Latex bead immobilisation in PDMS matrix for the detection of p53 gene point mutation and anti-HIV-1 capsid protein antibodies

Abstract

Two diagnostic chemiluminescent biochips were developed for either the detection of p53 gene point mutation or the serological detection of anti-HIV-1 p24 capsid protein. Both biochips were composed of 24 microarrays of latex beads spots (4×4) (150 μm in diameter, 800 μm spacing) entrapped in a poly(dimethylsiloxane) elastomer (PDMS). The latex beads, bearing oligonucleotide sequences or capsid protein, were spotted with a conventional piezoelectric spotter and subsequently transferred at the PDMS interface. The electron microscopy observation of the biochips showed how homogeneous and well distributed the spots could be. Point mutation detection on the codon 273 of the p53 gene was performed on the basis of the melting temperature difference between the perfect match sequence and the one base pair mismatch sequence. The hybridisation of a 20-mer oligonucleotide form the codon 273 including a one base pair mutation in its sequence on a biochip arrayed with non-muted and the muted complementary sequences, enabled a clear discrimination at 56°C between muted and wild sequences. Moreover, the quantitative measurement of the amount of muted sequence in a sample was possible in the range 0.4–4 pmol. Serological measurement of anti-HIV-1 p24 capsid protein on the biochip, prepared with 1-μm-diameter latex beads, enabled the detection of monoclonal antibodies in the range 1.55–775 ng mL⁻¹. Such a range could be lowered to 0.775 ng mL⁻¹ when using 50-nm-diameter beads, which generated a higher specific surface. The validation of the biochip for the detection of anti-HIV-1 capsid protein antibodies was performed in human sera from seropositive and seronegative patients. The positivity of the sera was easily discriminated at serum dilutions below 1:1,000.