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Technical Notes
Electrochemical Coding for Multiplexed Immunoassays of Proteins Guodong Liu, Joseph Wang,*,† Jeonghwan Kim, and M. Rasul Jan‡
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003 Greg E. Collins
Naval Research Laboratory, Chemistry Division, Washington D.C. 20375
An electrochemical immunoassay protocol for the simultaneous measurements of proteins, based on the use of different inorganic nanocrystal tracers is described. The multiprotein electrical detection capability is coupled to the amplification feature of electrochemical stripping transduction (to yield fmol detection limits) and with an efficient magnetic separation (to minimize nonspecific adsorption effects). The multianalyte electrical sandwich immunoassay involves a dual binding event, based on antibodies linked to the nanocrystal tags and magnetic beads. Carbamate linkage is used for conjugating the hydroxyl-terminated nanocrystals with the secondary antibodies. Each biorecognition event yields a distinct voltammetric peak, whose position and size reflects the identity and level, respectively, of the corresponding antigen. The concept is demonstrated for a simultaneous immunoassay of β2-microglobulin, IgG, bovine serum albumin, and C-reactive protein in connection with ZnS, CdS, PbS, and CuS colloidal crystals, respectively. These nanocrystal labels exhibit similar sensitivity. Such electrochemical coding could be readily multiplexed and scaled up in multiwell microtiter plates to allow simultaneous parallel detection of numerous proteins or samples and is expected to open new opportunities for protein diagnostics and biosecurity. As research moves into the era of proteomics, scientists are faced with the challenge of developing effective methods for identifying and quantitating proteins.1 Such new techniques are essential for the diagnosis of various disease states, for defense against biological threats, and for improving drug discovery. The ability to measure simultaneously multiple proteins in a single assay holds an enormous potential for meeting the growing * Corresponding author. E-mail:
[email protected]. Tel.: 505-646-2140. † Permanent address: Department of Chemical and Material Engineering, Arizona State University, Tempe, AZ 85287. ‡ Permanent address: Department of Chemistry, University of Peshawar, Pakistan. (1) Zhu, H.; Bilgin, M.; Snyder, M. Annu. Rev. Biochem. 2003, 72, 783.
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demands of these diagnostic and biodefense applications. Immunoassays are highly suitable for high-throughput screening, as they require minimal sample manipulations, are compatible with multiwell or microchip formats, and require small amounts of target analytes.2 Most efforts for multianalyte immunoassays have focused on multicolor fluorescent detection (in connection with different organic dyes).2 However, such multicolor fluorescencelinked immunoassays are often complicated by the requirement of an elaborate excitation and detection scheme and by the broad emission bands.3 Electrochemical immunoassays have evolved dramatically over the past two decades4 and are ideally suited for meeting the portability requirements of decentralized point-of-care testing or field detection of bioagents. A dual-analyte immunoassay using electrochemical detection of metal ion labels was proposed by Hayes et al.5 More recently, Karube and co-workers6 described an antibody-based array electrochemical biosensor for the simultaneous identification of multiple antigens. Here we report on an electrical immunoassay coding protocol for the simultaneous measurements of multiple proteins based on the use of different inorganic nanocrystal tracers. Colloidal nanocrystals have been used recently for the simultaneous fluorescent immunoassay of four toxins3 and for the electrical hybridization detection of multiple DNA targets.7 In our new bioassay (Figure 1), the target antigens are captured using magnetic beads conjugated with the corresponding antibodies (A, B). The bound antigens are then detected by reactions with a pool of nanocrystal-antibody pairs (C) and stripping voltammetric measurement of the corresponding metals (D). Each individual protein recognition event thus yields a distinct voltammetric peak, (2) (a) Swartzman, E. E.; Miraglia, S. J.; Mellentin-Michelotti, J.; Evangelista, L.; Yuan, P. M. Anal. Biochem. 1999, 271, 143. (b) Luminex100 IS total system, http: // luminexcorp.com. (3) Goldman, E. R.; Clapp, A. R..; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.; Medintz, I. L.; Mattoussi, H. Anal. Chem. 2004, 76, 684. (4) Cousino, M.; Jarbawi, T.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1997, 69, 544A. (5) Hayes, F. J.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1994, 66, 1860. (6) Kojima, K.; Hiratsuka, A.; Suzuki, H.; Yano, K.; Ikenbukuro, K.; Karube, I. Anal. Chem. 2003, 75, 1116. (7) Wang, J.; Liu, G.; Merkoci, A. J. Am. Chem. Soc. 2003, 125, 3214. 10.1021/ac049107l CCC: $27.50
© 2004 American Chemical Society Published on Web 11/02/2004
Figure 1. Multiprotein electrical detection protocol based on different inorganic colloid nanocrystal tracers. (A) Introduction of antibody-modified magnetic beads; (B) binding of the antigens to the antibodies on the magnetic beads; (C) capture of the nanocrystal-labeled secondary antibodies; (D) dissolution of nanocrystals and electrochemical stripping detection.
whose position and size reflect the identity and level, respectively, of the corresponding antigen. The new multianalyte electrical detection capability is coupled to high sensitivity (and fmol detection limits) and absence of nonspecific interactions. The attractive characteristics of the new multiprotein electrical immunoassay are reported in the following sections. EXPERIMENTAL SECTION Apparatus. Square-wave voltammetric stripping measurements were performed with an Autolab 12 (Eco Chemie), controlled by a PC, using a 1.5-mL electrochemical cell containing a glassy carbon disk working electrode (2-mm diameter), a Ag/ AgCl reference electrode (CH Instruments, Austin, TX), and a platinum wire counter electrode. The magnetic bead assays and separations were performed on a MCB 1200 Biomagnetic processing platform (Dexter, CA). All centrifugation steps were performed using a Micromax centrifuge (Thermo IEC). Reagents. All stock solutions were prepared using deionized and autoclaved water. Sodium acetate buffer (3 M, pH 5.2), TrisHCl buffer (1 M, pH 8.0), nitric acid, carbon disulfide, CdCl2, PbCl2, ZnCl2, CuCl2, petroleum ether, acetone, methanol, potassium hydroxide, lithium chloride, sodium hydroxide, and sodium chloride were purchased from Sigma. Hexadecanol, hexadecylamine (HDA), dithiolthreitol (DTT), 1,1-carbonyl diimidazole (CDI), anhydrous dioxane, toluene, and Tween 20 were purchased from Aldrich. Anti-mouse IgG biotin conjugate, mouse IgG, anti-mouse IgG, β2-microglobulin, anti-β2-microglobulin, C-reactive protein (CRP), polyclonal anti-human C-reactive protein, Immunoprobe biotiny-
lation kit, and albumin (from bovine serum, BSA) were purchased from Sigma. The anti-β2-microglobulin biotin conjugate and monoclonal mouse anti-human C-reactive protein were obtained from United States Biological (Swampscott, MA). Anti-BSA and antiBSA biotin conjugate were received from Molecular Probes Inc. (Eugene, OR) and Lab Version Inc. (Fremont, CA), respectively. Monoclonal anti-human C-reactive protein biotin conjugate was prepared by a standard protein biotinylation method with polyclonal anti-human C-reactive protein and the Immunoprobe biotinylation kit. Proactive streptavidin-coated magnetic microspheres (0.83-µm diameter, CM01N-Catalog No. 4725) were purchased from Bangs Laboratories (Fishers, IN). Preparation of Inorganic Nanocrystals and Their Bioconjugates. High-quality metal sulfide nanoparticles were produced using metal salts of hexadecyl xanthate (HDX) as precursors following the protocol of Pradhan and Efrima.8 The corresponding antibody-nanocrystal conjugates were prepared according to the procedure of Thompson and co-workers.9 Both protocols are summarized in Scheme 1 and are described below. Preparation of HDX (C16 Xanthate) Potassium Salts. The HDX capping agent was prepared first using a slightly modified literature procedure.10 Briefly, HDX potassium salts were prepared by mixing equimolar (0.04 mol) amounts of hexadecanol (9.69 g) and potassium hydroxide (2.24 g) and heating (and melting) the mixture at 150 °C. The resulting solution was suspended with (8) Pradhan, N.; Efrima, S. J. Am. Chem. Soc. 2003, 125, 2050. (9) Pathak, S.; Choi, S. K.; Arnheim, N.; Thompson, M. E. J. Am. Chem. Soc. 2001, 123, 4103. (10) Sawan, P.; Kovalev, E.; Klug, J.; Efrima, S. Langmuir 2001, 17, 2913.
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Scheme 1. Steps Involved in the Preparation of the Nanocrystal Tags and Their Antibody Conjugates
homogeneous stirring in 25 mL of toluene at 100 °C. Carbon disulfide (4.41 g) was added dropwise at 25 °C. The thick yellowish suspension was cooled and vigorously stirred for 1 h, and the solution was diluted with 100 mL of petroleum ether and stirred for additional 2 h. The product was filtered on glass funnel, and the solid was washed with petroleum ether and collected. The crude xanthate was then dried, filtered again with 20 mL cold deionized water, followed by drying in a vacuum oven and washing with petroleum ether. Finally, the HDX was vacuum-dried again. Preparation of Metal (Zn, Cd, Pb, Cu)-HDX. HDX (3.56 g) in 5 mL of methanol was mixed with equimolar M (Zn, Cd, Pb, Cu)chloride salt solution for 2 min. The mixture was centrifuged, and the resulting M-HDX was washed twice with methanol and then dried under vacuum. Preparation of Metal Sulfide Nanocrystals with a Capping Agent. In accordance to literature procedure,8 0.5 g of HDA (acting as a strong electron-donating monosurfactant), heated to 120 °C and cooled to 50 °C, was homogeneously mixed (by stirring) with 0.05 g of M-HDX, and the mixture was heated to 100 °C for 30 min. The temperature was gradually increased to 120 °C for 1.5 h (with the final 2 min at 140 °C). The annealed metal sulfide nanocrystals were slowly cooled to 70 °C. The resulting white (ZnS), yellowish (CdS), black (PbS), and brown (CuS) powders were precipitated by absolute methanol for flocculation, then washed twice with absolute methanol, and dried at room temperature followed by storage in the dark. Preparation of Nanocrystal-Antibody Conjugates. Carbamate linkage was used for conjugating the hydroxyl-terminated nanocrystal with the secondary antibody.9 Forty milligrams of the resulting nanocrystals was dispersed in anhydrous dioxane with 1 g of DTT. The suspension was refluxed for 12 h at 70 °C, followed by centrifugation and washing three times with anhydrous dioxane. The dried hydroxylated nanocrystals were redispersed in dioxane. The hydroxyl-modified nanocrystals were activated with CDI for 2 h at room temperature. Following the precipitation of the CDI-activated hydroxylated nanocrystals, the precipitate was washed twice with anhydioxane to remove excessive amount of CDI, and the products were dried under vacuum. The resulting powder was dissolved in dioxane. Then, the CDIactivated CdS, PbS, ZnS, and CuS nanocrystals were exposed to 100 µL of anti-IgG, anti-BSA, anti-β2-microglobulin, and polyclonal anti-human CRP (240 µM in 20 mM PBS, pH 7.4), respectively. The highly soluble mixture (pH 8.5, adjusted with NaOH) was stirred for 24 h and then centrifuged for 15 min at 14 000 rpm. The resulting antibodiy-nanocrystals were washed twice with dioxane and with PBS. The resulting conjugates were dispersed 7128
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in 0.1 M phosphate buffer. Based on steric considerations, only two to five antibodies can be linked to each nanocrystal. 11 Preparation of Antibody-Conjugated Magnetic Beads. The magnetic bead preparation was carried out on an MCB 1200 Biomagnetic processing platform using a modified procedure recommended by Bangs Laboratories (Tech Note 101).12 Briefly, 2.5 µL (i.e., 25 µg) of streptavidin-coated magnetic beads were transferred into a 1.5-mL centrifuge tube; Then, 95 µL of the TTL buffer (100 mM Tris-HCl, pH 8.0, 0.1% Tween, and 1 M LiCl) was added and mixed for 1 min with a speed of 0.5 rps. Application of the magnetic field (to the side of the centrifuge tube) attracted the beads to the sidewall. After the solution became clear, it was carefully removed with a pipet. The washing step was repeated once. The beads were then suspended in 21 µL of TTL buffer. Then, 4 µL of each biotinylated antibody (anti-IgG, anti-BSA, antiβ2-microglobulin, monoclonal anti-human C-reactive protein) was added to yield a final antibody concentration of 200 µg mL-1. The mixture was incubated for 30 min with gentle mixing. After magnetic separation, the antibody-magnetic beads were washed twice with 95 µL of TT buffer (250 mM Tris-HCl, 0.1% Tween 20) and suspended in 45 µL of TTL buffer. Sandwich Immunoassay. The selected amount of antigen (IgG, BSA, β2-microglobulin, C reactive protein) was then added and mixed for 30 min. Following their magnetic isolation, the magnetic beads bearing the immunocomplex were washed twice with 95 µL of TT and resuspended again in 40 µL of TTL buffer. Subsequently, 2.5 µL of each nanocrystal-antibody conjugate was added and mixed for 30 min. The resulting sandwich-conjugated microspheres were washed twice with 95 µL of TT and resuspended in 10 µL of 1 M HNO3 solution. After a 3-min mixing and a magnetic separation, the HNO3 solution (containing the dissolved Cd2+, Pb2+, Zn2+, and Cu2+) was transferred into 1 mL of the acetate buffer (0.1 M, pH 4.5) supporting electrolyte solution containing 10 ppm mercury(II). Control experiments were performed in a similar fashion but without adding the antigen. Electrochemical Detection. Stripping voltammetric measurements of the dissolved nanocrystals were performed (in a stirrer acetate buffer solution) using an in situ plated mercury film on a glassy carbon electrode, following a 1-min pretreatment at 0.6 V, using a 2-min accumulation at -1.4 V. Subsequent square-wave stripping was performed after a 15-s rest period (without stirring) from -1.2 to +0.1 V with step potential of 4 mV, amplitude of 20 mV, and frequency of 25 Hz. Baseline correction of the resulting voltammogram was performed using the “moving average” mode of the GPES (Autolab) software. RESULTS AND DISCUSSION In the current study, we used different antibody-conjugated inorganic nanocrystals to demonstrate the simultaneous detection of multiple proteins. The new electrical immunoassay involves a dual binding event, based on antibodies linked to the tagged spheres and magnetic beads (Figure 1). Each protein recognition thus yields a distinct voltammetric peak, whose position and size reflects the identity and level, respectively, of the corresponding antigen. The new concept is demonstrated for simultaneous immunoassays of β2-microglobulin, IgG, BSA, and CRP in con(11) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016. (12) Technote 101, Bangs Laboratories Inc., Fishers, IN, August 29, 1999.
Figure 2. Typical stripping voltammograms for (A) nanocrystallabeled antibodies and (B-F) magnetic bead-Ab-Ag-Ab-nanocrystal complexes. (A) Response for a solution containing dissolved ZnSanti-β2-microglobulin, PbS-anti-BSA, and CdS-anti-IgG conjugates. Five microliters of each nanocrystal-antibody conjugate solution dissolved in 20 µL of 1 M HNO3 and transferred into 1 mL of the supporting electrolyte solution. (B-D) Immunoassay stripping response for three different antigen targets (BSA, IgG, and β2microglobulin, respectively), each at 100 ng mL-1 (ppb) level. (E) Response for a sample mixture containing the three antigen targets present at the 100 ng mL-1 level. (F) Control experiment, as (E) but without the corresponding antigens. Amount of antibody-coated magnetic beads (in the 50-µL reaction solution), 25 µg; amount of nanocrystal-modified secondary antibody, 2.5 µL; sandwich assay with 30 min for each incubation step.
nection with ZnS, CdS, PbS, and CuS colloidal crystals, respectively. These proteins are often used for the clinical diagnosis of certain diseases. The method chosen for detecting the dissolved nanocrystal tracers is square-wave anodic stripping voltammetry (SWASV) as it combines the amplification feature of stripping voltammetry with the speed advantage of square-wave scanning.13 Figure 2 displays the typical SWASV response of the new multiantigen bioassay. A mixture of three antibody-conjugated nanocrystals yielded sharp and baseline-resolved peaks at -1.05 (ZnS-anti-β2-microglobulin), -0.65 (CdS-anti-IgG), and -0.48V (PbS-anti-BSA) (Figure 2A). Subsequently, we examined the response of the individual antigen and of a mixture of the three antigens (at 100 ng mL-1). As expected from Figure 2A, the individual antigens yielded welldefined peaks and resolved peaks (of similar sensitivity) at -0.50 (BSA) (B), -0.65 (IgG) (C), and -1.02 V (β2-microglobulin) (D) (vs Ag/AgCl). A sample mixture containing the three antigens exhibits three well-resolved signals of similar sensitivity [0.064 (β2 -microglobulin), 0.057 (IgG), and 0.063 (BSA) µA ng-1 mL] at -1.08 (β2 -microglobulin), -0.67 (IgG), and - 0.46 V (BSA) (E). In contrast, no response is observed for the corresponding control experiment without the corresponding antigens (F). The latter reflects the effective magnetic separation that (combined with the TTL buffer) eliminates nonspecific adsorption effects. The (13) Wang, J. Stripping Analysis; VCH Publishers: Deerfield Beach, 1985.
Figure 3. Stripping signals for increasing concentration of IgG (A) and BSA (B) in 25 ng mL-1 steps a-e. Dotted line shows the response for the control experiment without the antigens. Also shown (inset), the resulting calibration plots. Other conditions, as in Figure 2.
similar sensitivity of the different colloidal inorganic tracers is an attractive feature of the present protocol and reflects the similar metal contents of the different nanocrystals (i.e., control of the particle size via control of the metal-xanthate concentration and temperature during the preparation) and the similarity of their stripping signals. The size of the corresponding metal peaks provides the desired quantitative information on the target proteins. Figure 3 illustrates a dual protein quantitation from the stripping-voltammetric response for increasing IgG (A) and BSA (B) concentrations in 25 ng mL-1 steps (a-e). Well-defined peaks, proportional to the concentration of the corresponding protein, are observed. The resulting calibration plots (shown on top) are linear, with correlation coefficients of 0.990 (A) and 0.979 (B). Note also the flat baseline for the control experiment (without the antigens; dotted line). The high sensitivity (associated with the stripping voltammetric transduction) allows simultaneous monitoring of trace levels of both proteins. The favorable signal-to-noise characteristics of the response for the initial 25 ng mL-1 mixture (a) indicate a detection limit of ∼10 ng mL-1 (i.e., ∼3.3 (A) and ∼7.5 (B) fmol in the 50-µL samples). Similar detection limits were reported recently for analogous nanocrystal-based fluorescence immunoassays.3 Lower detection limits are expected in connection with longer deposition periods. The baseline correction capability (involving subtraction of the blank response) has been shown useful for addressing trace metal impurities in the supporting electrolyte solution (particularly zinc) and hence to enable such ultrasensitive protein measurements. A series of six repetitive measurements of a mixture containing 100 ng mL-1 IgG and BSA yielded a reproducible cadmium and lead peaks, respectively, with relative standard deviations of 6.9 and 9.2%. The degree of cross reactivity was examined by comparing the response for a 50 ng mL-1 IgG solution in the presence and absence of 5 µg mL-1 BSA. The large (100-fold) Analytical Chemistry, Vol. 76, No. 23, December 1, 2004
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Figure 4. Immunoassay stripping voltammograms for a sample mixture containing 100 ng mL-1 antigen targets (β2-microglobulin, IgG, BSA, C reactive protein) in the presence of ZnS-anti-β2-microglobulin, CdS-anti-IgG, PbS-anti-BSA, and CuS-polyclonal anti-human Creactive protein tags, respectively. Other conditions, as in Figure 2.
excess of BSA had a negligible effect upon the cadmium (IgG) signal, indicating minimal antibody-antigen cross reactivity (not shown; conditions as in Figure 2C, using anti-IgG conjugated magnetic beads and CdS-nanocrystals). These data reflect also the minimal nonspecific binding between the nanocrystal-labeled antibody and the coated magnetic beads. In designing mutliprotein electrical immunoassays based on multiple colloidal crystals tracers, it is essential to carefully consider the peak potentials of the corresponding metals. Common potential windows of ∼1.2 V can accommodate four to six resolved peaks for multiprotein detection. A pool of four nanocrystal-antibody pairs was used for demonstrating the simultaneous measurement of four proteins. Figure 4 displays the stripping voltammetric immunoassay response for a mixture containing 100 ng mL-1 β2-microglobulin, IgG, BSA, and CRP in connection with ZnS-, CdS-, PbS-, and CuS-labeled antibodies, respectively. Baseline-resolved peaks of nearly similar sensitivity and favorable signal-to-noise characteristics are observed for the four proteins. The wide flat baseline (e.g., between the Cd and Zn peaks or the Pb and Cu ones) indicates possible extension to (14) Wang, J.; Liu, G.; Rivas, G. Anal. Chem. 2003, 75, 4671. (15) Wang, J.; Xu, D.; Polsky, R. J. Am. Chem. Soc. 2002, 124, 4208.
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assay up to six proteins simultaneously. The concept could be further scaled up using parallel operations in 96- and 384-well microtiter plates, with each well carrying a simultaneous detection of four to six different proteins. In conclusion, we have introduced an effective and inexpensive multitarget electrochemical immunoassay based on the use of different colloidal nanocrystal tags. The multiprotein electrical detection capability has been coupled to the amplification feature of electrochemical stripping transduction (to yield fmol detection limits) and with an efficient magnetic separation (to minimize nonspecific binding effects). Up to five or six antigens are expected to be measured simultaneously in a single run (with greater number of analytes or samples when connected to an automated high-throughput multiwell operation). Higher degree of multiplexing could be achieved in connection with the encapsulation of various proportions of different nanocrystals into polymeric carrier beads.14 Further simplification could be achieved using a solidstate magnetic detection that obviates the need for dissolving the nanocrystals.15 Specific applications would require proper attention to potential nonspecific binding through a judicious choice of a blocking agent and wash steps. The low size, weight, and power requirements of electrical devices make them particularly attractive for various decentralized and field applications. The new electrical protein coding route should thus have a profound impact on biodiagnostics, biodefense, or environmental monitoring. ACKNOWLEDGMENT Financial support from the National Institutes of Health (Award R01A 1056047-01) and the National Science Foundation (Grant CHE 0209707) is gratefully acknowledged.
Received for review June 18, 2004. Accepted September 15, 2004. AC049107L
