Internally calibrated quantification of protein analytes in human serum by fluorescence immunoassays in disposable elastomeric microfluidic devices

Abstract

Herein we report on reliable reproducible quantification of protein analytes in human serum by fluorescence sandwich immunoassays in disposable PDMS microfluidic chips. The system requires 1,000 times less sample than typical clinical blood tests and is specifically shown to measure ferritin down to 250 pM in human serum. The in-built calibration method of spiking the serum with known concentrations of commercially available antigen avoids common sources of error and improves the reliability of the test results. The reported microfluidic system is an important new tool for fundamental scientific research, offering sensitive immunoassay measurements in small but complex biosamples. The system is also a further step towards comprehensive affordable "point-of-care" biomedical diagnostics.

Figure 1

(A) Microfluidic immunoassay chip. A 60-chamber PDMS chip bound to a 1-in.-wide epoxide slide was used for the experiments. The vertical cylinders are input ports for reagents and control pressure. The microchannel test matrix is visible in the middle. (B) Architectural diagram of the entire chip. Control channels (red) convey pressure to open and close microvalves, which steer reagents along flow channels (blue). Each intersection of flow channels in the central test matrix forms a microchamber where an immunoassay is constructed. Monoclonal antibodies flowing from inputs D1–5 to exhausts DE1–5 bind to the epoxide coating of the microchannel floor. Buffer flushes from input DB to exhausts DE1–5 and from input SB to exhausts SE1–6 passivate remaining epoxide groups. Samples are fed in parallel from inputs S1–6 to exhausts SE1–6 and pumped along closed circular 10-nL paths through the capture microchambers. Biotinylated polyclonal antibodies fed from inputs A1–5 to exhausts DE1–5 complete the sandwich immunoassays. Labeled streptavidin fed from input SA to exhausts DE1–5 binds to the biotinylated antibody. The detected fluorescence signal quantifies the captured antigen. (B, inset) Architectural diagram of a test column. Vertical and horizontal comb-like valve arrays enclose individual chambers. Each coliseum contains two test columns and each test column contains five chambers. Valve arrays 1, 2, and 3 pump the sample in a circle along each coliseum (e.g. here upward for actuation order 1,2,3) with a lap time of 20 s. (C) Fluorescence immunoassay spots. Strong fluorescence signal emanates from the capture chambers when the sought antigen is present in the sample and the sandwich immunoassays are properly completed. Darker color corresponds to higher fluorescence intensity.

Figure 2

Quantification of ferritin in human serum. The serum sample was spiked with known concentrations of commercially available ferritin. The endogenous ferritin concentration is calculated from the slope of the linear fit and the zero-spike value. Here, the chip result was 254±35 pM, while the clinical result was 198 pM.

Figure 3

Instrumental statistics. The results of 36 analogous measurements of ferritin in the same human serum sample are presented as a scatter plot (A) and a histogram (B). No systematic clustering among subsets of measurements was observed. The results showed agreement and reproducibility across different test lanes in the same chip as well as across different chips, thereby attesting to the overall reproducibility and reliability of the system.