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

Abstract

According to the World Health Organization (WHO), almost 2 billion people each year are infected worldwide with flu-like pathogens including influenza. This is a contagious disease caused by viruses belonging to the family Orthomyxoviridae. Employee absenteeism caused by flu infection costs hundreds of millions of dollars every year. To successfully treat influenza virus infections, detection of the virus during the initial development phase of the infection is critical, when tens to hundreds of virus-associated molecules are present in the patient's pharynx. In this study, we describe a novel universal diamond biosensor, which enables the specific detection of the virus at ultralow concentrations, even before any clinical symptoms arise. A diamond electrode is surface-functionalized with polyclonal anti-M1 antibodies, which then serve to identify the universal biomarker for the influenza virus, M1 protein. The absorption of the M1 protein onto anti-M1 sites of the electrode change its electrochemical impedance spectra. We achieved a limit of detection of 1 fg/ml in saliva buffer for the M1 biomarker, which corresponds to 5-10 viruses per sample in 5 minutes. Furthermore, the universality of the assay was confirmed by analyzing different strains of influenza A virus.

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 R_ct 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 work^,,–.