A combined experimental and theoretical study on the immunoassay of human immunoglobulin using a quartz crystal microbalance

Abstract

We investigate a immunoassay biosensor that employs a Quartz Crystal Microbalance (QCM) to detect the specific binding reaction of the (Human IgG1)-(Anti-Human IgG1) protein pair under physiological conditions. In addition to experiments, a three dimensional time domain finite element method (FEM) was used to perform simulations for the biomolecular binding reaction in microfluidic channels. In particular, we discuss the unsteady convective diffusion in the transportation tube, which conveys the buffer solution containing the analyte molecules into the micro-channel where the QCM sensor lies. It is found that the distribution of the analyte concentration in the tube is strongly affected by the flow field, yielding large discrepancies between the simulations and experimental results. Our analysis shows that the conventional assumption of the analyte concentration in the inlet of the micro-channel being uniform and constant in time is inadequate. In addition, we also show that the commonly used procedure in kinetic analysis for estimating binding rate constants from the experimental data would underestimate these rate constants due to neglected diffusion processes from the inlet to the reaction surface. A calibration procedure is proposed to supplement the basic kinetic analysis, thus yielding better consistency with experiments.

Keywords: Finite Element Method (FEM); Quartz Crystal Microbalance; basic kinetic analysis; biosensor; human IgG1.

Figure 1.

Sketch of the 3D model of the QCM device. Part 1 is the transportation tube conveying the analyte solution into the micro-channel. Part 2 is the micro-channel with the reaction surface.

Figure 2.

A typical result of the QCM experiment. There are four steps in the experimental process, as described in the text.

Figure 3.

The (Human IgG1)-(Anti-Human IgG1) protein pair binding curves. The supplemented volume of the Anti-Human IgG1 solution is: (A) 800 μL, (B) 500 μL and (C) 100 μL. For each case there are four concentrations of Anti-Human IgG1 solution (50, 25, 10 and 5 μg/mL). The error bar at each time point is marked according to 4∼5 replicates of the experimental raw data; namely picking the largest positive (negative) deviation value as our upper (lower) bound to the average value.

Figure 4.

Simulated binding reaction curves. The supplement volume of the Anti-Human IgG1 solution is (A) 800 μL (B) 500 μL and (C) 100 μL.

Figure 5.

The distribution of the analyte concentration at the outlet of the tube, which is the inlet of the micro-channel, and the corresponding binding reaction curves.

Figure 6.

The normalized experimental and simulated binding reaction curves for the 800 μL supplement volume. Here the concentrations of the Anti-Human IgG1 solution are (A) 50 μg/mL, (B) 25 μg/mL, (C) 10 μg/mL, and (D) 5 μg/mL.