SARS-CoV-2 detection using a nanobody-functionalized voltammetric device.

Background: An ongoing need during the COVID-19 pandemic has been the requirement for accurate and efficient point-of-care testing platforms to distinguish infected from non-infected people, and to differentiate SARS-CoV-2 infections from other viruses. Electrochemical platforms can detect the virus via its envelope spike protein by recording changes in voltammetric signals between samples. However, this remains challenging due to the limited sensitivity of these sensing platforms.
Methods: Here, we report on a nanobody-functionalized electrochemical platform for the rapid detection of whole SARS-CoV-2 viral particles in complex media such as saliva and nasopharyngeal swab samples. The sensor relies on the functionalization of gold electrode surface with highly-oriented Llama nanobodies specific to the spike protein receptor binding domain (RBD). The device provides results in 10 min of exposure to 200 µL of unprocessed samples with high specificity to SARS-CoV-2 viral particles in human saliva and nasopharyngeal swab samples.
Results: The developed sensor could discriminate between different human coronavirus strains and other respiratory viruses, with 90% positive and 90% negative percentage agreement on 80 clinical samples, as compared to RT-qPCR. Conclusions: We believe this diagnostic concept, also validated for RBD mutants and successfully tested on Delta variant samples, to be a powerful tool to detect patients' infection status, easily extendable to other viruses and capable of overcoming sensing-related mutation effects.

Introduction

Diagnosis of the highly contagious Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), remains largely based on reverse transcription PCR (RT-qPCR), which identifies the genetic material of the virus in the nasopharyngeal area or in saliva[1,2]. The advantage of RT-qPCR is high sensitivity[3,4] with limitations being assay time and cost-related issues[4,5]. Rapid electrochemical detection of SARS-CoV-2 based on isothermal rolling cycle amplification with probes that were functionalized with redox active labels was recently proposed by Chaibun et al. allowing in less than 2 h to detect 1 copy µL−1 of N and S genes[6]. Time-related issues can be further overcome by rapid antigenic tests where the presence of SARS-CoV-2 is detected using surface anchored antibodies that recognize and attach to the viral spike antigen[7-13]. Although faster and cheaper compared to RT-PCR assays, rapid antigenic tests are currently less sensitive[9,14]. The several positive aspects of antigenic tests being simple to implement, prompted us to look for an electrochemical alternative of high sensitivity, which can easily be mass-produced and implemented in clinical settings and as point of care devices. Indeed, electrical and electrochemical platforms own qualitative and quantitative sensing capacity all together in a user-friendly sensing format[8-11,15-19]. An original detection of COVID-19 using an organic electrochemical transistor was recently reported by the group of Inal and co-workers[10]. The combination of a solution-processable conjugated polymer as a transistor channel together with nanobody-SpyCatcher fusion protein surface receptors allows SARS-CoV-2 spike protein detection in nasopharyngeal swab samples of different viral loads. Nevertheless, electronic-based protein sensors translate still poorly into market products due to complex sensor design.

We report here a highly performing electrochemical COVID-19 detection approach with following key features: (1) the use of an engineered SARS-CoV-2 specific nanobody as surface receptor, (2) controlled non-fouling surface biofunctionalization, (3) the ability to sense the infectivity of a patient by using the spike protein (S1) as target, and (4) quantification via differentical pulse voltammetry (DPV) read out. We found VHH-72-13C to be most adapted surface receptor for the electrochemical “signal off” diagnostic assay using ferrocenemethanol as a redox mediator. Integration of VHH-72-13C onto a poly(ethylene)glycol-modified gold electrode via maleimide-thiol linkage chemistry reliably and specifically detects SARS-CoV-2 viral particles electrochemically with a limit of detection of LoD = 1.2 × 104 viral RNA copies mL−1, corresponding to a Ct value of 33 using cultured SARS-CoV-2 virus particles and correlates to about 2 ± 1 PFU mL−1. The sensing technology remains operational on recognizing RBD mutations including clades related to the Alpha, Beta and Delta variants.

The performance of the sensor was validated in a study on 80 patients (40 positive, 40 negative) unprocessed nasopharyngeal and saliva samples. The in vitro diagnostic device showed a 90% positive percentage agreement (PPA) and a 90% negative percentage agreement (NPA), as compared to RT-qPCR for nasopharyngeal samples. In the case of saliva, a 80% PPA and a 85% NPA were determined when compared to RT-qPCR for nasopharyngeal samples of the same patients. The portability of the sensor and its read out, which can be directly connected to a mobile telephone completes the electrical signal processing, making it user-friendly and operational in different situations and environments. This technology is broadly applicable and only limited by the availability of nanobodies targeting the antigen of interest.

Methods

Materials

3-mercaptopropionic acid (98%, Ref: M5801), 1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride) (EDC, Ref: E7750), N-hydroxysuccinimide (NHS, Ref. 130672), and ferrocenemethanol (97%, Ref. 335061) were purchased from Sigma Aldrich and used as-received. Phosphate saline solution (PBS, 1×, Ref. 10010-015) was obtained from Thermo Fisher scientific. Maleimide-PEGx-amine (MW 1 kDa, Ref. LV3811) was purchased from Interchim Uptima. MQ-water was used throughout the whole study.

Universal transport Medium (UTM) was obtained from Copan, Italy (UMT-RT). The formulation of UTM-RT medium includes protein for stabilization, antibiotics to minimize bacterial and fungal contamination, and a buffer to maintain a neutral pH.

Screen printed electrodes were obtained from IPM -Intelligent Pollutant Monitoring Denmark, AUH3600, via Hdts France and consist of a 4 mm diameter gold electrode, and a silver/silver chloride reference and carbon counter electrode. Disposable PD 10 Desalting Columns (ref. 17–0851-01.) were purchased from Cytiva, France.

Surface modification approaches

Direct immobilization of VHH-72-C13

The as-received Au electrodes were exposed to 20 µL of VHH-72-C13 (0.1 mg/mL) for 12 h at 4 °C. The surface was washed with MQ-water and dried with an air duster. The resulting modified surface was washed copiously with MQ-water to remove excess nanobody.

Immobilization of VHH-72-C13 and VHH-H11D4-13C via PEG units

The Au electrodes were exposed to 10 µL of an aqueous solution of 3-mercaptopropionic acid (25 mM) for 30 min at room temperature. The surface was washed with MQ-water and dried with an air duster. Then, the acid-terminated surface was activated with EDC/NHS (1:1 molar ratio, 15 mM) for 20 min, followed by immersion into NH2-PEG6-maleimide (10 µL, 0.1 mg/m, in PBS 1×) for 2 h at 4 °C and washing with MQ-water. The interface was then modified with the VHH-72-C13 or VHH-H11D4-13C nanobody (10 µL, 0.1 mg/mL, PBS 1×)) for another 2 h at 4 °C. The resulting modified surface was washed copiously with MQ-water to remove excess nanobodies and unreacted reagents, and then stored at 4 °C before use.

Immobilization of VHH-72-C13 via 3-mercaptoproponic acid

The as-received gold electrodes were functionalized with 20 µL of an aqueous solution of 3-mercaptopropionic acid (25 mM) for 30 min at room temperature. The surface was washed with MQ-water and dried with an air duster. The interface was treated with EDC/NHS (1:1 molar ratio, 15 mM) for 20 min, followed by the addition of VHH-72 (20 µL, 0.1 mg mL−1) for 2 h at room temperature. The VHH-72 modified electrode was washed with water, and stored at 4 °C for further use.

Surface modification with 6-(ferrocenyl) hexanethiol

Attachment of 6-(ferrocenyl) hexanethiol to Au-PEG6-MAL electrodes: Au-PEG6-MAL interfaces were coated with 200 µL of 6-(ferrocenyl) hexanethiol (100 µg mL−1) for 2 h followed by washing (three times) with ethanol and MQ water (three times).

Characterisation

X-ray photoelectron spectra (XPS) were recorded with an SPECSLAB II (Phoibos-Hsa 3500 150, 9 channels) SPECS spectrometer with Al Kα source (E = 1486.6 eV) operating at 12 kV, pass energy (Epass = 40 eV), 0.1 eV energy step and acquisition time of 1 s per point. The residual pressure inside the analysis chamber was ∼1 × 10−8 Torr. All XPS were referenced according to the adventitious C1s peak at 284.5 eV.

Electrochemical measurements were performed with a Sensite Smart smartphone potentiostat (Palmsense, The Netherlands, distributed by HDts in France). Differential pulse voltammograms (DPV) were recorded at the appropriate potential range using following DPV parameters: taqu = 3 s, Estep = 0.01 V, Epulse = 0.06 V, tpulse = 0.02 V, scan rate = 0.06 V s−1. The active surface area of naked gold was determined to be 0.127 cm2.

Working principle of the sensor

The sensors were incubated for 10 min in 200 µL of sample in the collection vial and the solution was agitated by hand gently to increase mass transport. Longer times did not result in further change of the electrochemical signal thereafter. The incubation step was followed by washing in PBS (1×). To this interface, 200 µL of fresh ferrocenemethanol (1 mM, PBS 1×) was added and a DPV was recorded. The difference of the maximal current before and after contact with the sample was used to discriminate between positive and negative samples. A current density difference of 2 µA was used as a cut-off value to differentiate between both cases.

In the case of analysis of saliva samples, the electrochemical measurements were performed immediately (5–15 min) after sample collection to minimize protease degradation of the samples.

Nanobody production

The full lenght sequence of VHH72-C13 is as follows

MKYLLPTAAAGLLLLAAQPAQVQLQESGGGLVCAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGSHHHHHH

The N-terminal PelB leader sequence is highlighted in grey, the cysteine mutation is in red and the C-terminal purification tag is bold.

VHH-72-C13 was ordered as synthetic codon-optimized genes in pET24 vector by Twist Biosciences for production in E. coli. Genes were fused with the N-terminal PelB leader sequence (MKYLLPTAAAGLLLLAAQPA) for periplasmic protein expression and with a C-terminal purification His-tag. pET24 plasmid contains an inducible T7 promoter with isopropyl β-d-1-thiogalactopyranoside (IPTG) and kanamycin resistance.

VHH-72-C13 was also synthetized as a gBlocks codon-optimized gene fragment by IDT Technologies for production with HEK293 mammalian cells and fused with a C-terminal purification His-tag. Gene fragment was introduced into pYD11 expression vector by using the In-Fusion HD cloning kit (Takara). pYD11 plasmid contains a CMV promotor and ampicillin resistance, an IgK signal peptide (METDTLLLWVLLLWVPGSTG) for VHH secretion and a human Fc-tag downstream the cloning site. All cloned sequences were verified by DNA sequencing from Eurofin Genomics. Plasmids were then amplified under antibiotic selection after transformation of E. coli RapidTrans TAM1 competent cells (Active Motif). Plasmids were purified by using a NucleoBond Xtra Maxi plus kit (Macherey-Nagel). The plasmid is not available via Addgene, but is available from the authors upon request.

VHH-72-C13 and VHH-H11D4-C13 were produced in Rosetta (DE3) pLysS E. coli cells (Novagen) cultured in Turbo Broth media (AthenaES) at 37 °C up to an OD600 of 0.6. Cells were then induced with 0.1 mM IPTG. At this stage, the temperature was decreased to 28 °C and cells grew for an additional 18 h. Cells were harvested by centrifugation (4000 g for 10 min) and the pellet was homogenized and frozen in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.1 mg/mL lysozyme, 1 mM phenylmethylsulfonyl fluoride). After thawing, DNAse I (20 μg/mL) and MgSO4 (1 mM) were added and cells were lysed by sonication. The pellet and soluble fractions were separated by centrifugation (16,000 g for 30 min). VHH-72-C13 were purified from the soluble fraction on immobilized metal ion affinity chromatography using a 5 mL HisTrap crude Ni2+-chelating column (GE Healthcare) equilibrated in buffer A (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole). VHH was eluted with buffer A supplemented with 250 mM imidazole and was further purified by a size exclusion chromatography (HiLoad 16/60 Superdex 75 prep grade, GE Healthcare) equilibrated in PBS buffer. Purity of the protein was monitored at all stages of the purification process using SDS-PAGE and visualized by Coomassie blue staining.

Biological experiments

Vero E6 cells

(ATCC CRL-1586) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% antibiotics (100 U mL−1 penicillin, and 100 μg mL−1 streptomycin), in a humidified atmosphere of 5% CO2 at 37 °C.

Virus titration

Vero E6 cells were plated in 96-well plates (2.5 × 105 cells/well) 24 h before performing the virus titration. A clinical isolate, obtained from a SARS-CoV-2 positive specimen, was cultured on Vero E6 cells. Infected cell culture supernatant was centrifuged for 10 min at 1500 rpm at 4 °C to obtain a virus suspension. The virus suspension was used undiluted and in serial ten-fold dilutions (10−1 to 10−9). Virus suspensions were distributed in 6 wells in DMEM supplemented with 10% FBS (Fetal Bovine Serum) to Vero E6 cells, 1% antibiotics (100 U mL−1 penicillin, and 100 μg mL−1 streptomycin), and 1% L-glutamine. The plates were incubated for 6 days in 5% CO2 atmosphere at 37 °C. The plates were examined daily using an inverted microscope (ZEISS Primovert) to evaluate the extent of the virus-induced cytopathic effect in cell culture. Calculation of estimated virus concentration was carried out by the Spearman and Karber[20,21] method and expressed as TCID50/mL (50% tissue culture infectious dose). TCID50/mL values were transformed to PFU/mL by using the formula PFU/mL = TCID50/mL × 0.7.

SARS-CoV-2 RT-PCR

A real-time RT-aPCR method developed by the French Reference Center for respiratory viruses (Institut Pasteur, Paris)[22] was used. This method is a duplex RT-PCR targeting two regions in the RdRp gene, namely IP2 and IP4. G6PDH RT PCR using primers G6PDH-6(GAAGGTGAAGGTCGGAGT), G6PDH-231(GAAGATGGTGATGGGATTTC) and the probe G6PDH-202(5′FAM-CAAGCTTCCCGTTCTCAGCC-3′BHQ) was additionally performed to monitor for specimen quality, extraction and PCR inhibition. Undetectable SARS-CoV-2 levels were set to Ct 40. Amplification was performed on 7500 Real-Time PCR System (Applied Biosystems, USA).

PCR of other viruses

Briefly, nucleic acid isolation from respiratory specimens was carried out with the STARMag Universal Cartridge Kit on the Microlab NIMBUS instrument (Seegene, Seoul, South Korea), and virus detection was done using the Allplex™ Respiratory assay (Panels 1, 2, and 3) (Seegene) on a CFX96 thermal cycler (Biorad, Marnes-la-Coquette, France).

Clinical study

Cor-Dial-1 study “Rapid Detection of COVID-19 by Portable and Connected Biosensor: Biological Proof of Concept” is a case control study that prospectively enrolled 200 participants from consultation (outpatient), hospitalisation and intensive care unit (mean age: 46 ± 22 years (min = 1 year: max = 95 years), sex ratio male/female:1.07) including the 100 first people with a positive diagnosis of COVID-19 and the 100 first people with a negative diagnosis of COVID-19 defined by RT-qPCR by the medical team (from August to December 2020). The same nasopharyngeal swabs were used for COVID-19 RT-PCR and for the viral sensor. The final diagnosis of COVID-19 was planned to be independently performed by the medical team (EF and JP) using the notion of infectious contagion, the clinical signs, the pulmonary CT scan and the RT-qPCR of SARS-CoV-2 and sometimes the serology and a second RT-qPCR test for negative cases suspected to be positive. However, in this series, all the RT-qPCR positive cases were considered COVID-19 and all the negative RT-qPCR were considered not to be COVID-19. Consequently, only percentage of concordance between this study and RT-qCR were calculated (no Cohen’s Kappa Coefficient for concordance) and the sensitivity and specificity of the sensor.

Our study (Ref Protocol: 2021/0063; Ref IDRCB 2021-A00387-34; Ref promotor: CHU of Lille: 21.02.11.57302) was approved by the independent ethics committee of Iles de France Paris IX on 7th of April 2021 (REF No. 2021/22) as a type 3. The study has been registered on ClinicalTrials.gov ID: NCT04780334. All patients provided written, informed consent before inclusion.

Statistics and reproducibility

Three technical replicates were taken on the same sample to validate the reproducibility of the approach. The mean (±SEM) was traced in all the experiments.