Detection of Multiple Proteins on One Spot by Laser Ablation

Jan 5, 2007 - has been successfully used for the detection of element- .... Laser ablation ICPMS (LA-ICPMS) is one of the most useful techniques for ...
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Anal. Chem. 2007, 79, 923-929

Detection of Multiple Proteins on One Spot by Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Application to ImmunoMicroarray with Element-Tagged Antibodies Shenghong Hu,†,‡ Sichun Zhang,*,† Zhaochu Hu,§ Zhi Xing,† and Xinrong Zhang*,†

Department of Chemistry, Key Laboratory for Atomic and Molecular Nanosciences of Education Ministry, Tsinghua University, Beijing 100084, People’s Republic of China, Faculty of Earth Sciences, State Key Laboratory of Geological Processes and Mineral Resources, China University of Geosciences, Wuhan 430074, People’s Republic of China, and Key Laboratory of Continental Dynamics, Northwest University, Xi’an 710069, People’s Republic of China

Inductively coupled plasma mass spectrometry (ICPMS) has been successfully used for the detection of elementtagged biomolecules with the advantage of multielement capability. However, this technique cannot be used for microarray detection due to the necessity to dissolve the elemental tags before introducing them to the plasma source. Here, we report the detection of multiple proteins on each spot of the immuno-microarray by laser ablation ICPMS. r-Fetoprotein IgG (AFP), carcinoembryonic antigen (CEA), and human IgG, as model proteins, have been detected on the basis of sandwich-type immunoreactions on a microarray with Sm3+-labeled AFP antibody, Eu3+-labeled CEA antibody, and Au-labeled goat-antihuman IgG (GAH) as labeled antibodies. The detection limits were 0.20, 0.14, and 0.012 ng mL-1 (3σ) with the RSD of 5.7%, 2.6%, and 2.3% at the concentration of 1.0 ng mL-1 for AFP, CEA, and human IgG, respectively. The present detection method permits detecting multiple analytes from each spot of microarray with a spatial resolution at micrometer range, which can alleviate the stress to fabricate high-density arrays. Furthermore, the substrate materials and immobilized proteins do not interfere with the detection. The present technique provides a new strategy for readout of microarray. The development of arrays of immobilized biological compounds in micrometer-sized spots on a surface has played an important role in modern biology and medicine, and is currently revolutionizing many aspects of biological detection and monitoring technologies.1-3 The high throughput demands of proteomics have further driven the development in the fabrication and detection of microarrays of a variety of proteins. Current detection * To whom correspondence should be addressed. Fax: (+86) 10-6277 0327. E-mail: [email protected] (S.Z.); [email protected] (X.Z.). † Tsinghua University. ‡ China University of Geosciences. § Northwest University. (1) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (2) Delehanty, J. B.; Ligler, F. S. Anal. Chem. 2002, 74, 5681-5687. (3) Kim, B. C.; Park, J. H.; Gu, M. B. Anal. Chem. 2005, 77, 2311-2317. 10.1021/ac061269p CCC: $37.00 Published on Web 01/05/2007

© 2007 American Chemical Society

strategies for protein microarrays are classified as label-free methods and labeled probe methods.4,5 Label-free methods allow obtaining direct information about proteins of interest without labeling molecules but suffered from the disadvantages of relatively difficult quantification for a trace amount of analytes. Labeled probe detection methods have evolved from clinical immunoassay protocols, incorporating fluorescence, chromogenic, radioactive, or other labeling strategies for detection of immobilized targets. Detection of captured targets in an array is mainly performed by fluorescence using charge coupled device cameras or laser scanners with confocal detection optics because fluorescently labeled detection methods are simple and stable to manipulate and provide highly sensitive and resolved results. However, the sample may have components that interfere with a selected fluorophore, and the autofluorescence of substratums can cause significant reduction in signal-to-noise ratios. Other tags, such as lanthanide chelate and quantum dots (QD), have been developed to overcome the shortage of organic tags.6-8 Although the existing detection methods contribute much for the readout of microarray, it is still a challenge to detect multiple proteins quantitively on one spot of microarray. The feasibility of the determination of multiple biological analytes with elemental tags by inductively coupled plasma mass spectrometry (ICPMS) has been demonstrated by our group9-11 and other groups12-15 in recent years. The advantages of using ICPMS to immunoassays with element-tagged antigen or antibody (4) Espina, V.; Woodhouse, E. C.; Wulfkuhle, J.; Asmussen, H. D.; Petricoin, E. F.; Liotta, L. A. J. Immunol. Methods 2004, 290, 121-133. (5) Hoheisel, J. D. Nat. Rev. Genet. 2006, 7, 200-210. (6) Nichkova, M.; Dosev, D.; Gee, S. J.; Hammock, B. D.; Kennedy, I. M. Anal. Chem. 2005, 77, 6864-6873. (7) Gao, X. H.; Nie, S. M. Anal. Chem. 2004, 76, 2406-2410. (8) Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884-1886. (9) Zhang, C.; Wu, F. B.; Zhang, X. R. J. Anal. At. Spectrom. 2002, 17, 13041307. (10) Zhang, C.; Zhang, Z.; Yu, B.; Shi, J.; Zhang, X. R. Anal. Chem. 2002, 74, 96-99. (11) Zhang, S. C.; Zhang, C.; Xing, Z.; Zhang, X. R. Clin. Chem. 2004, 50, 12141221. (12) Baranov, V. I.; Quinn, Z.; Bandura, D. R.; Tanner, S. D. J. Anal. At. Spectrom. 2002, 17, 1148-1152. (13) Quinn, Z. A.; Baranov, V. I.; Tanner, S. D.; Wrana, J. L. J. Anal. At. Spectrom. 2002, 17, 892-896.

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Figure 1. Scheme of laser ablation system and laser ablation sampling from a microarray. (a) Scheme of laser ablation system, and (b) scheme of laser ablation sampling for detecting multiple analytes on each spot of a microarray.

include low detection limits, large dynamic range, multiplexing potential, and better spectral resolution. In addition, this technique has low matrix effects from other components of the biological sample. However, it is still difficult to introduce elemental tags of an antibody immobilized on a microarray to ICPMS directly without breaking the conjugate of antibody-antigen by diluted nitric acid. Therefore, a sample introduction system for solid samples immobilized on the microarray is required to extend the technique to microarray format detection. Laser ablation ICPMS (LA-ICPMS) is one of the most useful techniques for quantitative determination of solid samples with a spatial resolution at micrometer range. It can offer powerful ability for determination of trace and ultratrace elements in solid samples.16-18 This technique has been successful applied to the determination of geological and environmental samples.19-21 Recently, it has been used for the analysis of biological and medical samples, in particular for the detection of phosphor-, (14) Baranov, V. I.; Quinn, Z.; Bandura, D. R.; Tanner, S. D. Anal. Chem. 2002, 74, 1629-1636. (15) Ornatsky, O.; Baranov, V. I.; Bandura, D. R.; Tanner, S. D.; Dick, J. J. Immunol. Methods 2006, 308, 68-76. (16) Mokgalaka, N. S.; Gardea-Torresdey, J. L. Appl. Spectrosc. Rev. 2006, 41, 131-150. (17) Vogel, A.; Venugopalan, V. Chem. Rev. 2003, 103, 577-644. (18) Gu ¨ nther, D.; Horn, I.; Haffendorf, B. Fresenius’ J. Anal. Chem. 2000, 368, 4-14. (19) Becker, J. S. Int. J. Mass Spectrom. 2005, 242, 183-195. (20) Pickhardt, C.; Dietze, H. J.; Becker, J. S. Int. J. Mass Spectrom. 2005, 242, 273-280. (21) Durrant, S. F.; Ward, N. I. J. Anal. At. Spectrom. 2005, 20, 821-829.

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seleno-, and metalloproteins22-24 in gels or gel blots after electrophoresis. For example, Che´ry and Gu¨nther22 reported the determination of protein-bound metals by LA-ICPMS after gel electrophoresis. In their works, LA-ICPMS was used for the identification of the elements, which were already associated with proteins. In contrast, the LA-ICPMS-based immunoassays provided a means to determine multiple proteins that did not necessarily contain a metal. Mu¨ller et al.25 reported that the proteins in gel blots had been detected by LA-ICPMS with the use of antibodies conjugated to gold clusters. Hutchinson et al.26 have developed a new strategy for detection and imaging β-amyloid in brain tissue by LA-ICPMS measurement of Ni- and Eu-tagged antibodies. These works have demonstrated that laser ablation can be used as a direct sample introducing method for ICPMS-based immunoassay with the advantages of high sensitivity, good linearity, and excellent spatial resolution. The ability for the determination of trace proteins on solid surface directly suggests that LA-ICPMS might be a potentially readout method for microarrays. (22) Che´ry, C. C.; Gu ¨ nther, D.; Cornelis, R.; Vanhaecke, F.; Moens, L. Electrophoresis 2003, 24, 3305-3313. (23) Becker, J. S.; Zoriy, M.; Krause-Buchholz, U.; Becker, J. S.; Pickhardt, C.; Przybylski, M.; Pompe, W.; Ro¨del, G. J. Anal. At. Spectrom. 2004, 19, 12361243. (24) Bandura, D. R.; Ornatsky, O.; Liao, L. J. Anal. At. Spectrom. 2004, 19, 96100. (25) Mu ¨ller, S. D.; Diaz-Bone, R. A.; Felix, J.; Goedeckeb, W. J. Anal. At. Spectrom. 2005, 20, 907-911. (26) Hutchinson, R. W.; Cox, A. G.; McLeod, C. W.; Marshall, P.; Harper, S. A.; Dawson, E. L.; Howlett, D. R. Anal. Biochem. 2005, 346, 225-233.

Table 1. Operating Parameters for LA-ICPMS instrument Agilent 7500a ICPMS

GeoLas laser ablation system (193 nm)

parameter

description

ICP RF power nebulizer gas flow auxiliary gas flow plasma gas flow sampling depth acquisition mode dwell time mass resolution isotope used laser wavelength

1350 W 0.65 L min-1 0.60 L min-1 15 L min-1 6 mm time-resolved analysis 10 ms 0.7 amu 147Sm, 151Eu, 197Au, 29Si 193 nm

laser output energy laser crater repetition frequency laser shot number laser sampling mode He carrier gas flow

190 mJ 30 or 90 µm 1 Hz 1 raster scanning mode 0.67 L min-1

The aim of the present work is to develop a detection method for microarrays based on LA-ICPMS with antibodies conjugated to different metal ions or nanoparticles. We have taken the R-fetoprotein IgG (AFP), carcinoembryonic antigen (CEA), and human IgG as model proteins to illustrate the potential application of LA-ICPMS for the detection of multiple analytes on one spot of a microarray. EXPERIMENTAL SECTION Apparatus and Materials. The scheme of laser ablation system is shown in Figure 1. The laser-ablation system of GeoLas 200M (MicroLas, Go¨ttingen, Germany) was used for this experiment. The system was equipped with a 193 nm ArF-excimer laser and a homogenizing, imaging optical system for getting a flat top beam onto the sample surface. The diameter of laser beam was varied between 4 and 120 µm by means of an aperture system. Helium was used as a carrier gas to enhance transport efficiency of ablated materials and mixed with argon before entering the plasma torch. The laser ablation system was coupled to a 7500a ICPMS (Agilent Technologies, Yokogawa, Japan) for the determination of elemental tags. The glass and polyethylene substrate with immobilized antibodies were fixed on microscope slides of laser cell. The ablation cell was mounted on an xyz-stage with a camera connected to the same computer to change the ablation site on the surface of substrate. The operating parameters of LAICPMS are given in Table 1. Eu3+-labeled AFP antibody (Eu3+-anti-AFP) and Sm3+-labeled PSA antibody (Sm3+-anti-PSA), used in the preliminary experiments, were purchased from the Jiangsu Institute of Nuclear Medicine (Wuxi, China). The standard solutions of AFP, CEA in the TRFIA (time-resolved fluorescent immunoassay) kits used to obtain the calibration curve were purchased from the same Institute. Anti-R-fetoprotein antibody (anti-AFP), anti-carcinoembryonic antigen (anti-CEA), R-fetoprotein (AFP), carcinoembryonic antigen (CEA), Sm3+-labeled monoclonal anti-R-fetoprotein antibody (Sm3+-anti-AFP), and Eu3+-labeled carcinoembryonic antigen antibody (Eu3+-anti-CEA) were purchased from this Institute. Rabbit anti-human IgG (RAH), human IgG, and Au nanoparticletagged goat-anti-human IgG (Au-GAH) were purchased from Bejing Biosynthesis Biothechnology Co. (Beijing, China). The conjugation density of labeled reagents is supplied by the

Figure 2. Transient signals of individual element-tagged antibodies obtained by LA-ICPMS from spots of dried droplets on a glass slide. (a) Eu3+-labeled anti-AFP, (b) Sm3+-labeled anti-PSA, and (c) colloidal Au nanoparticles labeled GAH. The volume of the droplets was 1 µL, and the concentration of the antibodies was 1.0 ng mL-1 for each labeled antibody. Laser conditions: single point with a laser crater of 30 µm in diameter.

manufacturer. One mole each of Eu3+-anti-AFP and Eu3+-anti-CEA were labeled with 7.3 and 7.5 mol of Eu3+ chelate, respectively. One mole each of Sm3+-anti-AFP and Sm3+-anti-PSA were labeled with 13 and 12.5 mol of Sm3+ chelate, respectively. One milliliter of Au nanoparticles was labeled with 25 µg of GAH. Deionized water was used to prepare solutions and wash the substrates in all of the experiments. Microarray slides with the printable area of 65 × 22 mm were purchased from CapitalBio Corp. (Product No. 420040, Beijing, China). The slide surface was covered by a thin layer of threedimensional polymer chain modified with aldehyde group. The Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

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Figure 3. Transient signals of mixing element-tagged antibodies obtained by LA-ICPMS from spots of dried droplets on a glass slide. (a) Eu3+-labeled anti-AFP and Sm3+-labeled anti-PSA, (b) Sm3+-labeled anti-PSA and colloidal Au-labeled GAH, (c) Eu3+-labeled anti-AFP and colloidal Au-labeled GAH, and (d) Eu3+-labeled anti-AFP, Sm3+-labeled anti-PSA, and colloidal Au-labeled GAH. The volume of the droplets was 1 µL, and the concentration of the antibodies was 0.50 ng mL-1 for each labeled antibody. Laser conditions: single point with a laser crater of 30 µm in diameter.

Figure 4. SEM pictures of laser ablation craters on glass slide. (a) SEM picture of craters with different diameter. The diameter were 40, 30, 20, 10, and 4 µm from the top to bottom row. (b) SEM picture of two adjacent laser ablation crater (30 µm).

protein molecules can diffuse into the polymer layer after spotting and were bound inside with the aldehyde groups on the polymer chain by covalent interaction.27,28 Protein microarrays consisting of 12 sub-arrays were generated for dealing multiple samples on one slide by the manufacturer. (27) Capitabio Corp., Microarray Slides; http://www.capitalbio.com.

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Immunoassay Procedures. The mixing monoclonal antibodies including anti-AFP, anti-CEA, and RAH were prepared in 40% glycerol and 0.01% SDS solution at a concentration of 0.10 mg mL-1. The mixed antibodies were spotted on the surface of slide (28) Nielsen, P. S.; Ohlsson, H.; Alsbo, C.; Andersen, M. S.; Kauppinen, S. J. Biotechnol. 2005, 116, 125-134.

Figure 5. SEM pictures of laser ablation crater on polyethylene slide by using different wavelength laser systems. (a) 193 nm ArF-excimer laser source, and (b) 266 nm Nd:YAG laser source.

Figure 6. The effect of laser repetition frequency on sample desorption efficiency. (a) The transient signals of Si and Eu by one pulse, (b) the transient signals of Si and Eu by two pulses, and (c) the transient signals of Si and Eu by five pulses. Table 2. Signal Intensities of Eu3+ in LA-ICPMS with Different Matrixes and Substrates ion intensity of transient signal/Cps (n ) 5) matrix human IgG Eu3+ + human IgG Eu3+ + human IgG + PSA Eu3+ + PSA + GAH

glass (mean ( SD)

polyethylene (mean ( SD)

200 ( 24 10 820 ( 848 11 200 ( 762

200 ( 24 25 370 ( 1395 24 830 ( 1442

10 320 ( 738

25 530 ( 1314

with approximately 0.20 µL for one spot, which corresponded to a diameter of about 1.5 mm. After spotting, the slide was placed in a chamber at room temperature and a relative humidity of 70% for 16 h to immobilize the antibodies. Next, 25 µL of blocking buffer containing 0.10 mol L-1 Tris-HCl and 3% BSA was added to each sub-array and incubated for 30 min at room temperature. After incubation, the slide was washed three times with PBS washing buffer containing 0.1% Tween-20 (PBS-T, pH 7.4) to remove unbound proteins, and dried under a stream of nitrogen gas. For capturing the proteins, an aliquot of 25 µL of AFP, CEA, and human IgG mixed solution was added to each sub-array. The slide was incubated at a relative humidity of 100% for 30 min at 37 °C. Next, it was washed with PBS washing buffer three times and dried under a stream of nitrogen gas. An aliquot of 25 µL of

the labeled antibodies (Sm3+-anti-AFP, Eu3+-anti-CEA, and AuGAH, 0.50 µg mL-1 for each) was added to each sub-array of the slide. After being incubated for 30 min at 37 °C and a relative humidity of 100%, the slide was washed and dried with the procedures mentioned above. Finally, the slide was placed in the ablation cell for detection by LA-ICPMS. Laser Ablation. Experiments were carried out using raster scanning mode and the time-resolved analysis (TRA) mode. A 30 or 90 µm laser beam was used across the surface of samples with an appropriate spacing and scanning rate. A typical run time was about 30 s for each spot. First, the blank signals with laser off were recorded for 15 s. Next, laser fire was used with one pulse to ablate the sample, followed by another 15 s for the transportation of ablated sample to plasma torch. The procedure was repeated for each sample spot. The position of carry gas inlet was placed a little under the substrate surface so as to improve the transportation efficiency and avoid spread of transient signal profile. RESULTS AND DISCUSSION Performance of LA-ICPMS for Element-Tagged Antibodies. The Sm3+-anti-PSA, Eu3+-anti-AFP, and Au-GAH were used for investigating a microarray detection method by LA-ICPMS. Dried droplet spots of 1 µL of element-tagged antibodies (1.0 ng Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

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Figure 7. The transient signals of the analytes obtained by LA-ICPMS from each spot of a microarray after sandwich immunoreactions. Inset: the linear regression curves of AFP, CEA, and human IgG. The concentrations of AFP, CEA, and human IgG were 0, 1.0, 10, 100, and 500 ng mL-1. The solution used for coating the microarray was a mixed solution of anti-AFP, anti-CEA, and RAH (0.10 mg mL-1 for each antibody). Labeled antibodies were a mixed solution of Sm3+-labeled anti-AFP, Eu3+-labeled anti-CEA, and Au nanoparticle-labeled RAH (0.50 µg mL-1 for each labeled antibody). Laser conditions: single point with a laser crater of 90 µm in diameter.

mL-1) were prepared individually on a piece of polyethylene substrate. The transient signals of them obtained by LA-ICPMS were shown in Figure 2a-c. All three element-tagged antibodies show clear signals and good repeatability with a RSD less than 5% for five parallel detections. The results indicated that LA-ICPMS could be used to detect antibodies labeled with metal ions or nanoparticles on the surface of slides. To demonstrate the application of LA-ICPMS for the detection of multiple analytes at one spot of microarray, a 4 × 4 array was fabricated with dual or ternary labeled antibodies at one spot. Dried droplets of 1 µL of mixed antibodies (0.50 ng mL-1) were prepared including a row of mixture of Eu3+-anti-AFP and Sm3+anti-PSA, mixture of Sm3+-anti-PSA and Au-GAH, mixture of Eu3+anti-AFP and Au-GAH, and mixture of Eu3+-anti-AFP, Sm3+-antiPSA, and Au-GAH. The transient signals of dual-analyte on one spot were obtained, as shown in Figure 3a-c. The results show that two antibodies labeled with different elemental tags can be detected at one spot with LA-ICPMS. Further experiment investigated the ability of LA-ICPMS for the detection of three elementtagged antibodies at one spot on a microarray. The signals of three element-tagged antibodies obtained by LA-ICPMS on each spot were given in Figure 3d. The results clearly show that the signals of multi-analyte can be obtained and the LA-ICPMS can be used to detect multiple antibodies with elemental tags on a microarray. 928

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Resolving Ability of LA-ICPMS Detection. In the detection of element-tagged antibody on a microarray by LA-ICPMS, the available density of sample spots depends on the sizes of laser ablation craters. The SEM pictures of craters formed by laser ablation are shown in Figure 4a. It can be seen that the craters vary significantly from 40 to 4 µm when the laser is focus on different diameter. For a 30 µm ablation crater, the space distance between centers of two adjacent craters is about 40 µm (Figure 4b), corresponding to a density of sample spots of 62 500 spots per square centimeter. The minimal availability ablation crater is 4 µm for a currently available new type of laser ablation system. Therefore, the detection of almost 5 × 105 sample spots on a square centimeter microarray could be achieved with the present detection method. The results indicate that the LA-ICPMS detection method not only permits detecting multiple analytes on one spot but also had a spatial resolution at micromolar range. Laser Wavelengths on Sampling Efficiency. The laser wavelength has a significant influence on the geometry of an ablation crater of LA-ICPMS. Figure 5 gives the SEM picture of ablation craters using ArF-excimer 193 nm and Nd:YAG 266 nm laser ablation system, respectively. The ablation crater profile of 193 nm laser ablation system exhibits a flat bottomed and straight welled crater, while that of 266 nm shows molten material around the crater rims and many particle depositions. A flat bottom and

straight wall crater implicate completely removing the samples on the surface of a solid substrate. Therefore, the labeled antibodies on the surface of a microarray could be removed entirely in the ablation crater by the 193 nm ArF-excimer laser system. Effects of Laser Pulse on Sampling Efficiency. For the microarrys with high sample density and high-throughput, it is essential that all samples in micrometer-sized spots are completely ablated and transported rapidly to the plasma to obtain a sharply transient signal. To verify the effect of laser repetition frequency on desorption and sampling efficiency, Eu3+-anti-AFP was immobilized on the glass substrate and ablated by different repetition frequency. Figure 6 showed the typical transient signal during the laser ablation. 29Si signal was used as a tracer to indicate the complete removal of samples from the glass surface. The signal intensity of Eu showed no detectable change by using different laser pulses, while the signal intensity of Si obviously increased. The results indicated that analyte on the surface of glass substrate was completely ablated with one laser pulse. Effects of Matrixes and Substrates on the Signal Intensity. To investigate the matrix effect, five samples were prepared by mixing the 1.0 µg mL-1 Eu3+ solution and antibodies, such as human IgG, anti-PSA, anti-AFP, and GAH. The results in Table 2 showed that there were no significant differences with various matrixes on the same substrate. Therefore, the LA-ICPMS-based microarray detection method can be used for effective quantitative analysis for multiple analytes with the presence of different antibodies or proteins. However, the signal intensities on the polyethylene substrate are 2 times higher than that on the glass substrate, as shown in Table 2. The higher signal on polyethylene substrate was because the size of each spot was smaller as compared to that on glass with the same volume solution, and we find that the same volume droplet on glass gives a spot with roughly 2 times area of that on polyethylene. Therefore, substrates have no significant effects on the detection of elemental tags with LA-ICPMS. Detection of Multiple Proteins in One Spot. Immunoreactions were performed with a common procedure of sandwich immunoassay on the microarray. AFP, CEA, and human IgG captured in one spot were detected by LA-ICPMS with element-

tagged antibodies, Sm3+-anti-AFP, Eu3+-anti-CEA, and Au-GAH. For calibrating purpose, a mixed solution of AFP, CEA, and human IgG with the concentration of 0, 1.0, 10, 100, and 500 ng mL-1 for each protein, which were used in the time-resolved fluorescent immunoassay (TRFIA) kits, was added to each sub-array and specially bound with the immobilized antibodies. When the immunoreactions were finished, the microarray was detected by LA-ICPMS. The obtained signals and calibration curves were shown in Figure 7. The correlation coefficients between signal intensities and the concentration of analytes were 0.992, 0.995, and 0.996 in the concentration range between 0 and 500 ng mL-1 for AFP, CEA, and human IgG, respectively. The limits of detection (LOD) were 0.20, 0.14, and 0.012 ng mL-1 (3σ) for the three proteins, respectively. At the concentration of 1.0 ng mL-1, the RSD values of the present method were 5.7%, 2.6%, and 2.3% for AFP, CEA, and human IgG, respectively. CONCLUSIONS In summary, we have developed a novel method for the determination of AFP, CEA, and human IgG on an immunomicroarray by using LA-ICPMS. Experimental results have proved that LA-ICPMS can detect multiple proteins from each spot of the microarray with a spatial resolution at micrometer range. The sensitivity of the present method could be further increased if all of the tags were nanoparticles instead of rare-earth ions. Because several analytes can be detected from one spot, the present readout method for microarray could alleviate the stress to fabricate high density arrays and might find broad applications in proteomics. Further development of this method may lay on the realization of large quantities of suitable nanoparticle labels. ACKNOWLEDGMENT This work is supported by grants from the National Natural Science Foundation of China (Nos. 20375022, 20575034, and 20575061).

Received for review July 13, 2006. Accepted November 23, 2006. AC061269P

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