Development of an Electrochemical Biosensor for Rapid and Effective Detection of Pathogenic Escherichia coli in Licorice Extract

Abstract

An aptamer-based electrochemical biosensor was successfully developed and applied in the rapid detection of pathogenic Escherichia coli (E. coli) in licorice extract. The thiolated capture probes were firstly immobilized on a gold electrode, and then the biotinylated aptamer probes for E. coli were introduced by hybridization with the capture probes. Due to the stronger interaction between the aptamer and the E. coli, a part of the biotinylated aptamers will dissociate from the capture probes in the presence of E. coli. The residual biotinylated aptamer probes can quantitatively bind with streptavidin-alkaline phosphatase. Subsequently, α-naphthyl phosphate substrate was catalytically hydrolyzed to generate electrochemical response, which could be recorded by a differential pulse voltammetry. The dependence of the peak current on the logarithm of E. coli concentration in the range from 5.0 × 10 2 colony forming units (CFU)/mL to 5.0 × 10 7 CFU/mL exhibited a linear trend with a detection limit of 80 CFU/mL. The relative standard deviation of 5 successive scans was 5.3%, 4.5% and 1.1% for 5.0 × 10 2 , 5.0 × 10 5 and 5.0 × 10 7 CFU/mL E. coli, respectively. In the detection of the licorice extract samples, the results obtained from the proposed strategy and traditional culture counting method were close to each other, but the time consumption was only~1/30 compared with the traditional method. These results demonstrate that the designed biosensor can be potentially utilized for rapid microbial examination in traditional Chinese medicine and relevant fields. Appl. Sci. 2019, 9, 295 2 of 15 bacterial colony morphology, color change, and biochemical reactions for quantitative detection. This detection method is economical and relatively simple to operate, but the detection time is generally 48-72 h or even longer, which cannot meet the needs for rapid detection. As an effective molecular biological detection technology, polymerase chain reaction (PCR) and the derived technology of PCR has been widely used in detecting E. coli [4,7]. PCR is based on the principle that DNA templates can denature at high temperatures, reactivate and extend (one cycle) at low temperatures by using appropriate DNA polymerase and primers. As the template DNA amplification product increases exponentially, increasing the number of cycles can magnify the specified DNA sequence by several million-fold in a short time, which greatly improves detection sensitivity [8]. However, PCR technology is costly and prone to produce false negatives [9], which limits the extensive application of PCR. In addition, enzyme-linked immunosorbent assay (ELISA), as a common immunological detection technology, has also been used in bacterial test [5,10]. ELISA technology undergoes a color change by the catalysis of the enzyme in the specific recognition process. Owing to the immune activity between antigen and antibody, ELISA method has superior specificity, high sensitivity and short detection time, and has been used in detection of different subtypes of E. col...

Flow Control Techniques for Enhancing the Bio-Recognition Performance of Microfluidic-Integrated Biosensors

“…S = r ads − r des (9) in which b m (mol/m 2 ) is the total surface concentration of active sites, b (mol/m 2 ) is the surface concentration of A, r ads (mol/(m 2 •s)) refers to the adsorption rate and r des (mol/(m 2 •s)) refers to the desorption rate of the targeted species. The Helmholtz-Smoluchowski [31] relation is used for relating the tangent component of the applied electric field with the electroosmotic velocity (Equation ( 10)).…”

“…The attention towards microfluidic-integrated biosensors is enhanced extra by its capability of detecting a low concentration of biomarkers [7,8]. It also has application in various fields such as real-time health monitoring, point-of-care (POC) disease diagnosis, significant data statistics in health management, food safety, and environmental monitoring [9][10][11].…”

QCM Sensor Arrays, Electroanalytical Techniques and NIR Spectroscopy Coupled to Multivariate Analysis for Quality Assessment of Food Products, Raw Materials, Ingredients and Foodborne Pathogen Detection: Challenges and Breakthroughs

“…Moreover, aptamers, single-stranded oligonucleotides, are also capable of binding to various food-borne pathogens with high affinity and selectivity. Electrochemical aptasensors have been reported recently for the detection of Salmonella enterica [271], Cronobacter sakazakii [272], and Escherichia coli [273]. Zhou et al have used T2 bacteriophage (a virus) as the biorecognition element on a screen-printed electrode for rapid and selective detection of Escherichia coli.…”

Special Issue on Nano-Biointerface for Biosensing

“…H. Wang and coworkers in their paper on the Development of an Electrochemical Biosensor for Rapid and Effective Detection of Pathogenic Escherichia coli in Licorice Extract [8], describe the development of an aptamer-based electrochemical biosensor and its application in the rapid detection of pathogenic Escherichia coli (E. coli) in licorice extract. For the biosensor fabrication, thiolated capture probes were firstly immobilized on a gold electrode, and then the biotinylated aptamer probes for E. coli were introduced by hybridization with the capture probes.…”