16S rRNA Monitoring Point-of-Care Magnetic Focus Lateral Flow Sensor

Abstract

The detection and profiling of pathogenic bacteria is critical for human health, environmental, and food safety monitoring. Herein, we propose a highly sensitive colorimetric strategy for naked eye screening of 16S ribosomal RNA (16S rRNA) from pathogenic agents relevant to infections, human health, and food safety monitoring with a magnetic focus lateral flow sensor (mLFS) platform. The method developed was demonstrated in model 16S rRNA sequences of the pathogen Escherichia coli O157:H7 to detect as low as 1 fM of targets, exhibiting a sensitivity improved by ∼5 × 10⁵ times compared to the conventional GNP-based colorimetric lateral flow assay used for oligonucleotide testing. Based on the grayscale values, semi-quantitation of up to 1 pM of target sequences was possible in ∼45 min. The methodology could detect the target 16S rRNA from as low as 32 pg/mL of total RNA extracted from pathogens. Specificity was demonstrated with total RNA extracted from E. coli K-12 MG1655, Bacillus subtilis (B. subtilis), and Pseudomonas aeruginosa (P. aeruginosa). No signal was observed from as high as 320 pg/mL of total RNA from the nontarget bacteria. The recognition of target 16S rRNA from 32 pg/mL of total RNA in complex matrices was also demonstrated. The proposed mLFS method was then extended to monitoring B. subtilis and P. aeruginosa. Our approach highlights the possibility of extending this concept to screen specific nucleic acid sequences for the monitoring of infectious pathogens or microbiome implicated in a range of diseases including cancer.

Scheme 1. Comparison between GNP-Based Colorimetric LFA and the Proposed mLFS Platform for the Detection of 16S rRNA

Figure 1

UV–vis spectra of mNPs, SH-PS-modified mNPs, casein-blocked SH-PS-modified mNPs, and magnetic probe immobilized with HRP (A). TEM image of mNPs (B) and its response to a magnet (C) where 0 min corresponds to the mNPs after sonication.

Figure 2

Detection results for TS at serial concentrations and the corresponding quantitative grayscale results.

Figure 3

Detection results for 16S rRNA in serial dilutions of total RNA extracted from E. coli O157:H7 at different levels of FITC-CS.

Figure 4

Specificity results with RNA extracted from E. coli K-12 MG1655, B. subtilis, and P. aeruginosa.

Figure 5

Detection results for serial concentrations of RNA extracted from E. coli O157:H7 diluted in the lettuce solution.

Figure 6

mLFS detection of B. subtilis with probes modified with SH-BSPS and BSCS-FITC and P. aeruginosa with probes modified with SH-PAPS and PACS-FITC.