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. 2023 Apr 29;1264:341300. doi: 10.1016/j.aca.2023.341300

Biopanning of specific peptide for SARS-CoV-2 nucleocapsid protein and enzyme-linked immunosorbent assay-based antigen assay

Pengxin Ma a,1, Junchong Liu a,1, Shuang Pang a,1, Wenhao Zhou b,1, Haipeng Yu a, Mingyang Wang a, Tao Dong a, Yanbo Wang a, Qiqin Wang c,∗∗, Aihua Liu a,∗
PMCID: PMC10148601  PMID: 37230729

Abstract

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide which triggered serious public health issues. The search for rapid and accurate diagnosis, effective prevention, and treatment is urgent. The nucleocapsid protein (NP) of SARS-CoV-2 is one of the main structural proteins expressed and most abundant in the virus, and is considered a diagnostic marker for the accurate and sensitive detection of SARS-CoV-2. Herein, we report the screening of specific peptides from the pIII phage library that bind to SARS-CoV-2 NP. The phage monoclone expressing cyclic peptide N1 (peptide sequence, ACGTKPTKFC, with C&C bridged by disulfide bonding) specifically recognizes SARS-CoV-2 NP. Molecular docking studies reveal that the identified peptide is bound to the “pocket” region on the SARS-CoV-2 NP N-terminal domain mainly by forming a hydrogen bonding network and through hydrophobic interaction. Peptide N1 with the C-terminal linker was synthesized as the capture probe for SARS-CoV-2 NP in ELISA. The peptide-based ELISA was capable of assaying SARS-CoV-2 NP at concentrations as low as 61 pg/mL (∼1.2 pM). Furthermore, the as-proposed method could detect the SARS-CoV-2 virus at limits as low as 50 TCID50 (median tissue culture infective dose)/mL. This study demonstrates that selected peptides are powerful biomolecular tools for SARS-CoV-2 detection, providing a new and inexpensive method of rapidly screening infections as well as rapidly diagnosing coronavirus disease 2019 patients.

Keywords: SARS-CoV-2 NP, SARS-COV-2 virus, Specific peptide, Phage display, ELISA

Graphical abstract

Image 1

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1. Introduction

The serious respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to spread rapidly around the world simultaneously with the pandemic outbreak of coronavirus disease 2019 (COVID-19) in late 2019. This has triggered unprecedented public-health issues and significantly weakened the global economy [1]. SARS-CoV-2, a new type of β-coronavirus [2], is a sphere-like particle with an envelope [3], which contains four major structural proteins: nucleocapsid (N)-, spike (S)-, envelope-, and membrane-protein [4]. The S protein, being a type I fusion protein, forms a trimer on the surface of the virus. The N protein (NP) is one of the main structural proteins expressed by SARS-CoV-2 and the most abundant in the virus [5]. NP mainly envelops the viral genome, which is used in virus replication [6], virus particle assembly, release, and interference with the cell cycle in the host. From the viewpoint of effective epidemic prevention, control, and treatment, NP is the key for enabling rapid and accurate diagnosis of people infected with the new coronavirus [7,8]. Currently, the most common detection method is nucleic acid detection. Although nucleic acid detection is highly sensitive, it suffers from drawbacks such as a lengthy detection time, expensive large-scale instruments, need for professional operators, and laboratories with strict biosafety conditions [9,10]. Furthermore, because the viral loads vary with samples, these tests may produce false negative results [[11], [12], [13]]. Viral antigen testing is a candidate method for diagnosing SARS-CoV-2 infection in its early stages [[14], [15], [16], [17]], which would facilitate controlling the epidemic because this method can be widely promoted and used to identify those who are at the highest risk of spreading the disease [18]. Among the four structural proteins of SARS-CoV-2, the mutation sites of variants are mainly concentrated in the S protein, whereas SARS-CoV-2 NP has sequence conservation [19], exceeding that of other coronavirus NP [20]. Thus, SARS-CoV-2 NP is of great interest as a marker for early diagnosis and detection of SARS-CoV-2 infection [21], enabling detection one day before clinical symptoms [6].

Phage display is an in vitro technique for selecting particular fusion peptides expressed on the phage surface [22,23]. After several rounds of biopanning with the target biomacromolecules, the most specific binding peptides are enriched from billions of peptides in the clonal library by affinity screening [24]. As a mature and powerful technology, phage display has been widely used in specific ligand screening [25], peptide drug development, biosensor development [[26], [27], [28]], bio-nanotechnology [[29], [30], [31]], antibody engineering [32], and targeted tumor imaging and therapy [33,34]. On account of their structural uniformity and stability, specific phage probes extracted from phage libraries have shown great potential as a new type of specific probe in immunoassay [[35], [36], [37], [38]].

Peptides offer several advantages over antibodies for certain biomedical applications. Compared with antibodies, peptides can be chemically synthesized at low cost and with good stability [39,40]. In addition, most of the peptides identified from natural sources or peptide library screening are sufficiently small in size and easy to modify [41,42]. Additionally, the smaller size of peptides compared with antibodies make the former less sterically hindered on the target surface [43].

To struggle for COVID-19, our goal is to discover useful peptides as molecular tools. In this study, phage clones from the C7C pIII phage display library are screened to identify those that bind to SARS-CoV-2 NP with high affinity and specificity. After affinity testing and antigenic epitope analysis by molecular docking, the corresponding specific peptide is synthesized and used as a capture probe to recognize SARS-CoV-2 NP for enzyme-linked immunosorbent assay (ELISA). Upon optimizing the conditions, the established ELISA method is used to detect SARS-CoV-2 NP with good sensitivity and accuracy, and is successfully applied in the analysis of SARS-CoV-2 virus.

2. Experimental section

2.1. Chemicals and reagents

The phage display loop-constrained heptapeptide (Ph.D.-C7C) library containing 2.8 billion independent random peptides was purchased from New England Biolabs (NEB, Beverly, MA, USA). The recombinant proteins of SARS-CoV-2 NP, Middle East respiratory syndrome coronavirus (MERS-CoV) NP, syndrome coronavirus (SARS-CoV) NP, SARS-CoV-2 spike 1 (S1), and SARS-CoV-2 spike receptor-binding domain (S-RBD) were obtained from Novoprotein Co., Ltd. (Shanghai, China). MERS-CoV S1 was obtained from ABclonal Tech Co., Ltd. (Wuhan, China). Horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody (HRP-anti-M13 mAb), human coronavirus (HCoV-OC43) NP, and human vascular endothelial growth factor 165 (VEGF165) were purchased from Sino Biological Co., Ltd. (Beijing, China). Mouse anti-SARS-CoV-2 NP monoclonal antibody (anti-SARS-CoV-2 NP mAb), goat anti-mouse immunoglobulin G (IgG)-HRP monoclonal antibody (IgG-HRP mAb), carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP) were kindly provided by HighTop Biotech Co., Ltd. (Qingdao, China). 3,3′,5,5′-Tetramethylbenzidine (TMB) was purchased from TCI Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) and human serum albumin (HSA) were obtained from Solarbio Life Sciences Co., Ltd. (Beijing, China).

2.2. Apparatus

The U-2910 spectrophotometer (Hitachi, Tokyo, Japan) and SPARK 10 M multi-function microplate reader (Tecan, Zurich, Switzerland) were used to record the UV–vis spectra and measure the absorbance, respectively.

2.3. Test for positive clones of chosen phages against SARS-CoV-2 NP

The phage bioscreening is described in the Supplementary Information. Phage ELISA was used to study the binding affinity between SARS-CoV-2 NP and the displayed peptides from the selected phage monoclones. Briefly, SARS-CoV-2 NP (1 μg/mL) was fixed in the wells of the microplates overnight. The wells were washed once with Tris-buffered saline (TBS) containing 0.5% Tween-20 (0.5% TBST), which was blocked with BSA for more than 1 h. Thereafter, the wells were washed 3 times with 0.5% TBST. The obtained phage monoclones were diluted stepwise, added to each well, and incubated for 2 h. Afterward, the wells were washed 6 times with 0.5% TBST to remove unbound phages. Subsequently, HRP-anti-M13 mAb was added and incubated with shaking for 1.5 h at room temperature (RT). Finally, color developing solution was added to the well and vibrated at RT to promote the color reaction; 2 M H2SO4 (50 μL) was added to terminate the color-developing reaction. The optical density at 452 nm (OD452nm) was recorded.

2.4. Molecular docking

To understand the mode of interaction between SARS-CoV-2 NP and the peptide, molecular docking was performed. The initial receptor structures were constructed based on the crystal structure of the SARS-CoV-2 NP N-terminal domain (NTD, Protein Data Bank (PDB) code: 7ACT) and C-terminal domain (CTD, PDB code: 7CE0) from PDB [44]. All non-standard groups (ligand and water) in the receptor structures were deleted. Molecular Operating Environment (MOE) was used to build and optimize the peptide structures. Molecular docking was carried out with the induced-fit docking protocol. In the SiteFinder module in MOE, the proteins (NTD and CTD) are regarded as the receptor and docking sites. The change in Gibbs free energy (ΔG) initial scoring methodology was used for conformational sampling, and the force field refinement with generalized-born volume integral/weighted surface area ΔG rescoring was used to screen the best docking pose [45]. The specificity test, dissociation constant assay, and peptide synthesis are described in the Supplementary Information.

2.5. Sandwich ELISA using peptide probe for SARS-CoV-2 NP

Briefly, 10 μg/mL of the specific peptide probe (100 μL) was fixed in microplates and shaken overnight. Further, the wells were incubated with 100 μL of SARS-CoV-2 NP with vibration for 3 h, followed by washing once with phosphate buffered saline (PBS) containing 0.05% Tween-20 (0.05% PBST). The wells were incubated with mouse anti-SARS-CoV-2 NP mAb (1:10,000) for 1.5 h with shaking and washed with 0.05% PBST once, then incubated with IgG-HRP mAb (1:15,000) with shaking for 1.5 h at RT. Further, the wells were color-developed. The OD 452nm value of each well was measured. The procedure for optimizing the ELISA conditions is described in the Supplementary Information.

2.6. Working curve for SARS-CoV-2 NP

Under the optimized ELISA conditions, that is, after 100 μL of peptide probe (10 μg/mL) was fixed in the microplates for 6 h at 37 °C, different concentrations of SARS-CoV-2 NP were added and incubated for 3 h at RT. This was followed by the addition of mouse anti-SARS-CoV-2 NP mAb (1:5000) and incubation at RT for 1.5 h. The system was then incubated with goat anti-mouse IgG-HRP mAb (1:15,000) with shaking at RT for 1.5 h. The color was developed and the OD 452nm values of the wells were recorded to construct the working curve.

2.7. Detection of SARS-CoV-2 virus

The preparation and subsequent treatment of SARS-CoV-2 virus was carried out at the Wuhan Institute of Virology (with biosafety level 3), Chinese Academy of Sciences. The virus was amplified and assayed by standard plaque formation on Vero E6 cells. Considering that exposure to SARS-CoV-2 virus or clinical samples carries a high risk of infection, heating is a simple method of inactivating the virus [46]. The SARS-CoV-2 virus was inactivated by heating for 30 min at 65 °C and further treated by mixing with 15% radio immunoprecipitation assay buffer lysis solution. The as-obtained aqueous samples were serially diluted with PBS buffer and detected under the optimized ELISA conditions.

3. Results and discussion

3.1. Biopanning of specific peptide for SARS-CoV-2 NP recognition

3.1.1. pⅢ phage library-based biopanning of SARS-CoV-2 NP specific phage monoclones

The detailed experimental protocol for the phage display can be found in the Supplementary Information. Herein, a pIII cyclic heptapeptide phage display library was used to pan phage clones that specifically bind to SARS-CoV-2 NP (Scheme S1). After four rounds of biopanning, the number of phages in each round of input and output was calculated by titration. The phage recovery rate (output phage/input phage) increased with the biopanning rounds (Table S1), indicating effective enrichment of the SARS-CoV-2 NP-binding phages.

3.1.2. Analysis of DNA sequence of randomly selected phage clones

As expected, the phages appearing in subsequent rounds of output have higher targeting affinity [26]. After four rounds of bioscreening, the amplified phage clone DNAs were extracted, and were subjected to electrophoresis using 1% agarose gel. Twenty-six phage clones were randomly chosen for amplification to extract DNA for sequencing, and ten sequences were obtained (Table S2). Focus was placed on the peptides with multiple repetitive sequences, namely phage-N1 (peptide N1 sequence: ACGTKPTKFC, 6/26), phage-N2 (peptide N2 sequence: ACPTTSTQYC, 5/26), phage-N3 (peptide N3 sequence: ACTDKASSSC, 5/26), phage-N4 (peptide N4 sequence: ACTPRSANYC, 3/26), and phage-N5 (peptide N5 sequence: ACLKTYWYNC, 2/26), because these peptides are more likely to have a higher affinity for SARS-CoV-2 NP.

3.1.3. Positive clones of chosen phages for targeting antigen

The binding characteristics of the above five phage monoclones were preliminarily evaluated. The phage monoclones are generally identified as positive clones of the target, given that their OD 452nm values are over 1-fold higher than that of the control (BSA). The results demonstrate that the phages with the N1, N4, or N5 peptide sequence have the ability to bind to SARS-CoV-2 NP with a significant difference compared to the control (P < 0.001), indicating that they are positive clones (Fig. 1 ). Although there is no criterion for judging positive clones, clones N1 and N5 may have higher affinity for NP than the other clones.

Fig. 1.

Fig. 1

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Phage-based ELISA to detect the interaction between 5 phage clones and sSARS-CoV-2 NP. Here ***P < 0.001 is indicated significantly different from the control (n = 3).

3.2. Specific binding of monoclonal phage to target antigen

The specificity of the selected phages for SARS-CoV-2 NP was further verified by using common coronavirus antigens, including severe acute respiratory SARS-CoV NP, MERS-CoV NP, SARS-CoV-2 S1, SARS-CoV-2 S-RBD, MERS-CoV S1, HCoV-OC43 NP, and other unrelated antigens such as VEGF165, HSA, AFP, CEA, and BSA as the controls. Notably, phage-N1 binds specifically to SARS-CoV-2 NP (Fig. 2 A). However, phage-N4 and phage-N5 can bind to either MERS-CoV NP, SARS-CoV NP, or HCoV-OC43 NP (Fig. 2B and C), suggesting that phages displaying N4 or N5 have limited selectivity. Thus, peptide N1 has the best specificity. Peptide N1 with a -GGGGGS linker at the C-terminal was synthesized for the follow-up experiments.

Fig. 2.

Fig. 2

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Specificity experiments on the different biopanned phage monoclones with various antigens. (A) phage-N1, (B) phage-N4, (C) phage-N5 (n = 3).

3.3. Dissociation constant (Kd) of phage-N1/SARS-CoV-2 NP complex

The binding curve for the association of peptide N1 with SARS-CoV-2 NP was constructed using phage ELISA (Fig. 3 ). The K d value was determined as 5.13 ± 0.56 nM by fitting the binding curve. This value is comparable to the K d value for the interaction of SARS-CoV-2 NP with commercial mAb such as Biospacific-2 (A03080041P, 2 nM) [47], suggesting that the selected N1 has good binding affinity for SARS-CoV-2 NP.

Fig. 3.

Fig. 3

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The binding curve of phage monoclonal-N1 with SARS-CoV-2 NP (n = 3).

3.4. Peptide binding to SARS-CoV-2 NP

Molecular docking was used to further elucidate the possible binding sites of the peptide with SARS-CoV-2 NP. Disulfide bonds are formed between two cysteines (Cys2-Cys10) to generate cyclic peptide N1 (Fig. S1). The structure of SARS-CoV-2 NP has not been completely resolved [48]. Through molecular docking, it was found that the docking with NTD is better than that with CTD (Table S3). For peptide N1, the optimal results of five predicted docking sites were obtained. Docking between the peptide and SARS-CoV-2 NP NTD had the highest score (the final score in Table S3), which suggests the lowest binding energy [45], indicative of the formation of the optimal peptide–protein complex. From the front view and side view of the molecular docking images, the peptide probe N1 is bound to the “pocket” region on SARS-CoV-2 NP (Fig. 4 A). The binding interfaces of the amino acid residues (Phe9, Pro6, and Lys5) of peptide N1 interact with the Thr49, Arg107, and Thr 54 residues of the SARS-CoV-2 NP (Fig. 4B), respectively, to form a hydrogen bond network [47,49]. In addition, the interaction between the hydrophobic amino acids (Trp, Phe, Val, Pro) is another possible reason for the high affinity [50,51]. Furthermore, Phe9 and Pro6 of peptide N1 are speculated to be the key amino acids.

Fig. 4.

Fig. 4

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Molecular docking results. The protein is expressed by white surface on which interacted amino acids are labeled as dark blue, while the peptide N1 are in cyan. (A) Front view and side view of the docking result of peptide N1 with SARS-CoV-2 NP NTD. (B) The interaction network between peptide N1 and SARS-CoV-2 NP NTD. The dashed yellow lines indicate hydrogen bonding. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.5. SARS-CoV-2 NP detection by peptide probe-based ELISA

Generally, traditional sandwich ELISA uses two kinds of antibodies. The first antibody captures the antigen that is identified by a second antibody. Herein, we replaced the original primary antibody with peptide probe N1, considering that peptide N1 can specifically bind to SARS-CoV-2 NP with good affinity. The peptide probes fixed to the wells of the microtiter plate can selectively capture SARS-CoV-2 NP, which is subsequently recognized by mouse anti-SARS-CoV-2 NP mAb and added goat anti-mouse IgG-HRP mAb. The addition of TMB and hydrogen peroxide, as the substrates of HRP, enabled color development in the wells (Scheme S2). Because the peptide probe was small, the presence of a blocking agent such as BSA would partially block the active sites of the peptide probe during the blocking process, thereby affecting the detection sensitivity. Therefore, a non-blocking condition was applied with 0.05% PBST to eliminate non-specific background signals. Here, the ELISA conditions were optimized, including the pH of the coating buffer, concentration of the peptide probe, antigen incubation time, anti-SARS-CoV-2 NP mAb dilution, and goat anti-mouse IgG-HRP mAb dilution.

3.5.1. Effect of coating buffers on ELISA

The selection of coating buffers is dependent on the type of microtiter plate and the immobilized proteins. To select a suitable coating buffer, PBS (pH 7.4), TBS (pH 7.5), TBS (pH 8.0), Na2CO3/NaHCO3 (CB, pH 8.6), and CB (pH 9.6) (each in 50 mM) were tested. Apparently, the OD 452nm value obtained with TBS (50 mM, pH 8.0) was higher than those obtained with the other buffers (Fig. 5 A), indicating that TBS (50 mM, pH 8.0) is the optimal coating solution for immobilizing the peptide probes.

Fig. 5.

Fig. 5

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Optimization of ELISA conditions. Effects of (A) different coating solutions with different pH buffer, (B) peptide probe concentrations, (C) peptide incubation condition, and (D) incubation time of antigen on OD452nm. (E) Both OD452nm and P/N ratio (red) as a function of anti-SARS-CoV-2 NP mAb dilution. (F) Both OD452nm value and P/N ratio (red) as a function of IgG-HRP mAb dilution (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.5.2. Effect of peptide dosage on ELISA

The final concentration of peptide N1 prepared in TBS (50 mM, pH 8.0) was varied in the experiments. The OD 452nm value increased when the peptide dosage was increased from 1 to 10 μg/mL. Thereafter, the OD 452nm decreased as the concentration of the peptide probe increased (Fig. 5B).

3.5.3. Effect of peptide coating condition on ELISA

Different incubation conditions for 10 μg/mL peptide N1 were tested. A relatively higher signal was observed at 37 °C after 6 h (Fig. 5C). These conditions were used in the subsequent experiments.

3.5.4. Effect of antigen incubation time on ELISA

The key feature of sandwich ELISA is that the peptide probe captures SARS-CoV-2 NP specifically. The incubation time of the antigen is an important factor. During 4 h incubation, the signal increased with the incubation time up to 3 h; thereafter, the signal declined slightly (Fig. 5D). Thus, 3 h of incubation was employed.

3.5.5. Effect of anti-SARS-CoV-2 NP mAb dilution

In this sandwich ELISA, SARS-CoV-2 NP is captured by the mouse anti-SARS-CoV-2 NP mAb, which is identified by the signal antibody, goat anti-mouse IgG-HRP. The positive-to-negative ratios (P/N) of 11.43 and 11.01 were favorable for capture by mouse anti-SARS-CoV-2 NP mAb with dilution ratios of 1:2500 and 1:5000, respectively (Fig. 5E). For better economic benefit, a dilution ratio of 1:5000 was selected.

3.5.6. Effect of goat anti-mouse IgG-HRP mAb dilution

To obtain the optimal amplification effect, we explored effect of the goat anti-mouse IgG-HRP mAb dilution ratio. The P/N ratio was optimal when the IgG-HRP mAb dilution was 1:15,000 (Fig. 5F). Thus, these conditions were used for subsequent experiments.

3.6. Calibration curve

Under the above optimized conditions, the OD 452nm was measured for SARS-CoV-2 NP standard solutions of different concentrations. The OD 452nm values increases linearly with increasing SARS-CoV-2 NP concentration within the range of 0.2–200 ng/mL, where the regression equation is y = 0.00536*x + 0.05796 (R 2 = 0.995) (Fig. 6 ). The calculated limit of detection (LOD) is 61 pg/mL (∼1.2 pM) (3σ/S). The analytical parameters of different methods for SARS-CoV-2 NP detection are summarized in Table S4. The LOD for SARS-CoV-2 NP was reported for the lateral flow immunoassay (2 ng/reaction) [52], magnetic particle spectroscopy platform (12.5 nM, ∼588 ng/mL) [53], double-antibody sandwich ELISA (0.78 ng/mL) [54], nanomechanical sensor platform (0.71 ng/mL) [55], microfluidic magneto immunosensor (0.23 ng/mL) [6], half-strip lateral flow assay (0.65 ng/mL) [56], ELISA (1.0 ng/mL) [57], and MagPlex fluid array assay (0.05 ng/mL) [58]. Apparently, the developed method has favorable sensitivity in detecting SARS-CoV-2 NP, which is superior to that of most methods. The advantage of using the peptide lies in its good stability and high selective affinity for the target [59]. Cyclization of the peptide readily generates a flat conformation, with the amino and carbonyl groups on the amide backbone forming a ring, which causes the amino acid side chain to orient outwardly. Conversely, the cyclic structure may limit the change that may occur during the peptide confirmation process, thereby maintaining the binding affinity and limiting possible degradation of the peptide by the protease [60]. We speculate that the good detection sensitivity is attributed to the excellent affinity of peptide probe N1 for the target antigen, which can be tightly captured by the probe. Therefore, the use of short peptides as biological recognition elements may become one of the most promising strategies.

Fig. 6.

Fig. 6

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The calibration curve for SARS-CoV-2 NP (n = 3). (The inset shows the enlarged image inside the circle).

3.7. Detection of SARS-CoV-2 virus

To assess the practicability of the as-developed ELISA method for real virus detection, SARS-CoV-2 virus was assayed after inactivation. The cut-off value was 50 TCID50 (median tissue culture infective dose)/mL real virus (Fig. 7 ), which was determined from the mean value of OD 452nm in the absence of the antigen plus three times the standard deviation. Recent studies reported detecting SARS-CoV-2 virus with different cut-off values: nanozyme-based chemiluminescence paper test (360 TCID50/mL) [61], Sanger sequencing-based assay (∼140 TCID50/mL) [62], gold cluster-based lateral flow immunoassay (54 TCID50/mL) [63], fluorescent microsphere immunochromatography (1000 TCID50/mL) [64]. Obviously, the proposed method has good sensitivity for the real virus. Thus, the as-developed method can be considered a safe diagnostic method for heating-inactivated clinical samples to avoid infection during analysis.

Fig. 7.

Fig. 7

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Sensitivity of the proposed ELISA for SARS-CoV-2 virus. The red dashed line is the cut-off value (the average of OD452 nm value for 0 TCID50/mL SARS-CoV-2 virus plus three times the standard deviation) (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

4. Conclusion

In summary, upon biopanning with the Ph.D.-C7C phage display library, a phage displaying cyclic peptide N1 was discovered. The chosen peptide N1 with linker -GGGGGS at the C-terminus was synthesized, which binds to SARS-CoV-2 NP with good specificity and high affinity. Molecular docking indicates that the identified peptide is bound to the “pocket” region on SARS-CoV-2 nucleocapsid NTD through formation of hydrogen bond network and hydrophobic interaction. By using the synthesized peptide as the SARS-CoV-2 NP capture probe, a direct ELISA was established, which is capable of detecting SARS-CoV-2 NP with a LOD of ∼1.2 pM. Moreover, this method could detect SARS-CoV-2 virus at limits as low as 50 TCID50/mL. Therefore, the as-developed peptide-based ELISA is expected to be a candidate method for the rapid detection of SARS-CoV-2 for screening infected patients.

CRediT authorship contribution statement

Pengxin Ma: Data curation, Methodology, Investigation, Writing – original draft. Junchong Liu: Data curation, Investigation, Writing – original draft. Shuang Pang: Data curation, Methodology, Investigation, Writing – original draft. Wenhao Zhou: Methodology, Investigation. Haipeng Yu: Investigation, Writing – original draft. Mingyang Wang: Investigation. Tao Dong: Investigation. Yanbo Wang: Investigation. Qiqin Wang: Validation, Investigation, Writing – review & editing. Aihua Liu: Conceptualization, Supervision, Project administration, Funding acquisition, Writing – review & editing.

Declaration of competing interest

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.

Acknowledgements

National Natural Science Foundation of China (Nos.22174081, 82173773, 21475144) is grateful for financial support of this work.

Handling Editor: Professor Chuck Henry

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2023.341300.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.docx (348.6KB, docx)

Data availability

The authors do not have permission to share data.

References

  • 1.Bojkova D., Klann K., Koch B., Widera M., Krause D., Ciesek S., Cinatl J., Munch C. Proteomics of SARS-CoV-2-infected host cells reveals therapy targets. Nature. 2020;583:469–472. doi: 10.1038/s41586-020-2332-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Lv Z., Deng Y.-Q., Ye Q., Cao L., Sun C.-Y., Fan C., Huang W., Sun S., Sun Y., Zhu L., Chen Q., Wang N., Nie J., Cui Z., Zhu D., Shaw N., Li X.-F., Li Q., Xie L., Wang Y., Rao Z., Qin C.-F., Wang X. Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody. Science. 2020;369:1505–1509. doi: 10.1126/science.abc5881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Kim D., Lee J.-Y., Yang J.-S., Kim J.W., Kim V.N., Chang H. The architecture of SARS-CoV-2 transcriptome. Cell. 2020;181:914–921. doi: 10.1016/j.cell.2020.04.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Yao H., Song Y., Chen Y., Wu N., Xu J., Sun C., Zhang J., Weng T., Zhang Z., Wu Z., Cheng L., Shi D., Lu X., Lei J., Crispin M., Shi Y., Li L., Li S. Molecular architecture of the SARS-CoV-2 virus. Cell. 2020;183:730–738. doi: 10.1016/j.cell.2020.09.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Cubuk J., Alston J.J., Incicco J.J., Singh S., Stuchell-Brereton M.D., Ward M.D., Zimmerman M.I., Vithani N., Griffith D., Wagoner J.A., Bowman G.R., Hall K.B., Soranno A., Holehouse A.S. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. Nat. Commun. 2021;12:1936. doi: 10.1038/s41467-021-21953-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Li J., Lillehoj P.B. Microfluidic magneto immunosensor for rapid, high sensitivity measurements of SARS-CoV-2 nucleocapsid protein in serum. ACS Sens. 2021;6:1270–1278. doi: 10.1021/acssensors.0c02561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Zhang Z., Pandey R., Li J., Gu J., White D., Stacey H.D., Ang J.C., Steinberg C.J., Capretta A., Filipe C.D.M., Mossman K., Balion C., Miller M.S., Salena B.J., Yamamura D., Soleymani L., Brennan J.D., Li Y. High-affinity dimeric aptamers enable the rapid electrochemical detection of wild-type and B.1.1.7 SARS-CoV-2 in unprocessed saliva. Angew. Chem. Int. Ed. 2021;60:2–11. doi: 10.1002/anie.202110819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Song Y., Song J., Wei X., Huang M., Sun M., Zhu L., Lin B., Shen H., Zhu Z., Yang C. Discovery of aptamers targeting the receptor-binding domain of the SARS-CoV-2 spike glycoprotein. Anal. Chem. 2020;92:9895–9900. doi: 10.1021/acs.analchem.0c01394. [DOI] [PubMed] [Google Scholar]
  • 9.Deng J., Tian F., Liu C., Liu Y., Zhao S., Fu T., Sun J., Tan W. Rapid one-step detection of viral particles using an aptamer-based thermophoretic assay. J. Am. Chem. Soc. 2021;143:7261–7266. doi: 10.1021/jacs.1c02929. [DOI] [PubMed] [Google Scholar]
  • 10.Wang D.M., He S.G., Wang X.H., Yan Y.Q., Liu J.Z., Wu S.M., Liu S.G., Lei Y., Chen M., Li L., Zhang J.L., Zhang L.W., Hu X., Zheng X.H., Bai J.W., Zhang Y.L., Zhang Y.T., Song M.X., Tang Y.G. Rapid lateral flow immunoassay for the fluorescence detection of SARS-CoV-2 RNA. Nat. Biomed. Eng. 2020;4:1150–1158. doi: 10.1038/s41551-020-00655-z. [DOI] [PubMed] [Google Scholar]
  • 11.Feng M., Chen J., Xun J.N., Dai R.X., Zhao W., Lu H.Z., Xu J., Chen L., Sui G.D., Cheng X.J. Development of a sensitive ommunochromatographic method using lanthanide fluorescent microsphere for rapid serodiagnosis of COVID-19. ACS Sens. 2020;5:2331–2337. doi: 10.1021/acssensors.0c00927. [DOI] [PubMed] [Google Scholar]
  • 12.Sitjar J., Liao J.-D., Lee H., Tsai H.-P., Wang J.-R., Liu P.-Y. Challenges of SERS technology as a non-nucleic acid or -antigen detection method for SARS-CoV-2 virus and its variants. Biosens. Bioelectron. 2021;181 doi: 10.1016/j.bios.2021.113153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wiersinga W.J., Rhodes A., Cheng A.C., Peacock S.J., Prescott H.C. Pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (COVID-19): a review. JAMA. 2020;324:782–793. doi: 10.1001/jama.2020.12839. [DOI] [PubMed] [Google Scholar]
  • 14.Liu J., Pang S., Wang M., Yu H., Ma P., Dong T., Zheng Z., Jiao Y., Zhang Y., Liu A. An ultrasensitive ELISA to assay femtomolar level SARS-CoV-2 antigen based on specific peptide and tyramine signal amplification. Sensor. Actuator. B Chem. 2023;387 doi: 10.1016/j.snb.2023.133746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Karakuş E., Erdemir E., Demirbilek N., Liv L. Colorimetric and electrochemical detection of SARS-CoV-2 spike antigen with a gold nanoparticle-based biosensor. Anal. Chim. Acta. 2021;1182 doi: 10.1016/j.aca.2021.338939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Yeh Y.-J., Le T.-N., Hsiao W.W.-W., Tung K.-L., Ostrikov K., Chiang W.-H. Plasmonic nanostructure-enhanced Raman scattering for detection of SARS-CoV-2 nucleocapsid protein and spike protein variants. Anal. Chim. Acta. 2023;1239 doi: 10.1016/j.aca.2022.340651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Chen P.-H., Huang C.-C., Wu C.-C., Chen P.-H., Tripathi A., Wang Y.-L. Saliva-based COVID-19 detection: a rapid antigen test of SARS-CoV-2 nucleocapsid protein using an electrical-double-layer gated field-effect transistor-based biosensing system. Sensor. Actuator. B Chem. 2022;357 doi: 10.1016/j.snb.2022.131415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Guglielmi G. Fast coronavirus tests are coming. Nature. 2020;585:496–498. doi: 10.1038/d41586-020-02661-2. [DOI] [PubMed] [Google Scholar]
  • 19.Carlson C.R., Asfaha J.B., Ghent C.M., Howard C.J., Hartooni N., Safari M., Frankel A.D., Morgan D.O. Phosphoregulation of phase separation by the SARS-CoV-2 N protein suggests a bophysical basis for its dual functions. Mol. Cell. 2020;80:1092–1103. doi: 10.1016/j.molcel.2020.11.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Robson B. COVID-19 Coronavirus spike protein analysis for synthetic vaccines, a peptidomimetic antagonist, and therapeutic drugs, and analysis of a proposed achilles' heel conserved region to minimize probability of escape mutations and drug resistance. Comput. Biol. Med. 2020;121 doi: 10.1016/j.compbiomed.2020.103749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Diao B., Wen K., Zhang J., Chen J., Han C., Chen Y.W., Wang S.F., Deng G.H., Zhou H.W., Wu Y.H. Accuracy of a nucleocapsid protein antigen rapid test in the diagnosis of SARS-CoV-2 infection. Clin. Microbiol. Infect. 2021;27:289.e1–289.e4. doi: 10.1016/j.cmi.2020.09.057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Smith G.P., Petrenko V.A. Phage display. Chem. Rev. 1997;97:391–410. doi: 10.1021/cr960065d. [DOI] [PubMed] [Google Scholar]
  • 23.Smith G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985;228:1315–1317. doi: 10.1126/science.4001944. [DOI] [PubMed] [Google Scholar]
  • 24.Anand T., Virmani N., Bera B.C., Vaid R.K., Vashisth M., Bardajatya P., Kumar A., Tripathi B.N. Phage display technique as a tool for diagnosis and antibody selection for coronaviruses. Curr. Microbiol. 2021;78:1124–1134. doi: 10.1007/s00284-021-02398-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Yin L., Luo Y.Z., Liang B., Wang F., Du M., Petrenko V.A., Qiu H.J., Liu A.H. Specific ligands for classical swine fever virus screened from landscape phage display library. Antivir. Res. 2014;109:68–71. doi: 10.1016/j.antiviral.2014.06.012. [DOI] [PubMed] [Google Scholar]
  • 26.Lang Q., Wang F., Yin L., Liu M., Petrenko V.A., Liu A. Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen. Anal. Chem. 2014;86:2767–2774. doi: 10.1021/ac404189k. [DOI] [PubMed] [Google Scholar]
  • 27.Wu F.-L., Lai D.-Y., Ding H.-H., Tang Y.-J., Xu Z.-W., Ma M.-L., Guo S.-J., Wang J.-F., Shen N., Zhao X.-D., Qi H., Li H., Tao S.-C. Identification of serum biomarkers for systemic lupus erythematosus using a library of phage displayed random peptides and deep sequencing. Mol. Cell. Proteomics. 2019;18:1851–1863. doi: 10.1074/mcp.RA119.001582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Qi H., Wang F., Petrenko V.A., Liu A. Peptide microarray with ligands at high density based on symmetrical carrier landscape phage for detection of cellulase. Anal. Chem. 2014;86:5844–5850. doi: 10.1021/ac501265y. [DOI] [PubMed] [Google Scholar]
  • 29.Han L., Liu P., Zhang H.J., Li F., Liu A.H. Phage capsid protein-directed MnO2 nanosheets with peroxidase-like activity for spectrometric biosensing and evaluation of antioxidant behaviour. Chem. Commun. 2017;53:5216–5219. doi: 10.1039/c7cc02049j. [DOI] [PubMed] [Google Scholar]
  • 30.Liu P., Han L., Wang F., Petrenko V.A., Liu A.H. Gold nanoprobe functionalized with specific fusion protein selection from phage display and its application in rapid, selective and sensitive colorimetric biosensing of staphylococcus aureus. Biosens. Bioelectron. 2016;82:195–203. doi: 10.1016/j.bios.2016.03.075. [DOI] [PubMed] [Google Scholar]
  • 31.Han L., Shao C., Liang B., Liu A. Genetically engineered phage-templated MnO2 nanowires: synthesis and their application in electrochemical glucose biosensor operated at neutral pH condition. ACS Appl. Mater. Interfaces. 2016;8:13768–13776. doi: 10.1021/acsami.6b03266. [DOI] [PubMed] [Google Scholar]
  • 32.Wang G., Yin P.X., Wang J., Ma P.X., Wang Y.B., Cai Y.Y., Qi H., Liu A.H. Specific heptapeptide screened from pIII phage display library for sensitive enzyme-linked chemiluminescence immunoassay of vascular endothelial growth factor. Sensor. Actuator. B Chem. 2021;333 [Google Scholar]
  • 33.Wang F., Liu P., Sun L., Li C., Petrenko V.A., Liu A. Bio-mimetic nanostructure self-assembled from Au@Ag heterogeneous nanorods and phage fusion proteins for targeted tumor optical detection and photothermal therapy. Sci. Rep. 2014;4:6808. doi: 10.1038/srep06808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Wang F., Xu L., Zhang Y., Petrenko V.A., Liu A. An efficient strategy to synthesize a multifunctional ferroferric oxide core@dye/SiO2@Au shell nanocomposite and its targeted tumor theranostics. J. Mater. Chem. B. 2017;5:8209–8218. doi: 10.1039/c7tb02004j. [DOI] [PubMed] [Google Scholar]
  • 35.Han L., Xia H.Q., Yin L., Petrenko V.A., Liu A.H. Selected landscape phage probe as selective recognition interface for sensitive total prostate-specific antigen immunosensor. Biosens. Bioelectron. 2018;106:1–6. doi: 10.1016/j.bios.2018.01.046. [DOI] [PubMed] [Google Scholar]
  • 36.Liu J., Ma P., Yu H., Wang M., Yin P., Pang S., Jiao Y., Dong T., Liu A. Discovery of a phage peptide specifically binding to the SARS-CoV-2 spike S1 protein for the sensitive phage-based enzyme-linked chemiluminescence immunoassay of the SARS-CoV-2 antigen. Anal. Chem. 2022;94:11591–11599. doi: 10.1021/acs.analchem.2c01988. [DOI] [PubMed] [Google Scholar]
  • 37.Han L., Wang D., Yan L., Petrenko V.A., Liu A. Specific phages-based electrochemical impedimetric immunosensors for label-free and ultrasensitive detection of dual prostate-specific antigens. Sensor. Actuator. B Chem. 2019;297 [Google Scholar]
  • 38.Wang M., Zheng Z., Zhang Y., Wang G., Liu J., Yu H., Liu A. An ultrasensitive label-free electrochemical impedimetric immunosensor for vascular endothelial growth factor based on specific phage via negative pre-screening. Anal. Chim. Acta. 2022;1225 doi: 10.1016/j.aca.2022.340250. [DOI] [PubMed] [Google Scholar]
  • 39.Liu R., Li X., Xiao W., Lam K.S. Tumor-targeting peptides from combinatorial libraries. Adv. Drug Deliv. Rev. 2017;110–111:13–37. doi: 10.1016/j.addr.2016.05.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Wolfe M., Webb S., Chushak Y., Krabacher R., Liu Y., Swami N., Harbaugh S., Chávez J. A high-throughput pipeline for design and selection of peptides targeting the SARS-Cov-2 Spike protein. Sci. Rep. 2021;11 doi: 10.1038/s41598-021-01225-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Saw P.E., Xu X., Kim S., Jon S. Biomedical applications of a novel class of high-affinity peptides. Acc. Chem. Res. 2021;54:3576–3592. doi: 10.1021/acs.accounts.1c00239. [DOI] [PubMed] [Google Scholar]
  • 42.Wang Y., Wang M., Yu H., Wang G., Ma P., Pang S., Jiao Y., Liu A. Screening of peptide selectively recognizing prostate-specific antigen and its application in detecting total prostate-specific antigen. Sensor. Actuator. B Chem. 2022;367 [Google Scholar]
  • 43.McGregor D.P. Discovering and improving novel peptide therapeutics. Curr. Opin. Pharmacol. 2008;8:616–619. doi: 10.1016/j.coph.2008.06.002. [DOI] [PubMed] [Google Scholar]
  • 44.Dinesh D.C., Chalupska D., Silhan J., Koutna E., Nencka R., Veverka V., Boura E. Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein. PLoS Pathog. 2020;16 doi: 10.1371/journal.ppat.1009100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Rahman N., Basharat Z., Yousuf M., Castaldo G., Rastrelli L., Khan H. Virtual screening of natural products against type II transmembrane serine protease (TMPRSS2), the priming agent of coronavirus 2 (SARS-CoV-2) Molecules. 2020;25:2271. doi: 10.3390/molecules25102271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Abraham J.P., Plourde B.D., Cheng L. Using heat to kill SARS-CoV-2. Rev. Med. Virol. 2020;30:e2115. doi: 10.1002/rmv.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Yamaoka Y., Miyakawa K., Jeremiah S.S., Funabashi R., Okudela K., Kikuchi S., Katada J., Wada A., Takei T., Nishi M., Shimizu K., Ozawa H., Usuku S., Kawakami C., Tanaka N., Morita T., Hayashi H., Mitsui H., Suzuki K., Aizawa D., Yoshimura Y., Miyazaki T., Yamazaki E., Suzuki T., Kimura H., Shimizu H., Okabe N., Hasegawa H., Ryo A. Highly specific monoclonal antibodies and epitope identification against SARS-CoV-2 nucleocapsid protein for antigen detection tests. Cell Rep. Med. 2021;2 doi: 10.1016/j.xcrm.2021.100311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Gransagne M., Aymé G., Brier S., Chauveau-Le Friec G., Meriaux V., Nowakowski M., Dejardin F., Levallois S., Dias de Melo G., Donati F., Prot M., Brûlé S., Raynal B., Bellalou J., Goncalves P., Montagutelli X., Di Santo J.P., Lazarini F., England P., Petres S., Escriou N., Lafaye P. Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein. J. Biol. Chem. 2022;298 doi: 10.1016/j.jbc.2021.101290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Gao B., Hu X., Xue H., Li R., Liu H., Han T., Ruan D., Tu Y., Zhao Y. Isolation and screening of umami peptides from preserved egg yolk by nano-HPLC-MS/MS and molecular docking. Food Chem. 2022;377 doi: 10.1016/j.foodchem.2021.131996. [DOI] [PubMed] [Google Scholar]
  • 50.Tao H.M., Cheng L., Liu L.H., Wang H., Jiang Z.J., Qiang X., Xing L.J., Xu Y.F., Cai X.Y., Yao J., Wang M., Qiu Z. A PD-1 peptide antagonist exhibits potent anti-tumor and immune regulatory activity. Cancer Lett. 2020;493:91–101. doi: 10.1016/j.canlet.2020.08.009. [DOI] [PubMed] [Google Scholar]
  • 51.Zak K.M., Kitel R., Przetocka S., Golik P., Guzik K., Musielak B., Dömling A., Dubin G., Holak T.A. Structure of the complex of human programmed Death 1, PD-1, and its ligand PD-L1. Structure. 2015;23:2341–2348. doi: 10.1016/j.str.2015.09.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Kim H.-Y., Lee J.-H., Kim M.J., Park S.C., Choi M., Lee W., Ku K.B., Kim B.T., Changkyun Park E., Kim H.G., Kim S.I. Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc fusion proteins. Biosens. Bioelectron. 2021;175 doi: 10.1016/j.bios.2020.112868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Wu K., Chugh V.K., Krishna V.D., di Girolamo A., Wang Y.A., Saha R., Liang S., Cheeran M.C.J., Wang J.-P. One-Step, Wash-free, nanoparticle clustering-based magnetic particle spectroscopy bioassay method for detection of SARS-CoV-2 spike and nucleocapsid proteins in the liquid phase. ACS Appl. Mater. Interfaces. 2021;13:44136–44146. doi: 10.1021/acsami.1c14657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Zhang L., Zheng B., Gao X., Zhang L., Pan H., Qiao Y., Suo G., Zhu F. Development of patient-derived human monoclonal antibodies against nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 for coronavirus disease 2019 diagnosis. Front. Immunol. 2020;11 doi: 10.3389/fimmu.2020.595970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Agarwal D.K., Nandwana V., Henrich S.E., Josyula V.P.V.N., Thaxton C.S., Qi C., Simons L.M., Hultquist J.F., Ozer E.A., Shekhawat G.S., Dravid V.P. Highly sensitive and ultra-rapid antigen-based detection of SARS-CoV-2 using nanomechanical sensor platform. Biosens. Bioelectron. 2021;195 doi: 10.1016/j.bios.2021.113647. 113647-113647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Grant B.D., Anderson C.E., Williford J.R., Alonzo L.F., Glukhova V.A., Boyle D.S., Weigl B.H., Nichols K.P. SARS-CoV-2 coronavirus nucleocapsid antigen-detecting half-strip lateral flow assay toward the development of point of care tests using commercially available reagents. Anal. Chem. 2020;92:11305–11309. doi: 10.1021/acs.analchem.0c01975. [DOI] [PubMed] [Google Scholar]
  • 57.Zhang L., Fang X., Liu X., Ou H., Zhang H., Wang J., Li Q., Cheng H., Zhang W., Luo Z. Discovery of sandwich type COVID-19 nucleocapsid protein DNA aptamers. Chem. Commun. 2020;56:10235–10238. doi: 10.1039/d0cc03993d. [DOI] [PubMed] [Google Scholar]
  • 58.Anderson G.P., Liu J.L., Esparza T.J., Voelker B.T., Hofmann E.R., Goldman E.R. Single-domain antibodies for the detection of SARS-CoV-2 nucleocapsid protein. Anal. Chem. 2021;93:7283–7291. doi: 10.1021/acs.analchem.1c00677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Vanova V., Mitrevska K., Milosavljevic V., Hynek D., Richtera L., Adam V. Peptide-based electrochemical biosensors utilized for protein detection. Biosens. Bioelectron. 2021;180 doi: 10.1016/j.bios.2021.113087. [DOI] [PubMed] [Google Scholar]
  • 60.Li C., Chen X., Zhang F., He X., Fang G., Liu J., Wang S. Design of cyclic peptide based glucose receptors and their application in glucose sensing. Anal. Chem. 2017;89:10431–10438. doi: 10.1021/acs.analchem.7b02430. [DOI] [PubMed] [Google Scholar]
  • 61.Liu D., Ju C., Han C., Shi R., Chen X., Duan D., Yan J., Yan X. Nanozyme chemiluminescence paper test for rapid and sensitive detection of SARS-CoV-2 antigen. Biosens. Bioelectron. 2020;173 doi: 10.1016/j.bios.2020.112817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Lim H.J., Park M.Y., Jung H.S., Kwon Y., Kim I., Kim D.K., Yu N., Sung N., Lee S.-H., Park J.E., Yang Y.-J. Development of an efficient sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations. PLoS One. 2021;16 doi: 10.1371/journal.pone.0260850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Oh H.-K., Kim K., Park J., Im H., Maher S., Kim M.-G. Plasmon color-preserved gold nanoparticle clusters for high sensitivity detection of SARS-CoV-2 based on lateral flow immunoassay. Biosens. Bioelectron. 2022;205 doi: 10.1016/j.bios.2022.114094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Zhang C., Zhou L., Du K., Zhang Y., Wang J., Chen L., Lyu Y., Li J., Liu H., Huo J., Li F., Wang J., Sang P., Lin S., Xiao Y., Zhang K., He K. Foundation and clinical evaluation of a new method for detecting SARS-CoV-2 antigen by fluorescent microsphere immunochromatography. Front. Cell. Infect. Microbiol. 2020;10 doi: 10.3389/fcimb.2020.553837. [DOI] [PMC free article] [PubMed] [Google Scholar]

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