Dark Field Microscopy-Based Biosensors for the Detection of E. coli in Environmental Water Samples

Abstract

Development of sensitive methods for the determination of E. coli bacteria contamination in water distribution systems is of paramount importance to ensure the microbial safety of drinking water. This work presents a new sensing platform enabling the fast detection of bacteria in field samples by using specific antibodies as the biorecognition element and dark field microscopy as the detection technique. The development of the sensing platform was performed using non-pathogenic bacteria, with the E. coli DH5α strain as the target, and Bacillus sp. 9727 as the negative control. The identification of the captured bacteria was made by analyzing the dark field microscopy images and screening the detected objects by using object circularity and size parameters. Specificity tests revealed the low unspecific attachment of either E. coli over human serum albumin antibodies (negative control for antibody specificity) and of Bacillus sp. over E. coli antibodies. The system performance was tested using field samples, collected from a wastewater treatment plant, and compared with two quantification techniques (i.e., Colilert-18 test and quantitative polymerase chain reaction (qPCR)). The results showed comparable quantification capability. Nevertheless, the present method has the advantage of being faster, is easily adaptable to in-field analysis, and can potentially be extended to the detection of other bacterial strains.

Dark-Field Microscopy: Recent Advances in Accurate Analysis and Emerging Applications

“…An automatic counting algorithm was also developed for the fast and robust imaging analysis of bacteria . By using size parameters and object circularity, Colpo et al sensitively detected E. coli in environmental water samples in a similar fashion …”

“…26 By using size parameters and object circularity, Colpo et al sensitively detected E. coli in environmental water samples in a similar fashion. 27 Aggregation. Sensing based on aggregation of probes is also simple and sensitive.…”

Principles and Recent Advances in Biosensors for Pathogens Detection

“…Another dark-field microscopic-based biosensor for detecting E. coli was developed using E. coli antibodies as a biorecognition platform in which the proposed pathogenic sensor exhibited excellent specificity toward the detection of E. coli in contaminated water samples. [127]…”

Optical Methods for Label-Free Detection of Bacteria

“…Compared with the labeling methods, which generally require a long incubation time, label-free approaches are much simpler, faster, and cost-effective, making them good candidates for rapid bacterial detection in clinical application. Efforts have been made in this direction, among which the optical methods, such as Raman spectroscopy and single-particle imaging approaches, are the most promising approaches due to their high sensitivity, simplicity, and low-cost for label-free detection of bacteria [ 40 , 41 , 42 ]. In this review, we describe the advantages and disadvantages of optical methods such as Raman spectroscopy, SPR, and dark-field microscopy for label-free detection of bacteria and their applications in clinical detection and drug resistance evaluation.…”