key: cord-0790249-nfit5kv0
authors: Zhao, Hui; Liu, Feng; Xie, Wei; Zhou, Tai-Cheng; OuYang, Jun; Jin, Lian; Li, Hui; Zhao, Chun-Yan; Zhang, Liang; Wei, Jia; Zhang, Ya-Ping; Li, Can-Peng
title: Ultrasensitive supersandwich-type electrochemical sensor for SARS-CoV-2 from the infected COVID-19 patients using a smartphone
date: 2020-09-14
journal: Sens Actuators B Chem
DOI: 10.1016/j.snb.2020.128899
sha: e8db5699cfe58f85eba0886f8822d3831f9cb67a
doc_id: 790249
cord_uid: nfit5kv0

The recent pandemic outbreak of COVID-19 caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a threat to public health globally. Thus, developing a rapid, accurate, and easy-to-implement diagnostic system for SARS-CoV-2 is crucial for controlling infection sources and monitoring illness progression. Here, we reported an ultrasensitive electrochemical detection technology using calixarene functionalized graphene oxide for targeting RNA of SARS-CoV-2. Based on a supersandwich-type recognition strategy, the technology was confirmed to practicably detect the RNA of SARS-CoV-2 without RNA amplification and reverse-transcription by using a portable electrochemical smartphone. The biosensor showed high specificity and selectivity during in silico analysis and actual testing. A total of 88 RNA extracts from 25 SARS-CoV-2-confirmed patients and eight recovery patients were detected using the biosensor. The detectable ratios (85.5% and 46.2%) were higher than those obtained using RT-qPCR (56.5% and 7.7%). The limit of detection (LOD) of the clinical specimen was 200 copies/mL, which is the lowest LOD among the published RNA measurement of SARS-CoV-2 to date. Additionally, only two copies (10 μL) of SARS-CoV-2 were required for per assay. Therefore, we developed an ultrasensitive, accurate, and convenient assay for SARS-CoV-2 detection, providing a potential method for point-of-care testing.

. As human-to-human transmission rapidly increased, COVID-19 has spread globally and poses a threat to public health in more than 200 countries. On March 11, 2020 , the World Health Organization (WHO) classified the COVID-19 outbreak as a pandemic [3] . As of 4 Sep 2020, more than 26,495,880 cases of COVID-19 have been confirmed around the world, resulting in 873,618 deaths [4] . Hence, early and accurate diagnostics is undoubtedly of vital importance to the containment of COVID-19 because it facilitates the control of infection sources and monitoring of illness progression.

Given that COVID-19 patients have nonspecific symptoms, SARS-CoV-2 detection is indispensable in accurate diagnosis. SARS-CoV-2 is a novel coronaviridae virus possessing a single-strand and positive RNA genome with ~3 kb length [5] . The genome comprises a 5′ untranslated region (UTR), replicase complex (ORF1ab), spike surface glycoprotein gene (S gene), small envelope gene (E gene), matrix gene (M gene), nucleocapsid gene (N gene), 3′UTR, and several unidentified non-structural open reading frames [5] . Although antibody-based serological test is rapid and convenient, the shortcomings of the technology limit its applicability. For example, generating an antibody against SARS-CoV-2 following symptom onset for detection takes a substantial amount of time. SARS-CoV-2 antibodies have potential crossreactivity with antibodies generated against other coronaviruses. Therefore, nucleic acid-based real-time reverse transcription PCR (RT-qPCR) assays are globally utilized as a golden standard for virus RNA detection. However, RT-qPCR has some drawbacks, such as expensive instruments and reagents, and need for trained personnel, and thus specimens needs to be shipped to reference laboratories. Currently, 11 nucleic-acid-J o u r n a l P r e -p r o o f based RT-qPCR detection kits have been approved by the China National Medical Products Administration (NMPA) for SARS-CoV-2 diagnostics [6] . False-negative results as high as 20% to 40% have been reported in China [7] . These results may be attributed to various factors, including sample source and quality, personnel operation, and test kit sensitivity. Undisputedly, detectable sensitivity is a crucial issue for the accurate diagnosis of COVID-19. According to the report by Wang et al. [8] , six commercial RT-qPCR kits approved by the China NMPA have poor limits of detection (LODs) and likely lead to false-negative results. Therefore, developing accurate and easy-to-implement methods for COVID-19 detection is necessary.

Electrochemical biosensors provide an alternative and reliable solution to clinical diagnosis due to their advantages, such as high sensitivity, low cost, user-friendliness, and robustness [9] . Especially, with the miniaturization and intelligent development of electrochemical device, electrochemical biosensors are considered useful in clinical diagnosis and point-of-care testing (POCT). In the field of nucleic acid biosensors, a supersandwich-type electrochemical biosensor has attracted considerable attention due to their high specificity and sensitivity [10] . This type of biosensor is composed of a capture probe (CP), target sequence, label probe (LP), and auxiliary probe (AP) [11] .

The 5′-and 3′-terminals of target sequence are complementary to CP and LP, respectively, and the 5′-and 3′-regions of AP have complementary sequences with two different LP areas [11, 12] . Therefore, sequence-specific detection can be achieved by using CP and LP, and AP hybridizes many times with LP to produce long concatamers, resulting in high sensitivity. However, in a traditional supersandwich-type J o u r n a l P r e -p r o o f electrochemical biosensor, each LP was labeled only one signal molecule and resulted low current signal. Therefore, we hypothesized that the sensitivity of the biosensor can be improved by facilitating of LP with signal molecules through other molecules or materials.

Host-guest recognition has attracted attention in the fabrication of electrochemical biosensors. Given that host-guest recognition motifs are specific and biorthogonal, they can form stable host-guest inclusion to increase the enrichment capability of guest molecules due to own more rigid and well-defined cavity [13] [14] [15] . Interesting macrocyclic host molecules, calixarenes, such as CX8, show excellent supramolecular recognition and enrichment capability for the electrochemical mediators of methylene blue and toluidine blue (TB) [16] [17] [18] [19] . Additionally, Au metal nanoparticles (NPs) have been widely used in improving biosensor sensitivity due to their various advantages, such as good conductivity, large surface area, and strong adsorption capability [20] .

Through the coordination of Au-S, probe functionalized sulfhydryl groups are immobilized with Au NPs anchored on material surfaces [17, 21] .

In the present study, we developed a supersandwich-type electrochemical biosensor based on p-sulfocalix [8] arene (SCX8) functionalized graphene (SCX8-RGO) to enrich TB for SARS-CoV-2 RNA detection (Scheme 1). We developed a plug-and-play method to achieve the sensitive, accurate, and rapid detection of SARS-CoV-2 samples from various clinical specimens without RNA amplification using an electrochemical biosensor equipped with a smartphone, providing a simple, low-cost and useful method for POCT. 

Graphite oxide was purchased from Nanjing XFNANO Materials Tech Co., Ltd (Nanjing, China). SCX8 was obtained from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). A Carbon-Three Electrode screen printing carbon electrode (SPCE) was purchased from Zensor Research & Development Co., Ltd (Beijing, China). TB was obtained from Aladdin Industrial Corporation (Shanghai, China). All the reagents were of analytical grade. All the aqueous solutions were prepared with diethyl pyrocarbonate, and the effect of RNAase on the mRNA stability was minimized by autoclaving all the sample tubes and glassware.

Differential pulse voltammetry (DPV) was performed with a smartphone equipped with a Sensit Smart electrochemical workstation from Palmsens (Netherlands). The morphologies of the prepared samples were characterized by JEM 2100 transmission electron microscopy (TEM, Tokyo, Japan). A Thermo Fisher Scientific Nicolet IS10 Fourier transform infrared (FTIR, Waltham, USA) Impact 410 spectrophotometer and a Q50 thermogravimetric analysis (TGA) instrument (New Castle, USA) were used for the FTIR study and TGA analysis, respectively. An ESCALAB 250 photoelectron spectrometer (Thermo-VG Scientific, USA) was used for X-ray photoelectron spectroscopy (XPS) analysis. A Bruker D8-advance X-ray diffractometer (Germany) J o u r n a l P r e -p r o o f was carried out X-ray powder diffraction (XRD) experiment. A Malvern Zetasizer Nano (Malvern, England) electrochemical workstation was used for the zeta potential measurements. QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) was used for qPCR experiment.

Primer pairs were synthesized according to the sequences provided by the Chinese Center for Disease Control and Prevention (CDC) and used to amplify ORF1ab gene in real-time PCR (qPCR). The sequences of CPs to ORF1ab gene were selected from the above PCR amplicon sequences. The sequences of the designed probes and primers are summarized in Table 1 . The primers and probes were synthesized by Tsingke Biotechnology (Beijing, China). In specificity analysis, we aligned the complete genomes of SARS-CoV-2 through the BLAST analysis of NCBI COVID resources (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and then a high conservation region was selected. The blast analyses were further performed using the genomes of the 39 respiratory pathogens listed in Table S1 .

For premix A preparation, Fe3O4 NPs were prepared and dissolved, and then PEG400, trisodium citrate, HAuCl4, and ascorbic acid were successively added for the production of Au@Fe3O4 nanocomposite. Then, 100 µL of 1 mg mL -1 Au@Fe3O4 dissolved in Buffer I (10 mM Tris-HCl containing 1 mM EDTA, 300 mM NaCl, and 10 J o u r n a l P r e -p r o o f mM TCEP, pH7.4) was incubated with 10 µL of 1 µM CP at 4 °C for 12 h. For the eliminate of non-specific binding and removal liquid supernatant, 10 µL of 1 mM hexane-1-thiol (HT) was added to the above mixture at room temperature for half an hour. Lastly, the precipitation was dissolved with 100 µL of Buffer II (10 mM Tris-HCl containing 1 mM EDTA, 300 mM NaCl, and 1 mM MgCl2, pH 7.4) and mixed slowly at 4 °C for 12 h.

For premix B preparation, graphene oxide (GO) and SCX8 aqueous solution were refluxed and dissolved through sonication. Then, HAuCl4 and TB solution were successively added for the production of Au@SCX8-RGO-TB nanocomposites. Next, 100 µL of 1 mg mL -1 Au@SCX8-RGO-TB dissolved in Buffer I was incubated with 10 µL of 1 µM LP at 4 °C for 12 h. After the supernatant was removed, the precipitate was added to 10 µL of 1 µM AP and 90 µL of Buffer II, and the resulting mixture was stored at room temperature before use.

Detection samples included artificial targets and clinical RNA samples. The sequences of artificial targets are listed in Table 1 . Given that RNA is easy to degrade, we synthesized the corresponding target sequences of single-strand DNA (ssDNA) according to the published RNA sequences of SARS-CoV-2 (GenBank No.

MN908947.3) for electrochemical detection. The annealed sequences were cloned to a pUC57 plasmid for copy number calculation.

All the clinical specimens used in this study were collected from the Second 

The mixture of 50 µL of premix A and 10 µL of detection samples were incubated for 1 h, and the supernatant was removed through magnetic separation. Then, 50 µL of premix B was added to the sediment and incubated for 2 h. The supernatant was removed by magnetic separation and washed with PBS (pH 7.2) three times. Finally, the resulting nanocomposite was dissolved in 50 µL of PBS and dropped on SPCE for electrochemical measurement. All the reactions were performed at room temperature.

J o u r n a l P r e -p r o o f

The measurements of SARS-CoV-2 were performed using a commercial 2019-nCOV ORF1ab/N nucleic acid detection kit (Tianlong, Xi'an, China). The reaction was set up according to the manufacturer's protocol. ORF1ab or N genes with a cycle threshold (Ct value) of <37 was considered positive samples. The copy number concentration of the plasmid with the ORF1ab fragment was calculated using the following formula: copies/mL = 6.02 × 10 23 × 10 -6 × concentration (ng/µL)/(fragment length × 660). Then, the 10-fold serial dilutions of the plasmid ranged from 10 3 to 10 9 copies/mL and subjected to qPCR. A standard curve was obtained.

Fisher's exact test was used in comparing the performance of the assays with SPSS 22.0 (IBM). A p-value of <0.05 was considered statistically significant and indicated that the sample is a positive sample.

For the characterization of Au@Fe3O4 particles, the morphology and microstructure of Au@Fe3O4 were analyzed through SEM. As shown in Figure 1A showed special characteristics at 38.2°, 44.4°, and 64.6° (Fig. 1C) , and the EDS result showed that the contents of Fe, O, and Au in the Au@Fe3O4 composite material were 64.9%, 33.2%, and 1.8%, respectively (Fig. 1D) . As shown in Fig. 1E , Au@Fe3O4 was negative-charged, which was attribute to the Au NPs on the Fe3O4 surface. The average particle size of Au@Fe3O4 was approximately ~388.3 nm (Fig. 1F) . All the results confirmed the successful loading of Au-NPs with Fe3O4.

The morphology of the RGO-SCX8-Au composite material was investigated through SEM, RGO-SCX8-Au was a single-layer sheet structure, and Au NPs were evenly distributed on its surface ( Fig. 2A) . As shown in Figure 2B , the FTIR spectrum revealed a stretching vibration of -OH (3440 cm -1 ), an oxygen-containing functional group C-O/C-C (1046 cm -1 ), and a conjugate C=C (1631 cm -1 ) in RGO material, and the peak vibration of -OH (3440 cm -1 ) and O-H bending (1400 cm -1 ) significantly enhanced in the RGO-SCX8 composite. Furthermore, the characteristic peak of CH2 (3190 cm -1 ) and typical peaks of -SO3at 1177 cm -1 were observed in the RGO-SCX8 composite, suggesting that SCX8 was successfully grafted onto the RGO. The TGA result showed that the lost mass of RGO was approximately 34.1% at 600 o C. By contrast, the RGO-SCX8 material lost approximately 64.5% mass at the same temperature (Fig. 2C) . Thus, the mass loss caused by the decomposition of SCX8 was 30.4% in the RGO-SCX8 composites, indicating that the RGO-SCX8 material was successfully prepared. The XPS patterns of RGO-SCX8-Au showed Au, C, and O were detected in the material (Fig. 2D ). As shown in Fig. 2E , the Zeta potential of the RGO-J o u r n a l P r e -p r o o f SCX8-Au composites was -28.9 mV, suggesting that the colloidal stability of RGO-SCX8-Au was well dispersed. All the above results demonstrated that the successful preparation of the RGO-SCX8-Au nanocomposites.

In this study, we designed and assembled the supersandwich-type biosensor for SARS-CoV-2 through the following procedures: i) the CPs labeled with thiol were immobilized on the surfaces of the Au@Fe3O4 nanoparticles and formed CP/Au@Fe3O4 nanocomposites; ii) the host-guest complexes (SCX8-TB) were immobilized on RGO to form Au@SCX8-TB-RGO-LP bioconjugate; iii) the sandwich structure of "CP-target-LP" produced; and iv) AP was introduced to form long concatamers (Scheme 1).

To monitor the assembling process of the modified SPCE electrode, we characterized the interface properties using electrochemical apparatus by EIS techniques. As shown in Figure 3A , the results of the impedance spectra showed that 

Given that COVID-19 is a person-to-person transmission disease, we synthesized the artificial target of ssDNA according to the sequences of SARS-CoV-2 RNA and explored the feasibility of the biosensor. As shown in Fig. 3B , a high electrochemical signal peak (DPV) was observed after incubation with the artificial target (10 -12 M), and DPV signal was extremely weak in the absence of a target, suggesting that the proposed electrochemical method is feasible for SARS-CoV-2 detection. The conditions for the SARS-CoV-2 biosensor assay on the ORF1ab gene were optimized. Specifically, 50 µL of premix A and 10 µL of target samples were incubated at room temperature for 1 h, then incubated with 50 µL of premix B at room temperature for 2 h (Fig. S1) . Finally, the electrochemical signal of TB was detectable in <10 sec by the portable smartphone (Scheme 1). Thus, the assay was easy-to-implement and rapid. demonstrating that electrochemical assay is more sensitive than RT-qPCR assay for SARS-CoV-2 determination.

The respiratory samples used for diagnosing COVID-19 were divided into upper respiratory samples (throat swab, oral swab, and oropharyngeal swab) and lower respiratory samples (e.g. sputum). In accordance with other studies [24, 25] , our results showed the sputum of lower respiratory sample was a reliable sample source for SARS-CoV-2 detection attributed to high viral load (11/11 for biosensor detection and 10/11

for RT-qPCR assay; Table 2 ). However, upper respiratory samples were broadly recommended for diagnosis because lower respiratory samples, especially for bronchoalveolar fluid and tracheal aspirates, have a high risk for aerosol generation [26] .

Therefore, developing a sensitive detection method for samples with a low viral load is of vital importance. Interestingly, compared with to RT-qPCR assay, our SARS-CoV-2 biosensor was superior to other assay in the detection of upper respiratory samples and other low-viral-load samples from feces, urine, and plasma (Table 2) .

To investigate detectable sensitivity, we analyzed the LODs of clinical specimens with the SARS-CoV-2 biosensor. First, the concentration of viral RNAs extracted from throat swabs were measured in copies per milliliter. The resulting calibration plot for log(copy numbers) vs. Ct values (Fig. S2 ) was used. Then, the diluted viral RNA samples were detected 10 times with the SARS-CoV-2 biosensor for each concentration. Finally, the lowest concentration level with a detection rate of 100% for positive results J o u r n a l P r e -p r o o f was regarded as the LOD of the SARS-CoV-2 biosensor. Consequently, the LOD of the proposed SARS-CoV-2 biosensor was confirmed to be 200 copies/mL (Table S2) . Other published assays for SARS-CoV-2 detection are listed in Table 3 . Intriguingly, our method has the lowest LOD and required the lowest number of copies per assay, providing an ultrasensitive assay. The proposed SARS-CoV-2 biosensor presented high sensitivity and specificity due to the following factors: i) the use of the supersandwichtype electrochemical biosensor improved binding specificity and increased signal enrichment ability; ii) several nanomaterials of high conductivity promoted signal intensity; and iii) the supermolecular recognition played an important role in the enrichment of electroactive molecule TB for improving sensitivity of the biosensor.

To ensure detection accuracy, we initially performed homology analyses of our designed CP sequences targeting SARS-CoV-2 in silico. After the alignment of 2291 the complete genomes of SARS-CoV-2 obtained from GenBank databases, the results showed the SARS-CoV-2 RNA sequences binding to CP were completely conserved (100%). A similar result was obtained by Wang et al [27] . Selectivity test was performed on an artificial one-mismatch target (1MT) and two-mismatch target (2MT) with the SARS-CoV-2 biosensor. Compared with the distinct current peak in the presence of the artificial target, the response was hardly detected incubation with 1MT, 2MT, or PBS (blank), respectively (Fig. 3B) . (Table S1) To the best of our knowledge, this work is the first to report the electrochemical detection of SARS-CoV-2 with a smartphone. Notably, the method does not require nucleic acid amplification and reverse transcription, samples are not needed to be transferred to laboratories, and no large-scale instrument and educated analysts are required. Thus, our proposed technology is a novel and plug-and-play diagnostic system, and the near-POC test remedies the shortcomings of PCR-based RNA assays.

The future development of this technology is to explore microfluidic-based cartridges for high-throughput diagnostics. 

The authors declare that they have no known competing financial interests or personal relationshipsthat could have appeared to influence the work reported in this paper. Table 1 Sequences of artificial target, probes, and RT-qPCR primers used in this study. 

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Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

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A new coronavirus associated with human respiratory disease in China

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Limits of detection of six approved RT-PCR kits for the novel SARScoronavirus-2 (SARS-CoV-2)

Biosensors: sense and sensibility

High specificity, electrochemical sandwich assays based on single aptamer sequences and suitable for the direct detection of smallmolecule targets in blood and other complex matrices

A simple and ultrasensitive electrochemical DNA biosensor based on DNA concatamers

An ultrasensitive supersandwich electrochemical DNA biosensor based on gold nanoparticles decorated reduced graphene oxide

Combining supramolecular chemistry with biology

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Supramolecular recognition of A-tracts DNA by calix[4]carbazole

Fluorescent dyes and their supramolecular host/guest complexes with macrocycles in aqueous solution

A comparison study of macrocyclic hosts functionalized reduced graphene oxide for electrochemical recognition of tadalafil

Fluorescent detection of tadalafil based on competitive host-guest interaction using psulfonated calix[6]arene functionalized grapheme

P-sulfonated calix[8]arene functionalized grapheme as a "turn on" fluorescent sensing platform for aconitine determination

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High specificity, electrochemical sandwich assays based on single aptamer sequences and suitable for the direct detection of small-molecule targets in blood and other complex matrices

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This work was supported by grants of the National Natural Science Foundation of 

The authors declare that they have no conflict of interest.J o u r n a l P r e -p r o o f