Integrated self-calibrationvia electrokinetic solvent proportioning for microfluidic immunoassays

Abstract

On-board generation of a set of calibration standards was demonstrated within a microfluidic device designed to perform immunoassay. Electrokinetic flow was used to proportionally mix the antibody (Ab) to bovine serum albumin (BSA) and a diluting buffer, to provide varying Ab concentrations for downstream mixing with fluorescently labeled BSA (BSA*). Mixing ratios were determined from electrical impedance modeling of the fluidic network using P-SPICE software, and peak heights for the labeled species were analyzed relative to the concentration calculated from the model. For dilution and separation of fluorescently labeled amino acids, a linear calibration curve was obtained for mixing ratios of 0.118 to 7.46. A linear calibration curve was obtained for the immunoassay calibration using dilution ratios between 0.197 and 5.077. Deviations were observed at larger extremes, possibly due to leakage effects at intersections. Peak height reproducibility was +/- 3% for the immunoassay, using diluted monoclonal Ab in mouse ascites fluid as the analyte. Recovery for on-chip calibration was 92 +/- 6% versus calibrants prepared off-chip, indicating a small bias.

Microfluidic chips for clinical and forensic analysis

“…The integration of the immunoassay separation step into a CE-based analysis rather than onto a surface circumvents the need for the multiple fluid handling steps required in heterogeneous, immunosorbent assays. Electrokinetic fluid handling also permits the generation of on-chip calibration standards through dilution of a stock solution with buffer at a T-junction [137]. Detection limits achieved using fluorescence can be very low, if proper consideration is given to optimization of the assay.…”

Online Preconcentration by Transient Isotachophoresis in Linear Polymer on a Poly(methyl methacrylate) Microchip for Separation of Human Serum Albumin Immunoassay Mixtures

“…10 Therefore, the reported applications of free solution electrophoresis in MCEIA of protein samples, unlike for small molecules, suffer from low resolution and low reproducibility. [11][12][13][14][15][16][17][18] The low resolution and reproducibility will, in turn, raise the detection limit (DL) in MCEIA. 19 To improve the resolution and increase the reproducibility of separation of proteins from their immunocomplexes, different strategies have been tried.…”

Recent advances in microfluidic technologies for biochemistry and molecular biology

“…Homogeneous immunoassays have been demonstrated in a number of studies. For example, Harrison and co-workers pioneered the development of microfluidic devices based on capillary electrophoresis (50,51). This approach allowed integration of 6 functional electrophoresis manifolds on a single chip and achieved simultaneous quantification of anti-estradiol and ovalbumin in less than 1 minute (50).…”