Aptasensor for the Detection of Moraxella catarrhalis Adhesin UspA2

Abstract

Innovative point-of-care (PoC) diagnostic platforms are desirable to surpass the deficiencies of conventional laboratory diagnostic methods for bacterial infections and to tackle the growing antimicrobial resistance crisis. In this study, a workflow was implemented, comprising the identification of new aptamers with high affinity for the ubiquitous surface protein A2 (UspA2) of the bacterial pathogen Moraxella catarrhalis and the development of an electrochemical biosensor functionalized with the best-performing aptamer as a bioreceptor to detect UspA2. After cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) was performed, next-generation sequencing was used to sequence the final aptamer pool. The most frequent aptamer sequences were further evaluated using bioinformatic tools. The two most promising aptamer candidates, Apt1 and Apt1_RC (Apt1 reverse complement), had K_d values of 214.4 and 3.4 nM, respectively. Finally, a simple and label-free electrochemical biosensor was functionalized with Apt1_RC. The aptasensor surface modifications were confirmed by impedance spectroscopy and cyclic voltammetry. The ability to detect UspA2 was evaluated by square wave voltammetry, exhibiting a linear detection range of 4.0 × 10⁴-7.0 × 10⁷ CFU mL^-1, a square correlation coefficient superior to 0.99 and a limit of detection of 4.0 × 10⁴ CFU mL^-1 at pH 5.0. The workflow described has the potential to be part of a sensitive PoC diagnostic platform to detect and quantify M. catarrhalis from biological samples.

Keywords: adhesins; aptamers; biosensor; cell-SELEX; detection; electrochemical; outer membrane proteins (OMPs); point-of-care (PoC); ubiquitous surface protein A2 (UspA2).

Figure 1

Illustration of a sedimentation assay. (a) Macroscopic analysis of auto-aggregation. Within a few minutes, Escherichia coli UspA2 (left tube) aggregates and settles at the bottom of the tube under static incubation, whereas the control culture of E. coli carrying the empty vector pASK IBA2 (E. coli IBA) (right tube) remains turbid. (b) Measurement of auto-aggregation. Culture samples were incubated statically and the OD_600nm value at the top of the culture tube was measured after 5 min of incubation. The reduction in turbidity at the top of the culture is a function of the OD_600nm value. Auto-aggregating bacteria settle at the bottom of the tube, resulting in a loss of turbidity (orange curve), whereas in negative control culture samples the reduction in turbidity is negligible (blue curve).

Figure 2

Sequence alignments and phylogenetic tree of the 20 aptamer sequences obtained after bioinformatic analysis. (A) Sequence alignments of the random region from the ten most frequent aptamer sequences and their reverse complement (RC) sequences obtained after NGS of the final aptamer pool of cell-SELEX. The regions of homology in the sequences are highlighted. (B) Phylogenetic relationship of the aptamer sequences using ‘Tree Builder’ in Geneious software. The relationship between the sequences was determined using a neighbor-joining model with no outgroups.

Figure 2

Sequence alignments and phylogenetic tree of the 20 aptamer sequences obtained after bioinformatic analysis. (A) Sequence alignments of the random region from the ten most frequent aptamer sequences and their reverse complement (RC) sequences obtained after NGS of the final aptamer pool of cell-SELEX. The regions of homology in the sequences are highlighted. (B) Phylogenetic relationship of the aptamer sequences using ‘Tree Builder’ in Geneious software. The relationship between the sequences was determined using a neighbor-joining model with no outgroups.

Figure 3

Secondary structures of finalized candidate aptamer sequences obtained from cell-SELEX. The Mfold web server was used to predict the secondary structures of Apt1 and Apt1_RC selected for in vitro characterization. To calculate the Gibbs free energy of the aptamer sequences, the constant primer regions were considered at 37 °C, 187 mM Na⁺ and 1.4 mM Mg²⁺. Random and primer regions are presented in blue and black, respectively.

Figure 4

Determination of the equilibrium dissociation constant $(K_d)$ of candidate aptamers Apt1 and Apt1_RC. The titration curves of each aptamer with E. coli UspA2 cells are shown. The aptamers were labeled with fluorescein (FAM) and fluorescence was assessed with a fluorescence spectrophotometer. $K_d$ (nM) values were calculated using GraphPad Prism 7, set to a non-linear fit model with one-site non-competitive binding to the fluorescent population ratio at the given aptamer concentrations. a.u = arbitrary units.

Figure 5

Illustration of the developed electrochemical biosensor to detect Escherichia coli UspA2. (A) Apt1_RC was immobilized on the gold electrode surface; (B) non-specific binding was blocked with 6-mercapto-1-hexanol (MCH); and (C) selective binding between E. coli UspA2 and Apt1_RC. (D) The electrochemical response is measured after each assembly step and during the analytical analysis of the performance of the aptasensor. Created with BioRender.com.

Figure 6

Electrochemical assays to assess the assembly of the aptasensor upon functionalization of the Au-SPE surface, 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 (CV) (a) and electrochemical impedance spectroscopy (EIS) represented by a Nyquist plot (b).

Figure 7

Electrochemical assays to assess the analytical response of the aptasensor in square wave voltammetry (SWV) (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. The aptasensors functionalized with Apt1_RC were incubated sequentially with increasing concentrations of inactivated Escherichia coli UspA2 cells in a buffer at pH 5.0 (a,b) or at pH 7.4 (c,d). The orange and blue plot lines (b,d) represent calibration curves performed on different days.