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.

Keywords: Y. enterocolitica; YadA; adhesin; aptamer; biosensor; cell-SELEX; cyclic voltammetry; impedance spectroscopy; square wave voltammetry.

Figure 1

Schematic illustration of the steps in the cell-SELEX process to isolate an aptamer pool with high-affinity to YadA adhesin over 8 cycles of evolution starting with a ssDNA random library. Created with BioRender.com (accessed on 22 July 2022).

Figure 2

Schematic illustration of the sequential steps in the construction and working of an electrochemical biosensor for detection of the YadA adhesin. (A) Immobilization of Apt1 on the gold surface; (B) Blocking the inactive sites using 6-mercapto-1-hexanol (MCH) to prevent non-specific binding; (C) Affinity binding between Apt1 and YadA and (D) measurement of the electrochemical response after each step. Created with BioRender.com (accessed on 22 July 2022).

Figure 3

Primary structure, phylogenetic tree and secondary structure of the selected aptamers: (a) Multiple sequence alignments of the random region from the ten most repeated oligonucleotide sequences obtained after NGS. (b) Phylogenetic analysis of the ten aptamer sequences was determined using the Tree Builder function in Geneious software. (c) Predicted secondary structure for aptamer candidate Apt1 that was selected for further in vitro characterization experiments. The presented predicted secondary structure was the one with lowest ΔG, i.e., the highest stability using the temperature 37 °C, 137 mM Na⁺, and 1.4 mM Mg²⁺, as calculated using the mfold web server. The randomized region is presented in blue.

Figure 4

Determination of the equilibrium dissociation constant of candidate aptamer Apt1. The binding curves of aptamer Apt1 with E. coli YadA and E. coli IBA cells (control), respectively, is shown. The cells were incubated with increasing concentrations of FAM-labelled aptamers and assessed by fluorescence spectrophotometer. Equilibrium dissociation constant (K_d) (nM) was calculated using GraphPad Prism 7, under the non-linear fit model, one-site non-competitive binding to fluorescent population ratio at given aptamer concentrations. a.u = arbitrary units.

Figure 5

Electrochemical assays for construction of the biosensor by Au surface modification, in 5.0 × 10⁻³ M [Fe (CN)₆ ]³⁻ and 5.0 × 10⁻³ M [Fe (CN)₆]⁴⁻ solution, prepared in phosphate buffer, pH 7.4. Cyclic voltammetry (a) and electrochemical impedance spectroscopy Nyquist plot (b).

Figure 6

Square wave voltammetry (SWV) measurements (a,c), and the corresponding calibration curves (b,d), in 5.0 × 10⁻³ M [Fe (CN)₆]³⁻ and 5.0 × 10⁻³ M [Fe (CN)₆]⁴⁻ in phosphate buffer, pH 7.4. For these studies, the aptasensors were immobilized with Apt1 and incubated sequentially with varying concentrations of E. coli YadA cells maintained at pH 5.0 (a,b) or at pH 7.4 (c,d).

Figure 7

Comparison of the calibration curves in 5.0 × 10⁻³ M [Fe (CN)₆]³⁻ and 5.0 × 10⁻³ M [Fe (CN)₆]⁴⁻ in phosphate buffer, pH 7.4, with different concentrations of Apt1 at pH 5.0 incubated at 37 °C (orange) or at room temperature (blue).

Figure 8

Selectivity behavior of the biosensor with Apt1 for E. coli YadA against P. putida and E. coli IBA after 30 min incubation and for the same redox probe previously tested. All bacterial strains were maintained at a concentration of 7.0 × 10⁶ CFU mL⁻¹. One-way ANOVA indicated that the differences between group means were not statistically significant and denoted ns for p > 0.05, the p value was 0.34.