Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica

Abstract

New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.

Computational Frontiers in Aptamer-Based Nanomedicine for Precision Therapeutics: A Comprehensive Review

“…Aptamers are a novel family of biorecognition elements with potential uses in therapeutic, diagnostic, and sensing fields shown in Figure . Their interaction with targets may enhance protein stability in addition to the resistance against denaturation. , Both the World Health Organisation and the National Institute of Health define biomarkers like any compound, structure, or procedure the fact that can be determined as the body while participating have an impact on or predict the likelihood of a condition or result. − Similarly, biomarkers are characteristics that are objectively determined and assessed as a representation of biological processes that are normal, pathogenic, or pharmacologic reactions to a therapeutic intervention. Biomarker discovery and validation is one of the most fascinating areas of biological research. , For this, a variety of proteomics techniques are used.…”

“… 167 , 168 Both the World Health Organisation and the National Institute of Health define biomarkers like any compound, structure, or procedure the fact that can be determined as the body while participating have an impact on or predict the likelihood of a condition or result. 169 − 171 Similarly, biomarkers are characteristics that are objectively determined and assessed as a representation of biological processes that are normal, pathogenic, or pharmacologic reactions to a therapeutic intervention. Biomarker discovery and validation is one of the most fascinating areas of biological research.…”

Aptamer-Based Point-of-Care Devices: Emerging Technologies and Integration of Computational Methods

“…It significantly decreased the detection time down to 30 min compared to the detection time of both traditional sputum microscopy and PCR (several hours or even days) [74]. Another electrochemical aptasensor was developed using the same Au-S interaction strategy with a screen-printed gold electrode to detect the Yersinia adhesin A (YadA) biomarker of Yersinia enterocolitica from Yersinia species, a species capable of causing diarrhea, mesenteric lymphadenitis, arthritis, and bacterial blood called sepsis [75,188]. The aptamer was selected using cell-SELEX, and likewise, the immobilization was performed through the Au-S method on a screen-printed gold electrode.…”

“…The aptamer was selected using cell-SELEX, and likewise, the immobilization was performed through the Au-S method on a screen-printed gold electrode. The device had a detection limit of 7.0 × 10 4 CFU•mL −1 and could exhibit a linear performance range of 7.0 × 10 4 CFU•mL −1 to 7.0 × 10 7 CFU•mL −1 [75].…”

Aptasensor for the Detection of Moraxella catarrhalis Adhesin UspA2

“…In this type of biosensor, an electrode is used as a solid support for the immobilization of biomolecules and electron movement [13]. These types of biosensors have been widely and successfully employed for the detection of various pathogenic moieties, including biosensors functionalized with aptamers [14][15][16][17][18].…”