key: cord-1054064-itc96ti1
authors: Liang, Chaolan; Liu, Bochao; Li, Jinfeng; Lu, Jinhui; Zhang, Enhui; Deng, Qitao; Zhang, Ling; Chen, Ruiai; Fu, Yongshui; Li, Chengyao; Li, Tingting
title: A nanoenzyme linked immunochromatographic sensor for rapid and quantitative detection of SARS-CoV-2 nucleocapsid protein in human blood
date: 2021-09-12
journal: Sens Actuators B Chem
DOI: 10.1016/j.snb.2021.130718
sha: 2a26c89cfa79f0552cab8630e6869e7623a36e9b
doc_id: 1054064
cord_uid: itc96ti1

The establishment of a simple, low-cost, high-sensitive and rapid immunoassay for detecting SARS-CoV-2 antigen in human blood is an effective mean of discovering early SARS-CoV-2 infection and controlling the pandemic of COVID-19. Herein, a smartphone based nanozyme linked immunochromatographic sensor (NLICS) for the detection of SARS-CoV-2 nucleocapsid protein (NP) has been developed on demand. The system is integrated by disposable immunochromatography assay (ICA) and optical sensor devices. Immunoreaction and enzyme-catalyzed substrate color reaction were carried out on the chromatographic strip in a device, of which the light signal was read by a photometer through a biosensor channel, and the data was synchronously transmitted via the Bluetooth to the app in-stored smartphone for reporting the result. With a limit of detection (LOD) of 0.026 ng/mL NP, NLICS had the linear detection range (LDR) between 0.05 and 1.6 ng/mL NP, which was more sensitive than conventional ICA. NLICS took 25 min for reporting results. For detection of NP antigen in clinical serum samples from 21 COVID-19 patients and 80 healthy blood donor controls, NLICS and commercial enzyme linked immunosorbent assay (ELISA) had 76.2% or 47.6% positivity, and 100% specificity, respectively (P=0.057), while a good correlation coefficient (r=0.99) for quantification of NP between two assays was obtained. In conclusion, the NLICS was a rapid, simple, cheap, sensitive and specific immunochromatographic sensing assay for early diagnosis of SARS-CoV-2 infection.

By the end of March 2021, the novel coronavirus pneumonia, named COVID-19, has been developed to a global pandemic, causing more than 3.7 million deaths. 1 The pathogen of COVID-19 is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 2 which is highly contagious, causing symptoms such as fever, dry cough, fatigue or progressive dyspnea through upper respiratory tract infection, or J o u r n a l P r e -p r o o f leading to death in severe cases. [3] [4] [5] [6] Several COVID-19 vaccines have been approved for emerging use in human vaccination, the efficacy requires being monitored. Therefore, it is very important to develop rapid and reliable methods for early detection of SARS-CoV-2 infected patients and asymptomatic virus carriers. [7] [8] [9] [10] Currently, the diagnosis for SARS-CoV-2 infection is primarily dependent on reverse transcription polymerase chain reaction (RT-PCR) and serological methods. [11] [12] [13] [14] RT-PCR is the most predominant method for detecting SARS-CoV-2. 15 Due to the limitations of sample preservation, testing reagents, requirement of the laboratory, professional operators and detection time (1-2 hours), RT-PCR is not suitable for rapid screening on-site. The high cost of RT-PCR also limits its popularization in many poor communities or health care systems. The serological testing of SARS-CoV-2 antibody is an auxiliary detection method for nucleic acid detection. [15] [16] [17] Some rapid methods such as lateral flow immunoassay for IgM/IgG antibody have been applied in clinic practice. However, the specific antibody against SARS-CoV-2 in blood is usually detected in 7-14 days of infection, which is not suitable for screening and diagnosis of COVID-19 in a window period of early infection. 18 Existing serological methods for antigen detection mainly include lateral flow immunoassay (LFIA) and double antibody sandwich ELISA. [19] [20] [21] [22] Most LFIAs were to detect the NP antigen in nasopharyngeal swab samples, and provided only qualitative rather than quantitative results. 23 The double antibody sandwich ELISA assay was time-consuming with complicated washing and incubation steps. 24 Therefore, there is an urgent need for a simple, fast, reliable and cheap test for quantitative detection of SARS-CoV-2 antigen.

In previous studies, applications based on nanozyme catalyzed color development have been reported. [25] [26] The nanozymes, such as Au@PtNPs, are nanomaterial with intrinsic enzyme mimicking activity, which have good catalytic activity and stability, presenting low cost and easy coupling, thus widely used in the various biosensors. [27] [28] [29] Compared with gold nanoparticles (AuNPs), the gold-platinum nanoparticles (Au@PtNPs) realized the signal amplification on color reaction, consequently the sensitivity was greatly improved. In comparison with J o u r n a l P r e -p r o o f stability, complicated preparation process, and lower sensitivity. [30] [31] [32] In the present study, a novel, rapid, sensitive, portable and low-cost nanoenzyme linked immunochromatographic sensor (NLICS) based on smartphone was developed for quantitative detection of the SARS-CoV-2 NP antigen in blood samples. Firstly, the NP antigen in blood sample was reacted with first specific monoclonal antibody (mAb1) coupled Au@PtNPs sprayed on conjugate pad of testing strip, and then migrated and captured by second mAb2 immobilized on the T-line (Test line) of NC membrane, forming a sandwich structure (Au@PtNPs-mAb1-NP-mAb2). Finally, the substrate solution was catalyzed by Au@PtNPs-mAb1-NP-mAb2 for color reaction.

The laser permeated through the reacted substrate filtration to reach the photometer, and the light was measured by the photometer, which was inversely proportional to the NP concentration in the sample. The results measured by a photometer were synchronously transmitted to a smartphone through Bluetooth connection for data processing and analysis. As a novel point of care test (POCT), the NLICS would be of great significance for rapid screening of suspected SARS-CoV-2 infection, especially in the early stages of viral infection. 

The smartphone based NLICS included an immunochromatographic assay (ICA) device and a result reading device. The ICA device was a coin sized U-shape strip to generate immunoreactive and colorimetric signal. The U-shaped ICA had a total size of 22 mm*11 mm*17 mm and was composed of eight components: shell, U-groove, silica gel stopple, optical fiber, sample pad, conjugate pad, NC membrane, and absorbent pad (Fig. S1A) . The U-groove and shell based on wedge-shaped structures were 3D-printed with UV light cured opaque photosensitive resin materials and could slide up and down after assembly. To prevent leakage, a silica gel stopple was pasted on the bottom of the U-groove, and an optical fiber with a diameter of 1.5 mm was inserted at the center of the silica gel stopple. The sample pad was 6 mm*15 mm glass fiber G-2 (pre-treated with 10 mmol/L Tris-HCl solution containing 0.1% Tween-20, pH 8.0). The conjugate pad was 6 mm*10 mm glass fiber SB06 (pre-treated with 10 mmol/L Tris-HCl solution containing 0.1% PVA, 2.5% trehalose, 2.5% sucrose, 0.5% sodium casein, 0.1% Tween-20, 0.03% sodium azide, pH8.0), sprayed with J o u r n a l P r e -p r o o f Au@PtNPs-mAb1. The 6 mm*25 mm NC membrane was placed in the middle of the U-groove, on which the T-line was coated with mAb2 (1 mg/mL). The sample pad, conjugate pad, NC membrane, and absorbent pad (6 mm*17 mm) were assembled sequentially with glue. In addition, the insertion plate and supporting plate specially designed for U-shaped ICA equipment were also fabricated by 3D-printing.

Result-reading device included an optical reading device and a handheld photometer for reading and transmitting results. The shell of the optical reading device was made of 3D printing with the total size of 46 mm*46 mm*73 mm, and the reading device consisted of switch, USB port, adjustable resistor and 450 nm laser 

According to the previously reported method, 33 the monoclonal antibodies to SARS-CoV-2 NP were produced and the paired antibodies were selected. Briefly, splenocytes of NP-immunized BALB/c mice and mouse myeloma cells SP2/0 were fused, and positive hybrioma cells were identified by indirect ELISA. Hybridoma cells were intraperitoneally injected into BALB/c mice to prepare ascites. The purified monoclonal antibodies were obtained through the purification of ascites with protein G agarose gel preloaded column. Then double antibody sandwich ELISA was used to select the paired antibodies and to determine the titer of antibodies.

Au@PtNPs were synthesized according to the method reported in the previous study. 34 Firstly, 13 nm AuNPs were synthesized. 100 mL of ultrapure water were added to a clean erlenmeyer flask, and heated to boiling with stirring, then 1 mL of 1% HAuCl 4 solution was added. And after boiling again, 4 mL of 1% sodium citrate were J o u r n a l P r e -p r o o f added quickly and continuously stirred for 30 min. When heating stopped, the stirring was continued to cool the AuNPs to room temperature and stored at 4℃. 597 μL of H 2 PtCl 6 (20 mmol/L) were added to 8.603 mL of AuNPs solution and heated to 90℃ by stirring. Then 800 μL of ascorbic acid (20 mmol/L) were slowly dropped into the mixture and heated for 30 min by stirring continuously. Stopped heating after 30 min, the mixture was cooled naturally to room temperature and stored at 4℃.

The antibody labeling was carried out in the laboratory according to the relevant literature with modification 35 . Briefly, 20 μL of 0.2 mol/L K 2 CO 3 were added to 1 mL of Au@PtNPs and mixed, then 20 μg mAb1 were added and incubated for 1h. After that, 100 μL of 10% BSA were added and blocked for 1h. Finally, the mixed solution was centrifuged at 10000 rpm for 15 min, and the Au@PtNPs-mAb1 were resuspended into 100 μL of preservation solution (0.1 mol/L PBS, 10% sucrose, 0.5% BSA, pH 7.4) and stored at 4℃. 

Statistical analysis was carried out by SPSS 18.0 software. The results were presented as mean ± standard deviation. The difference in detection sensitivity among two assays was calculated by Pearson's chi-squared test. P<0.05 was considered statistically significant.

The NLICS was designed ingeniously by combining ICA and smartphone devices.

The user only needs to complete the following operations: (1) to add the sample dilutions into the sample well of chromatographic device (Fig. 1A) ; (2) to push the chromatographic device into the supporting plate and to add TMB substrate solution for 10 min incubation, and then to add H 2 SO 4 solution for terminating reaction (Fig.   1B) ; (3) to turn on the power of laser in 10 min advance, to push the supporting plate into the optical channel, and to initiate the photometer and smartphone to read the illumination (Fig. 1C, D) . The sensor directly presents the data in digital form, and J o u r n a l P r e -p r o o f synchronously transmits it to smartphone via Bluetooth for result analysis and reporting (Fig. 1E ). In comparison with conventional ELISA, the NLICS is simple and rapid, it doesn't need multiple washing steps and additional equipment.

(Insert Fig.1) 

The NLICS contains two devices of immunochromatographic assay (ICA) and light signal reading. The chromatographic device is used for immunoassay. ICA device includes sample pad, conjugate pad, U-shaped NC membrane strip and absorbent pad ( Fig. 2A) . The overall size was similar to a RMB coin (Fig. 2B ). When the sample solution is added to sample pad, the liquid is sequentially flowed through the conjugate pad, NC membrane and absorbent paper due to capillary action. The sample pad and conjugate pad are pretreated with surfactant buffer solution (maintaining the appropriate ionic strength and pH value) or Au@PtNPs-mAb1 conjugates, respectively. The U-shaped ICA device is fixed and supported by the supporting plate, which can hold 7 U-shaped ICA devices. The NC membrane strip is separated between the conjugate pad and the absorbent pad by insertion plate to prevent TMB substrate and termination solutions being sucked back by the conjugate pad and the absorbent pad.

The reading device is used to read the light signal from immunoreaction in ICA device, which is assembled by a switch, USB port, adjustable resistor and 450 nm laser (Fig. 2C) . The laser, U-shaped ICA device, and optical fiber are located in the central axis to form the light sensing channel. The hand-held photometer receives the light signal and converts it into digital data. The total material cost for device manufacturing was $1.50 (Table S1) , of which some parts were fabricated by 3D-printing.

During the NLICS process for detecting SARS-CoV-2 NP antigens in blood, after the sample was added to sample pad, NP antigens flowed to the conjugate pad and reacted with the saturated Au@PtNPs-mAb1, and then migrated and bound to the immobilized mAb2 on T-line of NC membrane strip to form the sandwich structural J o u r n a l P r e -p r o o f complexes (Au@PtNPs-mAb1-NP-mAb2). When TMB substrate was added to U-shaped ICA device, the substrate was catalyzed for color reaction (Fig. 2D ). If NP antigens were not presence in the sample, Au@PtNPs-mAb1 conjugates would migrated through T-line of NC membrane strip to absorbent pad. When TMB was added, the substrate would not be catalyzed for color development. For measuring the substrate reaction and reporting the detection result, the laser was initiated in 10 min advance. The emission of 450 nm light passed through the filtration of reacted substrate solution and an optical fiber to the photometer (Fig. 2D) . The photometer displayed the light intensity and synchronously transmitted the data to smartphone for analysis via Bluetooth (Fig. 2E) . The application (app) in-sored smartphone is The stability of sensing system of NLICS was examined during 60 min working period (Fig. S2) . The temperature change of initiated laser was quickly increased during the initial 10 min and stably maintained within 3℃ (Fig. S2A) , while the light intensity of sensor was quickly decreased during the initial 10 min and then remained stable (less than 3% drift) up to 60 min (Fig. S2B) . The results suggested that NLICS had the satisfactory stability of sensing system, particularly after 10 min initiation of reading device.

In order to obtain the suitable NP antibody pairs, 5 mAbs were selected from 20 mAbs to NP by double-antibody sandwich ELISA. Among five mAbs paired with J o u r n a l P r e -p r o o f each other, 5E4-HRP/4G11 mAb pair had the highest S/CO value (Table S2 ). The mAbs 5E4 (mAb1) and 4G11 (mAb2) were designated as detection antibody or capture antibody in the assay, of which the titers of mAbs were identified as 2.44×10 -7 and 4.88×10 -7 , respectively (Fig. S3) . Finally, mAb 5E4 was selected to prepare Au@PtNPs-mAb1 conjugates, and mAb 4G11 was used to be immobilized on T-line of NC membrane strip for reacting with NP in NLICS.

With good catalytic activity, low cost, easy coupling and stability, Au@PtNPs were widely used in biosensor application. AuNPs with a diameter of 13 nm were evenly distributed under TEM observation, of which the particle surfaces were smooth (Fig. S4A) . By adding chloroplatinic acid to AuNPs with increasing concentration from 0.5% to 10%, the size of Au@PtNPs gradually increased and the color changed from wine red to brown black (Fig. S4A) . When AuNPs were saturated with 7% or 10% chlorogold acid, the particle size was about 40 nm, and the surfaces of Au@PtNPs were fully coated with spikes, forming "sea urchin" like protrusions (Fig. S4A) . By the full wavelength scanning of spectrum, the peak of AuNPs at 528 nm gradually flattened and finally disappeared with increasing of Au@PtNPs (Fig.   S4B ). These results indicated that the synthesized AuNPs and Au@PtNPs were successful.

The feasibility of Au@PtNPs catalysis was analyzed after the Au@PtNPs were added to the TMB substrate in microwells. The yellow color of substrate reaction deepened along with the increase of Au@PtNPs concentration (Fig.3A) , and accordingly OD 450 values were increased by measurement with a microplate reader (Fig. S4C) . In order to verify the catalytic activity, Au@PtNPs' catalytic speed and stability were tested at different concentrations in comparison with HRP in TMB substrate, respectively. The results showed that the Au@PtNPs presented highly catalytic activity in a dose and time dependent manner, even at an extremely low concentration of 0.5 pmol/L, while HRP had lowly detectable catalytic activity at a concentration > 5 nmol//L (Fig. 3B ). In addition, Au@PtNPs had good stability at J o u r n a l P r e -p r o o f room temperature remaining 95% activity over 15 days, but HRP lost 80% activity at room temperature after 7 days (Fig. S4D ).

The proper reacting conditions of NLICS were set up by optimizing the concentrations of H 2 O 2 and Au@PtNPs-mAb1, and reaction time. The absorbance ratio of positive to negative control (P/N) reached to the highest value when H 2 O 2 concentration was up to 2 mol/L, but decreased when H 2 O 2 concentration was > 2 mol/L (Fig. 3C) . Consequently, 2 mol/L H 2 O 2 was selected as working concentration in NLICS. Since the amount of Au@PtNPs-mAb1 saturated in the conjugate pad was critical to affect the assay's sensitivity, the eight dilutions of Au@PtNPs-mAb1 were compared (Fig. 3D) , in which 1:160 working dilution was determined for having the highest P/N value. The proper reaction time for the faster color development and higher P/N value was selected from 10 min substrate incubation in NLICS (Fig. 3E) .

The coefficient variations (CV) of intra-assay and inter-assay were examined for evaluation of NLICS precision or accuracy for detecting three concentrations (0.05, 0.4 and 1.6 ng/mL) of standard NP antigen. The intra-assay CV varied below 10% among 6 replicates in NLICS (Fig. 3F) , while the inter-assay CV ranged between 5.2% and 13% among 8 independent tests in NLICS (Fig. 3G) . The results suggested that the NLICS had the higher precision in detection of low, middle and high levels of SARS-CoV-2 NP antigen.

(Insert Fig.3) 

In this study, 100 μL of sample solutions with 0-51.2 ng/mL standard NP antigen were detected by loading to the sample well of the U-shaped ICA device of NLICS (Fig.   4A ). The OD 450 values were calculated by the algorithm (1) after termination of TMB substrate reaction (Fig. 4B ). In the algorithm (1) for OD 450 value, the I was the transmitted light intensity (lux) through the TMB substrate solution, and I0 was the light intensity value measured on the photometer and smartphone when only colorless J o u r n a l P r e -p r o o f water was added. In this experiment, the laser power was controlled by rotating the adjustable resistor, and the I0 value was adjusted to 70,000 lux. The transmitted light intensity was read by the photometer, and the results were synchronously transmitted to the smartphone through Bluetooth for analysis.

The light intensity values (lux) were converted to OD 450 values by formula calculation (Table S3 ). The results showed that OD 450 values were positively correlated with NP concentrations, the linear detection range (LDR) of NLICS for NP was 0.05-1.6 ng/mL (Fig. 4C ). The following line equation was used to calculate NP concentration in serum samples.

y=0.52271+0.86087x, and the fitting degree R 2 was 0.9954.

The limit of detection (LOD) of NLICS was determined as 0.026 ng/mL, calculated by substituting the mean + 3SD value of negative samples (n = 40) into the linear equation. Meanwhile, the traditional ELISA and ICA were used as controls to detect the standard NP dilutions. In traditional ELISA, the LDR was 0.05-12.8 ng/mL NP, with a LOD of 0.025 ng/mL (Fig. S5A ). In the traditional ICA, the T-line was clearly visible only when the NP concentration was greater than 0.1 ng/mL (Fig. S5B) .

The results suggested that NLICS was simpler and faster assay, which had similar sensitivity with ELISA but higher sensitivity than traditional ICA for detecting the NP standard in artificial samples.

(Insert Fig.4) 

In order to verify the specificity of NLICS, several proteins including SARS-CoV-2 NP, S and RBD proteins, H1NI hemagglutinin (HA) and neuraminidase (NA) proteins, hepatitis B surface antigen (HBsAg), and hepatitis C virus core protein (HCV core)

were tested in triplicate (Fig. 5) . The results showed that only NP (12.8 ng/mL) was reactive, while other proteins were not cross-reactive at concentration of 100 ng/mL in NLICS, suggesting the assay had high specificity to SARS-CoV-2 NP antigen.

(Insert Fig.5) J o u r n a l P r e -p r o o f (Table   1) . Notably, the sensitivity and accuracy of NLICS were higher than ELISA. In aspect of quantification of NP in clinical samples between NLICS and ELISA (Fig. 6A) , a strong correlation was observed with no significant difference ( Fig. 6B ; P < 0.01, r = 0.990). These results suggested that the NLICS was reliable, which was potential to be used for the point of care testing of SARS-CoV-2 infection in terms of high specificity, sensitivity and accuracy in clinical quantitative detection.

(Insert Fig.6 , Table 1 )

The viral nucleic acid testing (NAT) and antibody detection have been commonly 

The authors report no declarations of interest. The linear curve for detection of NP standard by NLICS. 

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Chaolan Liang: Conceptualization, Methodology, Formal analysis, Writing-original draft

Methodology, Investigation, Formal analysis Jinfeng Li: Resources, Methodology, Investigation

Methodology, Investigation, Validation Enhui Zhang: Investigation, Software

Qitao Deng: Investigation, Software

Formal analysis

Ruiai Chen: Resources

Chengyao Li: Supervision,Project administration, Funding acquisition, Writingreview & editing

Tingting Li: Supervision, Project administration, Funding acquisition,Writingreview & editing

The authors thank Mr. Ze Wu for his technical assistance in the laboratory, Shenzhen CDC for providing COVID-19 patients' serum samples,' and Guangzhou Blood Centers for providing healthy blood donor samples. 

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Highlights  SARS-CoV-2 lead to the pandemic of COVID-19 of which needs rapid, reliable, quantitative and easy diagnostic assays for antigen.  NLICS had 100% specificity, 76.2% sensitivity and 95.1% accuracy for detecting SARS-CoV-2 NP in clinical samples. Smartphone App and portable device were applied to quantitative detection. NLICS was a sensitive, rapid, portable, easy-operation, low-cost and safe immunoassay for early diagnosis of SARS-CoV-2 infection.