Micromachining Microcarrier-Based Biomolecular Encoding for

The number of distinct codes theoretically could be many thousands, depending on the coding element numbers. ... As proof of this statement, we demons...
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Anal. Chem. 2003, 75, 4125-4131

Micromachining Microcarrier-Based Biomolecular Encoding for Miniaturized and Multiplexed Immunoassay Zheng-liang Zhi,* Yasutaka Morita, Quamrul Hasan, and Eiichi Tamiya*

School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1, Asahidai, Tatsunokuchi, Ishikawa, 923-1292, Japan

Micromachining techniques, which originated in the microelectronics industry, have been employed to manufacture microparticles bearing an engraved dot-type signature for biomolecular encoding. These metallic microstructures are photolithographically defined and manufactured in a highly reproducible manner. In addition, the code introduced on the particle face is a straightforward visible feature that is easily recognizable with the use of optical microscopy. The number of distinct codes theoretically could be many thousands, depending on the coding element numbers. Such microparticles are, thus, with appropriate surface organic functionalizations, ideal for encoding biomolecular libraries and serving as a platform for developing high-throughput multiplexed bioassay schemes based on suspension array technology. As proof of this statement, we demonstrated that encoded microparticles tagged with antibodies to human immunoglobulin classes are capable, using imaging detection as the interrogating approach, of high sensitivity and high specificity, as well as multiplexed detection of the respective antigens in a microliter-sample volume. The advent of microarray technology represents a major advance in the development of miniaturized analytical tools, since it promises a high-throughput and cost-effective means for the multiplexing of microscale bioscreenings and bioassays. On-chip planar microarrays are among the most intensively investigated techniques for bioassays, especially in DNA hybridizations1 and, more recently, in antibody-based assays.2-8 However, microcarrier* To whom correspondence should be addressed. E-mail: [email protected] or [email protected]. (1) Gershon, D. Nature 2002, 416, 885-891. (2) Mitchell, P. Nat. Biotechnol. 2002, 20, 225-229. (3) Robinson, W. H.; DiGennaro, C.; Hueber, W.; Haab, B. B.; Kamachi, M.; Dean, E. J.; Fournet, S.; Fong, D.; Genovese, M. C.; Neuman de Vegvar, H. E.; Skriner, K.; Hirschberg, D. L.; Morris, R. I.; Muller, S.; Pruijn, G. J.; van Venrooij W. J.; Smolen, J. S.; Brown, P. O.; Steinman, L.; Utz, P. J. Nat. Med. 2002, 8, 295-301. (4) Joos, T. O.; Schrenk, M.; Ho¨pfl, P.; Kro¨ger, K.; Chowdhury, U.; Stoll, D.; Scho ¨rner, D.; Du ¨ rr, M.; Herick, K.; Pupp, S.; Sohn, K.; Ha¨mmerle, H. Electrophoresis 2000, 21, 2641-2650. (5) Silzel, J. W.; Cercek, B.; Dodson, C.; Tsay, T.; Obremski, R. J. Clin. Chem. 1998, 44, 2036-2043. (6) Wiese, R.; Belosludtsev, Y.; Powdrill, T.; Thompson, P.; Hogan, M. Clin. Chem. 2001, 47, 1451-1457. (7) Mezzasoma, L.; Bacarese-Hamilton, T.; Di Cristina, M.; Rossi, R.; Bistoni, F.; Crisanti, A. Clin. Chem. 2002, 48, 121-130. (8) Delehanty, J. B.; Ligler, F. S. Anal. Chem. 2002, 74, 5681-5687. 10.1021/ac034165c CCC: $25.00 Published on Web 07/12/2003

© 2003 American Chemical Society

based biomolecular encoding technology, also known as suspension array, recently has proven very promising as an alternative to planar microarray for a wide range of biomedical applications.9 The fact that suspension array is more promising is due to its inherent and unique characteristics, such as higher flexibility in array implementation, no requirement for a sophisticated highprecision spotting device, and occurrence of the binding events in bulk overcoming the typical difficulties associated with the planar surface. The encoded particles or beads also have great potential for other uses, for example, being combined with a flow cytometric system to make a highly multiplexed bioassay system for larger-scale, ultra-high-throughput screening.10 Therefore, such a technology is, complementary tosand in some implementations, may be superior tosexisting planar microarrays. The development of this technology relies fundamentally on the design and manufacture of microcarriers that integrate both bioprobes that have molecular recognition/binding abilities and intrinsic identity signatures. The former could normally be created through biomolecular coupling, while the latter could be created through encoding and decoding. An ideal encoding strategy depends not only on the number of unique codes that can be generated reliably and feasibly but also on the versatility of the microcarriers that can be used in various assays. Existing encoding approaches focus mainly on the incorporation of beads or particles with spectrally distinguishable fluorophores,11-13 fluorescent semiconductor quantum dots14 or colloids,15,16 and molecules with specific Raman and IR spectral features.17 The encoded microcarriers also have been manufactured as metallic rods with stripes18 or as porous silicon photonic crystals.19 The useful spectral range and the possible number of spectrally distinguishable labels, however, often limit the potential number of beads that can be encoded discretely. In addition, the validity (9) Braeckmans, K.; De Smedt, S. C.; Leblans, M.; Pauwel, R.; Demeester, J. Nat. Rev. Drug Discovery 2002, 1, 447-456. (10) Nolar, J. P.; Sklar, L. A. Trends Biotechnol. 2002, 20, 9-12. (11) Michael, K. L.; Taylor, L. C.; Schultz, S. L.; Walt, D. R. Anal. Chem. 1998, 70, 1242-1248. (12) Ferguson, J. A.; Boles, T. C.; Adams, C. P.; Walt, D. R. Nat. Biotechnol. 1996, 14, 1681-1685. (13) Szurdoki, F.; Michael, K. L.; Walt D. R. Anal. Biochem. 2001, 291, 219228. (14) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631-635. (15) Trau, M.; Battersby, B. J. Adv. Mater. 2001, 13, 975-979. (16) Battersby, B. J.; Bryant, D.; Meutermans, W.; Matthews, D.; Smythe, M. L.; Trau, M.; J. Am. Chem. Soc. 2000, 122, 2138-2139. (17) Fenniri, H.; Ding, L.; Ribbe, A. E.; Zyrianov, Y. J. Am. Chem. Soc. 2001, 123, 8151-8152.

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of the coding signatures is another serious concern, since the incorporated coding elements in some cases may be lost, photobleached, or interfered with spectrally by the analytical signals.13 In the case of multimetal microrods,18 the encoding scheme, while overcoming the main drawbacks of the other approaches, still suffers from a limitation: the nonuniformity of the rod sizes and striping patterns resulting from a membrane template with a wide pore-size variation of ∼100% used for the microstructures. Here we present a new encoding scheme and the use of micromachining techniques to fabricate magnetic microstructures bearing an engraved dot-type code. This manufacturing approach is superior to other existing methods: since the particle size and coding pattern are controlled by a photomask, structures can thus be produced in a highly reproducible and simple way. In addition, since this approach is straightforward, it does not require additional sophisticated chemistry for coding element incorporation. Theoretically, the number of codes that can be generated is unlimited, depending on the number of coding elements. Moreover, with proper surface modification, gold is easily compatible with available biomolecular coupling chemistries and fluorescence/ chemiluminescence-based detection techniques. Also, the incorporated Ni-poly(tetrafluoroethylene) (PTFE) layer renders the microparticles magnetic that could be potentially utilized in immunomagnetic capture and separation, which makes the process amenable to automation. We demonstrate here the utility of microfabricated-encoded microparticles for the development of high sensitivity and high specificity and multiplex immunoassays. EXPERIMENTAL SECTION Materials and Reagents. Negative photoresist XP SU-8 10, the developer, XP SU-8 release layer, and Nano Remover PG photoresist remover were obtained from Microchem Corp. (Newton, MA). SuperSignal ELISA Femto Maximum Sensitivity Substrate was obtained from Pierce. All the antigens (human IgA, IgG, IgM), antibodies (developed in goat), and peroxidaseconjugated reporter antibodies were obtained from Sigma Immunochemicals (St. Louis, MO). Metaflon FS Ni-PTFE dispersion electroplating solution containing nickel sulfamate (Ni2+ 100 g/L) and fine PTFE particles (diameter ∼0.2 µm, concentration 50 g/L) was obtained from C. Uyemura & Co. Ltd. (Osaka, Japan). Other chemicals used in this study were of reagent grade and purchased from locally available commercial sources. The photomasks were designed with Illustrator 9.01 (Adobe) and printed on a transparency film using a high-resolution laser-printer system. Particle Fabrication. Cr (20 nm) and then Cu (200 nm) were deposited by evaporation on cleaned glass slides (38 × 26 mm), used as the conducting seed layer and later also used as the sacrificial release layer. For contact photolithography, we used a Karl Suss MJB 3 mask aligner (Zeiss) in contact mode powered by a 350-W mercury UV light source. The metal-coated substrates were first spin-coated with a 2-µm XP SU-8 release layer according to the protocol provided by the manufacturer (Microchem Corp). The substrates were then spin-coated with ∼15 µm of SU-8 10 (18) Nicewarner-Pen ˜a, S. R.; Freeman, R. G.; Reiss, B. D.; He, L.; Pen ˜a, D. J.; Walton, I. D.; Cromer, R.; Keating, C. D.; Natan, M. J. Science 2001, 294, 137-141. (19) Cunin, F.; Schmedake, T. A.; Link, J. R.; Li, Y. Y.; Koh, J.; Bhatia, S. N.; Sailor, M. J. Nat. Mater. 2002, 1, 39-41.

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photoresist thick film and structured using standard photolithographic procedures. A 50 × 40 array of 200 × 100 µm-sized microwells with a ∼50-µm separation space between the wells was resist-patterned on a 10 × 8 mm area located on a Cu/Cr covered glass substrate. Two blocks of photoresist patterns with different coding signatures were designed on each slide. The photoresistpatterned substrates were cleaned with an O2 plasma treatment for 2 min. Ni-PTFE composite layer electrodeposition was followed by an electroplating bath at 50 °C for 30 min with a current density of ∼0.1 mA/mm2. The substrates were then deposited sequentially by metal evaporation on the top of the photoresist with 0.02 µm of Cr and 0.3 µm of Au. The metallized resist pattern on the substrate was subsequently removed by liftoff upon immersion in an SU-8 Remover PG solution at 80 °C for 10-20 min. The particles were finally freed from the underlying substrate by dissolving the sacrificial Cu layer with an etchant containing sodium hypochlorite (0.2 mol/L), ammonia (0. 7 mol/ L), and ammonium carbonate (2.6 mol/L). The pieces were washed with distilled water and stored in plastic tubes filled with ethanol until use. Multiplex Immunoassays for IgA, IgG, and IgM. The particles were coated with a self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHA) by soaking each set of particles for 24-48 h in a 1 mM solution of MHA dissolved in ethanol. The treated particles were washed with ethanol, transferred to a 0.5-mL plastic tube, and washed three times with distilled water. When we used 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC-NHS) activation, the particles were immersed in 2 mg/mL EDC and 1 mg/mL NHS in acetonitrile for 6 h with rotating agitation and then washed with distilled water. Four sets of particles were then treated with 50 µL of 2 µg/mL antibodies containing anti-IgA, anti-IgG, and antiIgM and 2% casein (as the negative control), respectively, in 10 mM phosphate-buffered saline (PBS), pH 7.4, for 3 h at ambient temperature or overnight at 4 °C with rotating agitation. The particles were subsequently treated with a 2% casein solution in PBS buffer containing 0.01% Tween 20 for 1 h to block the exposed particle surface. Finally, the particles were washed three times with 10 mM PBS, pH 7.4, containing 0.01% Tween 20. The sets, each containing 10-20 particles, were combined in a 0.5-mL plastic tube. Then, the particle mixture was withdrawn and washed. A 50-µL sample containing the analytessIgA, IgG, and IgM, diluted with 2% casein solution in 10 mM PBS bufferswas added and incubated for 40 min. After washing, a 50-µL mixture of three peroxidase-conjugated reporter antibodies with a concentration of 4 µg/mL eachsdiluted with 2% casein solution in 10 mM PBS bufferswas added for 40 min to form the sandwich immunocomplexes on the particles. The immunoreagent-treated particles were then washed three times. A magnet was used to assist heterogeneous separations for particle manipulation whenever necessary. Detection of Chemiluminescence. Chemiluminescence from the microscopic particle zones was imaged in a dark room with a MZFLIII microscope (Leica) equipped with a charge-coupled device (CCD) camera (AxioVision 3.0, Carl Zeiss Inc.) and AxioVision software. The saved 8-bit image data were analyzed quantitatively using IP LabSpectrumP software. Chemiluminescence was detected without illumination, and by taking another illuminated image for the particle distribution profile on a poly-

Figure 1. Pictorial illustrations of the fabrication protocol for encoded multilayer microparticles. SU-8 photoresist was first photolithographically patterned as the particle template on a Cu-coated glass substrate; Ni-PTFE layers (∼6 µm thick) were electrodeposited on the open areas of the photoresist-patterned microwells. After evaporation of Cr (20 nm) and Au (300 nm) from the whole chip surface, metallized SU-8 photoresist was liftoff using SU-8 Remover PG solution. Finally, the particles were released from the underlying substrate by dissolution of the Cu sacrificial layer. Only two types of the encoded particles are shown here as examples.

(dimethylsiloxane) (PDMS) measuring substrate, the individual particle signatures were identified. The hydrophobic interactions between the Ni-PTFE particle layer and the PDMS substrate promote adhesion of particles on the substrate with an orientation of the Au side facing up. In fact, more than 70% of the particles showed their Au side facing up and then the Ni-PTFE side facing down. This orientation was selected because the chemiluminescent signals generated on the Au side were somewhat higher than those generated on the Ni side. With the aid of a Pasteur pipet, further gentle manipulation of the solution and the particles over the substrate surface resulted in even higher ratios of individual particles facing Au side up and also in settled particles achieving good spatial resolution. To distribute the particles, a drop of PBS buffer was first placed onto the PDMS measuring substrate to define an area of ∼5 × 5 mm. Particle suspension was then dropped onto the defined area of the PDMS substrate. Then, the solution on the surface was withdrawn, leaving the spatially isolated particles to settle and stick on the substrate. The maximum density of the settled particles on the substrate was typically 150-200 pieces in a ∼5 × 5 mm imaging area captured by CCD camera. To initiate luminescent reaction, 0.05 mL of a freshly prepared mixture of the chemiluminescence substrates composed of luminol and H2O2 were pipetted and flooded onto the particles. The peroxidase-conjugated reporter antibodies associated by immunobindings on the particle surfaces generated localized chemiluminescent signals. Once the luminescent reaction initiated, the signal was essentially stable for about 3-5 min. Thus, the signal was amplified when the imaging was acquired with an integration time of 40 s (limitation of the CCD camera), immediately after starting of the reaction. A background signal (typically ∼12 arbitrary units) that was taken at a substrate area without immunocomplex formed particles was subsequently subtracted from each intensity value of the blue color emission derived from the gold particle surface. The targets bound to individual particles were identified by reading the encoding signature on the particle at any given location on the measuring substrate.

thermal evaporation, and wet etching. The typical microfabrication process is outlined in Figure 1. A prestudy revealed that electrodeposition of Ni-PTFE on a nonpatterned Cu seed film could provide a composite layer with a magnetic property and a hydrophobic nature, as confirmed by contact angles of 110-120° between a water droplet and the plated surface. Meanwhile, the Ni-PTFE composite layer has a good mechanical property and chemical stability. We used the electroplating-through-resist mask technique to replicate the resist patterns and build up the NiPTFE structures for the particles. In the photoresist-patterned particle template, a small resist dot code was photolithographically “written” in an area of each microwell in the resist pattern. The code was a symbol of 0-8 dots distributed in two rows, accompanied by an additional two dots fixed at the starting and ending points used to define the coding area. This coding scheme has the potential for 28 ) 254 types of particles. The level of multiplexing increases exponentially with the increase of coding numbers, if, for example, the coding element number increases to 10, the possible number of particles to be encoded is 1016. The coding resist patterns were transferred to the faces of the particles by means of Ni-PTFE electrodeposition and Au/Cr evaporation. These codes were used as the signatures to register and identify the biomolecules attached to the particle surface. This encoding scheme was used to fabricate an array of 50 × 40 rectangle particles with dimensions of 200 × 100 µm on a glass slide. The code pattern introduced on a particle face occupied an area of ∼80 × 50 µm. As can be seen in the pictures shown in Figure 1, the symbols are clearly distinguished after the structures were built up through Ni-PTFE electrodeposition and Au/Cr evaporation. The particles could be decoded by manual reading or, potentially, by pattern-recognition software. Using such types of particles, the microarray features generated are comparable in sizes to those normally used in planar protein microarrays (i.e., 100-200 µm).3,4 However, efforts to produce smaller pieces proved to be difficult with the laser-printer printed photomask (resolution ∼10 µm) used in this study. Nevertheless, according to previously reported works,21,22 fabrication of pieces with a size down to 10

RESULTS AND DISCUSSION Micromachining Encoded Microparticles. The encoded microparticles utilized as the biomolecular carrier were fabricated using a combination of micromachining techniques that include photolithography and “liftoff”, electroplating-through-mask,20 metal

(20) Romankiv, L. T.; O’Sullivan, E. J. M. Plating Techniques. In Handbook of Microlithography, Micromachining, and Microfabrication, Vol. 2, Micromachining, and Microfabrication; Rai-Chouhury, P., Ed.; SPIE Optical Engineering Press: Washington, DC, 1997; pp 199-290. (21) Clark, T. D.; Tien, J.; Duffy, D. C.; Paul, K. E.; Whitesides, G. M. J. Am. Chem. Soc. 2001, 123, 7677-7682.

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µm or even smaller should be feasible if a high-resolution metal photomask is used. The structural integrity of the manufactured microparticles, which is a critical requirement for the envisaged applications, was found to depend mainly on the thickness of the electrodeposited layer. In general, a uniform Ni-PTFE layer could be formed on a Cu substrate within 2 min of electroplating by maintaining a current density of ∼0.1 mA/mm2, but such a thin layer was found to be easily breakable during release from the sacrificial layer. Nevertheless, it was observed that a Ni-PTFE layer of ∼6 µm thick, which typically needed 30 min of electroplating time to complete, was essentially stable. The surface roughness of the electroplated layer was low, typically less than 0.2 µm. After evaporation of the Cr (20 nm) and Au (300 nm) layers and subsequent removal of the metallized SU-8 photoresist layer, the yields of the particles released from the underlying substrates were typically ∼95%. Several thousand copies of each type of encoded particles were produced with a single glass substrate. Optimization of the Microparticle-Based Chemiluminescent Immunoassay. Successful implementation of this approach for the multiplexed immunoassay requires solving the following two fundamental problems. The first problem is the luminescence quenching effect on the metallic particle surface. It is a commonly recognized phenomenon that a fluorescent or chemiluminescence-emitting molecule at a metal surface such as gold has a reduced luminescence efficiency because of quenching.23 Our experiments revealed that, even with a sandwich form of antibody/antigen/antibody-peroxidase of multiple protein layers covering the gold surface, the problem could not be eliminated effectively. Hence, we addressed this problem by passivating the gold surface with an ω-thiolated fatty acid SAM. The SAM was used here as an inert “spacer” to hold the luminescent molecules at a certain distance from the metal interface, thus improving luminescence efficiencies. A further benefit derives from the fact that the free carboxylic acid group of the SAM formed on the gold surface is easily available for covalent coupling (by cross-linking to reactive amine groups)24 or noncovalent coupling (by adsorption)25 to various biomolecules. Figure 2 shows that the chemiluminescent signal from a particlebased sandwich IgA immunoassay depends markedly on the alkyl chain length of the mercaptocarboxylic acid used in the SAM. It was observed that MHA gave the highest quantum yield of the chemiluminescent reaction; therefore, MHA was used in this study. Meanwhile, two approaches were tested and compared for coupling antibodies to the SAM of MHA: covalent coupling using EDC-NHS linking chemistry and physical adsorption. As shown in Figure 2, the latter gave a slightly higher signal for the same concentration of antigen, presumably because the adsorbed protein on the MHA layer could maintain the activity of the antibodies better. Additional assays were performed to compare the chemiluminescent efficiency on the SAM of MHA passivated gold particles to that of a commercially available ProteoChip (provided by Proteogen Inc.). The results obtained from two (22) Clark, T. D.; Ferrigno, R.; Tien, J.; Oaul, K. E.; Whitesides, G. M. J. Am. Chem. Soc. 2002, 124, 5419-5426. (23) Kuhn, H. J. Chem. Phys. 1970, 53, 101-108. (24) Dong, Y.; Shannon, C. Anal. Chem. 2000, 72, 2371-2376. (25) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702-1705.

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Figure 2. Effect of surface modification of the microfabricated particles with mercaptocarboxylic acid SAM on the chemiluminescent signal of IgA immunoassay and a comparison of the signals obtained with SAM passivated particles and with a ProteoChip. C16-ads, 16mercaptohexadecanoic acid (adsorption); C16-EDC, 16-mercaptohexadecanoic acid (EDC-NHS linkage); C8-ads, thiotic acid (adsorption); C3-ads, 3-mercaptpropionic acid (adsorption); ProteoChip, a slide glass available for protein immobilization, from Proteogen Inc. The concentrations of the antibodies used for the immobilization were optimized to be 2, 2, and 10 µg/mL for EDC-NHS linkage, adsorption, and ProteoChip, respectively.

concentration levels (0.01 and 0.1 µg/mL) of IgA are also shown in Figure 2. The chemiluminescent efficiency on the SAM of MHA passivated gold particles is comparable or even slightly better than the efficiency on the ProteoChip, suggesting that the quenching effects on the gold surface have been minimized or even eliminated. In this approximate comparison of chemiluminescence efficiency, the effect of the difference of protein feature sizes of the microparticle and of the ProteoChip (∼0.1 vs ∼0.5 mm) was not taken into account. The second problem is nonspecific adsorption of interfering proteins on the SAM modified gold surfaces. Normally, the level of nonspecific adsorption defines the limit of detection that can be reached for an immunoassay. The issue of nonspecific adsorption is even more critical to the performance of a multiplexed multianalyte immunoassay because of the increased potential of interferences and cross-talk between individual assays. In this study, we used antibody (antiIgA, antiIgG, and antiIgM)peroxidase conjugates as the protein probe to monitor nonspecific adsorption on the antibody-immobilized particles. We then compared two blocking agents, bovine serum albumin (BSA) and casein (sodium salt), for effectiveness in reducing nonspecific protein adsorptions in a particle-based immunoassay. It was observed that BSA blocking for the gold particles showed a slight or mediate nonspecific signal when an increased concentration of the antibody-peroxidase conjugate was applied, as can be seen in Figure 3a. In contrast, when 2% casein is applied as the blocking agent, the nonspecific signal from the antibody-peroxidase conjugates can be eliminated effectively, even at high conjugate concentrations. Elimination of nonspecific adsorption is very important for the assays, not only because it removes crosscontamination between different assays but also because it allows the use of high concentrations of antibody-peroxidase reporter

Figure 4. Time courses of immunobinding of the antibody-immobilized particles to the respective antigens in solution. The concentration of antibodies used for immobilization was 2 µg/mL. Ab, antibody; Ag, antigen. Inset represents the principle of the interaction studied. The binding was followed by a peroxidase-conjugated antibody-binding step for detection.

Figure 3. Effect of the blocking agents BSA and casein on the onparticle nonspecific adsorption. Peroxidase-conjugated antibodies were used as indicators of nonspecific proteins. (a) indicates that the use of casein as the blocking agent showed significant advantages over BSA in overcoming nonspecific adsorption. Error bars were omitted for clarity. (b) shows a comparison between the chemiluminescent signals obtained from a sample containing 1 ng/mL antigens and the signals obtained from the blank. A high-sensitivity CCD camera captured the signals.

conjugate to achieve better and faster binding and, therefore, higher sensitivity. To further test the efficiency of casein blocking, signals from the blank microparticles (without analyte treatment) and of those of the microparticles treated with 1 ng/mL antigens were compared. A high-sensitivity Hamamatsu Argus-50 CCD camera useful for photocounting was used to capture the chemiluminescent images with an integration time of 2 min. As can be seen in Figure 3b, the signals from samples containing 1 ng/mL antigens were significantly higher than signals from the blank samples, further confirming that a very low level of nonspecific protein adsorption on the particles had been achieved with the casein blocking scheme. The time course of the immunobinding of the particle-carried antibodies to the respective antigens in a sample solution was studied by varying the particle-sample incubation time for an assay. As can be seen in Figure 4, heterogeneous binding

reactions reached the maximum chemiluminescent signal within ∼40 min. This is a reasonably fast reaction, as compared to a typical time of 1-2 h for the conventional enzyme-linked immunosorbent assay (ELISA) methods and an on-chip microarray method.6 The shorter time for the binding steps was a direct consequence of the small sizes of the particles and, subsequently, the thorough mixing of the particles with the sample solution in the suspensions. Since this method was optimized to achieve the highest possible sensitivity, the incubation time should be ∼40 min. However, if high sensitivity is not importantsfor instance, in the case of rapid screeningsit would be easy to adapt a shorter incubation time, e.g., 10 min, for multiplexed immunoassays. Multiplexed Immunoassay on Microparticles. Using a model case of three independent human immunoglobulins (IgA, IgG, IgM), sandwich-type immunoassays were constructed with the antibody-immobilized, 100 × 200 µm sized microparticles, peroxidase-labeled reporter antibodies, and a luminol-based chemiluminescent detection system. For each assay, parameters such as concentration of antibodies used for immobilization, concentration of peroxidase-conjugated reporter antibodies, and incubation time for the immunobindings were optimized independently. Detailed values are mentioned in the Experimental Section. Calibration curves for the three analytes were obtained by plotting the average blue pixel intensity values (in triplicates) of the integrated particles against the concentration values of the antigens, as shown in Figure 5a. The representative calibration curves reflected a sigmoidal binding behavior, as was usually observed in a standard ELISA. IgA, IgG, and IgM could be determined over the range typically from ∼10 ng/mL to 1 µg/ mL. The detection limits, calculated as 3-fold standard deviation of the blanks, were as follows (in ng/mL): 3 for IgA, 3 for IgG, and 4 IgM; and IC50 (in µg/mL), 0.08 for IgA, 0.12 for IgG, and 0.12 for IgM. When the standard fluorescent ELISA formats (Figure 5b) were used, the detection limits were as follows (in ng/mL): 43 for IgA, 52 for IgG, and 122 for IgM; and IC50 (in µg/mL), 0.78 for IgA, 1.0 for IgG, and 1.2 for IgM. These very Analytical Chemistry, Vol. 75, No. 16, August 15, 2003

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Figure 5. (a) Representative calibration curves derived from the present microparticle-based assays for the analytes IgA, IgG, and IgM. Inset represents the sandwich immunoformat formed on gold particles and the principle of signal generation. POD, peroxidase. (b) Calibration curves for the same analytes obtained by standard fluorescent ELISA formats that used Amplex Red as the fluorogenic peroxidase substrate.

different results indicate that the present assays were about 1020-fold more sensitive. Also, these results are in direct contrast to a previous study in which microsphere-based flow cytometric assay with the FlowMetrix system for multianalyte immunoassays was ∼1 order of magnitude less sensitive than the assay using conventional ELISA.26 To test the specificity and multianalyte capability of the particlebased multiplexed assay, we combined all three sets of antibodyimmobilized particles and one set of the negative control immobilized with casein. The combined particles were then subjected to a treatment with a mixture of 0.1 µg/mL IgA and IgG and a mixture of 0.1 and 1 µg/mL of IgA, IgG, and IgM, respectively. As can be seen in Figure 6a, particles treated with a sample containing 0.1 µg/mL IgA and IgG gave rise to homogeneously and spatially resolved chemiluminescent signals from those particles tagged with antiIgA and antiIgG. The concentrations were determined, according to the calibration curves as shown in Figure (26) Dasso, J.; Lee, J.; Bach, H.; Mage, R. G. J. Immunol. Methods 2002, 263, 23-33.

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Figure 6. Demonstration of parallel, multianalyte immunoassay for IgA, IgG, and IgM with encoded particles using a luminol-based chemiluminescent detecting scheme. A negative control with caseinblocked particles was included in the assays. Case a shows the image for simultaneous detection of 0.1 µg/mL IgA and IgG. Case b shows the image for simultaneous detection of 0.1 µg/mL IgA, IgG, and IgM. Case c shows the image for simultaneous detection of 1 µg/mL IgA, IgG, and IgM. For all cases, the upper (for case c, the left) panels show optical microscope images of the location and individual registration of particles on a measuring PDMS substrate, while the lower (for case c, the right) panels show the chemiluminescent images associated with the on-particle immuno-binding events. The letters A, G, and M and the symbol (/) represent the positions of the particles tagged with antibodies of antiIgA, antiIgG, antiIgM, and casein, respectively.

5a, to be 0.11 and 0.097 µg/mL for IgA and IgG, respectively. Particles with immobilized antiIgM and casein showed no signal, indicating that no false positive or false negative signals were encountered in the assays. On the other hand, in the combined particles treated with a sample containing a 0.1 µg/mL concentration of all three analytes, a signal was detectable for all those particles with immobilized individual antibodies. The concentrations were determined to be 0.12, 0.10, and 0.095 µg/mL for IgA, IgG, and IgM, respectively. The control particles did not show any detectable signal (Figure 6b), indicating low nonspecific binding of the coexisting antigens or peroxidase-labeled reporter antibodies to the particles. In the experiment in which the particles were exposed to a sample containing IgA, IgG, and IgM at a concentration of 1 µg/mL each (which is at the high determination threshold), the concentrations determined were 0.90, 1.12, and 0.94 µg/mL for IgA, IgG, and IgM, respectively. The signal for the negative control particle was low, essentially ∼0, as shown in Figure 6c, indicating very low cross-interferences from coexisting assays/analytes even at high concentrations. Attempts to detect multiple analytes simultaneously by using fluorescent dye-labeled

microspheres had been reported previously, but only limited success was achieved, because of the interferences between the assays, especially when the targets were present in high concentrations.13 By contrast, in the experiment reported here, there was a great difference (1 µg/mL vs 0 ng/mL) in concentrations for different analytes, and the interference signals were not detectable. CONCLUSIONS By taking advantage of micromachining and microfabrication techniques, we developed a new approach for manufacturing encoded metallic microstructures used as versatile building blocks for miniaturized multiplex bioassays. The high flexibility and simple encoding/decoding scheme of this unique approach offer particular advantages in the implementation and use of assays. However, multiplexing, increasing of throughput, and miniaturization of analytical devices often compromise the specificity and sensitivity of a detection method, as is the case for microspherebased multiplexing/screenings assays.13,26 Nevertheless, the present approach is ideally compatible with the specificity and sensitivity (27) Vignali, D. A. J. Immunol. Methods 2000, 243, 243-255.

required for immunodiagnostic methods; as a result, it is significantly superior to the existing microsphere-based multiplexing systems and, subsequently, very promising for practical use. Finally, the fact that the size of the manufactured particles can be altered makes it conceivable that they can be further optimized and incorporated, as a new type of microcarrier, into a flow cytometric system27 for use in automated, massively multiplexed and ultrafast bioassays and screenings. ACKNOWLEDGMENT This work was partially funded by a Grant-in-Aid for Scientific Research Priority Area (A) of MEXT, and Research for Future Program from the Japan Society for Promotion of Sciences (JSPS). Z.-l.Z. gratefully acknowledges JSPS for a postdoctoral fellowship. We are thankful to Prof. R. B. DiGiovanni for critical reading of the manuscript. Received for review February 18, 2003. Accepted May 30, 2003. AC034165C

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