Quantification of protein interaction kinetics in a micro droplet

Abstract

Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip. C 2015 AIP Publishing LLC. [http://dx

Label-Free Immunoassay Using Droplet-Based Brewster’s Angle Straddle Interferometry

“…This phenomenon is discussed extensively in previous work where models of the binding curves under conditions that do not satisfy the premises of a simple Langmuir model are developed. 8,23 As can be seen in the fit of Figure 6, the effect is that one still observes Langmuir-like binding curves but where the fitted binding constant K d(fit) substantially overestimates the actual K d(ref) . Indeed, this is what we observe in Figure 6, where a fit to a simple Langmuir model is not too bad, but the estimate K d(fit) of 146 nM for human IgG−antihuman IgG and 164 nM for rabbit IgG−anti-rabbit IgG are around 4 times the literature values.…”

“…Optical detection of unlabeled target binding using surface plasmon resonance (SPR) is an approach that ameliorates some of these problems by using direct detection of unlabeled target molecules . Typically, SPR is performed on single targets in a fluidic environment with relatively complex optics, although it has been adapted with some compromise in sensitivity to use in imaging formats and made to work under static exposure to analytes without flow . While having helped to bring about many breakthroughs in clinical diagnostics and drug discovery, the complexity of ELISA and SPR has prevented them from making significant inroads into portable, point-of-care diagnostics or field testing applications. , Lateral flow assays have addressed some of the need for portable testing but are generally qualitative, are not easily adapted to multiplexing, and in many cases have considerably lower sensitivity.…”

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“…Although evaporation is a major concern for such systems, it can also be turned into a benefit. Controlled gradual evaporation of the deposited droplets yields an increase in analyte concentration that allows analysis of the encapsulated material inside droplets for obtaining variation in concentration, interaction kinetics, conductivity and osmotic pressure [20][21][22][23][24][25][26].…”

“…For dispensed droplet platforms, Yin and Tao et al used surface plasmon resonance sensing using the common prism-coupling configuration [59]. A droplet of the solution of interest was pipetted on the resonance sensing surface and protein interaction kinetics were studied.…”

Microfluidic Surface Plasmon Resonance Sensors: From Principles to Point-of-Care Applications

“…This benefit arises particularly when the analyte is a precious protein. Yin et al [ 65 ] demonstrated the SPR measurement of IgG and anti-IgG binding kinetics using a microdroplet of sub-μL volume. The amount of IgG in the sample volume was only 0.04 µg, 1/7000 times of the sample for a conventional flow-through SPR sensor.…”