A label-free nanowell-based impedance sensor for ten-minute SARS-CoV-2 detection

Abstract

This work explores label-free biosensing as an effective method for biomolecular analysis, ensuring the preservation of native conformation and biological activity. The focus is on a novel electronic biosensing platform utilizing micro-fabricated nanowell-based impedance sensors, offering rapid, point-of-care diagnosis for SARS-CoV-2 (COVID-19) detection. The nanowell sensor, constructed on a silica substrate through a series of microfabrication processes including deposition, patterning, and etching, features a 5 × 5 well array functionalized with antibodies. Real-time impedance changes within the nanowell array enable diagnostic results within ten minutes using small sample volumes (<5 μL). The research includes assays for SARS-CoV-2 spike proteins in phosphate-buffered saline (PBS) and artificial saliva buffers to mimic real human SARS-CoV-2 samples, covering a wide range of concentrations. The sensor exhibits a detection limit of 0.2 ng mL^-1 (1.5 pM) for spike proteins. Middle East respiratory syndrome (MERS-CoV) spike proteins are differentiated from SARS-CoV-2 spike proteins, demonstrating specificity.

Fig. 1. (a) Cross-sectional view of a single nanowell. (b) Equivalent circuit model. (c) Cross-sectional view of single nanowell adsorbing antibodies. (d) Cross-sectional view of the nanowell adsorbing target proteins. (e) Equivalent circuit of measurement platform using a lock-in amplifier.

Fig. 2. (a) Microfabrication procedures for nanowell sensors. Follow the arrows: first step: first 100 nm of the gold electrode after deposition and lift-off; second step: first 40 nm Al₂O₃ layer by ALD; third step: second 100 nm gold electrode using the same fabrication processes as the first layer; fourth step: second 40 nm Al₂O₃ layer by ALD; fifth step: zoom-in view of well-shaped arrays in the middle of overlapping area are exposed by multiple etchings for two Al₂O₃ layers and one gold layer; sixth step: finish connection setup with conductive wires and epoxy, and install PDMS fluidic cell. (b) From left to right: 1. View of a fabricated wafer; 2. Single nanowell sensor; 3. Microscope view of electrodes; 4. Microscope view of 5 × 5 well-shaped arrays.

Fig. 3. An example of the data analysis methods using 1000 ng mL⁻¹ SARS-CoV-2 spike protein samples. (a) Comparison of real-time impedance monitoring after pipetting 1× PBS, antibodies, and antigens. (b) Comparison of two analysis methods.

Fig. 4. Sensing of spike protein in saliva with antibody in 1× PBS solution. (a) Titration curve using updated data analysis method for SARS-CoV-2 spike proteins in saliva and antibodies in 1× PBS with 50–500 ng mL⁻¹ dynamic range and 200 ng mL⁻¹ (1.5 nM) detection limit; linear fit: (Z) = 8.36–6.11 × (C), R-square = 0.97. (b) Titration curve using previous data analysis method for SARS-CoV-2 spike proteins in saliva and antibodies in 1× PBS with 50–500 ng mL⁻¹ dynamic range and 200 ng mL⁻¹ (1.5 nM) detection limit; linear fit: (Z) = 6.74–5.2 × (C), R-square = 0.99, * Denotes negative control (C = 0 ng mL⁻¹) plotted at C = 10 ng mL⁻¹ for visualization purposes, N = 5 for C = 0 ng mL⁻¹, N = 1 for all other concentrations.

Fig. 5. Sensing of spike protein in saliva with antibody in 0.18× PBS solution. Negative control (C = 0 ng mL⁻¹) plotted at C = 0.01 ng mL⁻¹ for visualization purposes. (a) Data using updated data analysis method for SARS-CoV-2 spike proteins in saliva and antibodies in 0.18× PBS with 0.1–1000 ng mL⁻¹ dynamic range; linear fit: (Z) = 3.49–2.17 × log (C), R-square = 0.94. (b) Data using previous data analysis method for SARS-CoV-2 spike proteins in saliva and antibodies in 0.18× PBS with 0.1–1000 ng mL⁻¹ dynamic range; linear fit: (Z) = 3.49–2.11 × log (C), R-square = 0.95. *P ≤ 0.05; ***P ≤ 0.0001, N = 2 for C = 0 ng mL⁻¹, N = 5 for C = 50 ng mL⁻¹, N = 3 for all other concentrations.

Fig. 6. Comparisons between MERS-CoV spike proteins, SARS-CoV-2 spike proteins, and negative control using SARS-CoV-2 spike antibodies. Only the red SARS-CoV-2 spike protein curve gives a response to the SARS-CoV-2 antibodies, and MERS-CoV curves follow the same trends as the negative control curves, proving the device is capable of differentiating SARS-CoV-2 proteins from similar respiratory disease samples.