A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond

Figure 1. Schematic illustration of biosensing system. (A) Throat swab culture acquisition. (B) Boron-doped diamond electrode surface modification with polyclonal anti-M1 antibodies, which then serve to identify the universal biomarker for influenza virus, the M1 protein.

Figure 2. Preparation of surface-modified BDD electrode. (A) Cyclic voltammetry curves recorded for bare and modified BDD electrode in 1 mM K3Fe(CN)6/0.1 PBS with a scan rate of 50 mV/s. (B) Impedance spectra of bare and modified BDD electrode after incubation in different solutions recorded in 1 mM K3Fe(CN)6/0.1 PBS at E f = + 0.13 V vs. Ag/AgCl/0.1 M KCl. (C) List of values of elements calculated from the electric equivalent circuit (EEQC) for bare and modified BDD electrode incubated in different solutions. Samples BDD-aM1 – BDD electrode modified with anti-M1 antibody; BDD-aM1-BSA – electrode saturated with BSA; BDD-aM1-PBS – electrode incubated with PBS; BDD-aM1-NEG – electrode incubated with biological samples taken from a healthy man.

Figure 3. Detection of influenza biomarkers. (A) Impedance spectra of BDD-aM1-BSA electrode after incubating in solutions with different M1 concentrations recorded in 1 mM K3Fe(CN)6/0.1 PBS at E f = + 0.13 V vs. Ag/AgCl/0.1 M KCl. (B) List of values of elements calculated from the EEQC for BDD-aM1-BSA electrode incubated in different solutions of M1 protein. (C) Comparison of charge transfer resistance for two electrodes incubated with H1N1 virus (V1) and H3N2 virus (V2). Error bars show the standard deviations of measurements taken from three independent experiments. Electrochemical species: 1 mM K3Fe(CN)6 in 0.1 PBS. (D) List of values of elements calculated from the EEQC for BDD-aM1-BSA electrode incubated in V1 and V2 in 0.5% Triton X-100/0.1 PBS solution.

Figure 4. The impedance spectra registered for pristine BDD, BDD modified with aM1-BSA, incubated for different periods (5–25 min) in 8 pg/mL concentrated solution (A). The relation between the value of charge transfer resistance and the virus protein concentration (values taken from Table B, Fig. 3) (B).

Figure 5. Impedance spectra registered during validation of the biosensors in emulated throat conditions at the formal potential of Fe(CN)6 3−/4− redox species (after incubation with different interfering pathogens). The tables represent the set of Rct values obtained on the basis of EEQC analysis applied to the impedance spectra for the working electrode after incubation in the different electrolyte conditions. The concentration of S. Aureus, C. Albicans and influenza virus (V1) was kept at 106 cfu or pfu. Abbreviations: V1 - H1N1, SA – Streptococcus Aureus, CA- Candida Albicans.

Figure 6. Comparison of parameters of previously reported detection methods of influenza virus versus novel ultrasensitive biosensor presented in this work7,15,20–24.