User Configurable Microfluidic Device for Multiplexed Immunoassays

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Anal. Chem. 2009, 81, 1275–1279

User Configurable Microfluidic Device for Multiplexed Immunoassays Based on DNA-Directed Assembly Hendrik Schroeder,† Michael Adler,‡ Katrin Gerigk,§ Birgit Mu¨ller-Chorus,| Friedrich Go ¨ tz,§ and ,† Christof M. Niemeyer* Technische Universita¨t Dortmund, Fakulta¨t Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto Hahn Str. 6, 44227 Dortmund, Germany, Chimera Biotec GmbH, Emil-Figge Str. 76A, D-44227 Dortmund, Germany, Boehringer Ingelheim microParts, Hauert 7, D-44227 Dortmund, Germany, and Fachhochschule Gelsenkirchen, Neidenburger Str. 43, D-45877 GelsenKirchen, Germany We present a microfluidic device for multiplexed immunoassays based on DNA-directed immobilization. Because of the versatile building blocks used for this technique, it is possible to build up user configurable protein microarrays within the microfluidic system by means of DNAdirected self-assembly, which can be used for immunoassay applications. We demonstrate the performance of our system by parallel detection of cytokines in a multiplex immunoassay, employing silver deposition labeling and optical read-out. Quantitative immunological methods are essential for the analysis of biomarkers in biomedical diagnostics. While today’s immunoassays are routinely performed in microtiter plates, intensive research is currently devoted to the further miniaturization of this platform. To minimize sample volumes, reagent costs, and processing time and also to increase throughput by multiplexing of biological recognition events, protein microarrays are being developed,1-5 and this technology is on its way toward fully integrated microfluidic systems.6,7 However, this implementation is hampered by challenges arising from the development of lowcost manufacturing methods of the microfluidic chips, the configuration and loading of these chips with biomolecular content, their interfacing with the macroscopic world, and the minimization of nonspecific analyte/surface interactions. Moreover, the development of peripheral components for operation and read-out of the microfluidic chip is essential to eventually enable devices, * To whom correspondence should be addressed. E-mail: christof.niemeyer@ tu-dortmund.de. Fax: + 49 (0)231/755 7082. † Technische Universita¨t Dortmund. ‡ Chimera Biotec GmbH. § Fachhochschule Gelsenkirchen. | Boehringer Ingelheim microParts GmbH. (1) Phizicky, E.; Bastiaens, P. I.; Zhu, H.; Snyder, M.; Fields, S. Nature 2003, 422, 208–215. (2) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7, 55–63. (3) Yeo, D. S.; Panicker, R. C.; Tan, L. P.; Yao, S. Q. Comb. Chem. High Throughput Screen. 2004, 7, 213–221. (4) Sobek, J.; Bartscherer, K.; Jacob, A.; Hoheisel, J. D.; Angenendt, P. Comb. Chem. High Throughput Screen. 2006, 9, 365–380. (5) Jonkheijm, P.; Weinrich, D.; Schroeder, H.; Niemeyer, C. M.; Waldmann, H. Angew. Chem., Int. Ed. 2008, 47, 9618-9647. (6) Situma, C.; Hashimoto, M.; Soper, S. A. Biomol. Eng. 2006, 23, 213–231. (7) Henares, T. G.; Mizutani, F.; Hisamoto, H. Anal. Chim. Acta 2008, 611, 17–30. 10.1021/ac802228k CCC: $40.75  2009 American Chemical Society Published on Web 01/05/2009

suitable for point-of-care testing. One crucial technical problem for the integration of protein microarrays in microfluidic systems concerns the controlled immobilization of biomolecular capture probes, required for analyte binding and detection. We here report on the development of a microfluidic device, in which the hybridization of cDNA oligonucleotides is extensively utilized to (i) configure microfluidically addressable reaction vessels of the polymer chip with biomolecular capture probes and (ii) to facilitate low-cost optical read-out of the chip. We demonstrated the functionality of our device by parallel detection of cytokines, a family of desease related biomarkers, present in a variety of body fluids.8 As shown in Figure 1, the prototype of our system consists of a polymethylmetacrylate (PMMA) multiwell disposable chip and a microfluidic station. In this system, DNA-directed immobilization of capture antibodies is used to configure the chip, which allows for execution of the immunoassay (Figure 2). Subsequently, the chip is transferred to a CCD-camera based light transmission measurement system for postassay read-out (Figures 3 and 4). As shown in Figure 1, the fluidic system is based on a multiwell disposable chip (Figure 1d), which is designed to be sandwiched between the station base (Figure 1e) and station lid (Figure 1a). The station lid can accommodate an exchangeable fluidic structure (parts b or c of Figure 1), which, when assembled, is connected to the chip wells. Via this lid, individual or parallel bubble free filling of the wells is possible as well as synchronous washing of the wells in permanent flow. In the case of individual filling, sample liquids are applied to individual filling ports (Figure 1h) by a conventional pipet, while in the other case, a reservoir is being used to facilitate global filling of all wells with reagents or washing buffer. The liquids can be removed from the wells via an outlet in the well bottom, using the vacuum connection (Figure 1g), built in the station base (Figure 1e). All waste liquids are collected within the station base. Additional information on the fabrication process of the disposable chip can be found in Figure SI-1 in the Supporting Information.9 The PMMA chip, shown in Figure 1, contains 12 wells of 35 µL volume, each of which hosts an array of 4 × 4 capture oligonucleotides. The DNA arrays inside the wells are used for site-specific binding of complementary oligonucleotides, tethered (8) de Jager, W.; Rijkers, G. T. Methods 2006, 38, 294–303.

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Figure 1. Illustration of the microfluidic device. The fluidic station (f) is composed of a lid (a) with housing for an exchangeable microfluidic structure, containing ports (h) and reservoirs for either parallel (b) or individual filling (c) and the base of the fluidic station with an integrated waste reservoir (e). The microarray multiwell disposable slide (d) is sandwiched between the station base and station lid. Liquids can be removed from the wells by vacuum aspiration, using joint g.

Figure 2. Schematic drawing of multiplexed immunoassay performed in the wells of the disposable microarray. The different sandwich assays were assembled by site-specific DNA-directed immobilization to the dedicated capture probes cD-cG, illustrated in the scheme (see text for details).

to antibodies by means of covalent conjugates of streptavidin and single-stranded DNA. This linker system has previously been 1276

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developed by some of us10 to realize chip-based immunoassays,11,12 taking advantage of the DNA-directed immobilization (DDI) of

Figure 3. Illustration of the read-out platform (a) read-out platform with slide feeder; (b) opened readout platform; (c) scheme of the readout principle based on LED illumination, CCD readout, and a microarray sledge with piezomotors; and (d) illustration of a resulting raw image in the software module.

Figure 4. Results of multiplex immunoassay of biomarkers, prostata specific antigen (PSA), TNFR, Interleukin 6 (IL6) and Interleukin 23 (IL23): (a) schematic illustration of the capture positions and resulting greyscale images, observed in the experiment and (b) quantitative analysis of spiked solutions of the biomarkers.

antibody-DNA conjugates. The DDI method, in general, has proven to be a very efficient means for the immobilization of proteins to solid surfaces because it occurs under mild conditions, (9) Additional information on chip fabrication, results from multiplex immunoassays, and detailed experimental protocols are available in the Supporting Information.

thereby preventing delicate proteins from denaturation.13,14 Because sets of orthogonal oligonucleotides enable the parallel immobilization of different DNA-tagged proteins,15 the DDI method has been chosen here to realize configuration and loading (10) Niemeyer, C. M.; Sano, T.; Smith, C. L.; Cantor, C. R. Nucleic Acids Res. 1994, 22, 5530–5539.

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of the chip with capture antibody contents (Figure 2). As the consequence of the array-inside-well architecture, the chip is principally suited for holding up to 192 (16 spots per each well) different antibody probes. We here chose the parallel detection of up to four different biomarkers (see below) to demonstrate the suitability of our device for performing multiplexed immunoassays.16 As depicted in Figure 2, the configuration and operation of the device included several steps.9 In a preassay step, sets of four DNA benzophenone-modified capture oligonucleotides, denoted as cD, cE, cF, and cG, were arrayed inside the open microwell chip using a conventional piezo-driven microarray spotter (Nanoplotter 1.2, Gesim). The oligmers were then covalently attached to the PMMA surface by means of photocrosslinking.17 Subsequently, the chip was sealed with the lid unit, containing the microfluidic channels, and mounted on the aspiration chamber (Figure 1). The preconfigured DNA chip was then functionalized with a set of antibody-DNA conjugates using the microfluidic channels of the assembled device. Prior to that, the antibody-DNA conjugates were generated in separate reactions by mixing the covalent DNA-STV conjugates D, E, F, and G and biotinylated antibodies directed against the biomarkers prostata specific antigen (PSA), TNFR, Interleukin 6 (IL6), or Interleukin 23 (IL23), respectively (step I, in Figure 2). An equimolar mixture of the four antibody-DNA conjugates (25 nM in TETBS buffer) was applied to all wells of the chip using the microfluidic channel system, thereby allowing binding to the in-well arrayed DNA capture strands (step II). The fluids were removed by vacuum aspiration, and the chip was rinsed twice with TETBS buffer to remove unbound materials. Subsequently, a sample solution containing the biomarker was filled in, and it was incubated for 45 min to facilitate the antibody-antigen binding (step III). An (11) Wacker, R.; Niemeyer, C. M. ChemBioChem 2004, 5, 453–459. (12) Wacker, R.; Schroeder, H.; Niemeyer, C. M. Anal. Biochem. 2004, 330, 281–287. (13) Niemeyer, C. M. nanotoday 2007, 2, 42–52. (14) Microfluidic devices are often applied for the analysis of nucleic acid hybridization (Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. Rev. 2008, 108, 109–139. ) and have recently been used for DNA-directed immobilization of antibodies for cell capture experiments (Bailey, R. C.; Kwong, G. A.; Radu, C. G.; Witte, O. N.; Heath, J. R. J. Am. Chem. Soc. 2007, 129, 1959–1967). (15) Feldkamp, U.; Wacker, R.; Banzhaf, W.; Niemeyer, C. M. ChemPhysChem 2004, 5, 367–372. (16) The level of multiplexing depends on the availability of specific binders, which do neither cross-react with each other nor with other targets or contaminants present in the sample materials While more than 100-fold multiplexing has been reported for one-sided immunoassays (for examples, see Natarajan, S.; Hatch, A.; Myszka, D. G.; Gale, B. K. Anal. Chem. 2008, 80, 8561–8567. Lv, L. L.; Liu, B. C. Expert Rev. Proteomics 2007, 4, 505– 513. Hober, S.; Uhlen, M. Curr. Opin. Biotechnol. 2008, 19, 30-35, and references cited therein), the availability of compatible specific pairs of antibodies limits the level of multiplexing in two-sided (sandwich) immunoassays. Nonetheless, multiplexing levels of >20 have been reported (for examples, see: Schweitzer, B.; Roberts, S.; Grimwade, B.; Shao, W.; Wang, M.; Fu, Q.; Shu, Q.; Laroche, I.; Zhou, Z.; Tchernev, V. T.; Christiansen, J.; Velleca, M.; Kingsmore, S. F. Nat. Biotechnol. 2002, 20, 359–365. Nielsen, U. B.; Geierstanger, B. H. J. Immunol. Methods 2004, 290, 107–120. Gonzalez, R. M.; Seurynck-Servoss, S. L.; Crowley, S. A.; Brown, M.; Omenn, G. S.; Hayes, D. F.; Zangar, R. C. J. Proteome Res. 2008, 7, 2406–2414. The four antibody pairs used in this study, were previously tested for specific binding and lack of cross-reactivity by means of standard microplate ELISA (Adler, M.;Schroeder, H.;Wacker, R.;Niemeyer, C. M. Unpublished results). It was observed that none of the antibodies revealed significant affinity for any other antibody or antigen component used in our four-plex assay. (17) Dankbar, D. M.; Gauglitz, G. Anal. Bioanal. Chem. 2006, 386, 1967–1974.

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equimolar mixture of antibody-DNA detection conjugates (20 nM) was then allowed to bind to the immobilized biomarkers (step IV). These conjugates were previously assembled from the respective antibodies and covalent DNA-STV conjugate K bearing the oligonucleotide sequence K, which is not complementary and thus orthogonal to the sequences of the adaptors D-G. After incubation (60 min), excess materials were removed by washing and DNA-modified gold nanoparticles (DNA-AuNP, 24 nm diameter) were allowed to bind (step V). The nanoparticles were prepared by standard protocols,18 and they contained oligonucleotides with sequence cK, which is complementary to the antibody-DNA detection conjugates. The DNA-directed labeling of the immobilized immunocomplexes was then visualized by silver deposition (step VI, Figure 2), a standard technique, which was previously developed for array-based protein detection.18 Finally, the chip was transferred from the aspiration chamber to the read-out system to facilitate imaging and gray scale analysis of the silver stained spots. The in-house developed read-out system employs simple shadow printing as the imaging principle. As shown in Figure 3c, the chip is illuminated using a bright white LED. The light transmitted through the chip is recorded by a 1280 × 1024 pixel CMOS camera chip (Figure 3c). The size of the chip allows the capture of the complete image of the 4 × 4 silver stained spots in one well. The position of the spots is located by image analysis, and the grayscale value is determined by averaging over the pixels in the spot area. Measurement of all wells on a chip is achieved by moving the chip using an XY table, based on piezomotors.19 Since the exact position of the spot pattern within a well is located by image analysis, the XY table does not require an accurate position registration. The read-out system action is controlled by a microcontroller, which is also used to adjust LED intensity and camera settings. The system is connected by USB to a standard PC, where the image data is stored to facilitate data evaluation. Because of its simple optical and mechanical design, without the need for lenses and a high precision XY table, this readout system holds the potential for a portable, low-cost read-out system. Figure 4 shows results obtained for the detection of biomarkers, prostata specific antigen (PSA), TNFR, Interleukin 6 (IL6), or Interleukin 23 (IL23), using our microfluidic device. Initially, we investigated whether positional encoding of biomarker detection occurred at the correct spots of the in-well 4 × 4 capture arrays (for the whole set of arrays, see also Figure SI-1 in the Supporting Information). It is clearly evident from Figure 4a that silver-stained spots were only visible at those sites in which the complementary antibodies had been immobilized by DNA hybridization. Grayscale analysis of the silver-stained spots allowed us to determine the concentration of biomarkers present in the analyte solutions (Figure 4b). As determined from spiked solutions containing known concentrations of the various biomarker antigens, detection limits of less than 0.1 ng/mL, corresponding to approximately 1.7-3.8 pM of the corresponding cytokines, were observed. (18) Niemeyer, C. M.; Ceyhan, B. Angew. Chem., Int. Ed. 2001, 40, 3685–3688. (19) Schlu ¨ ter, M.; Magnussen, B.; van Vinckenroye, D. The ElliptecmotorApplications in Precision Positioning, Reduction of Vibrations, Force Detection and Low-Cost Driving Concepts. Posterpresentation on Conference Actuator 2006, Bremen, Germany, June 14-16, 2006, http://www. actuator.de/contents/pdf/programm/shorties/P033_schlueter_s.pdf.

In conclusion, the microfluidic device presented here can be applied for the facile in situ generation of protein microarrays, useful for multiplex immunological detection assays. We demonstrate here that the combination of top-down micromanufacturing with bottom-up assembly of biomolecules allows one to readily build up user-configurable microfluidic systems for multiplexed immunoassays. As demonstrated by parallel detection of different cytokines in a multiplex immunoassay, the performance of the system is at least similar to that of conventional microplate assays. However, it should be noted that, based on existing technologies, the current well geometries can easily be further miniaturized to further minimize sample and reagent volumes to less than one microliter per well. This would lead to a further reduction of sample volumes, reagent costs, and processing time. Because our microfluidic device as well as the read-out platform is excusively based on low-cost parts, we anticipate that this system is producable for moderate prices to eventually enable point-of-care testing

in the fields of clinical diagnostics, drug development, or biowarfare risk assessment. ACKNOWLEDGMENT We thank the government of North Rhine-Westphalia for financial support of this work through the PROTEOMICS project (Project FK 005-0407-0029). We also thank Holger Bartos and Gert Blankenstein for valuable discussions. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review December 2, 2008.

October

21,

2008.

Accepted

AC802228K

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