PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor

Abstract

A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100–1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids.

Nanoparticle-blockage-enabled rapid and reversible nanopore gating with tunable memory

“…Instead, it would be trapped in a position close to the nanopore due to a competition between electrophoretic force, electroosmotic drag force, and dielectrophoretic forces and partially block transport through the nanopore (see Fig. 1 C and SI Appendix , section S1 ) ( 25 , 26 ). The trapped nanoparticle can be quickly released by reversal of the applied bias.…”

Advances in quantifying microbial contamination in potable water: Potential of fluorescence‐based sensor technology

“…A wide range of technologies exists for detecting microbial contamination, and by extension E. coli , in potable waters. These include traditional laboratory‐based (e.g., Deshmukh et al, 2016) and molecular detection techniques (Campbell & Kleinheinz, 2020; Esfandiari et al, 2016; Kuo et al, 2021), Biosensors (sub‐categorized into electro‐chemical [Bigham, Dooley, et al, 2019; Thakur et al, 2018; Grossi et al, 2013; Velasquez‐Orta et al, 2017] and optical methods), optical detection techniques and fluorescence (Figure 1). These categories are based on the underlying measurement principles for each method illustrating the broad reach of techniques, some of which are widely and routinely used while others are currently at the more novel, proof‐of‐concept stage.…”

“…Currently, non‐fluorescence‐based field identification of E. coli is generally limited to proof‐of‐concept designs (e.g., Esfandiari et al, 2016; Gunda & Mitra, 2016; Thakur et al, 2018) owing to a lack of suitable methods. However, several fluorescence‐based studies have specifically targeted E. coli in the field (Baker et al, 2015; Bridgeman et al, 2015; Cumberland et al, 2012; Nowicki et al, 2019; Simoes et al, 2021; Sorensen, Vivanco, et al, 2018; Ward et al, 2021).…”

“…The scope and interconnectivity of methods that can be used to measure E. coli in water: Text in green denotes methods where their use in the field has been demonstrated in the literature; blue text indicates where potential field use has been identified but not formally demonstrated. Where a method is novel to a particular paper, numbers identify the following references: 1 Bigham, Casimero, et al (2019); 2 Esfandiari et al (2016); 3 Wildeboer et al (2010); 4 Gunda and Mitra (2016); 5 Sherchan et al (2018). …”

Construction of a point-of-care electrochemical biosensor for Escherichia coli 16S rRNA analysis based on MoS2 nanoprobes

“…This performance is comparable to or better than those in previous works, suggesting that the proposed biosensor has excellent analytical performance (Table S1 †). [26][27][28][29][30] In addition, the selectivity of this electrochemical sensor for different bacterial 16S rRNA detection was tested under the same concentration (1 pM, Fig. 3C).…”