FeCl3.6H2O, CoCl2.6H2O, Poly (acrylic acid), Hemin, TMB, HRP, EDC, NHS, BSA, DAB and hydrogen peroxide (H2O2) were purchased from Sigma-Aldrich Co. LLC. (Shanghai, China). Polyethylene glycol was purchased from Yeasen Biotech Co., Ltd.

Sodium acetate was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai,

activity evaluation of peroxidase-mimic

The peroxidase activity of Co-Fe@hemin nanozyme was measured according to a previously published procedure17. In brief, nanoparticles of different concentrations were added to 2 mL HAc/NaAc buffer (0.2 M, pH 3.6), then 100 µl TMB and 100 µl H2O2 (30% wt/vol) were added to the cuvette. The absorbance at 652 nm was measured for 1 min at the temperature of 37℃, and the rection-time curve was plotted. The specific activity (SA, U/mg) of nanozyme was calculated. The chemiluminescence catalytic activity was assessed by EnVision Multilabel plate reader (PerkinElmer) using a liquid auto-injection system and luminol substrate. The maximum chemiluminescent intensities catalyzed by Co-Fe@hemin nanozyme were measured at different pH, temperature pretreatment and different concentrations of H2O2, with HRP as positive control. Moreover, the intermediate reactive oxygen species in the reaction system were monitored by using electron spin resonance (ESR) spectroscopy and the spin trap agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).

The recombinant antigen is a receptor binding domain of the SARS-CoV-2 spike protein (S-RBD, located in the S1 subunit) 18. A cDNA sequence encoding S-RBD was expressed containing both N-terminal natural signal peptide as well as a 6×His tag at the C-terminus, after transfection into HEK293 cells. After 3 days, supernatants were collected and soluble protein was sequentially purified using HisTrap HP column and

Superdex 200 column. The concentration of S-RBD protein was determined using a BCA assay kit (Pierce). The purity of recombinant protein was analyzed by SDS-PAGE. Aliquoted protein was stored at -20℃~-80℃ until further use.

Anti-Spike antibodies were generated by sorting single memory B cells as previously reported19. Briefly, peripheral blood mononuclear cells from convalescent COVID-19 patients were incubated with His-tagged S-RBD at 100 nM before staining with antiCD3, anti-CD16, anti-CD235a, anti-CD19, anti-CD38, anti-CD27 and anti-His antibodies. Antigen-specific memory B cells were identified by the following markers: CD3-, CD16-, CD235a-, CD38-, CD19+, CD27+, hIgG+ and His+, and sorted into 96well PCR plates with single cell per/well. The genes encoding Ig VH and VL chains were amplified by 5’RACE and nested PCR. The variable regions of these genes were then cloned into the human IgG1 constant region to generate full-length monoclonal antibodies, which were then expressed and purified by conventional methods. The antibody binding affinity for S-RBD was evaluated by BIAcore 8K.

Anti-S-RBD antibody pairs were screening using ELISA method and nanozymebased colorimetric strips15. The preparation of nanozyme-based chemiluminescence test paper resembles the colloidal gold strip with further modifications. The nanozymebased chemiluminescence test paper was constituted of a PVC backing plate, an absorbing pad, a sample pad, a nitrocellulose membrane and a conjugate pad. First, the detection antibody of S-RBD (S-dAb) conjugated nanozyme chemiluminescent probes were prepared by chemical coupling of carboxyl group using EDC/NHS agents. The labeled nanozyme probes were stored in modified TB buffer containing 5% BSA. The nanozyme probes were dispensed on the pretreated conjugate pad using a BioDot dispensing equipment. The paired capture antibody of S-RBD (S-cAb) was immobilized on a nitrocellulose membrane at 1 mg/ml to form the test line (T-line), as well as the anti-human IgG antibody as the control line (C-line). Next, the above conjugate pad, nitrocellulose membrane, sample pad and absorbing pad were assembled on the PVC backing plate and followed by complete drying at 37℃ for 1~2 h. The paper boards were cut into 4 mm-wide strips. Finally, the strips were packed into clamp slots and stored at room temperature. The solid chemiluminescence substrate, excitant and dissolve buffer were stored in plastic ampoule bottles at room temperature.

In this study, rapid nanozyme-based chemiluminescence paper test of SARS-CoV-2 spike antigen was developed. Recombinant S-RBD protein of SARS-CoV-2 was used to evaluate the sensitivity of nanozyme-based chemiluminescence paper test. 100 µl of the sample containing S-RBD protein in dilution buffer was loaded onto the sample pad. The liquid sample flows through the conjugate pad and then the target analytes were recognized specifically by nanozyme probes to form immunocomplexes. Along with lateral flow, nanozyme complexes were captured by S-dAb and aggregated at T-line, presenting a brown color signal. Nanozyme probes uncombined with antigen bound with the control IgG antibody at C-line. The luminol substrate and excitant were dissolved and added onto the detection membrane after chromatography for 15min. The chemiluminescent signals of T-line and C-line were instantly captured by a smartphone camera or CCD imaging system (Clinx MiniChemi). In parallel, the sensitivity of ELISA detection for S-RBD antigen was evaluated by the double-antibody sandwich ELISA kit according to the manufacturer’s instructions.

The pseudo-SARS-CoV-2 were packaged by transfecting 293T cells with a HIV-1based retroviral vector coding SARS-CoV-2 spike S1 protein and luciferase reporter gene. The pseudovirus titer was determined as 1010 virus copies/ml. The spike S1 protein expressed by pseudovirus was quantified as 860 ng/ml. The serially diluted pseudovirus was detected by nanozyme-based chemiluminescence test paper according to the method described above. All virus-related operations were carried out inside a Class Ⅱ biological safety cabinet.

Co-Fe@hemin nanozyme is a type of nanomaterial that possesses peroxidasemimicking activity. To characterize the morphology and microstructure of CoFe@hemin nanozyme, we used both TEM and SEM analysis. As shown in Fig. 1A and B, the Co-Fe@hemin nanozymes resemble spherical particles, with a mean diameter of approximately 80nm. DLS analysis showed that the mean hydrated diameter of our nanozyme spheres was approximately 100 nm (Fig. 1C). In the UV-Vis spectrum, we detected two absorption peaks for nanozyme spheres, which are corresponding to the characteristic peaks of Co-Fe nanoparticles and hemin molecules (Fig. 1D). Using the absorption spectrum, we verified that the hemin modification was present on the surface of Co-Fe@hemin nanozyme. We then analyzed the surface carboxyl group modification of Co-Fe@hemin nanozyme using TG-DSC (Fig. 1E). Furthermore, element composition and content analysis by EDS indicated that the atomic ratio of Fe to Co in the nanozyme particle is 13:1 (Fig. 1F).

Peroxidase activity of our Co-Fe@hemin nanozyme was determined by the specific activity (SA, U/mg) according to the established procedures in literature17. TMB is one of chromogenic substrates for HRP. As shown in Fig. 2A, the catalytic activity of CoFe@hemin in TMB chromogenic reaction was 69.915 U/mg, which is higher than CoFe NPs (9.836 U/mg) and Fe3O4 NPs (5.40 U/mg, data not shown). Next, we evaluated the chemiluminescence catalytic properties of Co-Fe@hemin nanozyme for luminol substrate using a EnVision Multilabel plate reader. The chemiluminescence signals of in Fig. 2B, the maximum chemiluminescent intensity of Co-Fe@hemin was comparable to that of HRP, while showing a three-fold increase over than that of CoFe NPs. In comparison, the catalytic chemiluminescence activity of Fe3O4 NPs was found to be lower than expected.

Our Co-Fe@hemin nanozyme displayed high chemiluminescent catalytic activity (the maximum intensity exceeds 8E+7) at the pH range of 9 to 14, demonstrating that our nanozyme tolerate alkaline conditions (Fig. 2C). In comparison, natural HRP only achieved its maximum catalytic activity for luminol substrate at pH 8.5, and significantly decreased as the pH increased. As shown in Fig. 2D, temperature pretreatment at 4 ℃ ~100 ℃

for 2 h failed to affect the catalytic activity of CoFe@hemin nanozyme. In contrast, HRP was inactivated after heat pretreatment above 80℃. Together, these results clearly showed that our nanozyme is more robust than HRP, especially under extreme PH and temperature conditions.

Next, we investigated the reaction parameters of excitant H2O2 in Co-Fe@heminluminol catalytic system. As shown in Fig. 2E, the maximum chemiluminescent intensity increased with the concentration of H2O2 at the range of 0.2 µM to 2 mM, reaching the plateau phase above a concentration of 1 mM. Moreover, we monitored superoxide anions (O2∙−) signal in Co-Fe@hemin-H2O2 system under alkaline conditions using the ESR technique (Fig. 2F). Together, these findings indicated that luminol was oxidated by O2∙− radicals derived from the reaction of Co-Fe@hemin with H2O2, suggesting a possible mechanism of nanozyme-catalyzed chemiluminescence.

After successfully validating the different core parts of our novel testing platform, we next designed the nanozyme-based chemiluminescence paper test. Fig. 3A depicts the design of our nanozyme-based chemiluminescence paper test for SARS-CoV-2 SRBD antigen. Our paper test is based on the principle of a double antibody sandwich immunoassay. First, Co-Fe@hemin NPs labeled with S-dAb as the chemiluminescence probes were dispersed onto the conjugate pad. Along with the lateral flow of the sample, nanozyme probes combined with S-RBD antigen and S-cAb, forming the sandwich immunocomplexes. The nanozyme probes possess high peroxidase activity and thus catalyze luminol substrate in the presence of H2O2 under alkaline condition, resulting in chemiluminescence. This chemiluminescent signal is then captured and analyzed by a smartphone camera or CCD imaging system (Clinx MiniChemi). The detection can be completed within 16 min. The chemiluminescent intensity ratio of T-line to C-line is positively correlated with the concentrations of S-RBD antigen. Due to the small bonding surface of S-RBD antigen and sequence differences from other βcoronaviruses (such as HCoV-HUK1 and HCoV-OC43)20, it is unlikely that crossreactivity will occur for non-target antigen.

To identify the best paired antibodies against SARS-CoV-2 S-RBD antigen, we next screened a series of anti-spike antibodies using ELISA. Among the six antibodies tested, clones GH4, CB6, TP52 and RP01 exhibited high binding activity for recombinant SRBD protein (Fig. 3B). We then manufactured the nanozyme probes labeled with CB6 and RP01 clones respectively, and combined these probes with strips immobilized with GH4, RP01, TP52 and GH4, TP52 as capture antibodies. As shown in Fig. 3C, the CB6 and GH4 pair gave the best performance in S-RBD detection. However, all other clone pairings resulted in problematic combinations, suggesting that these antibodies recognize either identical or crossed antigenic epitopes. Thus, we chose CB6 as a detection antibody and GH4 as a capture antibody for our final antibody pairs in nanozyme-based chemiluminescence paper test.

Using the

CB6 and

GH4 pair, we manufactured the nanozyme-based chemiluminescence test strips to detect S-RBD antigen. To assess the sensitivity of our test, we prepared a serial dilution of S-RBD protein samples. Following a chromatography procedure at room temperature for 15 minutes, the color signals of Tline were observed by naked eyes at a concentration higher than 25 ng/ml. Next, we added the luminol substrate solution. The chemiluminescent signal was instantly captured using a CCD or smartphone camera in dark environment (Fig. 4A). Next, we analyzed the chemiluminescent signal intensity using the Image-Pro Plus software. The calibration curve of antigen test was plotted as shown in Fig. 4B. Our analysis showed that our test detected as little as 0.1 ng/ml of S-RBD protein. The detection limit for SRBD was defined as X+3SD (X: mean of the negative control, SD: standard deviation) value. The linearity of our test was identified as ranging from 0.2 to 100 ng/ml, with a correlation coefficient of 0.9855 (Fig. 4B). As the parallel test using ELISA, S-RBD antigen was detectable at 0.1 ng/ml, with a linear calibration curve ranging from 0.1 to 1.56 ng/ml (Fig. 4C). The sensitivity of our nanozyme-based chemiluminescence paper test of S-RBD protein was similar to that of ELISA, however, at least 32-fold enlargement in linearity range.

Moreover, the nanozyme-based

chemiluminescence paper test specifically recognized the SARS-CoV-2 S-RBD protein, whereas it failed to detect the spike proteins of other known coronaviruses, such as SARS-CoV, MERS-CoV, HCoV-HKU1 and HCoV-OC43 (Fig. 4D). While SARS-CoV-2 shares more than 60% similarities with SARS-CoV, MERS-CoV, HCoV-HKU1 as well as HCoV-OC43 (cause common flu) as identified by genome sequence analysis20, we clearly demonstrated here that our paper test is specific for SARS-CoV-2 spike antigen.

To verify the validity of our paper test in actual viral infection, we collected the pseudo-SARS-CoV-2 expressing spike protein (S1 subunit). The titer of pseudo-SARSCoV-2 stock was determined as 1.0E+10 virus copies/ml using real-time RT-PCR. Gradient dilution series of pseudovirus were detected by the nanozyme-based chemiluminescence test paper. As shown in Fig. 5A, the detection limit of chemiluminescence paper test for pseudo-SARS-CoV-2 was 7.81E+6 copy/ml (corresponding to 0.671 ng/ml S1 protein). The linearity of our paper test was ranging from 7.81E+6 to 2E+9 copy/ml (R2=0.9907) (Fig. 5B). In the parallel test, the sensitivity of ELISA reached 7.81E+6 copy/ml, with a relatively narrow linear range (from 3.91E+6 to 6.25E+7 copy/ml) (Fig. 5C).

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