IgG binding curves

Please see below for a selection of recent publication around the IRIS label-free target binding measurement technology and its applications. The result of this work is now implemented in the MX-100 system.

J. Needham, N. Lortlar Ünlü, and M. S. Ünlü, “Interferometric Reflectance Imaging Sensor (IRIS) for Molecular Kinetics with a Low-Cost, Disposable Fluidic Cartridge,” Biomimetic Sensors, Springer Methods in Molecular Biology, 2019

The determination of kinetic information and appropriate binding pairs is fundamental to the proper optimization and selection of ligands used in immunoassays, diagnostics, and therapeutics. However, the ability to estimate such parameters in a multiplexed and inexpensive format remains difficult and modification of the ligand is often necessary. Here, we detail the methods and materials necessary to evaluate hundreds of unlabeled ligands simultaneously using the interferometric reflectance imaging sensor (IRIS). The incorporation of a low-cost fluidic cartridge that integrates on the top of the sensor simplifies reagent handling considerably.

AY Ozkumur, FE Kanik, JT Trueb, C Yurdakul, and MS Ünlü, “Interferometric Detection and Enumeration of Viral Particles using Si-Based Microfluidics,” IEEE Journal of Selected Topics in Quantum Electronics 25 (1), 1-7 2018

Single-particle interferometric reflectance imaging sensor enables optical visualization and characterization of individual nanoparticles without any labels. Using this technique, we have shown end-point and real-time detection of viral particles using laminate-based active and passive cartridge configurations. Here, we present a new concept for low-cost microfluidic integration of the sensor chips into compact cartridges through utilization of readily available silicon fabrication technologies. This new cartridge configuration will allow simultaneous detection of individual virus binding events on a 9-spot microarray, and provide the needed simplicity and robustness for routine real-time operation for discrete detection of viral particles in a multiplex format.

M. S. Ünlü, "Interferometric Reflectance Imaging Sensor using Si-based Microfluidics," OSA Advanced Photonics 2018

There is a need for biological sensing and diagnostics tools with sensitivity compared to existing state-of-the-art technologies without complicated assays, sample preparation, and bulky equipment. Our platform technology, IRIS offers kinetic analysis of biomolecular binding and detection of proteins, nucleic acids, and individual biological nanoparticles in a simple assay format and with high sensitivity. We have shown that low-cost and disposable sensor chips and microfluidic cartridges compatible with this optical sensing technology can be manufactured using standard Si processing techniques

U. Aygun, O. Avci, E .Seymour, H. Urey, M. S. Ünlü, A. Yalcin Ozkumur, “Label-Free and High-Throughput Detection of Biomolecular Interactions Using a Flatbed Scanner Biosensor,” ACS sensors, Vol. 2 (10), pp. 1424-1429, 2017

Fluorescence based microarray detection systems provide sensitive measurements; however, variation of probe immobilization and poor repeatability negatively affect the final readout, and thus quantification capability of these systems. Here, we demonstrate a label-free and high-throughput optical biosensor that can be utilized for calibration of fluorescence microarrays. The sensor employs a commercial flatbed scanner, and we demonstrate transformation of this low cost (∼100 USD) system into an Interferometric Reflectance Imaging Sensor through hardware and software modifications. Using this sensor, we report detection of DNA hybridization and DNA directed antibody immobilization on label-free microarrays with a noise floor of ∼30 pg/mm2, and a scan speed of 5 s (50 s for 10 frames averaged) for a 2 mm × 2 mm area. This novel system may be used as a standalone label-free sensor especially in low-resource settings, as well as for quality control and calibration of microarrays in existing fluorescence-based DNA and protein detection platforms.

O. Avci, N. Lortlar Ünlü, A. Yalcin, and M. S. Ünlü, ”Interferometric Reflectance Imaging Sensor (IRIS)—A Platform Technology for Multiplexed Diagnostics and Digital Detection,” Sensors, Vol. 15 (7), pp. 17649-17665, 2015

Over the last decade, the growing need in disease diagnostics has stimulated rapid development of new technologies with unprecedented capabilities. Recent emerging infectious diseases and epidemics have revealed the shortcomings of existing diagnostics tools, and the necessity for further improvements. Optical biosensors can lay the foundations for future generation diagnostics by providing means to detect biomarkers in a highly sensitive, specific, quantitative and multiplexed fashion. Here, we review an optical sensing technology, Interferometric Reflectance Imaging Sensor (IRIS), and the relevant features of this multifunctional platform for quantitative, label-free and dynamic detection. We discuss two distinct modalities for IRIS: (i) low-magnification (ensemble biomolecular mass measurements) and (ii) high-magnification (digital detection of individual nanoparticles) along with their applications, including label-free detection of multiplexed protein chips, measurement of single nucleotide polymorphism, quantification of transcription factor DNA binding, and high sensitivity digital sensing and characterization of nanoparticles and viruses

C. Yu et al., “A High-Throughput Method to Examine Protein-Nucleotide Interactions Identifies Targets of the Bacterial Transcriptional Regulatory Protein Fur,” PloS ONE, Vol. 9(5), e96832, 2014

The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

X. X. Cheng, G. G. Daaboul, M. S. Ünlü, and K. Kerman, “LED-based interferometric reflectance imaging sensor for the detection of amyloid-b aggregation,” Analyst, Vol. 139, pp. 59-65, 2014

Self-aggregation of amyloid-β (Aβ) plays an important role in the pathogenesis of Alzheimer's disease (AD). Small molecule inhibitors of Aβ fibril formation reduce the Aβ-mediated neurotocixity. In this report, the interaction of amyloid-β (Aβ) with well-described modulators, (-)epigallocatechin-3-gallate (EGCG) and Zn(ii), was detected using a LED-based interferometric reflectance imaging sensor (LED-IRIS) in a high-throughput and real-time format. Nucleation-based fibril growth strategy was employed, as the "seeds" of Aβ were prepared in the presence of EGCG and Zn(ii). The seeds were then covalently immobilized on the chip surface. Using microfluidics, Aβ oligomers were exposed onto the seeds resulting in the elongation of fibrils, which was detected as the increase in the spot height. Monitoring the changes on the chip surface enabled to detect the efficacy of modulators to inhibit or facilitate the growth of Aβ fibrils. The proof-of-concept study reported here introduces a novel platform to facilitate the screening of small molecules towards the discovery of promising AD therapeutics.

S. Ahn, D. S. Freedman, P. Massari, M. Cabodi, M. S.Ünlü, “A Mass-Tagging Approach for Enhanced Sensitivity of Dynamic Cytokine Detection Using a Label-Free Biosensor,” Langmuir, Vol. 29 (17), pp. 5369-5376, 2013

Monitoring cytokine release by cells allows the investigation of cellular response to specific external stimuli, such as pathogens or candidate drugs. Unlike conventional colorimetric techniques, label-free detection of cytokines enables studying cellular secretions in real time by eliminating additional wash and labeling steps after the binding step. However, label-free techniques that are based on measuring mass accumulation on a sensor surface are challenging for measuring small cytokines binding to much larger capture agents (usually antibodies) because the relative signal change is small. This problem is exacerbated when the capturing antibodies desorb from the surface, a phenomenon that almost inevitably occurs in immunoassays but is rarely accounted for. Here, we demonstrate a quantitative dynamic detection of interleukine-6 (IL-6), a pro-inflammatory cytokine, using an interferometric reflectance imaging sensor (IRIS). We improved the accuracy of the quantitative analysis of this relatively small protein (21 kDa) by characterizing the antibody desorption rate and compensating for the antibody loss during the binding experiment. By correcting for protein desorption, we achieved an analytical limit of detection at 19 ng/mL IL-6 concentration. We enhanced the sensitivity by 7-fold by using detection antibodies that recognize a different epitope of the cytokine. We demonstrate that these detection antibodies, which we call “mass tags”, can be used concurrently with the target analyte to eliminate an additional wash and binding step. Finally, we report successful label-free detection of IL-6 in cell culture medium (with 10% serum) with comparable signal to that obtained in PBS. This work is the first to report quantitative dynamic label-free detection of small protein in a complex biological fluid using IRIS.

M. Cretich et al., “Interferometric silicon biochips for label and label-free DNA and protein microarrays,” Proteomics, Vol. 12, pp. 2963-2977, 2013

Protein and DNA microarrays hold the promise to revolutionize the field of molecular diagnostics. Traditional microarray applications employ labeled detection strategies based on the use of fluorescent and chemiluminescent secondary antibodies. However, the development of high throughput, sensitive, label-free detection techniques is attracting attention as they do not require labeled reactants and provide quantitative information on binding kinetics. In this article, we will provide an overview of the recent author's work in label and label-free sensing platforms employing silicon/silicon oxide (Si/SiO(2)) substrates for interferometric and/or fluorescence detection of microarrays. The review will focus on applications of Si/SiO(2) with controlled oxide layers to (i) enhance the fluorescence intensity by optical interferences, (ii) quantify with sub-nanometer accuracy the axial locations of fluorophore-labeled probes tethered to the surface, and (iii) detect protein-protein interactions label free. Different methods of biofunctionalization of the sensing surface will be discussed. In particular, organosilanization reactions for monodimensional coatings and polymeric coatings will be extensively reviewed. Finally, the importance of calibration of protein microarrays through the dual use of labeled and label-free detection schemes on the same chip will be illustrated.

S. Ahn et al., “Quantification of surface etching by common buffers and implications on the accuracy of label-free biological assays,” Biosensors and Bioelectronics, Vol. 36(1), pp. 222–229, 2012

High throughput analyses in biochemical assays are gaining popularity in the post-genomic era. Multiple label-free detection methods are especially of interest, as they allow quantitative monitoring of biomolecular interactions. It is assumed that the sensor surface is stable to the surrounding medium while the biochemical processes are taking place. Using the Interferometric Reflectance Imaging Sensor (IRIS), we found that buffers commonly used in biochemical reactions can remove silicon dioxide, a material frequently used as the solid support in the microarray industry. Here, we report 53 pm to 731 pm etching of the surface silicon oxide over a 12-h period for several different buffers, including various concentrations of SSC, SSPE, PBS, TRIS, MES, sodium phosphate, and potassium phosphate buffers, and found that PBS and MES buffers are much more benign than the others. We observe a linear dependence of the etch depth over time, and we find the etch rate of silicon dioxide in different buffers that ranges from 2.73±0.76 pm/h in 1M NaCl to 43.54±2.95 pm/h in 6×SSC. The protective effects by chemical modifications of the surface are explored. We demonstrate unaccounted glass etching leading to erroneous results with label-free detection of DNA microarrays, and offer remedies to increase the accuracy of quantitative analysis.

S. Ahn et al., “TATA binding proteins can recognize nontraditional DNA sequences,” Biophysical Journal, Vol. 103,   pp. 1510-1517, 2012

We demonstrate an accurate, quantitative, and label-free optical technology for high-throughput studies of receptor-ligand interactions, and apply it to TATA binding protein (TBP) interactions with oligonucleotides. We present a simple method to prepare single-stranded and double-stranded DNA microarrays with comparable surface density, ensuring an accurate comparison of TBP activity with both types of DNA. In particular, we find that TBP binds tightly to single-stranded DNA, especially to stretches of polythymine (poly-T), as well as to the traditional TATA box. We further investigate the correlation of TBP activity with various lengths of DNA and find that the number of TBPs bound to DNA increases >7-fold as the oligomer length increases from 9 to 40. Finally, we perform a full human genome analysis and discover that 35.5% of human promoters have poly-T stretches. In summary, we report, for the first time to our knowledge, the activity of TBP with poly-T stretches by presenting an elegant stepwise analysis of multiple techniques: discovery by a novel quantitative detection of microarrays, confirmation by a traditional gel electrophoresis, and a full genome prediction with computational analyses.

M. R. Monroe et al., “Multiplexed method to calibrate and quantitate fluorescence signal for allergen-specific IgE,”  Anal. Chem., 83 (24), pp 9485–9491, 2011

Using a microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of serum. However, variation of probe immobilization on microarrays hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics. To address this problem, we have developed a calibrated, inexpensive, multiplexed, and rapid protein microarray method that directly correlates surface probe density to captured labeled secondary antibody in clinical samples. We have identified three major technological advantages of our calibrated fluorescence enhancement (CaFE) technique: (i) a significant increase in fluorescence emission over a broad range of fluorophores on a layered substrate optimized specifically for fluorescence; (ii) a method to perform label-free quantification of the probes in each spot while maintaining fluorescence enhancement for a particular fluorophore; and (iii) a calibrated, quantitative technique that combines fluorescence and label-free modalities to accurately measure probe density and bound target for a variety of antibody–antigen pairs. In this paper, we establish the effectiveness of the CaFE method by presenting the strong linear dependence of the amount of bound protein to the resulting fluorescence signal of secondary antibody for IgG, β-lactoglobulin, and allergen-specific IgEs to Ara h 1 (peanut major allergen) and Phl p 1 (timothy grass major allergen) in human serum.

G. G. Daaboul et al., “LED-based Interferometric Reflectance Imaging Sensor for quantitative dynamic monitoring of biomolecular interactions,” Biosensors and Bioelectronics, Vol. 26, pp. 2221-2227, 2011

Since 1992, when the room temperature ionic liquids (ILs) based on the 1-alkyl-3-methylimidazolium cation were reported to provide an attractive combination of an electrochemical solvent and electrolyte, ILs have been widely used in electrodeposition, electrosynthesis, electrocatalysis, electrochemical capacitor, and lithium batteries. However, it has only been in the last few years that electrochemical biosensors based on carbon ionic liquid electrodes (CILEs) and IL-modified macrodisk electrodes have been reported. However, there are still a lot of challenges in achieving IL-based sensitive, selective, and reproducible biosensors for high speed analysis of biological and environmental compounds of interest. This review discusses the principles of operation of electrochemical biosensors based on CILEs and IL/composite-modified macrodisk electrodes. Subsequently, recent developments and major strategies for enhancing sensing performance are discussed. Key challenges and opportunities of IL-based biosensors to further development and use are considered. Emphasis is given to direct electron-transfer reaction and electrocatalysis of hemeproteins and enzyme-modified composite electrodes.

C. Pereira et al., “Synergetic Chemiluminescence and Label-Free Dual Detection for Developing a Hepatitis Protein Array,” Journal of Immunological Methods, Vol. 371, No. 1-2, pp. 159-164, 2011

A dual detection system for protein arrays is presented that combines label-free detection by optical interference with chemiluminescence. A planar protein array that targets hepatitis B surface antigen is developed. Surface densities for individual antibody spots are quantitated using optical interference prior to use. Target binding (10 ng/ml) is detected label-free. Target binding (1 ng/ml) is detected by both optical interference and chemiluminescence with the inclusion of secondary antibodies. Binding results using both methods are found to be directly proportion to the capture probe density measured initially. The dual detection system provides the analytical utility of optical interference detection with the established clinical utility of chemiluminescence detection.

M. Cretich et al., “Silicon biochips for dual label-free and fluorescence detection: Application to protein microarray development,” Biosensors and Bioelectronics, Vol. 26 (9), pp. 3938-3943, 2011.

A new silicon chip for protein microarray development, fabrication and validation is proposed. The chip is made of two areas with oxide layers of different thicknesses: an area with a 500 nm SiO2 layer dedicated to interferometric label-free detection and quantification of proteins and an area with 100 nm SiO2 providing enhanced fluorescence. The chip allows, within a single experiment performed on the same surface, label-free imaging of arrayed protein probes coupled with high sensitivity fluorescence detection of the molecular interaction counterparts. Such a combined chip is of high practical utility during assay development process to image arrays, check consistency and quality of the protein array, quantify the amount of immobilized probes and finally detect fluorescence of bioassays.

C. A. Lopez et al., “Label-free, multiplexed virus detection using spectral reflectance imaging,” Biosensors and Bioelectronics, Vol. 26 (8), Pages 3432-3437, 2011

We demonstrate detection of whole viruses and viral proteins with a new label-free platform based on spectral reflectance imaging. The Interferometric Reflectance Imaging Sensor (IRIS) has been shown to be capable of sensitive protein and DNA detection in a real time and high-throughput format. Vesicular stomatitis virus (VSV) was used as the target for detection as it is well-characterized for protein composition and can be modified to express viral coat proteins from other dangerous, highly pathogenic agents for surrogate detection while remaining a biosafety level 2 agent. We demonstrate specific detection of intact VSV virions achieved with surface-immobilized antibodies acting as capture probes which is confirmed using fluorescence imaging. The limit of detection is confirmed down to 3.5×105plaque-forming units/mL (PFUs/mL). To increase specificity in a clinical scenario, both the external glycoprotein and internal viral proteins were simultaneously detected with the same antibody arrays with detergent-disrupted purified VSV and infected cell lysate solutions. Our results show sensitive and specific virus detection with a simple surface chemistry and minimal sample preparation on a quantitative label-free interferometric platform.

R. S. Vedula et al., “Self-Referencing Substrates for Optical Interferometric Biosensors,” Journal of Modern Optics,  Vol. 57(16), pp. 1564–1569, 2010

Optical interference is a powerful technique for monitoring surface topography or refractive index changes in a thin film layer. Reflectance spectroscopy provides label-free biosensing capability by monitoring small variations in interference signature resulting from optical path length changes from surface-adsorbed biomolecules. Spectral reflectance data can be acquired either by broad wavelength illumination and spectroscopy at a single point, thus necessitating scanning, or by varying the wavelength of illumination and imaging the reflected intensity allowing for acquisition of a spectral image of a large field of view simultaneously. In imaging modalities, intensity fluctuations of the illuminating light source couple into the detected signal, increasing the noise in measured surface profiles. This article introduces a simple technique for eliminating the effects of illumination light power fluctuations by fabricating on-substrate self-reference regions to measure and normalize for the incident intensity, simplifying the overall platform for reflection or transmission-based imaging biosensors. Experimental results demonstrate that the sensitivity performance using self-referencing is equivalent or better than an optimized system with an external reference.

M. Cretich et al., “Allergen microarrays on high-sensitivity silicon slides,” Analytical and Bioanalytical Chemistry,      DOI 10.1007/s00216-0, 2010

We have recently introduced a silicon substrate for high-sensitivity microarrays, coated with a functional polymer named copoly(DMA-NAS-MAPS). The silicon dioxide thickness has been optimized to produce a fluorescence intensification due to the optical constructive interference between the incident and reflected lights of the fluorescent radiation. The polymeric coating efficiently suppresses aspecific interaction, making the low background a distinctive feature of these slides. Here, we used the new silicon microarray substrate for allergy diagnosis, in the detection of specific IgE in serum samples of subjects with sensitizations to inhalant allergens. We compared the performance of silicon versus glass substrates. Reproducibility data were measured. Moreover, receiver-operating characteristic (ROC) curves were plotted to discriminate between the allergy and no allergy status in 30 well-characterized serum samples. We found that reproducibility of the microarray on glass supports was not different from available data on allergen arrays, whereas the reproducibility on the silicon substrate was consistently better than on glass. Moreover, silicon significantly enhanced the performance of the allergen microarray as compared to glass in accurately identifying allergic patients spanning a wide range of specific IgE titers to the considered allergens.

E. Ozkumur et al., “Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches,” Biosensors and Bioelectronics, Vol. 25 (7), pp. 1789-1795, 2010

A novel method is proposed for direct detection of DNA hybridization on microarrays. Optical interferometry is used for label-free sensing of biomolecular accumulation on glass surfaces, enabling dynamic detection of interactions. Capabilities of the presented method are demonstrated by high-throughput sensing of solid-phase hybridization of oligonucleotides. Hybridization of surface immobilized probes with 20 base pair-long target oligonucleotides was detected by comparing the label-free microarray images taken before and after hybridization. Through dynamic data acquisition during denaturation by washing the sample with low ionic concentration buffer, melting of duplexes with a single-nucleotide mismatch was distinguished from perfectly matching duplexes with high confidence interval (>97%). The presented technique is simple, robust, and accurate, and eliminates the need of using labels or secondary reagents to monitor the oligonucleotide hybridization.

E. Ozkumur et al., “Spectral reflectance imaging for a multiplexed, high-throughput, label-free, and dynamic biosensing platform,” IEEE J. Select. Topics in Quantum Electron., Biophotonics, Vol. 16(3),  pp. 635-46, 2010

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.

E. Ozkumur et al., “Quantification of DNA and protein adsorption by optical phase shift,” Biosensors and Bioelectronics, Vol. 25, pp. 167-172, 2009

A primary advantage of label-free detection methods over fluorescent measurements is its quantitative detection capability, since an absolute measure of adsorbed material facilitates kinetic characterization of biomolecular interactions. Interferometric techniques relate the optical phase to biomolecular layer density on the surface, but the conversion factor has not previously been accurately determined. We present a calibration method for phase shift measurements and apply it to surface-bound bovine serum albumin, immunoglobulin G, and single-stranded DNA.

Biomolecules with known concentrations dissolved in salt-free water were spotted with precise volumes on the array surface and upon evaporation of the water, left a readily calculated mass. Using our label-free technique, the calculated mass of the biolayer was compared with the measured thickness, and we observed a linear dependence over 4 orders of magnitude. We determined that the widely accepted conversion of 1 nm of thickness corresponds to ~1 ng/mm2 surface density held reasonably well for these substances and through our experiments can now be further specified for different types of biomolecules. Through accurate calibration of the dependence of thickness on surface density, we have established a relation allowing precise determination of the absolute number of molecules for single-stranded DNA and two different proteins.

E. Özkumur et al., “Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications" PNAS, Vol. 105, pp. 7988-7992, 2008

Direct monitoring of primary molecular-binding interactions without the need for secondary reactants would markedly simplify and expand applications of high-throughput label-free detection methods. A simple interferometric technique is presented that monitors the optical phase difference resulting from accumulated biomolecular mass. As an example, 50 spots for each of four proteins consisting of BSA, human serum albumin, rabbit IgG, and protein G were dynamically monitored as they captured corresponding antibodies. Dynamic measurements were made at 26 pg/mm2 SD per spot and with a detectable concentration of 19 ng/ml. The presented method is particularly relevant for protein microarray analysis because it is label-free, simple, sensitive, and easily scales to high-throughput.


"Label-free technologies have been used throughout drug discovery for the detection and characterisation of molecular interactions. The implementation of automated systems, increased sensitivity and the development of new technologies have recently broadened the use of label-free methods from primary screening through to mechanism-of-action studies in lead optimisation" - Philip Rawlins, Drug Discovery World.

"The real-time analysis of biomolecular interactions (e.g. protein:protein, protein:peptide, and protein:small molecule and fragments) are of high importance to researchers across many scientific disciplines. Biomolecular interaction analysis facilitates the understanding of binding mechanisms and kinetics. This information is critical to both clone selection and characterization in the development and production of antibodies and biotherapeutic proteins. It is also a significant component in understanding the affinity of small molecule binding to a therapeutic target during lead optimization" - Technology Networks.