A universal nucleic acid sequence biosensor with nanomolar detection limits.

Abstract

A quantitative universal biosensor was developed on the basis of olignucleotide sandwich hybridization for the rapid (30 min total assay time) and highly sensitive (1 nM) detection of specific nucleic acid sequences. The biosensor consists of a universal membrane and a universal dye-entrapping liposomal nanovesicle. Two oligonucleotides, a reporter and a capture probe that can hybridize specifically with the target nucleic acid sequence, can be coupled to the universal biosensor components within a 10-min incubation period, thus converting it into a specific assay. The liposomal nanovesicles bear a generic oligonucleotide sequence on their outer surface. The reporter probes consist of two parts: the 3' end is complementary to the generic liposomal oligonucleotide, and the 5' end is complementary to the target sequence. Streptavidin is immobilized in the detection zone of the universal membranes. The capture probes are biotinylated at the 5' end and are complementary to another segment in the target sequence. Thus, by incubating the liposomal nanovesicles with the reporter probes, the target sequence, and the capture probes in a hybridization buffer for 20 min, a sandwich complex is formed. The mixture is applied to the membrane, migrates along the strip, and is captured in the detection zone via streptavidin-biotin binding. The biosensor assay was optimized with respect to hybridization conditions, concentrations of all components, and length of the generic probe. It was tested using synthetic DNA sequences and authentic RNA sequences isolated and amplified using nucleic acid sequence-based amplification (NASBA) from Escherichia coli, Bacillus anthracis, and Cryptosporidium parvum. Dose-response curves were carried out using a portable reflectometer for the instantaneous quantification of liposomal nanovesicles in the detection zone. Limits of detection of 1 fmol per assay (1 nM) and dynamic ranges between 1 fmol and at least 750 fmol (1-750 nM) were obtained. The universal biosensors were compared to specific RNA biosensors developed earlier and were found to match or exceed their performance characteristics. In addition, no changes to hybridization conditions were required when switching to the detection of a new target sequence or when using actual nucleic acid sequence-based amplified RNA sequences. Therefore, the universal biosensor described is an excellent tool for use in laboratories or at test sites for rapidly investigating and quantifying any nucleic acid sequence of interest.