Multichannel Immunosensor Platform for the Rapid Detection of SARS-CoV-2 and Influenza A(H1N1) Virus.

The coronavirus disease 2019 (COVID-19) can present a similar syndrome to an influenza infection, which may complicate diagnosis and clinical management of these two important respiratory infectious diseases, especially during the peak season of influenza. A rapid and convenient point-of-care test (POCT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus is of great importance for prompt and efficient control of these respiratory epidemics. Herein, a multichannel electrochemical immunoassay (MEIA) platform was developed based on a disposable screen-printed carbon electrode (SPCE) array for the on-site detection of SARS-CoV-2 and A(H1N1). The developed MEIA was constructed with eight channels and allowed rapid detection on a single array. On the SPCE surface, monoclonal antibodies against influenza A(H1N1) hemagglutinin (HA) protein or SARS-CoV-2 spike protein were coated to capture the target antigens, which then interacted with a horseradish peroxidase (HRP)-labeled detection antibody to form an immuno-sandwich complex. The results showed that the MEIA exhibited a broader linear range than ELISA and comparable sensitivity for A(H1N1) HA and SARS-CoV-2 spike protein. The detection results on 79 clinical samples for A(H1N1) suggested that the proposed MEIA platform showed comparable results with ELISA in sensitivity (with a positive rate of 100% for positive samples) but higher specificity, with a false-positive rate of 5.4% for negative samples versus that of 40.5% with ELISA. Thus, it offers great potential for the on-the-spot differential diagnosis of infected patients, which would significantly benefit the efficient control and prevent the spread of these infectious diseases in communities or resource-limited regions in the future.

Introduction

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has brought serious threats to global public health. As of April 16, 2021, more than 138 million infected cases with ∼2.9 million deaths worldwide have been reported by the World Health Organization (WHO). As a result, numerous countries worldwide have implemented significant blockades and mobility restrictions to stop the virus from spreading, causing serious damage to the global economy.[1]

Of particular concern, COVID-19 can cause similar symptoms to influenza. During the peak season of influenza, the coepidemics create an unprecedented burden on existing health systems.[2−6] Similar transmitting patterns of the influenza viruses have been established with COVID-19, comprising human-to-human communication and aerosols or airborne droplets.[7,8] Influenza outbreaks have occurred for decades, mainly caused by A-type influenza viruses, such as the A(H5N1) in 1997, A(H1N1) in 2009, and A(H7N9) in 2013.[9−13] Among these influenza viruses, the A(H1N1) subtype might have caused the longest impact to human society from 1918 to now. The symptoms of COVID-19 (fever, cough, nausea, vomiting, etc.) are similar to those of influenza initially, which may confuse the identification of influenza and COVID-19.[14] Besides, the SARS-CoV-2 virus will become endemic and coexist with mankind for a long time as did the A(H1N1) virus.[15] Therefore, a prompt, sensitive, affordable, miniature, easy-to-use point-of-care test (POCT) method is of great importance for timely differential diagnosis and treatment for SARS-CoV-2 and A(H1N1) infected cases.[16]

There are several diagnosis methods for testing SARS-CoV-2 and influenza A(H1N1) virus, such as virus culture, nucleic acid testing, and serological assays.[17−20] The most widely used test for the detection of SARS-CoV-2 and A(H1N1) virus relies on detecting viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which has played a great role in the diagnosis of influenza and COVID-19.[21,22] However, the RT-PCR method should not discriminate live or dead virus, which will bring out false-positive rates after amplification,[23] conversely. Meanwhile, COVID-19 patients with a low virus load of SARS-CoV-2 might be missed, resulting in false-negative results.[24] According to the WHO expenditures, a serological immunoassay for SARS-CoV-2 and A(H1N1) is a powerful complement to RT-PCR detection methods.[19]

In comparison with PCR-based nucleic acid detection, immunoglobulin and antigen detection could distinguish the virus state as live or dead because the PCR method is based on amplifying the nucleic acid by several orders of magnitude to produce a signal. Even if the nucleic acid of dead virus was degraded, it might also generate a large signal, resulting in it being difficult to distinguish between the dead and live viruses. However, for the immunoassay, because the surface protein of the dead virus has normally been denatured, the signal will be greatly reduced compared with a large signal for that of the live virus. Besides, the immunoassay method could be time-saving and available for point-of-care diagnosis, which complements the detection through viral RNA. For the assay of SARS-CoV-2, the nucleocapsid or spike protein is usually chosen as the biomarker, which is recognized via the platforms of lateral flow assay (LFA), ELISA, and field-effect transistor (FET);[17,25−27] while for A(H1N1), hemagglutinin protein (HA) is one of the good biomarkers. Currently, rapid antigen testing tools are mostly based on LFA platforms, which are cheap, easy to use, and suitable for rapid detection of antigens in the field but with relatively poor sensitivity.[28] Besides, ELISA has recently received emergency use authorization from the FDA. However, it lacks official performance validation with respect to sensitivity and specificity to influence its accuracy in screening COVID-19 cases.[29]

In recent years, a biosensor has presented great potential for point-of-care testing of pathogens because of its high sensitivity, portability, and automatic working mode.[11,30] For instance, colorimetric biosensors based on gold nanoparticles that take advantage of the color change are convenient on-the-spot assays with the naked eye, although the complexity of the procedures limits their applications on a large scale.[31] The surface plasmon resonance is label-free and real-time but normally depends on a unique machine and professional operation.[32] Among others, the FET biosensor is rapid and sensitive but less stable.[25] In comparison, an electrochemical biosensor is simple, cost-effective, rapid, and smart. Traditionally, the one-by-one electrochemical detection of the samples is time-consuming, whereas a multichannel assay can simultaneously detect multiple samples, quickly distinguishing between viral and nonviral infections, which is time-saving for further symptomatic treatment. During concurrent respiratory epidemics, the early differentiation between COVID-19 and influenza is an urgent need. Diagnosis and treatment will be delayed in a sequential detection for the respiratory pathogens. A sensitive and specific multiplex assay for simultaneous testing of the two pathogens will significantly improve the screening efficiency for efficient control of the two respiratory infectious diseases. Nevertheless, to date, a disposable multichannel electrochemical sensor for the simultaneous detection of SARS-CoV-2 and influenza virus has not been reported.

In this work, we developed a disposable multichannel electrochemical immunoassay (MEIA) platform for quantitative detection of two important respiratory pathogens, i.e., influenza A(H1N1) and SARS-CoV-2 viruses. The developed multichannel MEIA was constructed with eight channels and allowed simultaneous detection for the spike protein of SARS-CoV-2 and the HA protein of A(H1N1) based on an immunoenzymatic “sandwich” format. The SARS-CoV-2 spike protein and influenza A(H1N1) HA antibodies are immobilized on the working electrode arrays to capture these two pathogens, which then interact with a horseradish peroxidase (HRP)-labeled detection antibody to form an immuno-sandwich complex. The electrochemical responses were monitored using the amperometry method (Figure ) to demonstrate the immunocomplex formation. On the basis of the simple design and low-cost screen-printed carbon electrode (SPCE), the fabricated MEIA biosensing platform enables cost-effective mass production. Furthermore, the MEIA platform could perform with eight channels at the same time, which allows simultaneous detection of multiple respiratory viruses including A(H1N1) and SARS-CoV-2 at different scenarios, such as hospital, clinics, communities, or resource-limited regions.

Figure 1

Schematic diagram of multichannel MEIA for SARS-CoV-2 and influenza A(H1N1) virus. The antigen (SARS-CoV-2 or A(H1N1) virus) in the sample was recognized by the HRP-labeled detection antibody and then formed an immuno-sandwich complex with the capture antibody on the working electrode array. After adding a substrate of TMB and H2O2, the catalytic amperometric readout was recorded. The proposed immunosensor array was performed at −0.1 V. Blocking means blocking the immobilized electrode with bovine and casein.

Experimental Section

Materials

The PCR and UV Imager were purchased from Sigma-Aldrich. The Emstat Potentiostat (Palmsens) was obtained from Red Matrix. The SPCE array was from Zhejiang Nazhihui Biotechnology Co., Ltd. A/Beijing/501/2009(H1N1) (A(H1N1/BJ501)), A(H3N2), influenza B virus, adenovirus, and EV71 were preserved in our laboratory. The DL2000 Marker was purchased from Takara. A(H1N1) influenza virus-specific capture antibody and HRP-labeled detection antibody, A(H7N9), SARS-CoV-2 (2019-nCoV) spike S1-His recombinant protein, SARS-CoV-2 (2019-nCoV) spike protein antibody, SARS-CoV-2 (2019-nCoV) spike protein antibody (HRP), goat anti-mouse IgG secondary antibody (HRP), SARS-CoV-2 (2019-nCoV) spike ELISA kit, MERS-CoV, HCoV-NL63, SARS-CoV, and pseudovirus of SARS-CoV-2 were purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. Casein (CAS) was purchased from Solarbio. Bovine serum albumin (BSA) was bought from Sigma-Aldrich. Tris-HCl was from Amersco. Tween-20 was purchased from China National Pharmaceutical Company. K-blue substrate solution was obtained from Neogen. All chemicals from commercial sources were of analytical reagent grade. All solutions were prepared using Milli-Q water (18 MΩ cm resistivity) from a Millipore system.

Fabrication of the MEIA for A(H1N1) and SARS-CoV-2

First, 5 μL of capture antibodies with a certain concentration for A(H1N1) virus or SARS-CoV-2 spike protein were dropped on the SPCE array surface and incubated overnight at 2–8 °C. After rinsing with phosphate-buffered saline (PBS), each electrode was then incubated for 1.5 h at room temperature with 50 μL of blocking solution containing 1% BSA and 1% CAS to block any nonspecific binding sites on the SPCE and finally stored at 2–8 °C until use. The fabricated electrodes were characterized by the electrochemical impedance spectroscopy (EIS) method (Figure S2).

Assay of A(H1N1) and SARS-CoV-2

Ten μL of A(H1N1)/BJ501 diluent or 5 μL of SARS-CoV-2 spike protein were casted onto the as-prepared electrodes and incubated at 37 °C for 1 h. After rinsing with PBS, 5 μL of HRP-labeled detection antibodies for A(H1N1)/BJ501 or SARS-CoV-2 spike protein were then dropped on the corresponding working electrodes, followed by incubation at 37 °C for 45 min. Next, 80 μL of TMB substrate was added to react with HRP (labeled on the detection antibody) to generate an electrochemical signal. Finally, the reactions in the eight channels were measured simultaneously by an amperometric measurement at −0.1 V to record the current signal at 60 s. The immunoassay procedure on the surfaces of the electrodes was characterized by the EIS method (Figure S2).

Optimizing the MEIA Performance

To improve the performance of MEIA, we investigated and optimized the experimental conditions, including the amounts of capture antibody and detection antibody, which is vital for immunoassay. For A(H1N1), the capture antibody was serially diluted (0, 25, 50, 100, 200, 500, 1000, and 2000 μg/mL) and coated onto the SPCE, and then goat anti-mouse IgG secondary antibody (HRP) was used as the detection antibody to screen the optimal concentration of the capture antibody in the experiment. For the amount of detection antibody of A(H1N1), an eight-channel capture antibody-coated SPCE array was incubated with 10 μL of A(H1N1)/BJ501 chicken embryo allantoic fluid (a titer of 16) on each working electrode at 37 °C for 60 min. After that, 10 μL of HRP-labeled detection antibody gradient dilution (10, 40, 60, 80, 100, and 150 μg/mL) was dropped on each working electrode of MEIA at 37 °C for 30 min, followed by washing with phosphate-buffered saline/Tween (PBST) three times and rinsing with PBS. Then 80 μL of TMB substrate was dropped onto each SPCE, and an amperometric measurement was conducted at −0.1 V. Similarly, the appropriate concentrations of capture antibody and detection antibody for SARS-CoV-2 spike protein were also investigated.

Evaluating the Performance of the MEIA

A series of A(H1N1) HA protein solutions ranging from 4 to 64 unit/mL and SARS-CoV-2 spike protein ranging from 0.15 to 100 ng/mL were prepared in dilution solution and tested for the dynamic range and detection limit of the MEIA. Besides, we compared the specificity of the MEIA with the commercial ELISA. The established biosensing platform was evaluated using the HA proteins of A(H1N1), A(H3N2), A(H7N9), influenza B virus, adenovirus, and EV71 virus at the same dilution level. Furthermore, blank, MERS-CoV (50 ng/mL), HoCV-NL63 (50 ng/mL), SARS-CoV (50 ng/mL), and SARS-CoV-2 (50 ng/mL) were also tested.[33]

Clinical Sample Tests

To examine the validity and reliability of the proposed MEIA, 79 serum samples preserved in our laboratory were tested, including influenza B, adenovirus, EV71, negative control (NC), and A(H1N1). In addition, a series of concentrations (0, 29, 143, and 286 pfu/mL) of pseudovirus were employed as stimulating specimens to test the performance of the fabricated biosensing platform.

Results and Discussion

MEIA Platform Strategy

We previously designed an electrochemical immunosensor with an interfacial assembly strategy.[34] The aim of this study is to construct a multichannel, rapid, specific, and sensitive electrochemical immunosensing platform based on SPCE for the simultaneous POCT detection of influenza A(H1N1) virus and SARS-CoV-2. The MEIA platform employed a sandwich immunoassay configuration (Figure ), which involved capture antibodies immobilized on SPCE and HRP-labeled detection antibodies to form an immunocomplex with the target antigens, i.e., A(H1N1) HA protein and SARS-CoV-2 spike protein. The substrate buffers containing TMB and H2O2 were catalyzed by HRP, inducing a charge change on the electrode surface so that the immuno-reaction on the SPCE can be recorded by the amperometric method; the response signals were proportional to the concentrations of the target analytes. The simple proposed MEIA allows simultaneous on-the-spot detection of multiple respiratory viruses.

Although the RT-PCR technique has the advantages of high sensitivity and accuracy, its usage is limited because it is time-consuming, has complex sample processing, and demands complicated equipment and skilled personnel. Moreover, RNA viruses are easily degraded, so they need to be stored in a standardized manner after obtaining patient samples and tested as soon as possible. In comparison, antigen testing does not require rigorous laboratory conditions and can be used in early screening and diagnosis, which has great potential for large-scale screening conducted by community hospitals as a complement to RT-PCR. Our proposed disposable MEIA, established for the quantification of A(H1N1) HA protein and SARS-CoV-2 spike protein, can be performed at different settings, such as hospitals, clinics, communities, etc. The whole detection process can be accomplished within 2 h.

Identification of Influenza A(H1N1) Virus

The titer of chicken embryo allantoic fluid of A(H1N1) influenza virus collected for the experiment was tested as 27 or 128 HA unit/mL by hemagglutination assay.[35] The cultured chicken embryo allantoic fluid of A(H1N1) virus was identified by PCR. The length of the target fragment was 198 bp. The result of gel electrophoresis analysis demonstrated that the product by PCR displayed a unique clear band near 200 bp between 100 bp and 250 bp, and the position of the chicken embryo cultured allantoic fluid was consistent with that of the seed virus, indicating that the cultured chicken embryo allantoic fluid contained influenza A(H1N1) virus as described in Figure S1.[36]

Optimization Performance Assay

The amounts of capture antibody and detection antibody for both A(H1N1) and SARS-CoV-2 were investigated for improving the performance of the biosensing platform. According to Figure A, the response current increased greatly along with the capture antibody concentration in the range of 0–500 μg/mL. The signal no longer obviously increased above the concentration of 500 μg/mL, probably because of the saturated adsorption of the capture antibody on the working electrode surface. Therefore, the appropriate capture antibody concentration was selected as ∼450 μg/mL with consideration of the operation method. Similarly, the optimizing capture antibody concentration of SARS-CoV-2 spike protein was chosen as ∼22 μg/mL in this experiment (Figure B). Meanwhile, it was obviously seen in Figure C that, when the concentration of the detection antibody for A(H1N1) was 60–80 μg/mL, the response signal rose greatly with the increase of the concentration of the detection antibody. Conversely, >80 μg/mL, the electrical signal decreased significantly, indicating that the conduction block of the sandwich complex was bound to the electrode for A(H1N1). Thus, we have chosen ∼80 μg/mL as the concentration of the detection antibody for A(H1N1) in the next experiments. Furthermore, the optimal concentration of the detection antibody (HRP) for SARS-CoV-2 spike protein was chosen as ∼25 μg/mL in the following experiments (Figure D).

Figure 2

Optimization of the concentrations of the capture antibodies of A(H1N1) HA protein (A) and SARS-CoV-2 spike protein (B), and the detection antibodies of A(H1N1) HA protein (C) and SARS-CoV-2 spike protein (D). The error bars show the standard deviations.

Evaluation of the MEIA Performance

The performance of the as-fabricated MEIA was further evaluated. Under the optimized concentration conditions of capture antibody and detection antibody mentioned earlier, the calibration curves for A(H1N1) and SARS-CoV-2 were established, respectively. A series of different concentrations (0, 1, 2, 4, 8, 16, 32, and 64 HA unit/mL) of A(H1N1) were determined via the amperometric method (Figure A). The current versus concentration curve is shown in Figure B. There was a wider linear relationship between the response current and A(H1N1) HA protein concentration in the range of 4–64 unit/mL (the regression equation is y = −114.81 exp(−lgx/942.22) + 114.87 (R2 = 0.9988)) compared with ELISA (1–16 unit/mL) (Figure C). Its limit of detection is 1.12 unit/mL, which was similar to that for ELISA (0.532 unit/mL). Likewise, the amperometry performance of the as-fabricated MEIA was evaluated in the range of 0–100 ng/mL of SARS-CoV-2 spike protein (Figure D), where the current response proportionally increased to the concentration of the SARS-CoV-2 spike protein in the range of 0.15–100 ng/mL with a LOD of 150 pg/mL. The regression equation was y = 3.07 × 10–9 exp(−lgx/84.32) – 3.15 × 10–9 (R2 = 0.9990) (Figure E), which was 10 times wider than that with ELISA (0.156–10 ng/mL) (Figure F). Compared with the ELISA analysis, the proposed MEIA exhibited good performance for detecting A(H1N1) HA protein and SARS-CoV-2 spike protein with higher sensitivity and broader linearity, meeting clinical requirements. On the one hand, the antibody pairs could recognize the specific binding sites for A(H1N1) HA protein and SARS-CoV-2 spike protein. On the other hand, the surface of the working carbon electrodes with a rough interface might load more capture antibodies, resulting in more immunocomplex formation and higher response current.[37] The MEIA method generated a response current signal during the measurement by the electron transfer between the electrode and the reaction product, which is not affected by the color of the reaction product.

Figure 3

Amperometric (I–t) response curves of the MEIA for A(H1N1) HA protein (A) and SARS-CoV-2 spike protein (D). The dose–response curves of A(H1N1) assay (MEIA) (B) and (ELISA) (C). The dose–response curves of SARS-CoV-2 spike protein assay (MEIA) (E) and (ELISA) (F). Serial amounts of A(H1N1) are 0, 1, 2, 4, 8, 16, 32, and 64 HA unit/mL (MEIA). Serial amounts of A(H1N1) are 0, 0.5, 1, 2, 4, 8, and 16 HA unit/mL (ELISA). Serial amounts of SARS-CoV-2 spike protein are 0, 0.15, 0.3, 0.625, 1.25, 2.50, 5.0, 10, 25, 50, and 100 ng/mL (MEIA). Serial amounts of SARS-CoV-2 spike protein are 0, 0.156, 0.312, 0.625, 1.25, 2.50, 5.0, and 10 ng/mL (ELISA). The current signal was recorded at 60 s. The error bars show the standard deviations.

Next, to study the specificity of this proposed MEIA, we investigated the response of the MEIA platform through examining its cross-reaction with other viral antigens in comparison with the target analytes. We tested the responses of the established MEIA platform to the HA protein of A(H1N1), A(H3N2), A(H7N9), influenza B virus, adenovirus, and EV71 virus at the same concentration dilution. As shown in Figure A, the current response signal of A(H1N1) was much higher than those of A(H3N2), A(H7N9), influenza B virus, adenovirus, and EV71 virus, which were near to the signal of the blank. No cross-reactivity signal was produced when testing A(H1N1) against the other five pathogens, exhibiting a significant difference (p < 0.001) (Figure A). The result demonstrated the specific recognition ability of the antibody pairs for A(H1N1), as well as the good blocking effects of 1% bovine and 1% casein toward nonspecific sites for A(H1N1) on the surface of the carbon electrodes. Moreover, we also investigated the repeatability and accuracy of the MEIA platform for A(H1N1). It was shown that the RSD values of the three levels of samples (1.5, 5, and 11 unit/mL) were all <5%, indicating good repeatability (Table S1). The recovery tests were also performed with different known titers of A(H1N1) (10, 32, and 46 unit/mL) added into human saliva samples. The results showed that the recovery values were obtained between 95–100%, meeting the clinical requirements (Table S2).

Figure 4

Verification of the specificity of the MEIA. The respective signal responses of the MEIA to the targets of EV71, adenovirus, influenza B virus, A(H3N2), A(H7N9), and A(H1N1) virus (A) and to that of MERS-CoV, HoCV-NL63, SARS-CoV, and SARS-CoV-2 at the same concentration level (B). The blank represents the negative control. (***) p < 0.001 indicates an extremely significant difference. The error bars show the standard deviations.

ß-Coronaviruses (CoVs) have brought out three zoonotic outbreaks, including SARS-CoV in 2003, MERS-CoV in 2012, and the sudden SARS-CoV-2 in late 2019. MERS-CoV, HoCV-NL63, SARS-CoV, and SARS-CoV-2 at the same concentration level (50 ng/mL) were also determined. The current response of SARS-CoV-2 was much higher than that of the other three CoVs. No cross-reactivity was found when testing the SARS-CoV-2 spike protein against MERS-CoV and HoCV-NL63, while SARS-CoV has moderate interference, exhibiting a significant difference (p < 0.001) (Figure B). Both highly pathogenic SARS-CoV-2 and SARS-CoV are highly homologous and originated from bats, spike proteins of SARS-CoV-2 share ∼76% of amino acid sequences with SARS-CoV, and the amino acid sequence of the receptor-binding domain (RBD) of SARS-CoV-2 is ∼74% homologous to that of SARS-CoV,[33] which is consistent with the commercial ELISA kit.

To further verify the performance of the MEIA platform, 79 serum samples (influenza B, adenovirus, EV71, NC, and A(H1N1) influenza virus) were tested by the MEIA and the results were compared with ELISA. The cutoff values (COV) in the two strategies are calculated to be 0.072 μA and 0.098 (OD450), respectively. From the comparison between the MEIA and ELISA, we can see that the data points of negative samples and blanks via the MEIA method were all near the COV line (Figure A), while some negative samples and blank data points in the ELISA method exceeded the COV line (Figure B). This showed that there were obvious false-positive results in ELISA. The results of the two methods are calculated according to the respective COV analysis to calculate the positive detection rate and sensitivity (Table S3). In the MEIA, all 42 positive samples responded 100% positive, which was the same with ELISA. This demonstrated that the antibody pairs can recognize the specific binding sites for A(H1N1) HA protein, as well as for SARS-CoV-2 spike protein. The results showed that the positive detection rates of negative samples of influenza B virus, adenovirus, and EV71 virus and NC were 33.3%, 12.5%, 75%, and 20% in ELISA, respectively, while they were 0%, 0%, 16.7%, and 0% in the MEIA, respectively. In the MEIA, a total of 2 of 37 negative samples responded positive, yielding a 5.4% false-positive rate. Meanwhile, a total of 15 of 37 negative samples responded positive in ELISA, yielding a higher 40.5% false-positive rate. This is because the detection signals of some samples were slightly higher than the baseline in ELISA. It seemed that the ELISA method could not distinguish samples in the gray zone. According to the receiver operating characteristic (ROC) curve for the detection of A(H1N1), although the sensitivities in the MEIA and ELISA were both 100%, the specificity has a striking improvement (94.6% for the former, and 59.5% for the latter) (Figure C and D). These results suggested that the specificity and accuracy of the MEIA were better than those of ELISA.

Figure 5

Responses of the MEIA to the clinical pathogen samples or pseudovirus. Various pathogen samples determined by the MEIA (A) and ELISA (B). ROC curve for detecting various pathogen samples by the MEIA (C) and ELISA (D). Different concentrations (0, 29, 143, and 286 pfu/mL) of SARS-CoV-2 pseudovirus were tested by the MEIA (E) and ELISA (F). NC, negative control. The COV of the MEIA and ELISA were defined as the baselines. (***) p < 0.001 and (**) 0.001< p < 0.01 indicate extremely significant differences. The error bars show the standard deviations.

SARS-CoV-2 pseudovirus without replication capability, a substitute for SARS-CoV-2, was employed for evaluating the proposed MEIA in our study. A series of different concentrations (0, 29, 143, and 286 pfu/mL) of pseudovirus were tested in the MEIA and ELISA. It was shown in Figure E and F that the response current signals of three concentration levels of pseudovirus (29, 143, and 286 pfu/mL) were obviously differentiated from that of the blank control in both the MEIA and ELISA (p < 0.01), indicating that the proposed MEIA technology performed equivalently to ELISA. Pseudovirus is similar to the SARS-CoV-2 particle, which makes it suitable to mimic SARS-CoV-2 virus. Therefore, it is used as a quality control material for evaluating the performance of SARS-CoV-2 kits because of the difficulty in obtaining patient samples and the risk of the virus spreading.[38] We also utilized this pseudotype system to evaluate the performance of the MEIA and ELISA. The results showed that the MEIA is a potential tool for a POCT for the two respiratory viruses.

Conclusions

We developed a disposable, cost-effective MEIA platform based on SPCE to detect SARS-CoV-2 and A(H1N1). The MEIA approach exhibits good performance with a linear range of 4–64 unit/mL and a limit of detection of 1.12 unit/mL for A(H1N1), as well as with a linear range of 0.15–100 ng/mL (10 times wider than that of ELISA) and and LOD of 0.15 ng/mL for SARS-CoV-2 spike protein. The proposed immunoassay method also presents comparable or even higher specificity in comparison with the commercial ELISA kit. For 79 clinical samples including A(H1N1), the MEIA showed 100% positive rate to positive samples, in accordance with ELISA; however, it presented a 5.4% false-positive rate to negative samples, much lower than the 40.5% false-positive rate in ELISA, suggesting its higher specificity. As for different concentrations of SARS-CoV-2 pseudovirus, the response signal of the MEIA method was equivalent to that of ELISA. The results suggest that the proposed MEIA could meet urgent needs in point-of-care testing of SARS-CoV-2 and A(H1N1). Besides, the developed MEIA is easy to use, low cost, and does not require rigorous laboratory conditions for early screening at a large scale. It could facilitate the diagnosis of infected patients, which would benefit prompt decision making for the efficient control and prevention of communicable diseases related to public health events in the community or even a resource-limited region. In summary, this MEIA biosensing platform has a great potential to perform large-scale on-site screening and differential diagnosis of respiratory infectious diseases including influenza A(H1N1) and SARS-CoV-2 in the future.