Advection‐Enhanced Kinetics in Microtiter Plates for Improved Surface Assay Quantitation and Multiplexing Capabilities

Abstract

Surface assays such as ELISA are pervasive in clinics and research and predominantly standardized in microtiter plates (MTP). MTPs provide many advantages but are often detrimental to surface assay efficiency due to inherent mass transport limitations. Microscale flows can overcome these and largely improve assay kinetics. However, the disruptive nature of microfluidics with existing labware and protocols has narrowed its transformative potential. We present WellProbe, a novel microfluidic concept compatible with MTPs. With it, we show that immunoassays become more sensitive at low concentrations (up to 9× signal improvement in 12× less time), richer in information with 3–4 different kinetic conditions, and can be used to estimate kinetic parameters, minimize washing steps and non‐specific binding, and identify compromised results. We further multiplex single‐well assays combining WellProbe's kinetic regions with tailored microarrays. Finally, we demonstrate our system in a context of immunoglobulin subclass evaluation, increasingly regarded as clinically relevant.

Microscale hydrodynamic confinements: shaping liquids across length scales as a toolbox in life sciences

“…Some further applications include using parallel HFCs for diffusion based separation of surface bound species, 126 rapid sequential micro-immunohistochemistry 127 and multiplex analysis and quantitation in surface assays. 128…”

Ultrafast Electrothermal Flow‐Enhanced Magneto Biosensor for Highly Sensitive Protein Detection in Whole Blood

“…The ability to achieve rapid protein detection with high analytical sensitivity in whole blood is hampered by inefficient mass transport and slow protein binding kinetics in the complex liquid matrix [15] . Various techniques have been demonstrated to enhance mass transport and kinetics in surface binding assays, such as the use of microfluidic flows to confine the sample to the sensor surface [16] or continuously refresh the sensor with fresh analyte [17] . While these methods are capable of increasing the analytical sensitivity and reducing the assay time, they require complicated fluidic systems or result in increased sample/reagent consumption.…”

“…[15] Various techniques have been demonstrated to enhance mass transport and kinetics in surface binding assays, such as the use of microfluidic flows to confine the sample to the sensor surface [16] or continuously refresh the sensor with fresh analyte. [17] While these methods are capable of increasing the analytical sensitivity and reducing the assay time, they require complicated fluidic systems or result in increased sample/reagent consumption. Alternatively, direct current (DC) electrokinetics [18] or alternating current (AC) electrokinetics [19][20][21] has been shown to be an effective technique for manipulating and separating biomolecules in small volume samples.…”

Efficient Pathogen Capture and Sensing Promoted by Dynamic Deformable Nanointerfaces

“…Surface assay such as enzyme linked immunosorbent assay and solid‐phase extraction are pervasive in clinical diagnostics, environmental monitoring, drug screening, etc. [ 1 ] The efficient interaction between ligand on solid surface and analyte in liquid phase is the first step to facilitate the successive recognition, adsorption of analyte, or signal transduction. The surface kinetics govern the first step, which is mainly limited by either mass transport or the reaction rate.…”