4-(Dimethylamino)butyric Acid Labeling for Electrochemiluminescence

Jie Wu , Xue Zhang , Tongjie Yao , Jing Li , Hao Zhang and Bai Yang. Langmuir 2010 26 .... Liping Qiu , Zhifa Shen , Zai-Sheng Wu , Guo-Li Shen , Ruqi...
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Anal. Chem. 2005, 77, 3525-3530

4-(Dimethylamino)butyric Acid Labeling for Electrochemiluminescence Detection of Biological Substances by Increasing Sensitivity with Gold Nanoparticle Amplification Xue-Bo Yin, Bin Qi, Xuping Sun, Xiurong Yang,* and Erkang Wang*

State Key Laboratory of Electroanalytical Chemistry, Changchun Insititute of Applied Chemistry, Chinese Academy of Science, Changchun, 130022, China

4-(Dimethylamino)butyric acid (DMBA) labeling combined with gold nanoparticle amplification for electrochemiluminescence (ECL) determination of a biological substance (bovine serum albumin (BSA) and immunoglobulin G (IgG) as models) was presented. After DMBA, an analogue of tripropylamine, was tagged on the (anti)analytes, an ECL signal related to the content of the analytes was generated when the analyte tagged with DMBA was in contact with tris(2,2′-bipyridine)ruthenium (Ru(bpy)32+) solution and a potential was applied. To improve the adsorption capacity, a gold nanoparticle layer was first combined into the surface of the 2-mm-diameter gold electrode. For the determination of BSA, avidin was covalently conjugated to a self-assembled monolayer of 3-mercaptopropanoic acid on the gold nanoparticle layer. Biotinylated BSA-DMBA was then immobilized on the gold nanoparticle layer of the gold electrode via the avidin-biotin reaction. IgG was tested via a typical sandwich-type immobilization method. ECL signals were generated from the electrodes immobilized with BSA or IgG by immersing them in a 1 mmol L-1 Ru(bpy)32+ solution and scanning from 0.5 to 1.3 V versus Ag/AgCl. With gold nanoparticle amplification, the ECL peak intensity was proportional to the concentration over the range 1-80 and 5-100 µg/mL for BSA and IgG consuming 50 µL of sample, respectively. A 10- and 6-fold sensitivity enhancement was obtained for BSA and IgG over their direct immobilization on an electrode using DMBA labeling. The relative standard deviations of five replicate determinations of 10 µg/mL BSA and 20 µg/ mL IgG were 8.4 and 10.2%, respectively. High biocompatibility and low cost were the main advantages of the present DMBA labeling technique over the traditional Ru(bpy)32+ labeling. A rapid, highly specific method of detecting and quantifying biochemical and biological substances is increasingly needed.1,2 These materials can be determined by a binding method with a * To whom correspondence should be addressed. Fax: (86)431 5689711. E-mail: [email protected]. (1) Mikkelsen, S. R. Electroanalysis 1996, 8, 15-19. (2) Wang J. Anal. Chim. Acta 2002, 469, 63-71. 10.1021/ac0503198 CCC: $30.25 Published on Web 04/12/2005

© 2005 American Chemical Society

high degree of specificity which characterizes many biochemical and biological systems, such as antigen-antibody systems, nucleic acid hybridization techniques, and protein-ligand systems. Some techniques, including electrochemical,1,2 optical,3 electrochemiluminescence (ECL),4-11 and photoelectrochemical methods,12 have been used as detection methods. Among them, ECL based on tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy)32+) is characterized by good spatial and temporal resolution and has attracted much attention in biomaterial analysis.8,9 Moreover, Ru(bpy)32+ ECL has high efficiency over a wide range of buffer pH with high sensitivity and selectivity.4,8,9 The conventional procedures for DNA assays and immunoassays based on ECL used generally Ru(bpy)32+ labels.6,7,10 As given in previous works,6,10 the procedure possesses some limitations. Ruthenium labeling at multiple sites may result in the loss of biological activity of the molecules and the precipitation of target analytes, e.g., protein.10,13 The synthesis of the label and the labeling procedure are complex and the whole procedure often needs low temperature and a light-tight environment. To overcome the drawback to some extent, Zhou et al.10,13 proposed a multilabeling technique at a single site, i.e., using the dendritic molecule containing three Ru(bpy)32+ units to increase the signal of the target analyte and decrease the bond sites to the analyte. Labeling of bovine serum albumin (BSA) with the dendrimer at one NH2 position was demonstrated with no subsequent loss of biological activity or precipitation of the BSA-dendrimer complex.10 But the preparation of dendrimer is more complex than that of the commonly used Ru(bpy)32+ label. Therefore, searching of the stable, easy to preparation, and highly biocompatible labels is of critical importance for the analysis of biological substance. (3) Christodoulides, N.; Tran, M.; Floriano, P. N.; Rodriguez, M.; Goodey, A.; Ali, M.; Neikirk, D.; McDevitt, J. T. Anal. Chem. 2002, 74, 3030-3036. (4) Lee, W. Y. Mikrochim. Acta 1997, 127, 19-39. (5) Fa¨hnrich, K. A.; Pravda, M.; Guilbault, G. G. Talanta 2001, 54, 531-559. (6) Miao, W. J.; Bard, A. J. Anal. Chem. 2003, 75, 5877-5885. (7) Namba, Y.; Usami, M.; Suzuki, O. Anal. Sci. 1999, 15, 1087-1093. (8) Knight, A. W.; Greenway G. M. Analyst 1995, 120, 2543-2747. (9) Xu, X. H.; Jeffers, R. B.; Gao, J.; Logan B. Analyst 2001, 126, 1285-1292. (10) Zhou, M.; Roovers, J.; Robertson, G. P.; Grover, C. P. Anal. Chem. 2003, 75, 6708-6717. (11) Richter, M. M. Chem. Rev. 2004, 104, 3003-3036. (12) Dong, D.; Zheng, D.; Wang, F. Q.; Yang, X. Q.; Wang, N.; Li, Y. G.; Guo, L. H.; Cheng, J. Anal. Chem. 2004, 76, 499-501. (13) Zhou, M.; Roovers, J. Macromolecules 2001, 34, 244-252.

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If tripropylamine (TPA), the most important coreactant in traditional ECL, can be tagged on the biomaterials, an ECL signal will be generated when a potential is applied to the electrode in contact with Ru(bpy)32+ solution. Morita and Konishi14,15 found that N-(3-aminopropyl)pyrrolidine, 2-(2-aminoethyl)-1-methylpyrrolidine, N-methyl-L-proline, 3-(diethylamino)propionic acid, and 4-(dimethylamino)butyric acid (DMBA) displayed strong enhancement to Ru(bpy)32+ ECL and applied these compounds to label carboxylic acids and amine followed by high-performance liquid chromatography-ECL detection. In principle, the tertiary amine derivatives above should be good labels in terms of stability and biocompatibility for biological material analysis, e.g., protein. The previous work16 illustrated also that the tertiary amines, as labels, have some advantages over Ru(bpy)32+ and conventional fluorophore (e.g., fluorescein), such as high label efficiency, good biocompatibility, and so on. In this work, DMBA was used as ECL labels for proteins analysis (BSA and immunoglobulin G (IgG) as models) using a 2-mm-i.d. gold electrode. But the primary works indicated that a lower sensitivity was resulted in because of the low adsorption capacity of the gold electrode, while a high background noise was obtained using a large-area electrode. So, a gold nanoparticle was used to improve the adsorption capacity and, thus, enhance the detection sensitivity. After BSA tagged with DMBA via a biotinavidin reaction or IgG via a sandwich-type method was immobilized on the gold electrode, ECL related to the concentration of BSA or IgG was obtained when the electrode was in contact with Ru(bpy)32+ solution and scanned from 0.5 to 1.3 V versus Ag/AgCl. EXPERIMENTAL SECTION Reagents. All the reagents employed were prepared freshly unless otherwise stated. Double-distilled water (DDW) was used throughout. DMBA, N-hydroxysuccinimide (NHS), 3-mercaptopropanoic acid (3-MPA), and tris(2,2′-bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)3Cl2‚6H2O) were purchased from Sigma-Aldrich (St. Louis, MO). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), avidin, BSA, and NHS-LCbiotin from Pierce (Rockford, IL) were used without further purification. Mouse IgG and goat anti-mouse IgG (anti-IgG) were obtained from Pierce and Sigma, respectively. 1,3-Propanedithiol (Alfa Aesar) was used to tag the gold nanoparticle on the surface of gold electrode. A 20 mM Ru(bpy)22+ stock solution dissolved in DDW was stored in refrigerator. The working solution was prepared fresh by diluting the stock solutions with 0.1 M phosphate salt solution (pH 7.5) and degassed ultrasonically for 10 min just before use. DMBA was dissolved in DDW directly at a concentration of 10 mM as stock solution. The solutions containing EDC and NHS were prepared fresh or added into the targeted solution directly for immobilization or labeling. Instrumentation. The electrochemical measurement for ECL experiments was carried out with model CH800 voltammetric analyzer (CH Instruments, Austin, TX). A three-electrode system was employed with Pt wire as counter electrode, Ag/AgCl/KCl (14) Morita, H.; Konishi, M. Anal. Chem. 2002, 74, 1584-1589. (15) Morita, H.; Konishi, M. J. Liq. Chromatogr. Relat. Technol. 2002, 25, 24132423. (16) Waguespack, B. L.; Lillquist, A.; Townley, J. C.; Bobbitt, D. R. Anal. Chim. Acta 2001, 441, 231-241.

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(sat) as reference electrode, and 2-mm-diameter gold disk as working electrode. The scan rate was fixed at 50 mV s-1. The gold electrode was fabricated by sealing a 1-cm length of gold wire into a poly(tetrafluoroethylene) (PTFE) tube using epoxy, with one end protruding out of the tip. The gold wire was connected with a copper wire through graphite powder filled into the PTFE tube. The new gold disk electrode was polished with 0.3- and 0.05-µm R-Al2O3 powder on fine abrasive paper. Because the ECL performance of a gold electrode depended on its pretreatment history, the electrode was repolished with 0.05-µm R-Al2O3 powder and washed with ethanol and DDW thoroughly before each experiment. The ECL emission was detected with a model MCDR-A chemiluminescence analyzer (Xi’An Remax Science & Technology Co. Ltd., Xi’An, China) and the voltage of the photomultiplier tube (PMT) used in the chemiluminescence analyzer was set at 600 V in the process of detection. Only the ECL corresponding to the first cycle of cyclic voltammetry was recorded due to the nonregeneration of the tertiary amine label and the oxidation of thiolayers, which resulted in the analyte loss from the electrode. Preparation of Gold Nanoparticles. Gold nanoparticles with a diameter of ∼12 nm were prepared by the sodium citrate reduction of AuCl4+ ions according to a well-known method.17 In brief, 100 mL of solution containing 0.01 g of HAuCl4‚3H2O was brought to reflux and then 3 mL of 1% sodium citrate solution was introduced while stirring. The solution was then kept boiling for another 40 min and left to cool to room temperature. The resulting gold sol contains ∼12-nm particles according to TEM.18 Biotinylation with Biotin-LC-NHS and Labeling with DMBA of BSA. BSA, as a model of protein, was used in the present DMBA labeling and ECL detection because it contains 59 lysines and 30-35 primary amines capable of being labeled and is commonly employed as a protein standard in bioanalytical assays.10 BSA was labeled with DMBA and biotinylated with biotin-LC-NHS as following: 2 mg of biotin-LC-NHS was dissolved in 50 µL of dry DMF and then added into the 5-mg BSA solution in 1 mL of 0.1 M phosphate buffer (pH 7.5). After incubation of 1 h for the BSA biotinylation at 37 °C, the reagent unreacted with BSA was removed via dialysis at 4 °C for 2 days with 0.1 M phosphate buffer (pH 7.5). The phosphate buffer was replaced every 8 h. The dialysis cassette (Pierce) with molecular weight cutoff of 7000 was used throughout. For labeling biotinlyted BSA with DMBA, about 2 mg of DMBA, 1 mg of NHS, and 2 mg of EDC were added to the biotinylated BSA solution and mixed for 1 h at 37 °C to label the biotinylated BSA with DMBA. The unreacted reagents were removed via dialysis for 2 days with 0.1 M phosphate buffer (pH 7.5). The final solution was diluted to 5 mL with 0.1 M phosphate buffer solution (pH 7.5) and stored at ∼4 °C prior to use. Because of the good biocompatibility of DMBA, no precipitation was observed in the whole pretreatment procedure. Preparation of Biotinylated Anti-IgG and DMBA Labeling Anti-IgG. A 2-mg sample of biotin-LC-NHS.was dissolved in 50 µg of dry DMF and then added into the 5-mg anti-IgG solution in 1 mL of 0.1 M phosphate buffer (pH 7.5). After 1-h mixing for the anti-IgG biotinylation at 37 °C, the biotin unreacted with anti-IgG was removed via dialysis at 4 °C for 2 days with 0.1 M phosphate (17) Frens G. Nat. Phys. Sci. 1973, 241, 20-22. (18) Lu L.; Wang H.; Xi S.; Zhang H. J. Mater. Chem. 2002, 12, 156-158.

buffer (pH 7.5). Anti-IgG labeled with DMBA was prepared as following: 100 µL containing 20 mg mL-1 DMBA, 10 mg mL-1 NHS, and 20 mg mL-1 EDC was added to 1 mg mL-1 anti-IgG solution and mixed for 1 h at 37 °C. The unreacted reagents were removed via dialysis for 2 days with 0.1 M phosphate buffer (pH 7.5). Biotinylated anti-IgG and DMBA labeling anti-IgG were used to determine IgG via sandwich-type immobilization. Direct Immobilization of BSA via Avidin-Biotin Reaction and IgG via Sandwich Mode on an Au Electrode. After the gold electrode was polished with R-Al2O3 powder and cleaned with ethanol and DDW thoroughly, the clean electrode was immersed in a 1 M 3-MPA aqueous solution for preparation of thiol selfassembled monolayers (SAM) on the surface of the gold electrode for ∼24 h. The newly formed Au/thio-SAM was rinsed with ethanol and DDW and dried with a N2 stream. And then, a 10 µL of 1 mg mL-1 avidin in 0.1 M phosphate buffer (pH 7.5) containing 20 mg mL-1 EDC and 10 mg mL-1 NHS was dropped on the gold electrode precoated with 3-MPA thiol monolayer to form the Au/ thio-SAM/avidin layer for 40 min at 37 °C. After the Au/SAM/ avidin layer and biotinylated BSA-DMBA solution were prepared, 10 µL of biotinylated BSA-DMBA solution at different concentrations obtained from the dilution of the biotinylated BSA-DMBA solution prepared above was dropped on the electrode covered with SAM/avidin layer. The biotion-avidin reaction was finished at 37 °C for 40 min. After being washed with DDW and ethanol and dried with a N2 stream, the electrode above was in contact with 1 mM Ru(bpy)32+ solution and scanned from 0.5 to 1.3 V versus Ag/AgCl, and hence, an ECL signal was obtained. For IgG immobilization, the formation of the Au/SAM/avidin layer was similar to that in the BSA immobilization above. A 10µL sample of 1 mg mL-1 biotinylated anti-IgG phosphate solution (pH 7.5) was dropped on the Au/SAM/avidin surface, and the biotin-avidin reaction took place at 37 °C for 40 min. The redundant biotinylated anti-IgG was removed through washing with ethanol and DDW and the electrode was dried with a N2 stream. The 10-µL aliquots of different concentrations of IgG were used as samples for IgG determination and dropped on the Au/ SAM/avidin/biotinylated anti-IgG surface. After antibody-antigen reaction carried out at 37 °C for 40 min, the matrix was removed by washing with ethanol and DDW and drying with a N2 stream. A 10-µL aliquot of 1 mg mL-1 anti-IgG tagged with DMBA was used to couple IgG. The unreacted anti-IgG was removed by washing with ethanol and DDW and drying with a N2 stream. The flowcharts and the schematic diagrams of the pretreated electrodes for BSA and IgG immobilization are illustrated in Figure 1A and B, respectively. Immobilization of BSA via Avidin-Biotin Reaction and IgG via Sandwich Mode on an Au Electrode after Au Nanoparticle Amplification. Liu et al.19,20 applied the gold nanoparticle amplification in a quartz crystal microbalance to improve the DNA analyses. For gold nanoparticle amplification in this work, a gold nanoparticle layer was first immobilized on the gold electrode through 1,3-propanedithiol. To this end, the gold electrode was immersed in 5 mmol L-1 1,3-propanedithiol in ethanol and was incubated at room temperature for 20 h. And (19) Liu T.; Tang J.; Zhao H. Q.; Deng Y. P.; Jiang L. Langmuir 2002, 18, 56245626. (20) Liu T.; Tang J.; Jiang L. Biochem. Biophys. Res. Commun. 2002, 295, 1416.

Figure 1. Flowcharts (A) and schematic diagrams (B) of the processes for the electrode pretreatment of immobilization of BSA (a) and IgG (b) via avidin-biotin reaction on the gold electrode.

Figure 2. Schematic diagrams of immobilization of BSA (a) and IgG (b) on the gold electrode with gold nanoparticle amplification.

then, 20 µL of a gold nanoparticle colloid solution formed above was dropped on the surface of the gold electrode containing 1,3propanedithiol monolayer. After 4 h, a gold nanoparticle layer was assembled on the surface of gold electrode. In case of immobilization of BSA via avidin-biotin reaction, the following procedures for the immobilization of 3-MPA, avidin reacting with 3-MPA, avidin-biotin reaction between biotinylated BSA-DMBA and avidin were similar to that of the direct immobilization of BSA on gold electrode. For determination of IgG, the processes from immobilization of 3-MPA to ECL measurement were consistent with that of the direct immobilization of IgG on gold electrode. Because of the high adsorption capacity due to gold nanoparticle amplification, the volume for analysis of BSA and IgG can be increased to 50 µL, compared with the 10 µL in their direct immobilization on gold electrode. The schematic diagrams of the pretreated electrodes for immobilization of BSA and IgG via gold nanoparticle amplification were showed in Figure 2. RESULT AND DISCUSSION ECL Detection. First, we used detection conditions similar to that of the capillary electrophoresis-ECL system in our previous work, 21 i.e., 5 mM Ru(bpy)32+ in 0.1 M phosphate salt (21) Yin, X. B.; Qiu, H.; Sun, X.; Yan, J.; Liu, J.; Wang, E. Anal. Chem. 2004, 76, 3846-3850.

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Figure 3. Electrochemiluminescence (A) and cyclic voltammogram (B) intensity versus potential. (a) On the bare gold electrode; (b) on the gold electrode with thio self-assembly monolayer; (c) after avidin immobilized on the gold electrode via self-assembly thio monolayer; and (d) after 50 µg/mL biotinylated BSA-DMBA immobilized on the gold electrode via avidin-biotin reaction. 1 mM Ru(bpy)32+ in 0.1 M phosphate solution (pH 7.5) and scan rate of 50 mV s-1.

Figure 4. Electrochemiluminescence profiles (A) and the cyclic voltammograms (B) versus potential obtained from (a) 20 µg/mL biotinylated BSA-DMBA, (b) 50 µg/mL biotinylated BSA-DMBA, (c) 80 µg/mL biotinylated BSA-DMBA, and (d) 100 µg/mL biotinylated BSA-DMBA immobilized on the gold electrode via avidin-biotin reaction. The other conditions as shown in Figure 3.

solution (pH 7.5) and 850 V of the voltage of PMT of model MCDR-A . But the background noise is very high because the area of the working electrode in the present work (2-mm-diameter Au disk) is much larger than that in the previous work (300-µmdiameter Pt disk21). After investigation, we found the voltage of the PMT at 600 V combined with the concentration of Ru(bpy)32+ at 1 mM gave a suitable sensitivity, a low background noise. Determination of BSA via the Avidin-Biotin Reaction and the DMBA Labeling. The labeling and immobilization of BSA on the gold working electrode via the avidin-biotin reaction was described in the Experimental Section and Figure 1Aa, and the schematic diagram of the treated electrode was illustrated in Figure 1Ba. Figure 3 is the ECL and EC22 profile related to the process of electrode pretreatment. It can be found that the ECL profile after the formation of thio SAM was essentially the same as that on the bare gold electrode although an oxidation peak of thio appeared on the cyclic voltammogram, indicating that 3-MPA had no ECL enhancement to that of Ru(bpy)32+ (Figure 3a and b). Once avidin was attached to the thio SAM on the gold electrode, a higher ECL intensity than that from the bare gold electrode was obtained (Figure 3Ac). Figure 4 showed the ECL profiles and the CVs versus potential of 20, 50, 80, and 100 µg mL-1 BSA tagged with DMBA on the gold electrode. When the concentration of BSA was above 50 µg mL-1, two ECL peaks (the first and second peaks in Figure 4A) appeared. As a tertiary amine derivative, DMBA has an electrochemical and ECL property similar to that of TPA. The mechanism of Ru-

(bpy)32+/DMBA is also similar to that of the Ru(bpy)32+/TPA system. Two kinds of reaction routes were concerned with the ECL of Ru(bpy)32+/TPA systems4,11,23 that is related to the concentration ratio of Ru(bpy)32+/TPA near the working electrode. When the concentration of Ru(bpy)32+ is higher than that of TPA, the ECL intensity reaches its maximum at a potential value of 1.14 V versus Ag/AgCl, 4,11,23 in the potential region of the direct oxidation of Ru(bpy)32+. That corresponded to the second peak in Figure 4A. But when the concentration of TPA was much higher than that of Ru(bpy)32+ at the surface of the electrode, another ECL peak at 0.82 V versus Ag/AgCl appeared.11,23,24 That was related to the first ECL peak in Figure 4A. Thus, when the concentration of DMBA immobilized on the working electrode was high enough and Ru(bpy)32+ was in bulk solution, there should be two obvious ECL peaks corresponding to the oxidation of DMBA and Ru(bpy)32+, which was proved by the produced ECL profiles beyond 50 µg mL-1 as shown in Figure 4A. We thought that the two ECL peaks were concerned with the oxidation of DMBA and Ru(bpy)32+, respectively. Figure 5 was the ECL and CV along with increasing the number of CVs, where decreased ECL signals were observed with increasing numbers of CVs. The profile of the fifth cyclic voltammogram and the corresponding ECL was consistent with that on a bare gold electrode (Figure 5c). In contrast to the Ru(bpy)32+ label, DMBA is not regenerable in the ECL procedure.

(22) Notes: the current in cyclic voltammogram may be from the oxidation of all compounds attached on the gold electrode, such as, 3-MPA, BSA, and DMBA and Ru(bpy)32+ in bulk solution.

(23) Miao, W. J.; Choi, J. P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 1447814485. (24) Li, F.; Zu, Y. Anal. Chem. 2004, 76, 1768-1772.

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Figure 5. Electrochemiluminescence profiles (A) and the cyclic voltammograms (B) versus potential of Au/thio-SAM/50 µg/mL biotinylated BSA-DMBA immobilized on the gold electrode from (a) the first cyclic voltammetry, (b) the second cyclic voltammetry, and (c) the fifth cyclic voltammetry. The other conditions as shown in Figure 3.

Moreover, an oxidation of the thio layer was also observed that may result in the analyte loss from the electrode surface. Thus, only the first cycle of cyclic voltammetry was performed and the corresponding ECL was recorded with the present DMBA labeling technique. Determination of BSA via DMBA Labeling after Gold Nanoparticle Amplification. The immobilization and labeling of BSA on the gold working electrode via avidin-biotin reaction after gold nanoparticle amplification was described as shown in the Experimental Section, and the schematic diagram of the treated electrode was illustrated in Figure 2a. Figure 6 showed the ECL and EC profiles related to the process of electrode pretreatment for determination of 10 µg mL-1 BSA. From (a) to (d), they were the ECL and CV profiles of bare gold electrode, the gold electrode immobilized gold nanoparticle, the gold electrode combined with avidin via gold nanoparticle, and the electrode illustrated in Figure 2a, respectively. In contrast to the direct immobilization of BSA on the electrode surface, an obvious oxidation current of gold nanoparticle was observed above 1.10 V as shown in Figure 6Bb. As for the ECL profiles, no peaks related to the oxidation of DMBA were found at all the BSA concentrations tested. It may be due to the extended distance between DMBA and the surface of the working electrode after gold nanoparticle amplification. Another difference between with and without gold nanoparticle amplification was that a small ECL peak was obviously observed near 0.5 V after nanoparticle amplification. But the small peak was related to the reduction of gold nanoparticle and not to the BSA concentration. Analytical Performance. In contrast to the Ru(bpy)32+ label, the tertiary amine label is not regenerated, so multiplex cyclic

Figure 6. Electrochemiluminescences (A) and cyclic voltammograms (B) versus potential. (a) On the bare gold electrode; (b) on the gold electrode tagged gold nanoparticle via1, 3-propanedithiol; (c) after avidin immobilized on the gold nanoparticle; and (d) after 10 µg/mL biotinylated BSA-DMBA immobilized on the gold electrode via gold nanoparticle amplification. The other conditions as shown in Figure 3.

voltammetric results in a decreased ECL signals gradually. Moreover, the oxidation of the thio layer may have resulted in the analytes loss from the surface of the electrode. So, only the ECL signal of the first cycle of CV was recorded in the work. For the determination of BSA via direct immobilization on the gold electrode surface, a dynamic range was 10-120 µg/mL. The calibration function and the correlation coefficient were E ) 34.875C + 644.35 and 0.9965, respectively, where E was ECL intensity (counts) and C was the concentration of BSA (µg mL-1). The leveling off of ECL response with BSA concentration above 120 µg/mL suggested that the avidin immobilized was saturated by biotinylated BSA-DMBA. Because the analytes were immobilized on the electrode, most of the analyte can make contact with the surface of the electrode and thus the high sensitivity was obtained compared with the consumption of 10 µL of sample. The relative standard deviation (RSD) of five replicate determinations of 50 µg/mL BSA was 6.3%. Although the ECL peak (the first peak in Figure 4) corresponding to the direct oxidation of DMBA can be used to quantify the contents of BSA, its sensitivity was lower than that using the second peak. Only BSA with a concentration above 50 µg mL-1 can be detected using the first peak, as shown in Figure 4. The sensitivity was further improved by the gold nanoparticle amplification and the increase in loading capability, and a dynamic range of 1-80 µg/mL was obtained with consumption of 50 µL of sample because of the 10 000 counts of maximum scale of MCDR-A chemiluminescence analyzer. The Analytical Chemistry, Vol. 77, No. 11, June 1, 2005

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calibration function and the correlation coefficient were E ) 131.21C + 634.55 and 0.9962, respectively, where E was ECL intensity (counts) and C was the concentration of BSA (µg mL-1). The RSD of five replicate determinations of 10 µg/mL BSA with gold nanoparticle amplification was 8.4%. It is important that we did not observe the precipitation of BSA during the procedures of BSA tagging with DMBA and dialysis because of the good biocompatibility of DMBA at the present condition. On the other hand, the character of a small molecule and good solubility of DMBA makes it easy entering the interior of BSA. Application. The present method of DMBA labeling and gold nanoparticle amplification was also used in IgG immunoassay. The analysis procedures of IgG sandwich-type immobilization with and without gold nanoparticle amplification were shown in the Experimental Section and Figures 1 and 2. The ECL and CV profiles of IgG sandwich-type immobilized on the gold electrode were similar to that in Figure 3. But no ECL peak related to the oxidation of DMBA within the all IgG concentrations tested was observed for the determination of IgG directly immobilized on the electrode. The ECL and CV profiles of IgG sandwich-type immobilization after gold nanoparticle amplification were similar to that in Figure 6. Compared with 30-150 µg/mL of directly immobilizing IgG on the surface of the working electrode, a dynamic range of 5-100 µg/mL IgG was obtained with IgG immobilized on the surface of the working electrode after gold nanoparticle amplification because of the 10 000 counts of maxi-

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mum scale of the MCDR-A chemiluminescence analyzer. The RSD of five replicate determinations of 20 µg/mL IgG with gold nanoparticle amplification was 10.2%. CONCLUSION This work proved the possibility of tertiary amine derivatives as ECL labels and gold nanoparticle amplification to improve the adsorption capacity for protein ECL analysis. As the alternative to the Ru(bpy)32+ label, DMBA has higher biocompatibility. Moreover, there are a lot of commercial and low-cost choices (the tertiary amine derivatives as given in ref 14) that can potentially be used as ECL labels. Combining with gold nanoparticle amplification, a 10- and 6-fold sensitivity improvement was obtained for determination of BSA and IgG over their direct immobilization of the electrode with DMBA labeling, respectively. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China with Grants 20299030, 20335040, and National Key Basic Research Program 2001CB5102. X.-B.Y. acknowledges the support of the China Postdoctoral Science Foundation for this project. Received for review February 21, 2005. Accepted March 22, 2005. AC0503198