Capacitive immunosensors were made by coupling monoclonal antibodies to thioctic acid, which had self-assembled on a gold electrode. Surface areas that were not covered were plugged with 1-dodecanethiol to make the layer dense and insulating. Cyclic voltammetry showed that the hexacyanoferrate redox reactions were blocked by this procedure. The capacitance of the electrode was evaluated from the current transients obtained when a potentiostatic step was applied. The immunosensor was placed in a flow system, and a capacitance decrease could be observed after injection of an unlabeled antigen. It was linear over almost three decades when plotted vs the logarithm of the antigen concentration. Human chorionic gonadotropin hormone could be determined in the range 1 pg/mL-1 ng/mL, with a detection limit of 0.5 pg/mL (15 10(-15) M). A similar response was obtained with immobilized F(ab)2 fragments. No cross-reactivity was observed with the thyrotropic hormone, which has one chain in common with gonadotropin. Monoclonal antibodies toward interleukin-2 immobilized on the immunosensor gave also a response over 1 pg/mL-1 ng/mL, with a detection limit of 1 pg/mL. An immunosensor with monoclonal antibodies toward human albumin gave a calibration curve with lower slope than the other proteins but still with a detection limit of 1 pg/mL.
This preliminary study was performed to prove the feasibility of a direct capacitive DNA biosensor for detection of nucleic acids. Two different methods for immobilization of the oligonucleotide probes were used. The ®rst type of sensor was composed of a gold rod with a self-assembled monolayer of a 26-base long oligonucleotide probe, modi®ed with an SH-group at the 5 H -end. Coverage studies showed that only around 20% of the surface was covered, probably due to the bulky nature of the probes. Hybridization studies performed in a¯ow-through cell showed selectivity towards a DNA sample containing single stranded fragments of cytomegalo virus (CMV) possessing a complementary sequence. As few as 25 molecules could be detected at sample concentrations of 0.2 attomolar with an injection volume of 250 mL. Controls with fragments of double-stranded CMV and single-stranded hepatitis B virus and tyrosinase mRNA gave all lower responses. The other type of sensor was modi®ed by covalent immobilization of a phosphorylated 8-base long oligonucleotide probe to a self-assembled monolayer of cysteamine. This biosensor also showed selectivity against single stranded fragments of CMV and also in this case as few as 25 molecules could be detected.
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