Label-Free Electrochemical Sensor for Rapid Bacterial Pathogen Detection Using Vancomycin-Modified Highly Branched Polymers

Abstract

Rapid point of care tests for bacterial infection diagnosis are of great importance to reduce the misuse of antibiotics and burden of antimicrobial resistance. Here, we have successfully combined a new class of non-biological binder molecules with electrochemical impedance spectroscopy (EIS)-based sensor detection for direct, label-free detection of Gram-positive bacteria making use of the specific coil-to-globule conformation change of the vancomycin-modified highly branched polymers immobilized on the surface of gold screen-printed electrodes upon binding to Gram-positive bacteria. Staphylococcus carnosus was detected after just 20 min incubation of the sample solution with the polymer-functionalized electrodes. The polymer conformation change was quantified with two simple 1 min EIS tests before and after incubation with the sample. Tests revealed a concentration dependent signal change within an OD₆₀₀ range of Staphylococcus carnosus from 0.002 to 0.1 and a clear discrimination between Gram-positive Staphylococcus carnosus and Gram-negative Escherichia coli bacteria. This exhibits a clear advancement in terms of simplified test complexity compared to existing bacteria detection tests. In addition, the polymer-functionalized electrodes showed good storage and operational stability.

Keywords: AMR; bacteria pathogen detection; electrochemical impedance spectroscopy (EIS); highly branched polymers; label-free; point of care diagnostics.

Scheme 1

Test principle of the EIS-based sensor for detection of Gram-positive bacteria with vancomycin-modified HB-PNIMAM polymers immobilized on gold electrodes (a), which undergoes a coil-to-globule phase transition upon binding to Gram-positive bacteria (b) and thus causes a reduction in the R_ct value in the EIS Nyquist plot (c); the figure was created with BioRender.com (accessed on 1 February 2021).

Figure 1

Molar mass distributions of the HB-PNIPAM-Van polymers obtained by size exclusion chromatography in methanol with refractive index and viscometric detection.

Figure 2

(a) Functionalization of gold screen-printed electrodes with three different types of vancomycin-modified highly branched polymers (HB-PNIPAM-A, B, C; see Table 1) shown by increased R_ct values after polymer immobilization on electrodes that were tested before and after (with baseline, grey bars) and only after polymer attachment (without baseline, red bars) compared to the R_ct values of bare, unmodified gold electrodes (blue bars), respectively; data are presented as mean ± standard error of the mean (SEM); n = 3. (b) EIS Nyquist plot of an unmodified, bare gold electrode (blue line + symbol) and an electrode tested after functionalization with the HB-PNIPAM-Van-C polymer without an initial baseline EIS measurement (red line + symbol); the green lines show the fitted data using the Randles equivalent circuit model.

Figure 3

Bode plot EIS spectra of the same electrodes as shown in Figure 2b with the phase shift (dark blue) and absolute values of the impedance (light blue) of the unmodified, bare gold electrode plotted against the tested frequencies and the phase shift (red) and absolute values of the impedance (orange) of an electrode tested after functionalization with the HB-PNIPAM-Van-C polymer without an initial baseline EIS measurement.

Figure 4

Storage stability of vancomycin-modified polymer HB-PNIPAM-Van-C functionalized electrodes demonstrated by stable R_ct values detected on a single set of polymer functionalized electrodes retested four times over 13 days (red bars); a set of bare, unmodified gold electrodes was re-tested over the same period of time for comparison (grey bars); data are presented as mean ± SEM; n = 3.

Figure 5

Temperature-induced R_ct signal change ratios of electrodes immobilized with three different vancomycin-modified highly branched polymers tested after incubation at different temperatures with EIS measurements; the mean R_ct values for each measurement are expressed as a ratio relative to the first RT measurement; data are presented as mean ± SEM; n = 4.

Figure 6

(a) R_ct signal change ratio after incubation of vancomycin-modified highly branched polymer HB-PNIPAM-Van-C functionalized electrodes with S. carnosus and E. coli suspensions in PBS (OD₆₀₀ = 0.2) and with PBS buffer alone for 2 h in a 15 mL reaction tube with the electrodes covered with 3.5 mL bacteria solution at 21 °C (RT), 30 °C, 35 °C, respectively; results presented as a ratio of mean R_ct for an electrode after incubation with the target relative to the same value before incubation; data are presented as mean ± SEM; significance was determined using ANOVA multiple comparison tests; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 6. (b) EIS Nyquist plots of vancomycin-modified highly branched polymer HB-PNIPAM-Van-C functionalized electrodes before and after incubation with S. carnosus (green: before; blue: after) and E. coli (orange: before; red: after) at 35 °C, respectively. The dash-dotted lines at the matched colours represent the fitted data based on the Randles equivalent circuit.

Figure 7

EIS Bode plots of vancomycin-modified highly branched polymer HB-PNIPAM-Van-C functionalized electrodes before and after incubation with S. carnosus (a) and E. coli (b) at 35 °C, respectively.

Figure 8

(a) R_ct signal change ratio after incubation of vancomycin-modified highly branched polymer HB-PNIPAM-Van-C functionalized electrodes with S. carnosus and E. coli suspensions in PBS (OD₆₀₀ = 0.1) and with PBS buffer alone for 20 min or 2 h in the measurement cell with 80 µL sample volume or in a 15 mL reaction tube with the electrodes covered with 3.5 mL bacteria solution at 30 °C, respectively; results presented as a ratio of mean R_ct for an electrode after incubation with the target relative to the same value before incubation; data are presented as mean ± SEM; significance was determined using ANOVA multiple comparison tests; * p < 0.05; n = 4. (b) Nyquist plot EIS spectra of HB-PNIPAM-Van-C functionalized electrodes tested with S. carnosus (green: before incubation with S. carnosus; blue: after incubation) and E. coli (orange: before incubation with E. coli; red: after) (OD₆₀₀ = 0.1, 2 h incubation in measurement cell). The dash-dotted lines at the matched colours represent the fitted data based on the Randles equivalent circuit.

Figure 9

EIS Bode plots of vancomycin-modified highly branched polymer HB-PNIPAM-Van-C functionalized electrodes before and after incubation with S. carnosus (a) and E. coli (b) at 30 °C for 2 h in the measurement cell, respectively.

Figure 10

R_ct signal change ratio after 2 h incubation with different concentrations of S. carnosus Gram-positive bacteria (bacterial suspensions with an OD₆₀₀ of 0, 0.002, 0.02, 0.1, 0.2, 0.4 at 35 °C, respectively); data are presented as mean ± SEM; n = 4.