Electrochemical Aptamer-Based Biosensor for Detecting Pap31, a Biomarker for Carrion's Disease

Abstract

Carrion's disease, caused by infection with the bacterium Bartonella bacilliformis (B. bacilliformis), is effectively treated with antibiotics, but reaches fatality rates of ~90% if untreated. Current diagnostic methods are limited, insufficiently sensitive, or require laboratory technology unavailable in endemic areas. Electrochemical aptamer-based (E-AB) biosensors provide a potential solution for this unmet need, as these biosensors are portable, sensitive, and can rapidly report the detection of small molecule targets. Here, we developed an E-AB biosensor to detect Pap31, a biomarker of Carrion's disease and an outer membrane protein in B. bacilliformis. We identified an aptamer with Pap31-specific binding affinity using a magnetic pull-down assay with magnetic bead-bound Pap31 and an aptamer library followed by electrophoretic mobility shift assays. We incorporated the Pap31-binding aptamer into a DNA oligonucleotide that changes conformation upon binding Pap31. The resultant Pap31 E-AB biosensor produced a rapid, significant, and target-specific electrical current readout in the buffer, demonstrating an apparent K_D of 0.95 nM with a limit of detection of 0.1 nM, and no significant signal change when challenged with off-target proteins. This proof-of-concept Pap31 biosensor design is a first step toward the development of more rapid, sensitive, and portable diagnostic tools for detecting Carrion's disease.

Keywords: E-AB; aptamer; carrion’s disease; electrochemical biosensor; neglected tropical disease.

Figure 1

Schematic of the Pap31 E-AB biosensor. In the unbound state, the aptamer (black curvy line) possesses relatively high levels of conformational flexibility and electron transfer between the gold-plated electrode (yellow disc) and methylene blue redox group (blue ball). Upon Pap31 binding, the aptamer adopts a more rigid conformation and relatively low levels of electron transfer. Together, this results in a signal-off mechanism. Created in BioRender. Bonham, A. (2024) BioRender.com/s17v834.

Figure 2

Pap31-specific aptamer binding and conformation switching. (a) Pap31 protein binding of the selected Pap31-specific aptamer via Pap31 titration in EMSAs, as described in Section 2.3. Aptamer binding affinity assessed via bound spot intensity and fit to a Langmuir isotherm model displayed robust and reproducible Pap31 affinity, with a K_D^app of 10 ± 2.6 nM. (b,c) Secondary structure prediction and optimization of this aptamer were performed to create a structure with two isoenergetic states: (b) a disrupted binding interface (i.e., Pap31 non-binding state) and (c) an intact Pap31-binding motif (i.e., Pap31 binding state) (images generated with RNAcanvas software, https://rna2drawer.app/, accessed on 2 October 2024 [23]).

Figure 3

The Pap31-binding E-AB biosensor sensitively and specifically detected Pap31. (a) Raw current values of a prepared biosensor in buffer (“Blank”) challenged with the addition of 1 uM Pap31 (“Pap31”) (top panel) and alternatively challenged with the addition of buffer added in the same manner as used for Pap31 addition, but with no Pap31 protein included (“Buffer”) (bottom panel). (b) Titration of Pap31 protein against the biosensor equilibrated in buffer showed signal-off dose-responsive current changes, with a K_D^app of 0.95 ± 0.29 nM. (c) This signal-off current change was not observed when the biosensor was challenged with non-target proteins ENOX2 (a secreted human protein biomarker; p-value < 0.03) or BSA (a major component of blood; p-value < 0.003) or serial addition of buffer vehicle only (p-value < 0.006). * p ≤ 0.05; ** p ≤ 0.01.