On-chip micro-biosensor for the detection of human CD4⁺ cells based on AC impedance and optical analysis

The current study was undertaken to fabricate a small micro-electrode on-chip to rapidly detect and quantify human CD4⁺ cells in a minimal volume of blood through impedance measurements made with simple electronics that could be battery operated implemented in a hand held device. The micro-electrode surface was non-covalently modified sequentially by incubation with solutions of protein G′, human albumin, monoclonal mouse anti-human CD4, and mouse IgG. The anti-human CD4 antibody served as the recognition and capture molecule for CD4⁺ cells present in human blood. The binding of these biomolecules to the micro-electrodes was verified by impedance and cyclic voltammetry measurements. An increase in impedance was detected for each layer of protein adsorbed onto the micro-electrode surface. This process was shown to be highly repeatable. Increased impedance was measured when CD4 ⁺ cells were captured on the micro-electrode, and the impedance also increased as the number of captured cells increased. Fluorescence microscopy of captured cells immunolabeled with anti-human CD4, CD8, and CD19 antibodies, and the nuclear label DAPI, confirmed that only CD4⁺ cells were captured. The results were highly dependent on the specimen preparation method used. We conclude that the on-chip capture system can efficiently quantify the number of CD4⁺ cells.