Minute-scale detection of SARS-CoV-2 using a low-cost biosensor composed of pencil graphite electrodes

Abstract

COVID-19 has led to over 3.47 million deaths worldwide and continues to devastate primarily middle- and low-income countries. High-frequency testing has been proposed as a potential solution to prevent outbreaks. However, current tests are not sufficiently low-cost, rapid, or scalable to enable broad COVID-19 testing. Here, we describe LEAD (Low-cost Electrochemical Advanced Diagnostic), a diagnostic test that detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within 6.5 min and costs $1.50 per unit to produce using easily accessible and commercially available materials. LEAD is highly sensitive toward SARS-CoV-2 spike protein (limit of detection = 229 fg⋅mL^-1) and displays an excellent performance profile using clinical saliva (100.0% sensitivity, 100.0% specificity, and 100.0% accuracy) and nasopharyngeal/oropharyngeal (88.7% sensitivity, 86.0% specificity, and 87.4% accuracy) samples. No cross-reactivity was detected with other coronavirus or influenza strains. Importantly, LEAD also successfully diagnosed the highly contagious SARS-CoV-2 B.1.1.7 UK variant. The device presents high reproducibility under all conditions tested and preserves its original sensitivity for 5 d when stored at 4 °C in phosphate-buffered saline. Our low-cost and do-it-yourself technology opens new avenues to facilitate high-frequency testing and access to much-needed diagnostic tests in resource-limited settings and low-income communities.

Keywords: ACE2; COVID-19; graphite; point of care; sensor.

Fig. 1.

LEAD, a rapid and low-cost electrochemical biosensor. (A) Schematic representation of graphite electrodes used in LEAD. (B) AuNPs-cys functionalization on graphite electrodes after modification with glutaraldehyde. (C) Modification of AuNPs-cys with ACE2 using EDC and NHS to enable amide bond formation and BSA for surface blockage. The analytical response of LEAD in the presence of SARS-CoV-2 was based on current suppression due to selective binding of viral SP with the ACE2-functionalized electrode, which partially blocked the electrodic area, leading to decreased peak current of the redox probe ([Fe(CN)₆]^3−/4− solution). (D) Cost and detection time comparison between LEAD and existing FDA-approved antigen, serological, and molecular tests (47).

Fig. 2.

AuNPs-cys and GPE characterization studies. (A) UV-vis spectrum obtained for HAuCl₄ solution (yellow color with a maximum absorbance band at 309 nm) and AuNPs-cys formation (wine color and absorbance band at 532 nm). (B) SEM image of AuNPs-cys dispersion with a histogram inset. The spherical AuNPs-cys present a mean diameter of 14.13 ± 0.18 nm. (C) SEM image of the bare GPE electrode polished showing a flat surface containing carbon sheets. (D) SEM image of the GPE electrode modified with GA and AuNPs-cys, which present a high AuNPs-cys distribution throughout the electrode, thus facilitating ACE2 immobilization and SARS-CoV-2 SP detection.

Fig. 3.

Functionalization steps and electrochemical characterization of LEAD. (A) Schematic representation of stepwise functionalization steps of LEAD. (B) CVs recorded for each modification step of the GPE surface in a solution of 5.0 mmol⋅L⁻¹ [Fe(CN)₆]^−3/−4 containing 0.1 mol⋅L⁻¹ KCl as the supporting electrolyte at a scan rate of 50 mV⋅s⁻¹. (C) Nyquist plots were obtained using the same conditions as in A. (Inset) A zoomed view of the plots in high-frequency regions. The following experimental conditions were used for these experiments: frequency range from 1 × 10⁵ Hz to 0.1 Hz and 10-mV amplitude. All measurements were performed at room temperature.

Fig. 4.

Kinetic study of the interaction between SARS-CoV-2 SP and LEAD. (A) Calibration curves built using SP at concentrations ranging from 1 × 10⁻¹² g⋅mL⁻¹ to 1 × 10⁻¹⁰ g⋅mL⁻¹ and using different incubation times (ranging between 1 and 7 min). Increased analytical sensitivities were achieved after 5 min (6.94 × 10⁻³ ± 1.00 × 10⁻³) and 7 min (5.42 × 10⁻³ ± 6.30 × 10⁻³). Thus, 5 min was chosen as the optimal incubation time for LEAD. All measurements were recorded in triplicate, and each measure was done using a different sensor. (B) Baseline-corrected SWVs for the 5.0 mmol⋅L⁻¹ [Fe(CN)₆]^3−/4− redox probe after the electrode incubation with different concentrations of SP ranging from 1 × 10⁻¹⁴ to 1 × 10⁻⁹ g⋅mL⁻¹. (C) Linear regression for the analytical curve constructed using the suppression current signal. Conditions: frequency, 80 Hz; amplitude, 70 mV, and step, 8 mV. All experiments were carried out in triplicate (n = 3), using 5.0 mmol⋅L⁻¹ [Fe(CN)₆]^3−/4− containing 0.1 mol⋅L⁻¹ KCl as the supporting electrolyte after exposure of the electrode to 50 µL of standard SP solution for 5 min. (D) Baseline-corrected SWV plots for tittered-inactivated viral solutions at concentrations ranging from 10² to 10⁶ PFU⋅mL⁻¹ in VTM. (E) Linearized correlation between the ΔI values and concentration of inactivated virus in solution. The analytical curve was carried out in triplicate measurements using different LEAD devices. (F) Stability study under different storage conditions: 25 °C (black circles), −20 °C (red circles), and stored dry at 4 °C (blue circles) and stored at 4 °C in PBS medium (pH = 7.4) (magenta circles) over 7 d. Sensitivity values were obtained by analytical curves in the concentration range from 1 × 10⁻¹² g⋅mL⁻¹ to 1 × 10⁻⁹ g⋅mL⁻¹ of SP. All experiments were carried out in triplicate (n = 3).

Fig. 5.

Cross-reactivity studies of LEAD using other coronaviruses and noncoronavirus strains. Baseline-corrected SWVs recorded at optimized experimental conditions before (black lines) and after incubation of the electrode with (A) SARS-CoV-2, (B) SARS-CoV-2 UK variant B, and possible interfering viruses such as (C) H₁N₁-A/California/2009, (D) Influenza-B/Colorado, (E) herpes simplex virus-2, and (F) MHV. The following conditions were used for all experiment: frequency of 80 Hz, amplitude of 70 mV, and step potential 8 mV. All experiments were performed in 5.0 mmol⋅L⁻¹ [Fe(CN)₆]^−3/−4 in 0.1 mol⋅L⁻¹ KCl. The specificity studies were carried out using the following viral strains: MHV at 10⁸ PFU⋅mL⁻¹ (coronavirus); H₁N₁, A/California/2009; Influenza B, B/Colorado; HSV2, and herpes simplex virus-2 (all at 10⁵ PFU⋅mL⁻¹). The analysis of the contagious SARS-CoV-2 B.1.1.7 UK variant showed that the variant presented higher interaction between SP and ACE2 compared to SARS-CoV-2. Each virus was incubated in a final volume of 50 µL for 5 min. All viruses were in stored VTM and experiments were performed at room temperature.