Abstract
A microfluidic assay for the detection of promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) fusion protein was developed. This microfluidic-based system can be used for rapid personalized differential diagnosis of acute promyelocyte leukemia (APL) with the aim of early initiation of individualized therapy. The fusion protein PML-RARα occurs in 95% of acute promyelocytic leukemia cases and is considered as diagnostically relevant. The fusion protein is formed as a result of translocation t(15,17) and is detected in the laboratory by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). Diagnostic methods require many laboratory steps with specialized staff. The developed microfluidic assay includes a sandwich enzyme-linked immunosorbent assay (ELISA) system for PML-RARα on surface of magnetic microparticles in a microfluidic chip. A rapid detection of PML-RARα in cell lysates is achieved in less than one hour. A biotinylated PML-antibody on the surface of magnetic streptavidin coated microparticles is used as capture antibody. The bound translocation product is detected by a RARα antibody conjugated with horseradish peroxidase and the substrate QuantaRed. The analysis is performed in microfluidic channels which involves automated liquid processing with stringent washing and short incubation times. The results of the developed assay show that cell lysates of PML-RARα-positive cells (NB-4) can be clearly distinguished from PML-RARα-negative cells (HL-60, MV4-11).
Highlights
In microfluidics, liquids are processed in structures in the submillimeter range [1,2]
We have developed a method to detect the fusion protein promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) in cell lysates by a sandwich enzyme-linked immunosorbent assay (ELISA) on the surface of magnetic particles in a microfluidic chip in less than one hour
The developed bead-based sandwich ELISA uses anti-PML and anti-RARα antibodies against regions of PML-RARα which occur in all forms of PML-RARα fusion proteins
Summary
Liquids are processed in structures in the submillimeter range [1,2]. The behavior of liquids in microfluidics differs fundamentally from the behavior in the macroscopic range, because frictional forces dominate inertial forces, resulting in laminar flows and avoiding turbulent flows [2]. Small quantities of liquids can be moved against the force of gravity [2]. This technology is used in diagnostics and biological research [3]. An increased surface-to-volume ratio increases molecular diffusion and heat transfer, whereas the molecular reaction remains unchanged [4]. This results in faster and more sensitive diagnostics [4]
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