Rapid Real-Time Antimicrobial Susceptibility Testing with Electrical Sensing on Plastic Microchips with Printed Electrodes

Abstract

Rapid antimicrobial susceptibility testing is important for efficient and timely therapeutic decision making. Due to globally spread bacterial resistance, the efficacy of antibiotics is increasingly being impeded. Conventional antibiotic tests rely on bacterial culture, which is time-consuming and can lead to potentially inappropriate antibiotic prescription and up-front broad range of antibiotic use. There is an urgent need to develop point-of-care platform technologies to rapidly detect pathogens, identify the right antibiotics, and monitor mutations to help adjust therapy. Here, we report a biosensor for rapid (<90 min), real time, and label-free bacteria isolation from whole blood and antibiotic susceptibility testing. Target bacteria are captured on flexible plastic-based microchips with printed electrodes using antibodies (30 min), and its electrical response is monitored in the presence and absence of antibiotics over an hour of incubation time. We evaluated the microchip with Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) as clinical models with ampicillin, ciprofloxacin, erythromycin, daptomycin, gentamicin, and methicillin antibiotics. The results are compared with the current standard methods, i.e. bacteria viability and conventional antibiogram assays. The technology presented here has the potential to provide precise and rapid bacteria screening and guidance in clinical therapies by identifying the correct antibiotics for pathogens.

Keywords: antibiotic resistant pathogen; antibiotic susceptibility test; electrical sensing; flexible electronics; screen-printed electrodes.

Figure 1

Experimental design. (A) Schematic diagram of the biosensor with a microfluidic channel. (B) Photograph of a biosensor with interdigitated electrodes, PMMA microfluidic channel, integrated heater, and temperature sensor. (C) Antibiotic susceptibility measurement using label-free electrical sensing on a biosensor without surface modification. (D) On-chip bacteria capture and isolation from whole blood using affinity-based sensing and measurement of bacterial antibiotic susceptibility using electrical sensing.

Figure 2

Electrical characterization of a screen printed heater on a flexible plastic substrate. (A) Plot shows a linear relationship between applied voltage and temperature with R² of 0.954 in an empty microchannel. (B) Plot shows the response of the heater for different voltages. (C) Temperature stability (37 °C) of the heater for 14 h at constant voltage of 9 V in an empty channel. Error bars represent standard error of the mean (n = 3), p < 0.005.

Figure 3

Electrical response of E. coli in the presence of ampicillin, ciprofloxacin, and erythromycin over a period of 1 h. (A) Real time impedance measurement of E. coli in the presence of ampicillin. (B) Statistical analysis of the effect of ampicillin on E. coli (p = 0.0055, n = 3). (C) Viability test results of E. coli in the presence of ampicillin after an overnight incubation by measuring their OD at 600 nm wavelength, (p < 0.0001, n = 3). (E) Real time impedance measurement of E. coli in the presence of ciprofloxacin. (F) Statistical analysis of the effect of ciprofloxacin on E. coli (p = 0.1915, ns, n = 3). (G) Viability test results of E. coli in the presence of ciprofloxacin (p = 0.0850, n = 3). (I) Real time impedance measurement of E. coli in the presence of erythromycin. (J) Statistical analysis of the effect of erythromycin on E. coli (p = 0.0055, n = 3). (K) Viability test results of E. coli in the presence of erythromycin (p = 0.0850, n = 3). (D, H, and L) Antibiotic susceptibility of E. coli against ampicillin, ciprofloxacin, and erythromycin, respectively, using the disc diffusion method (24 h).

Figure 4

Electrical response of MRSA in the presence of daptomycin, gentamicin, and methicillin over a period of 1 h. (A) Real time impedance measurement of MRSA in the presence of daptomycin. (B) Normalized impedance magnitude of MRSA-spiked samples in the presence of daptomycin at t = 30 min (p = 0.05, n = 3). (C) Viability test results of MRSA in the presence of daptomycin after an overnight incubation by measuring their OD at 600 nm wavelength, (p < 0.0001, n = 3). (E) Real time impedance measurement of MRSA in the presence of gentamicin. (F) Normalized impedance magnitude of MRSA-spiked samples in the presence of gentamicin at t = 30 min (p = 0.1915, ns, n = 3). (G) Viability test results of MRSA in the presence of gentamicin (p = 0.0850, n = 3). (I) Real time impedance measurement of MRSA in the presence of methicillin. (J) Normalized impedance magnitude of MRSA-spiked samples in the presence of methicillin at t = 30 min (p = 0.0055, n = 3). (K) Viability test results of MRSA in the presence of methicillin (p = 0.0850, n = 3). (D, H, and L) Antibiotic susceptibility of MRSA against daptomycin, gentamicin, and methicillin, respectively, using the disc diffusion method (24 h).

Figure 5

On-chip bacteria capture and antibiotic susceptibility testing. (A) Photograph of the fabricated microchip: (i) surface modified microchip loaded with MRSA-spiked whole blood (10⁶ CFU/mL) (incubated for 30 min) and (ii) microchip washed with PBS. (B) Histogram plot indicating the bacterial load and recovery (>43%) for the two spiked blood samples used in the study (sample 1, 10⁸ CFU/mL; sample 2, 10⁷ CFU/mL) (n = 3). Ten micrograms per microliter anti-MRSA antibody was used for chip functionalization. (C) Electrical response of the captured MRSA (10⁵ to 10⁷ CFU/mL) in the presence of gentamicin (0.1–100 μg/mL). Error bars represents standard error of mean (n = 3). MRSA-spiked whole blood without antibiotic was used as control.

Figure 6

Growth of microorganisms in different media was monitored by measuring their impedance magnitude over a period of 1 h. Electrical response of E. coli (A) and MRSA (B) during bacterial growth in LB broth, 1× PBS, and purified and unpurified urine.