Spectral Reflectance Imaging for a Multiplexed ... - Semantic Scholar
IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 16, NO. 3, MAY/JUNE 2010
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Spectral Reflectance Imaging for a Multiplexed, High-Throughput, Label-Free, and Dynamic Biosensing Platform ¨ Emre Ozkumur, Student Member, IEEE, Carlos A. Lopez, Ayc¸a Yalc¸ın, Student Member, IEEE, ¨ u, Fellow, IEEE John H. Connor, Marcella Chiari, and M. Selim Unl¨ (Invited Paper)
Abstract—There are a number of emerging optical biosensing techniques utilizing interferometric and resonant characteristics of light. We have recently demonstrated an interferometric technique, the spectral reflectance imaging biosensor (SRIB) that uses optical wave interference to detect changes in the optical path length as a result of capture of biological material on the microarray surface without the need for labels and secondary reagents. In this paper, we review the principles and performance of the SRIB technique in the context of label-free biosensors and demonstrate its high-throughput, quantitative and calibrated, versatile, and dynamic (kinetic) capabilities. A unique aspect of the SRIB system is that the measurement technique is independent of surface conformation and allows for utilization of novel polymeric coatings for surface binding, thus providing a versatile and high-density platform. We present experimental results on multiplexed antibody/antigen arrays and DNA hybridization in real time, as well as specific binding of whole virus particles. The simplicity of the overall system, its high sensitivity and compatibility with glass surface chemistries, and a linear dynamic range of nearly four orders of magnitude makes SRIB a promising platform for multiplexed detection of different biological analytes in a complex sample, with potential impact in research and clinical applications. Index Terms—Biosensing, high-throughput, interferometry, label-free, microarrays, quantitative detection.
I. INTRODUCTION IVING organisms function as a result of a vast network of macromolecular interactions, including those between antigen–antibody, receptor–ligand, virus–cell, and protein– DNA. Proper cellular function and survival is largely the collective effect of continuous reversible binding events between
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Manuscript received July 16, 2009; revised October 21, 2009; accepted November 3, 2009. Date of publication January 15, 2010; date of current version June 4, 2010. This work was supported in part by the National Institute of General and Medical Sciences under Grant National Institutes of Health R21 GM 074872-02, in part by the U.S. Army Research Laboratory under Grant DAAD17-99-2-0070, and in part by the National Science Foundation OISE0601631 (International Research Experiences for Students). ¨ ¨ u are with the ElecE. Ozkumur, C. A. Lopez, A. Yalc¸ın, and M. S. Unl¨ trical and Computer Engineering Department, Boston University, Boston, MA 02215 USA (e-mail: [email protected]; [email protected]; [email protected]; [email protected]). J. H. Connor is with the Department of Microbiology, Boston University School of Medicine, Boston, MA 02118 USA (e-mail: [email protected]). M. Chiari is with the Istituto di Chimica del Riconoscimento Molecolare, National Research Council, 20131 Milano, Italy (e-mail: marcella.chiari@ icrm.cnr.it). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/JSTQE.2009.2037438
these biological entities. Therefore, knowing the binding affinities between biomolecules and detecting the presence, absence, or amount of biomolecules are essential for understanding and controlling both cell physiology and disease progression. For this reason, research in medical and life sciences benefits greatly from improvements in the biosensing technologies. Spectral reflectance imaging biosensor (SRIB) was introduced recently as a promising technique for performing labelfree, dynamic, and high-throughput biosensing [1], [2]. Surface mass sensitivity of ∼5 pg/mm2 and concentration sensitivity of approximately nanograms per milliliter of antibody in buffer was demonstrated before. So far, only binding of the antibodies to the spotted antigens was demonstrated with this system. In this paper, after a background on high-throughput and label-free sensing, a detailed description of the technique is given. Then, to demonstrate the potential of utilizing SRIB for a variety of applications, proof-of-principle experiments for detecting antibodies, antigens, oligonucleotides, and whole viruses are shown. A. Motivation for High-Throughput Detection Experiments in the fields of medicine, biology, and drug discovery drive a demand for improved biosensors. As the investigated networks are extremely complex and involve a vast number of interactions, scientists are interested in simple and high-throughput analytical methods, such as microarrays. Microarrays were first introduced for RNA expression level studies in the mid 1990s. The small volumes needed to immobilize the probe single-stranded DNA (ssDNA) and small amounts of mRNA extract needed for slide incubation, as well as the high density of the arrays were a major advancement in the speed of expression profiling [3]. Introduction of in situ synthesis of DNA strands on glass surfaces for DNA microarray fabrication has enabled analysis of millions of spots on a single microscope slide [4]. The significance of high-throughput platforms has been demonstrated by the success of gene arrays in the analysis of nucleic acids for many important applications, including medical diagnostics and cancer research [5]–[9]. This success has further provided impetus for extending the use of microarray platforms to perform interaction analysis of DNA and protein transcription factors (TFs) [10], [11], as well as proteins with other proteins [12], [13]. Protein microarrays have received great
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interest from the biological and medical research communities [14]–[18] and their applications have expanded to potential use in diagnostics [19]–[21] and biohazard detection [22]. Despite this excitement, protein microarrays face many challenges that are not encountered by DNA microarrays [23]. First, proteins are more complex and demonstrate a higher degree of variability than their nucleic acid counterparts, and thus, cannot be synthesized as easily. Furthermore, handling and storing proteins always require more attention. Therefore, a significant research effort across various disciplines is addressing these challenges. Two examples are the “nucleic-acid-programmable protein array” (NAPPA) and “DNA array to protein array” (DAPA) techniques, which have been recently introduced [24], [25]. These two techniques rely on a similar idea in which proteins are synthesized in situ from coding double-stranded DNA and captured on the surface in the vicinity of DNA spots to form the protein array. Biomolecular interactions greatly depend on the conformation of the interacting molecules and this poses another challenge for protein microarrays. Immobilization on solid surfaces may cause them to denature, thus affecting their affinity for other proteins. DNA arrays that retain optimal protein binding activity are also desirable for more sensitive detection of TF interactions. Better and simpler surface functionalization techniques under investigation allow higher densities and more functional immobilization of biomolecules on the surface [26]–[28]. Toward this goal, Chiari et al. have introduced a novel polymeric coating that increases the immobilization density and functionality of the spotted proteins and nucleic acid probes [29], [30]. Yalc¸ın et al. demonstrated that the volume of this polymeric coating increases by swelling in aqueous environments, thus possessing a 3-D structure that mimics a solution-like state [31]. While a significant amount of research is performed to improve the microarray deposition methods, there are efforts to improve the detection systems as well. Label-free imaging methods are under investigation for more reliable and accurate detection of microarray experiments. B. Motivation for Label-Free Detection Traditional microarray imaging commonly employs tags (mainly, fluorescent molecules) that are attached to the captured analytes to create a detectable signal. The binding of target molecules to the immobilized probes are visualized by imaging the surface with a fluorescence scanner. Despite its popularity, it is generally acknowledged that this method has inherent drawbacks, and that for many, if not for most applications, label-free detection would be highly desirable. Even though fluorescence detection generally performs with superior sensitivity, there are applications for which label-free detection sensitivity is sufficient and label-free detection is required [16], [23], [32]–[34]. One of the drawbacks of labeled detection is that labels might modify the interaction affinities of the macromolecules to which they are attached. In some cases, special attention is paid to attach the label to a designated epitope on the protein in order to minimize the effect on binding kinetics. But, frequently, proteins are labeled nonspecifically, and the fluorescent labels may inter-
fere with the binding sites. This is a significant problem when studying small molecules and peptides, as the amino acids involved in binding are more likely to be affected. The effect is clear for larger molecules as well. A recent study by Sun et al. [35] shows how the properties of fairly simple and widely used proteins are affected by labeling. In this study, streptavidin– peptide and antibody–antigen reactions were monitored with a label-free technique, and it was observed that when the target is labeled with Cy3 molecules, the detected reaction kinetics change significantly. To avoid complications that result from directly labeling the target molecules, sandwich assays are often employed [16], [36]. After the first incubation with target molecules that are not labeled, the array is incubated with labeled secondary probe molecules that are also specific to the target. However, the second incubation should not interfere with the first one, namely, two different probes that are specific for two distinct nonoverlapping epitopes on the same target protein are required for the sandwich assay to work properly [37]. Otherwise, a competition reaction occurs that can create false-negative signals. Also, the secondary probe should not have any specificity for the spotted probes. If this is not satisfied, a false positive may occur when the secondary probes bind to spotted molecules without the initially captured target. In summary, the detection of analytes through secondary probes is intrinsically complex, as it requires multiple layers of interacting components that provide specificity without interfering with one another. Label-free measurements can provide kinetic information about the reactions, which is not possible with fluorescent techniques. Even though quantification of captured biological mass is possible in fluorescence measurements using on-chip calibration procedures [38], the bleaching of fluorescent molecules avoid real-time data acquisition to characterize the biomolecular interaction thermodynamics. Since the photoluminescence ability of dyes decay over time through excitation, a phenomenon called photobleaching occurs and prevents a linear response during long exposure times. Labeled detection methods are widely used because of their sensitivity and the historical lack of viable alternatives. However, depending on the application, the challenges they present can be significant and difficult to overcome when a quantitative measure of binding is required. In such cases, label-free detection has major advantages over the labeled detection methods. Besides the advantages listed in this section, label-free methods eliminate at least one chemical step from the assay process. Thus, they are time- and cost-efficient, and reduce experimental variability due to user error. C. Current and Emerging Biosensing Platforms Surface plasmon resonance (SPR) has been the leading technology among label-free detection methods. Traditional SPR has been limited to a macroformat with very limited throughput (four or eight channels). However, recent advancements in the technology show that higher throughput is, indeed, possible. Imaging SPR, presented by Shumaker–Parry and Campbell, is capable of imaging interactions for 120 features, in real time,
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with 3-pm height sensitivity [39], [40]. In contrast to regular SPR in imaging SPR, both the angle and the wavelength are fixed to the high linear region of system response. The reflection is imaged to a charge-coupled device (CCD) camera, and molecular accumulation on the surface is detected as an intensity change on the camera [41]. This technique is currently going through modifications and adjustments to improve the sensitivity and throughput further [42]. Campbell and Kim, in their review article, discuss a commercially available system that is capable of imaging >1300 spots with ∼3-pm noise floor for each spot, with a time resolution of 1 s. [43]. Although SPR has been quite successful, some of its shortcomings may be prohibitively difficult to overcome, depending on the application of interest. First, imaging SPR has a limited linear dynamic range. As a result, similar initial mass densities and surface uniformity have to be maintained for all the immobilized probes [44]. For the same reason, multiple layers of interactions are hard to monitor with the same sensitivity. Improvements are under investigation to increase the dynamic range of SPR microscopy [45]. Also, the requirement of gold surfaces increases the cost of substrates. Furthermore a change in the optical properties of the buffer affects the signal (known as “bulk” or “matrix” effect); therefore, buffer-dependent association/dissociation experiments may be problematic to carry out. The system temperature should also be well regulated during measurements, which requires welldesigned temperature-control systems to be used for long experiments, thus the instrumentation is further complicated. As an alternative to SPR, methods that employ interferometric, ellipsometric, and resonant characteristics of light have become popular for biosensing, as they offer simple and sensitive detection. Using molecular interferometric imaging (MI2) [46], [47] real-time detection on a 40-spot array was shown with a noise floor of 15–20 pm per spot. Another technique, the biomolecular interaction detector (BIND), has been commercialized by SRU Biosystems. Using BIND, very high sensitivities were achieved and small molecules (103 spots with 0.01-ng/mm2 mass sensitivity with the current system. The data acquisition takes less than 30 s; therefore, kinetic characterization of interactions is also possible. SRIB presents an alternative to current state-of-the-art assay technologies, with the added advantages of using an inexpensive substrate for probe immobilization, reduced dependence on bulk effects, large linear dynamic range, and quantitative binding determinations. REFERENCES [1] E. Ozkumur, J. W. Needham, D. A. Bergstein, R. Gonzalez, M. Cabodi, J. M. Gershoni, B. B. Goldberg, and M. S. Unlu, “Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications,” Proc. Nat. Acad. Sci. USA, 2008, vol. 105, no. 23, pp. 7988– 7992. [2] E. Ozkumur, A. Yalc¸in, M. Cretich, C. A. Lopez, D. A. Bergstein, B. B. ¨ u, “Quantification of DNA and protein Goldberg, M. Chiari, and M. S. Unl¨ adsorption by optical phase shift,” Biosens. Bioelectron., vol. 25, no. 1, pp. 167–172, 2009.
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[48] B. Cunningham, P. Li, B. Lin, and J. Pepper, “Colorimetric resonant reflection as a direct biochemical assay technique,” Sens. Actuators B, Chem., vol. 81, no. 2/3, pp. 316–328, 2002. [49] B. Lin, J. Qiu, J. Gerstenmeier, P. Li, H. Pien, J. Pepper, and B. Cunningham, “A label-free optical technique for detecting small molecule interactions,” Biosens. Bioelectron., vol. 17, no. 9, pp. 827–834, 2002. [50] I. D. Block, L. L. Chan, and B. T. Cunningham, “Photonic crystal optical biosensor incorporating structured low-index porous dielectric,” Sens. Actuators B, Chem., vol. 120, no. 1, pp. 187–193, 2006. [51] P. Y. Li, B. Lin, J. Gerstenmaier, and B. T. Cunningham, “A new method for label-free imaging of biomolecular interactions,” Sens. Actuators B, Chem., vol. 99, no. 1, pp. 6–13, 2004. [52] C. J. Choi and B. T. Cunningham, “A 96-well microplate incorporating a replica molded microfluidic network integrated with photonic crystal biosensors for high throughput kinetic biomolecular interaction analysis,” Lab Chip, vol. 7, no. 5, pp. 550–556, 2007. [53] J. Piehler, A. Brecht, and G. Gauglitz, “Affinity detection of low molecular weight analytes,” Anal. Chem., vol. 68, no. 1, pp. 139–143, 1996. [54] H. M. Schmitt, A. Brecht, J. Piehler, and G. Gauglitz, “An integrated system for optical biomolecular interaction analysis,” Biosens. Bioelectron., vol. 12, no. 8, pp. 809–816, 1997. [55] C. Hanel and G. Gauglitz, “Comparison of reflectometric interference spectroscopy with other instruments for label-free optical detection,” Anal. Bioanalytical Chem., vol. 372, no. 1, pp. 91–100, 2002. [56] G. Gauglitz, “Multiple reflectance interference spectroscopy measurements made in parallel for binding studies,” Rev. Sci. Instrum., vol. 76, no. 6, pp. 062224-1–062224-10, 2005. [57] J. P. Landry, X. D. Zhu, and J. P. Gregg, “Label-free detection of microarrays of biomolecules by oblique-incidence reflectivity difference microscopy,” Opt. Lett., vol. 29, no. 6, pp. 581–583, 2004. [58] X. D. Zhu, J. P. Landry, Y. S. Sun, J. P. Gregg, K. S. Lam, and X. W. Guo, “Oblique-incidence reflectivity difference microscope for label-free highthroughput detection of biochemical reactions in a microarray format,” Appl. Opt., vol. 46, no. 10, pp. 1890–1895, 2007. [59] D. A. Bergstein, E. Ozkumur, A. C. Wu, A. Yalcın, J. R. Colson, J. W. Needham, R. J. Irani, J. M. Gershoni, B. B. Goldberg, C. DeLisi, M. F. Ruane, and M. S. Unlu, “Resonant cavity imaging: A means toward highthroughput label-free protein detection,” IEEE J. Sel. Topics Quantum Electron., vol. 14, no. 1, pp. 131–139, Jan./Feb. 2008. [60] V. S. Y. Lin, K. Motesharei, K. S. Dancil, M. J. Sailor, and M. R. Ghadiri, “A porous silicon-based optical interferometric biosensor,” Science, vol. 278, no. 5339, pp. 840–843, 1997. [61] A. Yalcin, K. C. Popat, J. C. Aldridge, T. A. Desai, J. Hryniewicz, N. Chbouki, B. E. Little, O. King, V. Van, S. Chu, D. Gill, M. Anthes-Washburn, M. S. Unlu, and B. B. Goldberg, “Optical sensing of biomolecules using microring resonators,” IEEE J. Sel. Topics Quantum Electron., vol. 12, no. 1, pp. 148–155, Jan./Feb. 2006. [62] C. C. Striemer, T. R. Gaborski, J. L. McGrath, and P. M. Fauchet, “Chargeand size-based separation of macromolecules using ultrathin silicon membranes,” Nature, vol. 445, no. 7129, pp. 749–753, 2007. [63] J. E. Lugo, M. Ocampo, A. G. Kirk, D. V. Plant, and P. M. Fauchet, “Electrochemical sensing of DNA with porous silicon layers,” J. New Mater. Electrochem. Syst., vol. 10, no. 2, pp. 113–116, 2007. [64] D. A. Markov, K. Swinney, and D. J. Bornhop, “Label-free molecular interaction determinations with nanoscale interferometry,” J. Amer. Chem. Soc., vol. 126, no. 50, pp. 16659–16664, 2004. [65] D. J. Bornhop, J. C. Latham, A. Kussrow, D. A. Markov, R. D. Jones, and H. S. Sørensen, “Free-solution, label-free molecular interactions studied by back-scattering interferometry,” Science, vol. 317, no. 5845, pp. 1732– 1736, 2007. [66] C. L. Baird and D. G. Myszka, “Current and emerging commercial optical biosensors,” J. Mol. Recog., vol. 14, no. 5, pp. 261–268, 2001. [67] M. A. Cooper, “Label-free screening of bio-molecular interactions,” Anal. Bioanalytical Chem., vol. 377, no. 5, pp. 834–842, 2003. [68] A. Brecht and G. Gauglitz, “Recent developments in optical transducers for chemical or biochemical applications,” Sens. Actuators B, Chem., vol. 38, no. 1–3, pp. 1–7, 1997. [69] P. I. Nikitin, B. G. Gorshkov, E. P. Nikitin, and T. I. Ksenevich, “Picoscope, a new label-free biosensor,” Sens. Actuators B, Chem., vol. 111, pp. 500– 504, 2005. [70] C. M. Joo and J. F. de Boer, “Spectral-domain optical coherence reflectometric sensor for highly sensitive molecular detection,” Opt. Lett., vol. 32, no. 16, pp. 2426–2428, 2007. [71] P. Yeh, Optical Waves in a Layered Media. New York: Wiley, 1988.
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¨ Emre Ozkumur (S’04) was born in Usak, Turkey, in 1982. He received the B.S. degrees in physics and electrical and electronics engineering from Koc University, Istanbul, Turkey, in 2004, and the M.S. and Ph.D. degrees in electrical and computer engineering from Boston University, Boston, MA, in 2007 and 2009, respectively. He is currently a Postdoctoral Researcher with Boston University. His research interests include label-free interferometric optical biosensors, and DNA and protein microarrays. ¨ Dr. Ozkumur is a member of the International Society for Optical Engineering and IEEE Lasers and Electro-Optics Society.
Carlos A. Lopez was born in Tuxtla Gutierrez, Mexico, in 1977. He received the B.S. degree in bioengineering: biotechnology from the University of California, San Diego, in 2001 and the Ph.D. degree in biomedical engineering from Boston University, Boston, MA, in 2007. He is currently a second-year Postdoctoral Research Associate with the Optical Characterization and Nanophotonics Laboratory, Boston University, where he is involved in multiplexed virus and cytokine detection capabilities of the SRIB platform. His research interests include work on cell-based and polymeric drug delivery methods and tissue engineering.
Ayc¸a Yalc¸ın (S’04) was born in Istanbul, Turkey, in 1982. She received the B.S. degree in electrical and electronics engineering from Bilkent University, Ankara, Turkey, in 2003, the M.S. degree in photonics, and the Ph.D. degree in electrical and computer engineering from Boston University, Boston, MA, in 2005 and 2009, respectively. She is currently a Postdoctoral Researcher with Boston University. Her research interests include label-free optical microresonator biosensors, interferometric techniques in fluorescence spectroscopy, and DNA and protein microarrays. Dr. Yalc¸ın is a member of the International Society for Optical Engineering and IEEE Lasers and Electro-Optics Society.
John H. Connor received the B.A. degree in chemistry from Swarthmore College, Swarthmore, PA, in 1994, and the Ph.D. degree in pharmacology from Duke University, in 1999. He was a Postdoctoral Fellow at Wake Forest University, Winston-Salem, NC. In 2006, he joined the faculty of Boston University School of Medicine, Boston, MA, where he is currently an Assistant Professor of microbiology. His research interests include understanding viral pathogenesis and the virus/host interface.
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Marcella Chiari received a B.S. in chemistry and pharmaceutical techniques in 1982 and a Ph.D. degree in clinical biochemistry in 1990, both from the University of Milan, Italy. She is currently a Senior Research Scientist with the Italian National Research Council, Milan, Italy, where she leads the Analytical Microsystem Laboratory, Institute of Molecular Recognition. She has world-wide recognized experience in development of hydrophilic linear polymers. She is involved in the area of protein and DNA microarray research. Her research interests include the development of innovative polymeric coatings for glass slides. She has been engaged in development of a number of new hydrophilic acrylic monomers and polymers to be used in capillary electrophoresis as DNA sieving matrices and capillary coatings.
¨ u¨ (M’90–SM’95–F’07) received the M. Selim Unl B.S. degree from Middle East Technical University, Ankara, Turkey, in 1986, and the M.S.E.E. and Ph.D. degrees in electrical engineering from the University of Illinois, Urbana-Champaign, in 1988 and 1992, respectively. Since 1992, he has been with the Department of Electrical and Computer Engineering, Boston University, Boston, MA, where he is currently a Professor of electrical and computer engineering, biomedical engineering, and physics, an Associate Dean for research and graduate programs in engineering, as well as the Associate Director of Center for Nanoscience and Nanobiotechnology. His research laboratories— Optical Characterization and Nanophotonics (www.bu.edu/OCN)—are located in the Photonics Center. His current research interests include nanophotonics and biophotonics, research and development of photonic materials, semiconductor optoelectronic devices, high-resolution microscopy and spectroscopy of semiconductor and biological materials, and biological sensing and imaging. ¨ u was the Chair of IEEE Laser and Electro-Optics Society (LEOS), Dr. Unl¨ Boston Chapter, winning the LEOS Chapter-of-the-Year Award during 1994– 1995. He is an Associate Editor for IEEE JOURNAL OF QUANTUM ELECTRONICS and Vice President of LEOS for membership and regional activities – Americas. He has been selected as a LEOS Distinguished Lecturer for 2005– 2007 and Australian Research Council Nanotechnology Network Distinguished Lecturer for 2007. He was the recipient of the National Science Foundation Research Initiation Award in 1993, United Nations TOKTEN award in 1995 and 1996, and both the National Science Foundation CAREER and Office of Naval Research Young Investigator Awards in 1996. He was the former Chair of the IEEE/LEOS technical committee on Photodetectors and Imaging and currently, the Chair of IEEE/LEOS Nanophotonics committee.
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Spectral Reflectance Imaging for a Multiplexed, High-Throughput, Label-Free, and Dynamic Biosensing Platform ¨ Emre Ozkumur, Student Member, IEEE, Carlos A. Lopez, Ayc¸a Yalc¸ın, Student Member, IEEE, ¨ u, Fellow, IEEE John H. Connor, Marcella Chiari, and M. Selim Unl¨ (Invited Paper)
Abstract—There are a number of emerging optical biosensing techniques utilizing interferometric and resonant characteristics of light. We have recently demonstrated an interferometric technique, the spectral reflectance imaging biosensor (SRIB) that uses optical wave interference to detect changes in the optical path length as a result of capture of biological material on the microarray surface without the need for labels and secondary reagents. In this paper, we review the principles and performance of the SRIB technique in the context of label-free biosensors and demonstrate its high-throughput, quantitative and calibrated, versatile, and dynamic (kinetic) capabilities. A unique aspect of the SRIB system is that the measurement technique is independent of surface conformation and allows for utilization of novel polymeric coatings for surface binding, thus providing a versatile and high-density platform. We present experimental results on multiplexed antibody/antigen arrays and DNA hybridization in real time, as well as specific binding of whole virus particles. The simplicity of the overall system, its high sensitivity and compatibility with glass surface chemistries, and a linear dynamic range of nearly four orders of magnitude makes SRIB a promising platform for multiplexed detection of different biological analytes in a complex sample, with potential impact in research and clinical applications. Index Terms—Biosensing, high-throughput, interferometry, label-free, microarrays, quantitative detection.
I. INTRODUCTION IVING organisms function as a result of a vast network of macromolecular interactions, including those between antigen–antibody, receptor–ligand, virus–cell, and protein– DNA. Proper cellular function and survival is largely the collective effect of continuous reversible binding events between
L
Manuscript received July 16, 2009; revised October 21, 2009; accepted November 3, 2009. Date of publication January 15, 2010; date of current version June 4, 2010. This work was supported in part by the National Institute of General and Medical Sciences under Grant National Institutes of Health R21 GM 074872-02, in part by the U.S. Army Research Laboratory under Grant DAAD17-99-2-0070, and in part by the National Science Foundation OISE0601631 (International Research Experiences for Students). ¨ ¨ u are with the ElecE. Ozkumur, C. A. Lopez, A. Yalc¸ın, and M. S. Unl¨ trical and Computer Engineering Department, Boston University, Boston, MA 02215 USA (e-mail: [email protected]; [email protected]; [email protected]; [email protected]). J. H. Connor is with the Department of Microbiology, Boston University School of Medicine, Boston, MA 02118 USA (e-mail: [email protected]). M. Chiari is with the Istituto di Chimica del Riconoscimento Molecolare, National Research Council, 20131 Milano, Italy (e-mail: marcella.chiari@ icrm.cnr.it). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/JSTQE.2009.2037438
these biological entities. Therefore, knowing the binding affinities between biomolecules and detecting the presence, absence, or amount of biomolecules are essential for understanding and controlling both cell physiology and disease progression. For this reason, research in medical and life sciences benefits greatly from improvements in the biosensing technologies. Spectral reflectance imaging biosensor (SRIB) was introduced recently as a promising technique for performing labelfree, dynamic, and high-throughput biosensing [1], [2]. Surface mass sensitivity of ∼5 pg/mm2 and concentration sensitivity of approximately nanograms per milliliter of antibody in buffer was demonstrated before. So far, only binding of the antibodies to the spotted antigens was demonstrated with this system. In this paper, after a background on high-throughput and label-free sensing, a detailed description of the technique is given. Then, to demonstrate the potential of utilizing SRIB for a variety of applications, proof-of-principle experiments for detecting antibodies, antigens, oligonucleotides, and whole viruses are shown. A. Motivation for High-Throughput Detection Experiments in the fields of medicine, biology, and drug discovery drive a demand for improved biosensors. As the investigated networks are extremely complex and involve a vast number of interactions, scientists are interested in simple and high-throughput analytical methods, such as microarrays. Microarrays were first introduced for RNA expression level studies in the mid 1990s. The small volumes needed to immobilize the probe single-stranded DNA (ssDNA) and small amounts of mRNA extract needed for slide incubation, as well as the high density of the arrays were a major advancement in the speed of expression profiling [3]. Introduction of in situ synthesis of DNA strands on glass surfaces for DNA microarray fabrication has enabled analysis of millions of spots on a single microscope slide [4]. The significance of high-throughput platforms has been demonstrated by the success of gene arrays in the analysis of nucleic acids for many important applications, including medical diagnostics and cancer research [5]–[9]. This success has further provided impetus for extending the use of microarray platforms to perform interaction analysis of DNA and protein transcription factors (TFs) [10], [11], as well as proteins with other proteins [12], [13]. Protein microarrays have received great
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interest from the biological and medical research communities [14]–[18] and their applications have expanded to potential use in diagnostics [19]–[21] and biohazard detection [22]. Despite this excitement, protein microarrays face many challenges that are not encountered by DNA microarrays [23]. First, proteins are more complex and demonstrate a higher degree of variability than their nucleic acid counterparts, and thus, cannot be synthesized as easily. Furthermore, handling and storing proteins always require more attention. Therefore, a significant research effort across various disciplines is addressing these challenges. Two examples are the “nucleic-acid-programmable protein array” (NAPPA) and “DNA array to protein array” (DAPA) techniques, which have been recently introduced [24], [25]. These two techniques rely on a similar idea in which proteins are synthesized in situ from coding double-stranded DNA and captured on the surface in the vicinity of DNA spots to form the protein array. Biomolecular interactions greatly depend on the conformation of the interacting molecules and this poses another challenge for protein microarrays. Immobilization on solid surfaces may cause them to denature, thus affecting their affinity for other proteins. DNA arrays that retain optimal protein binding activity are also desirable for more sensitive detection of TF interactions. Better and simpler surface functionalization techniques under investigation allow higher densities and more functional immobilization of biomolecules on the surface [26]–[28]. Toward this goal, Chiari et al. have introduced a novel polymeric coating that increases the immobilization density and functionality of the spotted proteins and nucleic acid probes [29], [30]. Yalc¸ın et al. demonstrated that the volume of this polymeric coating increases by swelling in aqueous environments, thus possessing a 3-D structure that mimics a solution-like state [31]. While a significant amount of research is performed to improve the microarray deposition methods, there are efforts to improve the detection systems as well. Label-free imaging methods are under investigation for more reliable and accurate detection of microarray experiments. B. Motivation for Label-Free Detection Traditional microarray imaging commonly employs tags (mainly, fluorescent molecules) that are attached to the captured analytes to create a detectable signal. The binding of target molecules to the immobilized probes are visualized by imaging the surface with a fluorescence scanner. Despite its popularity, it is generally acknowledged that this method has inherent drawbacks, and that for many, if not for most applications, label-free detection would be highly desirable. Even though fluorescence detection generally performs with superior sensitivity, there are applications for which label-free detection sensitivity is sufficient and label-free detection is required [16], [23], [32]–[34]. One of the drawbacks of labeled detection is that labels might modify the interaction affinities of the macromolecules to which they are attached. In some cases, special attention is paid to attach the label to a designated epitope on the protein in order to minimize the effect on binding kinetics. But, frequently, proteins are labeled nonspecifically, and the fluorescent labels may inter-
fere with the binding sites. This is a significant problem when studying small molecules and peptides, as the amino acids involved in binding are more likely to be affected. The effect is clear for larger molecules as well. A recent study by Sun et al. [35] shows how the properties of fairly simple and widely used proteins are affected by labeling. In this study, streptavidin– peptide and antibody–antigen reactions were monitored with a label-free technique, and it was observed that when the target is labeled with Cy3 molecules, the detected reaction kinetics change significantly. To avoid complications that result from directly labeling the target molecules, sandwich assays are often employed [16], [36]. After the first incubation with target molecules that are not labeled, the array is incubated with labeled secondary probe molecules that are also specific to the target. However, the second incubation should not interfere with the first one, namely, two different probes that are specific for two distinct nonoverlapping epitopes on the same target protein are required for the sandwich assay to work properly [37]. Otherwise, a competition reaction occurs that can create false-negative signals. Also, the secondary probe should not have any specificity for the spotted probes. If this is not satisfied, a false positive may occur when the secondary probes bind to spotted molecules without the initially captured target. In summary, the detection of analytes through secondary probes is intrinsically complex, as it requires multiple layers of interacting components that provide specificity without interfering with one another. Label-free measurements can provide kinetic information about the reactions, which is not possible with fluorescent techniques. Even though quantification of captured biological mass is possible in fluorescence measurements using on-chip calibration procedures [38], the bleaching of fluorescent molecules avoid real-time data acquisition to characterize the biomolecular interaction thermodynamics. Since the photoluminescence ability of dyes decay over time through excitation, a phenomenon called photobleaching occurs and prevents a linear response during long exposure times. Labeled detection methods are widely used because of their sensitivity and the historical lack of viable alternatives. However, depending on the application, the challenges they present can be significant and difficult to overcome when a quantitative measure of binding is required. In such cases, label-free detection has major advantages over the labeled detection methods. Besides the advantages listed in this section, label-free methods eliminate at least one chemical step from the assay process. Thus, they are time- and cost-efficient, and reduce experimental variability due to user error. C. Current and Emerging Biosensing Platforms Surface plasmon resonance (SPR) has been the leading technology among label-free detection methods. Traditional SPR has been limited to a macroformat with very limited throughput (four or eight channels). However, recent advancements in the technology show that higher throughput is, indeed, possible. Imaging SPR, presented by Shumaker–Parry and Campbell, is capable of imaging interactions for 120 features, in real time,
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with 3-pm height sensitivity [39], [40]. In contrast to regular SPR in imaging SPR, both the angle and the wavelength are fixed to the high linear region of system response. The reflection is imaged to a charge-coupled device (CCD) camera, and molecular accumulation on the surface is detected as an intensity change on the camera [41]. This technique is currently going through modifications and adjustments to improve the sensitivity and throughput further [42]. Campbell and Kim, in their review article, discuss a commercially available system that is capable of imaging >1300 spots with ∼3-pm noise floor for each spot, with a time resolution of 1 s. [43]. Although SPR has been quite successful, some of its shortcomings may be prohibitively difficult to overcome, depending on the application of interest. First, imaging SPR has a limited linear dynamic range. As a result, similar initial mass densities and surface uniformity have to be maintained for all the immobilized probes [44]. For the same reason, multiple layers of interactions are hard to monitor with the same sensitivity. Improvements are under investigation to increase the dynamic range of SPR microscopy [45]. Also, the requirement of gold surfaces increases the cost of substrates. Furthermore a change in the optical properties of the buffer affects the signal (known as “bulk” or “matrix” effect); therefore, buffer-dependent association/dissociation experiments may be problematic to carry out. The system temperature should also be well regulated during measurements, which requires welldesigned temperature-control systems to be used for long experiments, thus the instrumentation is further complicated. As an alternative to SPR, methods that employ interferometric, ellipsometric, and resonant characteristics of light have become popular for biosensing, as they offer simple and sensitive detection. Using molecular interferometric imaging (MI2) [46], [47] real-time detection on a 40-spot array was shown with a noise floor of 15–20 pm per spot. Another technique, the biomolecular interaction detector (BIND), has been commercialized by SRU Biosystems. Using BIND, very high sensitivities were achieved and small molecules (103 spots with 0.01-ng/mm2 mass sensitivity with the current system. The data acquisition takes less than 30 s; therefore, kinetic characterization of interactions is also possible. SRIB presents an alternative to current state-of-the-art assay technologies, with the added advantages of using an inexpensive substrate for probe immobilization, reduced dependence on bulk effects, large linear dynamic range, and quantitative binding determinations. REFERENCES [1] E. Ozkumur, J. W. Needham, D. A. Bergstein, R. Gonzalez, M. Cabodi, J. M. Gershoni, B. B. Goldberg, and M. S. Unlu, “Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications,” Proc. Nat. Acad. Sci. USA, 2008, vol. 105, no. 23, pp. 7988– 7992. [2] E. Ozkumur, A. Yalc¸in, M. Cretich, C. A. Lopez, D. A. Bergstein, B. B. ¨ u, “Quantification of DNA and protein Goldberg, M. Chiari, and M. S. Unl¨ adsorption by optical phase shift,” Biosens. Bioelectron., vol. 25, no. 1, pp. 167–172, 2009.
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¨ OZKUMUR et al.: SPECTRAL REFLECTANCE IMAGING
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¨ Emre Ozkumur (S’04) was born in Usak, Turkey, in 1982. He received the B.S. degrees in physics and electrical and electronics engineering from Koc University, Istanbul, Turkey, in 2004, and the M.S. and Ph.D. degrees in electrical and computer engineering from Boston University, Boston, MA, in 2007 and 2009, respectively. He is currently a Postdoctoral Researcher with Boston University. His research interests include label-free interferometric optical biosensors, and DNA and protein microarrays. ¨ Dr. Ozkumur is a member of the International Society for Optical Engineering and IEEE Lasers and Electro-Optics Society.
Carlos A. Lopez was born in Tuxtla Gutierrez, Mexico, in 1977. He received the B.S. degree in bioengineering: biotechnology from the University of California, San Diego, in 2001 and the Ph.D. degree in biomedical engineering from Boston University, Boston, MA, in 2007. He is currently a second-year Postdoctoral Research Associate with the Optical Characterization and Nanophotonics Laboratory, Boston University, where he is involved in multiplexed virus and cytokine detection capabilities of the SRIB platform. His research interests include work on cell-based and polymeric drug delivery methods and tissue engineering.
Ayc¸a Yalc¸ın (S’04) was born in Istanbul, Turkey, in 1982. She received the B.S. degree in electrical and electronics engineering from Bilkent University, Ankara, Turkey, in 2003, the M.S. degree in photonics, and the Ph.D. degree in electrical and computer engineering from Boston University, Boston, MA, in 2005 and 2009, respectively. She is currently a Postdoctoral Researcher with Boston University. Her research interests include label-free optical microresonator biosensors, interferometric techniques in fluorescence spectroscopy, and DNA and protein microarrays. Dr. Yalc¸ın is a member of the International Society for Optical Engineering and IEEE Lasers and Electro-Optics Society.
John H. Connor received the B.A. degree in chemistry from Swarthmore College, Swarthmore, PA, in 1994, and the Ph.D. degree in pharmacology from Duke University, in 1999. He was a Postdoctoral Fellow at Wake Forest University, Winston-Salem, NC. In 2006, he joined the faculty of Boston University School of Medicine, Boston, MA, where he is currently an Assistant Professor of microbiology. His research interests include understanding viral pathogenesis and the virus/host interface.
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IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 16, NO. 3, MAY/JUNE 2010
Marcella Chiari received a B.S. in chemistry and pharmaceutical techniques in 1982 and a Ph.D. degree in clinical biochemistry in 1990, both from the University of Milan, Italy. She is currently a Senior Research Scientist with the Italian National Research Council, Milan, Italy, where she leads the Analytical Microsystem Laboratory, Institute of Molecular Recognition. She has world-wide recognized experience in development of hydrophilic linear polymers. She is involved in the area of protein and DNA microarray research. Her research interests include the development of innovative polymeric coatings for glass slides. She has been engaged in development of a number of new hydrophilic acrylic monomers and polymers to be used in capillary electrophoresis as DNA sieving matrices and capillary coatings.
¨ u¨ (M’90–SM’95–F’07) received the M. Selim Unl B.S. degree from Middle East Technical University, Ankara, Turkey, in 1986, and the M.S.E.E. and Ph.D. degrees in electrical engineering from the University of Illinois, Urbana-Champaign, in 1988 and 1992, respectively. Since 1992, he has been with the Department of Electrical and Computer Engineering, Boston University, Boston, MA, where he is currently a Professor of electrical and computer engineering, biomedical engineering, and physics, an Associate Dean for research and graduate programs in engineering, as well as the Associate Director of Center for Nanoscience and Nanobiotechnology. His research laboratories— Optical Characterization and Nanophotonics (www.bu.edu/OCN)—are located in the Photonics Center. His current research interests include nanophotonics and biophotonics, research and development of photonic materials, semiconductor optoelectronic devices, high-resolution microscopy and spectroscopy of semiconductor and biological materials, and biological sensing and imaging. ¨ u was the Chair of IEEE Laser and Electro-Optics Society (LEOS), Dr. Unl¨ Boston Chapter, winning the LEOS Chapter-of-the-Year Award during 1994– 1995. He is an Associate Editor for IEEE JOURNAL OF QUANTUM ELECTRONICS and Vice President of LEOS for membership and regional activities – Americas. He has been selected as a LEOS Distinguished Lecturer for 2005– 2007 and Australian Research Council Nanotechnology Network Distinguished Lecturer for 2007. He was the recipient of the National Science Foundation Research Initiation Award in 1993, United Nations TOKTEN award in 1995 and 1996, and both the National Science Foundation CAREER and Office of Naval Research Young Investigator Awards in 1996. He was the former Chair of the IEEE/LEOS technical committee on Photodetectors and Imaging and currently, the Chair of IEEE/LEOS Nanophotonics committee.
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