Internally calibrated quantification of VEGF in human plasma by fluorescence immunoassays in disposable elastomeric microfluidic devices

Abstract

Herein we report on a proof of principle for the reproducible quantification of Vascular Endothelial Growth Factor (VEGF) in human plasma by fluorescence sandwich immunoassays using disposable polydimethylsiloxane (PDMS) microfluidic chips. The system requires 100 times less sample than typical clinical blood tests, while its current quantification limit is established at 4pM. The in-built calibration method of spiking the plasma with known concentrations of commercially available antigen avoids common sources of error and improves the reliability of the test results. The demonstrated technique is important for immunoassay applications in fundamental scientific research and "point-of-care" (POC) biomedical diagnostics. In particular, the system is immediately applicable to microfluidic quantification of VEGF in human plasma in cancer studies.

Figure 1

(A) Microfluidic immunoassay chip. A 60-chamber PDMS chip bound to a one-inch-wide epoxide slide was used for the experiments. The vertical cylinders are input ports for reagents, samples, and control pressure. The microchannel test matrix is visible in the middle. (B) Architectural diagram of the 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 a sandwich immunoassay is constructed. Fluorophore labels produce signal that is used to quantify the captured antigen.

Figure 1

(A) Microfluidic immunoassay chip. A 60-chamber PDMS chip bound to a one-inch-wide epoxide slide was used for the experiments. The vertical cylinders are input ports for reagents, samples, and control pressure. The microchannel test matrix is visible in the middle. (B) Architectural diagram of the 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 a sandwich immunoassay is constructed. Fluorophore labels produce signal that is used to quantify the captured antigen.

Figure 2. Quantification of VEGF in human plasma

The plasma sample was spiked with known concentrations of commercially available analog. The endogenous VEGF concentration is calculated from the slope of the linear fit and the zero-spike net signal. Here, the linear fit (R=0.9757, p=0.0045) indicated a concentration of 2.16 ± 0.28 pM, while the clinical result was 1.6 pM.

Figure 3. Instrumental Performance

The results of 39 analogous measurements of VEGF in the same human plasma 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 of the results.

Figure 3. Instrumental Performance

The results of 39 analogous measurements of VEGF in the same human plasma 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 of the results.