Microarrays 2014, 3, 282-301; doi:10.3390/microarrays3040282 OPEN ACCESS
microarrays ISSN 2076-3905 www.mdpi.com/journal/microarrays Article
Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection Ingeborg Hospach 1,*, Yvonne Joseph 2, Michaela Kathrin Mai 1, Nadejda Krasteva 1 and Gabriele Nelles 1 1
2
Materials Science Laboratory, Sony Deutschland GmbH, Hedelfinger Strasse 61, 70327 Stuttgart, Germany; E-Mails: [email protected] (M.K.M.); [email protected] (N.K.); [email protected] (G.N.) Institute of Electronic and Sensor Materials, Technische Universität Bergakademie Freiberg, Gustav-Zeuner-Strasse 3, 09599 Freiberg, Germany; E-Mail: [email protected]
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +49-0-711-5858-837; Fax: +49-0-711-5858-99-837. External Editor: Ulrich Certa Received: 6 June 2014; in revised form: 15 November 2014 / Accepted: 1 December 2014 / Published: 9 December 2014
Abstract: Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Optimization of the array preparation was done in regard of spot homogeneity and spot size. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα) and Interleukin 6 (IL-6) were chosen as the first targets of interest. First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D). The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As a first step towards real sample studies, a proof of principle of cytokine detection has been established.
Microarrays 2014, 2
283
Keywords: microarray; protein; antibody; cytokines; spotting; hydrogel; biosensor; fluorescence readout
1. Introduction Breath analysis is an interesting and motivating field of research; most recently, it has been found that dogs can smell lung cancer with high probability if trained accordingly [1]. Furthermore, lung diseases like asthma and chronic obstructive pulmonary disease (COPD) show similar somatic symptoms: the airways are inflamed but different medication is required. To distinguish between the diseases, classical diagnostic methods like imaging, spirometry, analysis of bronchoalveolar lavage (BAL), and invasive bronchoscopy are currently applied [2–4]. The vision for tomorrow, however, will be as in many medical fields the point-of-need diagnosis, a non-invasive breath analysis in order to spare patients invasive examinations, to increase the specificity of analysis, and to reduce costs. Critical high concentrations of nitrogen monoxide (NO) in breath, a potential marker for asthma, are already detected by chemical gas sensors, but the gaseous fraction of the breath varies strongly depending on personal habits [5,6]. On the other hand, and similar to other body fluids, like blood, urine, and saliva, monitoring of the cytokine pattern in exhaled breath condensate (EBC) can contribute to the identification and diagnosis of diseases [7–11]. Depending on the kind of disease, the body reacts with a certain distinct immune response, and thus different cytokines are present in specific amounts and different cytokine patterns are characteristic, as described for COPD and asthma in Barnes et al. [8]. The challenge of EBC analysis will be that the characteristic cytokines are only present in very low concentrations (250 µm. In the literature, lithographic technologies to fabricate arrays with spot sizes down to 50 nm are undergoing development in order to reduce sample volume, incubation time, and possibly the LoD [15]. However, in this case it would be necessary to change experimental parameters and conventional fluorescent scanners or microscopes, which would be another topic of investigation. Our low spot to spot distance is of advantage, considering the diffusion and kinetics of proteins in a microarray immunoassay. Short diffusion lengths were further realized by using the LifterSlipTM. Analogous considerations like microfluidics or similar approaches should be taken into account for further developments. For the alternative amine and epoxy functionalized surfaces, it was also possible
Microarrays 2014, 2
299
to bind antibodies covalently; however further investigations were omitted because of their drawbacks. On these surfaces the spots were ~3× bigger, the antibody could not be as densely packed, and fluorescent intensities of spotted antibodies were already very low after antibody washing, and minimal higher antibody density could only be achieved if the concentration of antibody was increased significantly. Additionally, the stability of the fabricated antibody microarray on the 3D hydrogel could be given at least for 1 month, meaning no significant quality loss could be observed. Further, we investigated different fluorescence dyes, of which APC and especially PE showed very high fluorescent intensities; however, as they are themselves large proteins they might influence the wetting properties, the coverage, as well as the antibody-antigen binding process, i.e., the biological activity. The FITC label was smaller but on the other hand not suitable due to strong bleaching. Stable and small labels, ideally with inert properties could reduce the effects of a label to a minimum, reducing steric hindrances and other systematic errors within the protein-protein binding. To prove the function of the fabricated antibody microarray, the analyte was detected by either direct cytokine labeling or by a second labeled antibody. In both cases the amount of cytokine detected by a single antibody spot was in the low pg-range, and this without further optimization of the LoD. Apart from the detectable mass, the sample volume was also of importance and the interesting question was: what is the absolute mass detected by a single spot within a microarray? Achieved attomolar sensitivity of one single spot is comparable to the golden standard method for cytokine detection, the ELISA, however, the microarray has the potential to be geometrically optimized towards even higher sensitivities, independently from the fact that high sensitivity is given by the perfect antibody-antigen partner. To further improve the LoD in the future, different signal amplification techniques as for example reactions catalyzed by horseradish peroxidase (HRP) as known from the standard ELISA, or PCR-based amplification techniques can be considered in combination with an appropriate blocking procedure to further reduce background signal and unspecific binding. The vision is still to have a pattern of relevant and validated biomarker molecules, which can possibly give more information regarding a disease or therapy monitoring. In clinical diagnostics there are still many challenges to overcome in order to develop tools for clinically relevant and validated biomarkers. It is unknown how many discovered biomarkers fail in the validation process, and a single biomarker may lack in specificity and fail FDA approval [15]. If thinking about a diagnostic device, 100 spots/mm2 can already result in thousands of assays on an area of a microscopic slide. The vision to be very sensitive, to avoid sample pre-treatment, and to detect sample volume and concentration independently might become possible within a small volume microfluidic device with negligible non-specific binding, also for whole sample of body fluids. A mixture of miniaturized ELISA and antibody microarray for low LoDs could be the next generation immunoassays, reducing sample volume, unspecific binding, and different kinds of cross-reactions in multiplexing [13]. Acknowledgments The work was supported by Michael Hulko. The authors want to thank all colleagues for the fruitful discussions.
Microarrays 2014, 2
300
Author Contributions Ingeborg Hospach, Yvonne Joseph, Nadejda Krasteva, and Michaela Kathrin Mai conceived and designed the experiments, Ingeborg Hospach and Yvonne Joseph, performed the experiments and analyzed the data. Ingeborg Hospach wrote the manuscript, supported by Yvonne Joseph, Nadejda Krasteva, Michaela Kathrin Mai and Gabriele Nelles. All authors studied and discussed the literature and the experimental results. Conflicts of Interest The authors declare no conflict of interest. References 1.
Ehmann, R.; Boedeker, E.; Friedrich, U.; Sagert, J.; Dippon, J.; Friedel, G.; Walles, T. Canine scent detection in the diagnosis of lung cancer: Revisiting a puzzling phenomenon. Eur. Respir. J. 2012, 39, 669–676. 2. Adelroth, E. How to measure airway inflammation: Bronchoalveolar lavage and airway biopsies. Can. Respir. J. 1998, 5, 18A–21A. 3. Patel, I.S.; Roberts, N.J.; Lloyd-Owen, S.J.; Sapsford, R.J.; Wedzicha, J.A. Airway epithelial inflammatory responses and clinical parameters in COPD. Eur. Respir. J. 2003, 22, 94–99. 4. Gizycki, M.J.; Hattotuwa, K.L.; Barnes, N.; Jeffery, P.K. Effects of fluticasone propionate on inflammatory cells in COPD: An ultrastructural examination of endobronchial biopsy tissue. Thorax 2002, 57, 799–803. 5. Kharitonov, S.A.; Barnes, P.J. Biomarkers of some pulmonary diseases in exhaled breath. Biomarkers 2002, 7, 1–32. 6. Kharitonov, S.A.; Barnes, P.J. Exhaled Markers of Pulmonary Disease. Am. J. Respir. Crit. Care Med. 2001, 163, 1693–1722. 7. Bozza, F.A.; Salluh, J.I.; Japiassu, A.M.; Soares, M.; Assis, E.F.; Gomes, R.N.; Bozza, M.T.; Castro-Faria-Neto, H.C.; Bozza, P.T. Cytokine profiles as markers of disease severity in sepsis: A multiplex analysis. Crit. Care 2007, 11, R49, doi:10.1186/cc5783. 8. Barnes, P.J. The cytokine network in asthma and chronic obstructive pulmonary disease. J. Clin. Investig. 2008, 118, 3546–3556. 9. Casado, B.; Iadorola, P.; Luisettt, M.; Kussmann, M. Proteomics-based diagnosis of chronic obstructive pulmonary disease: The hunt for new markers. Expert Rev. Proteomics 2008, 5, 693–704. 10. Horváth, I.; Hunt, J.; Barnes, P.J. On behalf of the ATS/ERS Task Force on Exhaled Breath Condensate. Exhaled breath condensate: Methodological recommendations and unresolved questions. Eur. Respir. J. 2005, 26, 523–548. 11. Borrill, Z.L.; Roy, K.; Singh, D. Exhaled breath condensate biomarkers in COPD. Eur. Respir. J. 2008, 32, 472–486.
Microarrays 2014, 2
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12. Cretich, M.; Galati, C.; Renna, L.; Condorelli, G.G.; Gagni, P.; Chiari, M. Characterization of a new fluorescence-enhanceing substrate for microarrays with femtomolar sensitivity. Sens. Actuators B 2014, 192, 15–22. 13. Pla-Roca, M.; Leulmi, R.F.; Tourekhanova, S.; Bergeron, S.; Laforte, V.; Moreau, E.; Gosline, S.J.C.; Bertos, N.; Hallett, M.; Park, M.; et al. Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples. Mol. Cell. Proteomics 2012, 11, doi:10.1074/mcp.M111.011460. 14. Seurynck-Servoss, S.L.; Baird, C.L.; Rodland, K.D.; Zangar, R.C. Surface chemistries for antibody microarray. Front. Biosci. 2007, 12, 3956–3964. 15. Huang, R.-P.; Burkholder, B.; Jones, V.S.; Jiang, W.-D.; Mao, Y.-Q.; Chen, Q.-L.; Shi, Z. Cytokine Antibody Arrays in Biomarker Discovery and Validation. Curr. Proteomics 2012, 9, 55–70. 16. Ekins, R. Ambient Analyte Assay. In The Immunoassay Handbook, 3rd ed.; Wild, D., Ed.; Elsevier Ltd.: Oxford, UK, 2005; pp. 48–62. 17. Ekins, R.P.; Chu, F.W. Multianalyte Microspot Immunoassay-Microanalytical “Compact Disk” of the Future. Clin. Chem. 1991, 37, 1955–1967. 18. Zeiss—Objektive für die Forschung. Available online: http://microscopy.zeiss.com/microscopy/ de_de/service-support/glossar/objektive-fuer-die-forschung.html (accessed on 18 February 2014). 19. Smith, G.P. Kinetics of Amine Modification of Proteins. Bioconjugate Chem. 2006, 17, 501–506. 20. Seurynck-Servoss, S.L.; White, A.M.; Baird, C.L.; Rodland, K.D.; Zangar, R.C. Evaluation of Surface Chemistries for Antibody Microarrays. Anal. Biochem. 2007, 371, 105–115. 21. Templin, M.F.; Stoll, D.; Schrenk, M.; Traub, P.C.; Vöhringer, C.F.; Joos, T.O. Protein microarray technology. Trends Biotechnol. 2002, 20, 160–166. 22. Hartmann, M.; Roeraade, J.; Stoll, D.; Templin, M.F.; Joos, T.O. Protein microarrays for diagnostic assays. Anal. Bioanal. Chem. 2009, 393, 1407–1416. 23. Kusnezow, W.; Syagailo, Y.V.; Rüffer, S.; Baudenstiel, N.; Gauer, C.; Hoheisel, J.D.; Wild, D.; Goychuk, I. Optimal Design of Microarray Immunoassays to Compensate for Kinetic Limitations. Mol. Cell. Proteomics 2006, 5, 1681–1696. 24. Hartmann, M. Microfluidic Methods for Protein Microarrays. Doctoral Thesis, Division of Analytical Chemistry, Department of Chemistry, School of Chemical Science and Engineering, Royal Institute of Technology, KTH, Stockholm, Sweden, 2010. 25. Chen, A.; Vu, T.; Stybayeva, G.; Pan, T.; Revzin, A. Reconfigurable microfluidics combined with antibody microarrays for enhanced detection of T-cell secreted cytokines. Biomicrofluidics 2013, 7, 024105, doi:10.1063/1.4795423. 26. Khademhosseini, A. Chips to Hits: Microarray and microfluidic technologies for high-throughput analysis and drug discovery. Expert Rev. Mol. Diagn. 2005, 5, 843–846. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
microarrays ISSN 2076-3905 www.mdpi.com/journal/microarrays Article
Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection Ingeborg Hospach 1,*, Yvonne Joseph 2, Michaela Kathrin Mai 1, Nadejda Krasteva 1 and Gabriele Nelles 1 1
2
Materials Science Laboratory, Sony Deutschland GmbH, Hedelfinger Strasse 61, 70327 Stuttgart, Germany; E-Mails: [email protected] (M.K.M.); [email protected] (N.K.); [email protected] (G.N.) Institute of Electronic and Sensor Materials, Technische Universität Bergakademie Freiberg, Gustav-Zeuner-Strasse 3, 09599 Freiberg, Germany; E-Mail: [email protected]
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +49-0-711-5858-837; Fax: +49-0-711-5858-99-837. External Editor: Ulrich Certa Received: 6 June 2014; in revised form: 15 November 2014 / Accepted: 1 December 2014 / Published: 9 December 2014
Abstract: Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Optimization of the array preparation was done in regard of spot homogeneity and spot size. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα) and Interleukin 6 (IL-6) were chosen as the first targets of interest. First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D). The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As a first step towards real sample studies, a proof of principle of cytokine detection has been established.
Microarrays 2014, 2
283
Keywords: microarray; protein; antibody; cytokines; spotting; hydrogel; biosensor; fluorescence readout
1. Introduction Breath analysis is an interesting and motivating field of research; most recently, it has been found that dogs can smell lung cancer with high probability if trained accordingly [1]. Furthermore, lung diseases like asthma and chronic obstructive pulmonary disease (COPD) show similar somatic symptoms: the airways are inflamed but different medication is required. To distinguish between the diseases, classical diagnostic methods like imaging, spirometry, analysis of bronchoalveolar lavage (BAL), and invasive bronchoscopy are currently applied [2–4]. The vision for tomorrow, however, will be as in many medical fields the point-of-need diagnosis, a non-invasive breath analysis in order to spare patients invasive examinations, to increase the specificity of analysis, and to reduce costs. Critical high concentrations of nitrogen monoxide (NO) in breath, a potential marker for asthma, are already detected by chemical gas sensors, but the gaseous fraction of the breath varies strongly depending on personal habits [5,6]. On the other hand, and similar to other body fluids, like blood, urine, and saliva, monitoring of the cytokine pattern in exhaled breath condensate (EBC) can contribute to the identification and diagnosis of diseases [7–11]. Depending on the kind of disease, the body reacts with a certain distinct immune response, and thus different cytokines are present in specific amounts and different cytokine patterns are characteristic, as described for COPD and asthma in Barnes et al. [8]. The challenge of EBC analysis will be that the characteristic cytokines are only present in very low concentrations (250 µm. In the literature, lithographic technologies to fabricate arrays with spot sizes down to 50 nm are undergoing development in order to reduce sample volume, incubation time, and possibly the LoD [15]. However, in this case it would be necessary to change experimental parameters and conventional fluorescent scanners or microscopes, which would be another topic of investigation. Our low spot to spot distance is of advantage, considering the diffusion and kinetics of proteins in a microarray immunoassay. Short diffusion lengths were further realized by using the LifterSlipTM. Analogous considerations like microfluidics or similar approaches should be taken into account for further developments. For the alternative amine and epoxy functionalized surfaces, it was also possible
Microarrays 2014, 2
299
to bind antibodies covalently; however further investigations were omitted because of their drawbacks. On these surfaces the spots were ~3× bigger, the antibody could not be as densely packed, and fluorescent intensities of spotted antibodies were already very low after antibody washing, and minimal higher antibody density could only be achieved if the concentration of antibody was increased significantly. Additionally, the stability of the fabricated antibody microarray on the 3D hydrogel could be given at least for 1 month, meaning no significant quality loss could be observed. Further, we investigated different fluorescence dyes, of which APC and especially PE showed very high fluorescent intensities; however, as they are themselves large proteins they might influence the wetting properties, the coverage, as well as the antibody-antigen binding process, i.e., the biological activity. The FITC label was smaller but on the other hand not suitable due to strong bleaching. Stable and small labels, ideally with inert properties could reduce the effects of a label to a minimum, reducing steric hindrances and other systematic errors within the protein-protein binding. To prove the function of the fabricated antibody microarray, the analyte was detected by either direct cytokine labeling or by a second labeled antibody. In both cases the amount of cytokine detected by a single antibody spot was in the low pg-range, and this without further optimization of the LoD. Apart from the detectable mass, the sample volume was also of importance and the interesting question was: what is the absolute mass detected by a single spot within a microarray? Achieved attomolar sensitivity of one single spot is comparable to the golden standard method for cytokine detection, the ELISA, however, the microarray has the potential to be geometrically optimized towards even higher sensitivities, independently from the fact that high sensitivity is given by the perfect antibody-antigen partner. To further improve the LoD in the future, different signal amplification techniques as for example reactions catalyzed by horseradish peroxidase (HRP) as known from the standard ELISA, or PCR-based amplification techniques can be considered in combination with an appropriate blocking procedure to further reduce background signal and unspecific binding. The vision is still to have a pattern of relevant and validated biomarker molecules, which can possibly give more information regarding a disease or therapy monitoring. In clinical diagnostics there are still many challenges to overcome in order to develop tools for clinically relevant and validated biomarkers. It is unknown how many discovered biomarkers fail in the validation process, and a single biomarker may lack in specificity and fail FDA approval [15]. If thinking about a diagnostic device, 100 spots/mm2 can already result in thousands of assays on an area of a microscopic slide. The vision to be very sensitive, to avoid sample pre-treatment, and to detect sample volume and concentration independently might become possible within a small volume microfluidic device with negligible non-specific binding, also for whole sample of body fluids. A mixture of miniaturized ELISA and antibody microarray for low LoDs could be the next generation immunoassays, reducing sample volume, unspecific binding, and different kinds of cross-reactions in multiplexing [13]. Acknowledgments The work was supported by Michael Hulko. The authors want to thank all colleagues for the fruitful discussions.
Microarrays 2014, 2
300
Author Contributions Ingeborg Hospach, Yvonne Joseph, Nadejda Krasteva, and Michaela Kathrin Mai conceived and designed the experiments, Ingeborg Hospach and Yvonne Joseph, performed the experiments and analyzed the data. Ingeborg Hospach wrote the manuscript, supported by Yvonne Joseph, Nadejda Krasteva, Michaela Kathrin Mai and Gabriele Nelles. All authors studied and discussed the literature and the experimental results. Conflicts of Interest The authors declare no conflict of interest. References 1.
Ehmann, R.; Boedeker, E.; Friedrich, U.; Sagert, J.; Dippon, J.; Friedel, G.; Walles, T. Canine scent detection in the diagnosis of lung cancer: Revisiting a puzzling phenomenon. Eur. Respir. J. 2012, 39, 669–676. 2. Adelroth, E. How to measure airway inflammation: Bronchoalveolar lavage and airway biopsies. Can. Respir. J. 1998, 5, 18A–21A. 3. Patel, I.S.; Roberts, N.J.; Lloyd-Owen, S.J.; Sapsford, R.J.; Wedzicha, J.A. Airway epithelial inflammatory responses and clinical parameters in COPD. Eur. Respir. J. 2003, 22, 94–99. 4. Gizycki, M.J.; Hattotuwa, K.L.; Barnes, N.; Jeffery, P.K. Effects of fluticasone propionate on inflammatory cells in COPD: An ultrastructural examination of endobronchial biopsy tissue. Thorax 2002, 57, 799–803. 5. Kharitonov, S.A.; Barnes, P.J. Biomarkers of some pulmonary diseases in exhaled breath. Biomarkers 2002, 7, 1–32. 6. Kharitonov, S.A.; Barnes, P.J. Exhaled Markers of Pulmonary Disease. Am. J. Respir. Crit. Care Med. 2001, 163, 1693–1722. 7. Bozza, F.A.; Salluh, J.I.; Japiassu, A.M.; Soares, M.; Assis, E.F.; Gomes, R.N.; Bozza, M.T.; Castro-Faria-Neto, H.C.; Bozza, P.T. Cytokine profiles as markers of disease severity in sepsis: A multiplex analysis. Crit. Care 2007, 11, R49, doi:10.1186/cc5783. 8. Barnes, P.J. The cytokine network in asthma and chronic obstructive pulmonary disease. J. Clin. Investig. 2008, 118, 3546–3556. 9. Casado, B.; Iadorola, P.; Luisettt, M.; Kussmann, M. Proteomics-based diagnosis of chronic obstructive pulmonary disease: The hunt for new markers. Expert Rev. Proteomics 2008, 5, 693–704. 10. Horváth, I.; Hunt, J.; Barnes, P.J. On behalf of the ATS/ERS Task Force on Exhaled Breath Condensate. Exhaled breath condensate: Methodological recommendations and unresolved questions. Eur. Respir. J. 2005, 26, 523–548. 11. Borrill, Z.L.; Roy, K.; Singh, D. Exhaled breath condensate biomarkers in COPD. Eur. Respir. J. 2008, 32, 472–486.
Microarrays 2014, 2
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12. Cretich, M.; Galati, C.; Renna, L.; Condorelli, G.G.; Gagni, P.; Chiari, M. Characterization of a new fluorescence-enhanceing substrate for microarrays with femtomolar sensitivity. Sens. Actuators B 2014, 192, 15–22. 13. Pla-Roca, M.; Leulmi, R.F.; Tourekhanova, S.; Bergeron, S.; Laforte, V.; Moreau, E.; Gosline, S.J.C.; Bertos, N.; Hallett, M.; Park, M.; et al. Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples. Mol. Cell. Proteomics 2012, 11, doi:10.1074/mcp.M111.011460. 14. Seurynck-Servoss, S.L.; Baird, C.L.; Rodland, K.D.; Zangar, R.C. Surface chemistries for antibody microarray. Front. Biosci. 2007, 12, 3956–3964. 15. Huang, R.-P.; Burkholder, B.; Jones, V.S.; Jiang, W.-D.; Mao, Y.-Q.; Chen, Q.-L.; Shi, Z. Cytokine Antibody Arrays in Biomarker Discovery and Validation. Curr. Proteomics 2012, 9, 55–70. 16. Ekins, R. Ambient Analyte Assay. In The Immunoassay Handbook, 3rd ed.; Wild, D., Ed.; Elsevier Ltd.: Oxford, UK, 2005; pp. 48–62. 17. Ekins, R.P.; Chu, F.W. Multianalyte Microspot Immunoassay-Microanalytical “Compact Disk” of the Future. Clin. Chem. 1991, 37, 1955–1967. 18. Zeiss—Objektive für die Forschung. Available online: http://microscopy.zeiss.com/microscopy/ de_de/service-support/glossar/objektive-fuer-die-forschung.html (accessed on 18 February 2014). 19. Smith, G.P. Kinetics of Amine Modification of Proteins. Bioconjugate Chem. 2006, 17, 501–506. 20. Seurynck-Servoss, S.L.; White, A.M.; Baird, C.L.; Rodland, K.D.; Zangar, R.C. Evaluation of Surface Chemistries for Antibody Microarrays. Anal. Biochem. 2007, 371, 105–115. 21. Templin, M.F.; Stoll, D.; Schrenk, M.; Traub, P.C.; Vöhringer, C.F.; Joos, T.O. Protein microarray technology. Trends Biotechnol. 2002, 20, 160–166. 22. Hartmann, M.; Roeraade, J.; Stoll, D.; Templin, M.F.; Joos, T.O. Protein microarrays for diagnostic assays. Anal. Bioanal. Chem. 2009, 393, 1407–1416. 23. Kusnezow, W.; Syagailo, Y.V.; Rüffer, S.; Baudenstiel, N.; Gauer, C.; Hoheisel, J.D.; Wild, D.; Goychuk, I. Optimal Design of Microarray Immunoassays to Compensate for Kinetic Limitations. Mol. Cell. Proteomics 2006, 5, 1681–1696. 24. Hartmann, M. Microfluidic Methods for Protein Microarrays. Doctoral Thesis, Division of Analytical Chemistry, Department of Chemistry, School of Chemical Science and Engineering, Royal Institute of Technology, KTH, Stockholm, Sweden, 2010. 25. Chen, A.; Vu, T.; Stybayeva, G.; Pan, T.; Revzin, A. Reconfigurable microfluidics combined with antibody microarrays for enhanced detection of T-cell secreted cytokines. Biomicrofluidics 2013, 7, 024105, doi:10.1063/1.4795423. 26. Khademhosseini, A. Chips to Hits: Microarray and microfluidic technologies for high-throughput analysis and drug discovery. Expert Rev. Mol. Diagn. 2005, 5, 843–846. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
