 0.19 4.1 ( 0.21
rabbit-anti-human IgG and goat-anti-human IgG concentration, 5 ng/ mL; human IgG and goat IgG concentration, 10 µg/mL.
aerosol formation or the formation of ions in the plasma. The matrix effect of colloidal Au nanoparticle-labeled antibody was investigated prior to coupling to the immunoreaction. The preformance of Au solution, colloidal Au nanoparticle solution, and colloidal Au nanoparticle-labeled antibody solution in the ICP torch were examined. Table 2 indicated no difference was observed by comparing the results obtained from these solutions, indicating that the Au signal was not sensitive to the organic matrix. Immunoreaction. The immunoreaction was conducted by using the procedure for the sandwich type. The scheme is shown in Figure 2. Human IgG was immobilized on the solid phase followed by using 1% BSA to block the nonspecific binding site. After the blocking step, the rabbit-anti-human IgG and colloidal Au-labeled goat-anti-rabbit antibody were added to form a sandwich complex of human IgG-rabbit-anti-human IgG-colloid goldlabeled goat-anti-rabbit IgG. Since the antigen-antibody complex can be dissociated under some extreme physiochemical conditions such as high temperature, low pH, and strong ionic strength, 1% HNO3 was employed to get the measurable signals of Au eluted from the solid surface after completion of the immunoassay in this experiment. Specificity. The specific recognition of antigenic species was studied. Goat-anti-rabbit IgG labeled with colloidal Au nanoparticles was used for immune recognition. Human IgG immobilized on the plate served as the capture antibody. The results in Table 3 showed that the Au signal only increased after immunoreaction of rabbit-anti-human antibody with human IgG coated on the plate. The Au signal obtained from goat-anti-human IgG, however, was slightly higher than that from the blank, suggesting that only rabbit-anti-human IgG was specifically captured by the substrate as well as bound with colloidal Au goat-anti-rabbit IgG. Since colloidal Au nanoparticles with extremely high particle density
Figure 3. Dependence of Au intensity on rabbit-anti-human IgG concentration. Table 4. Intra-assay and Interassay for Rabbit-anti-human IgG Determination rabbit-anti-human IgG concn (ng/mL)
mean ( SD
RSD (%)
2.5 10 50
Intra-assay (n ) 6) 2.34 ( 0.18 10.3 ( 0.58 48.5 ( 2.1
7.7 5.6 4.3
2.5 10 50
Interassay (n ) 5) 2.41 ( 0.16 10.5 ( 0.78 49.5 ( 3.6
6.6 7.4 7.3
have strong adsorption activity, it is necessary to determine the extent of the nonspecific binding (NSB) of colloidal Au-labeled antibody in the assay. The results in Table 3 showed that the Au signal from NSB was less than 5% by adding colloidal gold-labeled goat-anti-rabbit IgG to either the blank microtiter plate or the plate coated by using goat IgG, indicating an acceptable NSB level. Analytical Performance. The dilution test of colloidal Au goatanti-rabbit IgG showed that the detection limit of the colloidal Au goat-anti-rabbit IgG was 0.008 ng/mL by series dilution. After immunoreaction, the detection limit for the rabbit-anti-human IgG was 0.4 ng/mL (3σ). The dependence of Au intensity on rabbitanti-human IgG concentration was shown in Figure 3. A good linear relationship between Au intensity and rabbit-anti-human IgG in the concentration range between 0.8 and 50 ng/mL was obtained (r2 ) 0.9846). The departure from linearity was observed when the concentration of rabbit-anti-human IgG was up to 100 ng/mL. The reproducibility of an assay was expressed in term of values for a within-batch (intra-assay) and a between-batch (interassay)
Figure 4. Correlation of ICPMS and ELISA for the determination of rabbit-anti-human IgG.
relative standard deviation (RSD) in the presented paper. The obtained mean values, standard deviation (SD), and RSD by replicate analyses (n ) 6) in the intra-assay and in the interassay (n ) 5) are reported in Table 4. The RSD values were all below 10%, indicating an acceptable level of precision. Finally, a correlation of results for rabbit-anti-huamn IgG serum by ICPMS and ELISA was investigated. The results of comparative studies were shown in Figure 4. It can be seen that relatively good correlation was obtained (r2 ) 0.9528) between these methods. CONCLUSION A novel nonisotopic method using immunoreaction coupled to ICPMS has been developed. Studies have shown that the immunoassays may be successful by detecting nanoparticlelabeled antibody with ICPMS. Compared with other nonisotopic methods, the proposed detection has a wide choice of label and, thus, may expand the range of application. The ICPMS-based nonisotopic method demonstrated here may open up new possibilities for biological assays and clinical diagnoses. Furthermore, it has potential to be applicable to simultaneous determination of several biological or clinical analytes of interest by selecting proper nanoparticles of inorganic elements and labeling them to the biological molecules. ACKNOWLEDGMENT This work is supported by National Natural Science Foundation of China (20075014) Received for review March 23, 2001. Accepted September 25, 2001. AC0103468
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