Rapid, Economical Detection of Helicobacter pylori Using Gold Colloidal Nanoparticle Biosensors

Abstract

Nucleic acid tests (NAT), the gold standard diagnostic technology, play a crucial role in the prevention of infectious diseases. However, PCR, the current state-of-the-art NAT, is expensive, slow, and requires dedicated infrastructure and facilities. Therefore, there exists an urgent need to create alternative molecular diagnostic technologies. We describe the use of a gold colloidal nanobiosensor detection system that can specifically and sensitively detect the 16S rRNA gene of the worldwide gastric pathogen Helicobacter pylori. We demonstrate the systematic identification of oligonucleotide probe sequences according to secondary structure, binding energy, and homology search criteria. We selected three probe sequences that were used to evaluate the detection of a 120 nt synthetic analyte. Detection of this analyte resulted in a visual color change in the solution to a limit of detection (LOD) of 10 nM and by spectrophotometric means to 1 nM. Furthermore, we demonstrated that the system could detect clinical samples of H. pylori with a LOD of 5 × 10⁵ copies/mL. The system displayed no cross-reactivity with potentially confounding bacterial pathogens. Importantly, we also demonstrated the ability of the detection system to detect clinical samples of H. pyloriwithout the requirement of a separate DNA extraction, allowing for a one-step detection system. In summary, we have created a simple-to-use, economical, rapid, sensitive, and specific alternative to PCR that could be useful in resource-limited settings to control the spread of infectious diseases.

Figure 1

Scheme for the validation of the H. pylori assay.

Figure 2

Oligonucleotides selection. (a) Alignment of different H. pylori strains on their 16S rRNA gene. (b) Table containing the different selected oligonucleotides with their calculated binding energy with and without T10 spacer, self-binding energy, and secondary structure energy.

Figure 3

120 nt short target detection. (a) Secondary structure of the 120 nt short target. (b) Image, (c) UV–vis spectra, and (d) absorbance ratio (540/750 nm) of the limit of detection assay using the 120 nt short target mimicking targeted region of the 16S rRNA gene.

Figure 4

Detection of cultured samples from different patients infected with different bacteria, (a) absorbance ratio of assays containing extracts from cultures of H. pylori obtained from different patients, (b) absorbance ratio of assays containing heat inactivated cultures of H. pylori obtained from different patients, (c) absorbance ratio of assays containing different concentrations of cultured extracts from different bacteria, and (d) absorbance ratio of assays containing different concentrations of cultured H. pylori extract.