Microchip immunoaffinity electrophoresis of antibody–thymidine kinase 1 complex

Abstract

Thymidine kinase-1 (TK1) is an important cancer biomarker whose serum levels are elevated in early cancer development. We developed a microchip electrophoresis immunoaffinity assay to measure recombinant purified TK1 (pTK1) using an antibody that binds to human TK1. We fabricated poly(methyl methacrylate) microfluidic devices to test the feasibility of detecting antibody (Ab)-pTK1 immune complexes as a step towards TK1 analysis in clinical serum samples. We were able to separate immune complexes from unbound antibodies using 0.5X phosphate buffer saline (pH 7.4) containing 0.01% Tween-20, with 1% w/v methylcellulose that acts as a dynamic surface coating and sieving matrix. Separation of the antibody and Ab-pTK1 complex was observed within a 5 mm effective separation length. This method of detecting pTK1 is easy to perform, requires only a 10 μL sample volume, and takes just 1 minute for separation.

Pressure-actuated microfluidic devices for electrophoretic separation of pre-term birth biomarkers

“…Laser-induced fluorescence detection was performed in a system with 488 nm laser excitation (JDSU, Shenzhen, China), and a photomultiplier tube (PMT; Hamamatsu, Japan), for point detection or a CCD camera (CoolSnap HQ; Photometrics, Tucson, AZ) for plug capture imaging. [6,15] The on-chip pump and valves were actuated by external pressure applied through a 10 valve solenoid manifold (Clippard Instrument, Cincinnati, OH) controlled with LabView software (National Instruments, Austin, TX) through a home-built electronic circuit. Plastic tubing was filled with the colored dye solution and on-chip connections were made thereafter.…”

“…[14] Most studies have used electrokinetic injection for reasons such as device fabrication ease, automated and simple operation, and established theoretical models. [11,15–17] However, electrokinetic injection also has several downsides: it is ineffective with either low- or high-conductivity samples, analyte loading is biased by electrophoretic mobilities for short injection times (tens of seconds, depending on device layout and analytes), and it becomes increasingly difficult to implement effectively as integrated designs become more complex. [11,14,18]…”

Safe and cost-effective rapid-prototyping of multilayer PMMA microfluidic devices

“…Due to its low cost, optical transparency and the availability of a range of processing methods, poly(methyl methacrylate) (PMMA) has been adopted for many microfluidic applications (Das et al 2015;Pagaduan et al 2015). Compared to silicon-based materials used for microfluidic applications, PMMA is less fragile and does not require expensive manufacturing techniques.…”

Recent advances in microfluidic sample preparation and separation techniques for molecular biomarker analysis: A critical review

“…Although this work utilized a novel signal generating mechanism, further improvements in analyte concentration are needed for potential clinical applications. Pagaduan et al [55] reported a µCE device for determination of thymidine kinase 1 (TK1), a cancer biomarker. A microchip immunoaffinity assay was reported for measuring Ab-TK1 complex after separating it from the unbound mAb.…”