Evaluation of electropolymerized molecularly imprinted polymers (E-MIPs) on disposable electrodes for detection of SARS-CoV-2 in saliva

Abstract

We investigate electropolymerized molecularly imprinted polymers (E-MIPs) for the selective recognition of SARS-CoV-2 whole virus. E-MIPs imprinted with SARS-CoV-2 pseudoparticles (pps) were electrochemically deposited onto screen printed electrodes by reductive electropolymerization, using the water-soluble N-hydroxmethylacrylamide (NHMA) as functional monomer and crosslinked with N,N'-methylenebisacrylamide (MBAm). E-MIPs for SARS-CoV-2 showed selectivity for template SARS-CoV-2 pps, with an imprinting factor of 3:1, and specificity (significance = 0.06) when cross-reacted with other respiratory viruses. E-MIPs detected the presence of SARS-CoV-2 pps in <10 min with a limit of detection of 4.9 log₁₀ pfu/mL, suggesting their suitability for detection of SARS-CoV-2 with minimal sample preparation. Using electrochemical impedance spectroscopy (EIS) and principal component analysis (PCA), the capture of SARS-CoV-2 from real patient saliva samples was also evaluated. Fifteen confirmed COVID-19 positive and nine COVID-19 negative saliva samples were compared against the established loop-mediated isothermal nucleic acid amplification (LAMP) technique used by the UK National Health Service. EIS data demonstrated a PCA discrimination between positive and negative LAMP samples. A threshold real impedance signal (Z^Re) ≫ 4000 Ω and a corresponding charge transfer resistance (R_CT) ≫ 6000 Ω was indicative of absence of virus (COVID-19 negative) in agreement with values obtained for our control non-imprinted polymer control. A Z^Re at or below a threshold value of 600 Ω with a corresponding R_CT of <1200 Ω was indicative of a COVID-19 positive sample. The presence of virus was confirmed by treatment of E-MIPs with a SARS-CoV-2 specific monoclonal antibody.

Keywords: Biosensor; COVID-19; Electrochemical impedance spectroscopy; Electrochemical polymerization; Molecularly imprinted polymers; SARS-CoV-2.

Graphical abstract

Fig. 1

Schematic illustration of the proof-of-concept components, detection principle and detection procedure of COVID-19 E-MIPs. Initially, the SARS-CoV-2 pseudoparticle (pps) imprinted MIP is electrodeposited onto a disposable screen-printed electrode (SPE). In the presence of SARS-CoV-2 pps the anodic/cathodic peak currents to an external redox marker (ferricyanide) decrease and EIS spectra show a corresponding change, leading to qualitative and quantitative approaches to in-situ SARS-CoV-2 detection.

Fig. 2

Typical CVs illustrating the current density (j) changes in a 10 scan electropolymerization process (∼5 min, RT, 22 ± 2 °C) of SARS-CoV-2 pps (5.6 log₁₀ pfu/mL) imprinted MIP (A) and NIP (B) layers in synthetic saliva at a scan rate of 50 mV s⁻¹. CV characterisations (3rd scan) using 5 mM potassium ferricyanide solution containing 0.5 M KCl at a scan rate of 50 mV s⁻¹ of: MIP (C) and NIP (D) layers before (SPE) and after polymer modifications and following elution, pps rebinding and mAb addition. Rebinding (2 min, RT 22 ± 2 °C) conducted using 50 μL of 5.6 log₁₀ pfu/mL SARS-CoV-2 pps and then subjected to 50 μL mAb CR3022 (2 min, RT 22 ± 2 °C, 0.9 mg/mL). SPE represents the bare (unmodified) electrode. SEM scans of SPE before (E) and after polymerization (F) demonstrating the presence of polymer modification and topography change. Scans were conducted using a Thermo Fisher Quattro S scanning electron microscope at 5 kV and SPEs were measured in triplicate using 3 different sites.

Fig. 3

Factor analysis (A) and PCA (B) for polymer formation and SARS-CoV-2 pps capture (∼5 min, RT, 22 ± 2 °C) in the ‘qualitative’ approach. Each data point represents triplicate inputs collected from CV data using the current change in the first 5 cycles as variables. Groupings illustrate SARS-CoV-2 pps titres imprinted using saliva, based on log₁₀ pfu/mL: Neg = 0; Low = 4.5–4.7; Mid = 5.0–5.3; High = 5.5–5.6.

Fig. 4

(A) Nyquist plots for SPEs comparing the E-MIP and E-NIP layers, following SARS-CoV-2 pps rebinding and coupling with SARS-CoV-2 mAb CR3022; data fitted with Randles equivalent circuit, data represents mean, n = 3. (B) Schematic representation of the SPE modification stages corresponding to responses seen in Nyquist plots in Fig. 4A. (C) Nyquist plots for SARS-CoV-2 pps rebinding; data fitted with Randles equivalent circuit, data represents mean, n = 3. (D) Calibration curve for SARS-CoV-2 pps rebinding (2min, RT 22 ± 2 °C) using R_CT data as input, insert shows LDR following axis titles, data represents mean ± SD, n = 3.

Fig. 5

(A) PCA for polymer formation using ‘qualitative’ approach on 24 LAMP confirmed COVID-19 positive (red) and negative (blue) human saliva samples. (B) Typical Nyquist plots for SPEs comparing positive (MIP) and negative (NIP) confirmed saliva samples before and after treatment with 50 μL mAb CR3022 (0.9 mg/mL, 2 min, RT, 22 ± 2 °C). Fitted with Randles equivalent Circuit, data represents mean, n = 10. (C) Typical CV characterisations (3rd scan) of representative positive (MIP) and negative (NIP) sample depositions before and after 50 μL antibody treatment (0.9 mg/mL, 2 min, RT, 22 ± 2 °C). Schematic representations of each stage of treatment of positive and negative samples given on the right-hand side.