Bio-impedance sensing device (BISD) for detection of human CD4⁺ cells

The aim of this work is to develop a small on-chip bio-impedance sensing device (BISD) to rapidly detect and quantify cells with a specific phenotype in a heterogeneous population of cells (e.g., human CD4⁺ cells in blood, for monitoring HIV-infected individuals) using a minimal sample volume and minimal preparation. The transducers, gold/titanium microelectrodes (100×100μm and 80×80μm), have been fabricated on glass substrates. The microelectrode surface is non-covalently modified sequentially with protein G', human albumin, monoclonal anti-human CD4 antibody, and mouse IgG. The anti-human CD4 antibody binds CD4⁺ cells present in human blood. The basic function of the microelectrodes was characterized using electrochemical cyclic voltammetry before and after protein G' deposition. The binding of biomolecules, protein G' and antibodies, as well as cells was detected by precisely measuring the electrical current, as a function of frequency (1-8 kHz), that flowed between the microelectrode and the much larger reference electrode. This current was measured using a specially designed very low-noise amplifier based on instrument-grade operational amplifiers. This measurement was plotted as impedance, using the constant-amplitude voltage applied between the two electrodes. We have conducted a series of AC impedance and output voltage measurements with various sizes of gold microelectrodes with adsorbed protein layers, as well as with the CD4⁺ cells. When a sample of human peripheral blood mononuclear cells (PBMCs) was incubated on the biosensor, an increase in impedance was observed; this increase was due to the presence of CD4⁺ cells, which cause a decrease in the current flow. Incubation of the captured cells with FITC-labeled anti-human CD4 antibody verified that all captured cells were CD4⁺, demonstrating the selectivity of the BISD system. The BISD system is a promising tool for the detection of CD4⁺ cells in HIV-infected individuals, and for the detection and quantification of antigen:antibody or receptor:ligand interactions.