From Laboratory Immunoassays To Immunosensors With An Integrated Microfluidic Flow System

Key Findings

TMB Quantification Using Chronoamperometry

In this configuration, TMB, which is oxidized by horseradish peroxidase (HRP) during an immunoassay, can be quantified by reducing it back to its initial chemical state using chronoamperometry. The optimal background current was observed at 330 mV vs. Ag/AgCl, which allowed for a strong net signal. This signal, defined as the difference between the faradaic and background currents, was sufficient to ensure the sensitive quantification of TMB.

Optimizing Flow Cell Design

Conventional macroscopic flow cells tend to require large sample volumes and high flow rates, which consume significant amounts of reagents. In contrast, a microfluidic flow cell was integrated and drastically reduced reagent consumption. Typical sample volumes in plate-based immunoassays are around 100 – 300 μL, and this miniaturized flow cell design ensures that similar sample volumes are used efficiently. Fast flow rates allow for high signal intensities within a short measurement time. [4]

3D Chip Architecture for High-Flow Precision

To meet these demands, a 3D chip architecture was designed (Figure 2). This miniaturized flow cell features a screen-printed electrode (SPE) with a central inlet channel for sample injection and four outlet channels that merge into one. The vertical injection design ensures efficient flow, especially for high flow rates, and helps remove bubbles that could interfere with the detection process. The system showed exceptional stability at flow rates between 500 – 600 μL∙min−1, consuming only 250 μL per sample with a rapid detection time of 30 seconds.

Testing with Magnetic Bead-Based Immunoassay (MBBA)

The integrated 3D flow cell was tested using a previously optimized magnetic bead-based immunoassay (MBBA) for indirect diclofenac (DCF) detection, constructed with the selective anti-DCF antibody F01G21.1. Briefly, A solution containing Diclofenac, Diclofenac-magnetic particles, and HRP-labeled antibodies is prepared. The Diclofenac competes with the magnetic particles for antibody binding. After magnetic separation, the number of antibodies bound to the particles is inversely proportional to the Diclofenac concentration in the sample (i.e., more Diclofenac results in fewer antibodies bound, as shown in Figure 4.a). The HRP protein converts the TMB substrate into its oxidized form, a common electrochemical marker, with signal amplification achieved through reoxidation of HRP by H₂O₂. Finally, the oxidized TMB is acidified, forming a yellow species with a distinct electrochemical response.

As expected, increasing the flow rate from 100 – 600 μL∙min−1 led to an increase in signal intensity (Figure 3.a). However, excessively high flow rates caused bubble formation, leading to a decreased signal-to-noise ratio (SNR). An optimal flow rate of 500 μL∙min−1 was found, providing both high signal intensity and reproducibility (Figure 3.b).