Rapid amplification-free microbial detection system for water and clinical samples

Overview

This technology offers a rapid, amplification-free method for detecting viable microbes in aqueous and clinical samples, delivering results in approximately 5 minutes at sensitivities of ≤100 CFU/mL. The approach targets species-specific ribosomal RNA sequences, enabling direct identification of living pathogens without the need for PCR or other nucleic acid amplification steps. This dramatically reduces time-to-result compared to conventional culture-based methods (which take 24–48 hours) while maintaining high specificity. The platform is designed for portability, with planned integration into a handheld, battery-powered reader and single-use test strips suitable for field deployment.

Potential applications include water quality monitoring, clinical diagnostics (including point-of-care testing for urinary tract infections), biodefense screening, food safety, and environmental surveillance. The technology addresses critical needs in settings where rapid, on-site pathogen detection is essential and laboratory infrastructure is unavailable or impractical.

Technical specifications

Detection principle:

• Charge-neutral peptide nucleic acid (PNA) oligomers serve as capture probes conjugated to microbeads
• When negatively charged target rRNA hybridizes to the PNA-microbead conjugates, the complexes become mobile in a low-voltage electric field
• Directed migration through a sub-micron pore in a glass membrane produces a measurable step reduction in ionic current, which serves as the detection signal
• No nucleic acid amplification is required, eliminating associated reagents, time, and contamination risks

Key features and demonstrated performance:

• Detection of specific bacterial rRNA at concentrations as low as 10⁻¹⁹ M, corresponding to approximately 1 CFU/100 mL
• Single-base mismatch resolution demonstrated with anthrax lethal factor DNA sequence detection
• N. gonorrhoeae detected in urine at 10–100 CFU/mL against background flora with approximately 98% sensitivity and 100% specificity
• Hybridization achieved in approximately 1 minute following rapid RNA extraction and 0.1-µm filtration
• MEMS-fabricated glass membranes with single sub-micron pores enable miniaturization and low-cost manufacturing
• Multiplexing capability through design of multiple PNA probe sequences to detect several similar microbes simultaneously

Planned prototype components:

• Single-use sampling/lysis/filtration device delivering 4–5 filtered drops onto a test strip
• Single-use lateral flow test strip pre-loaded with buffer and PNA-beads, with integrated glass chip detector
• Repeated-use, battery-powered handheld reader incorporating user interface, low-voltage detection electronics, and wireless communication

Technology readiness level

The core detection technology has been validated through multiple proof-of-concept demonstrations. Bacterial rRNA detection at 10⁻¹⁹ M has been achieved, and clinical validation with N. gonorrhoeae in urine samples has demonstrated high sensitivity and specificity. The MEMS fabrication process for glass membranes with sub-micron pores has been developed. As finalists in the US Army xTechSearch competition, the team demonstrated integration of the glass chip detector with a lateral flow assay format.

The current development phase focuses on building and testing integrated prototypes, including the single-use sampling/lysis/filtration device, the integrated test strip, and the handheld reader. The technology is advancing toward a deployable system suitable for field and clinical use, with remaining work centered on device integration, user interface development, and wireless communication capabilities.