Highly sensitive and selective surface plasmon resonance biosensor for the detection of SARS-CoV-2 spike S1 protein

Abstract

The reality that the coronavirus disease 2019 (COVID-19) is still raging around the world and making a comeback with a strong presence has highlighted the need for rapid and sensitive quantitative detection methods of viral RNA, antibody and antigen for widespread tracking and screening applications. Surface plasmon resonance (SPR) detection technology has achieved rapid development and become a standard measurement method in the fields of biosensing, biomedicine, biochemistry and biopharmaceuticals due to its advantages of high sensitivity, fast response and no need for labelling. Here, we report a sandwiched structure-based SPR biosensor for detecting a specific viral antigen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike S1 protein. The sensor combines a Ti₃C₂-MXene nanosheet modified sensing platform and polydopamine (PDA)–Ag nanoparticle (AgNP)/anti-SARS-CoV-2 spike S1 protein nanoconjugate signal enhancers, exhibiting a wide linear range of 0.0001 to 1000 ng mL⁻¹ with a low detection limit of 12 fg mL⁻¹ (S/N = 3). In the analysis of artificial saliva and human serum samples, the proposed SPR biosensor exhibits good reproducibility and high specificity, which indicates its potential for application in complex bodily fluids. The exploitation of the MXene-based SPR biochip for recognizing the SARS-CoV-2 antigen provides an accessible and rapid way for COVID-19 diagnosis, and promotes the application of 2D nanomaterial-based sensing chips in clinical diagnosis and disease screening. Significantly, the proposed method possesses general applicability that can be reprogrammed to detect any protein antigen if a corresponding specific nanobody is available.

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1. Introduction

The coronavirus disease 2019 (COVID-19) is still circulating globally and the number of cases continues to increase. Research on developing new detection technologies for achieving rapid, accurate, sensitive, and low-cost screening is of great significance for responding to the threat of COVID-19.^1–4 Currently, there are three main approaches for diagnosing COVID-19: nucleic acid detection, antibody detection and antigen detection.^5–7 The genes of the single-stranded RNA virus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly similar to SARS-CoV and other coronaviruses. Reverse transcription-polymerase chain reaction (RT-PCR) for measuring SARS-CoV-2 is the gold standard for detecting COVID-19. However, RT-PCR measurement is tedious and expensive, and RNA degrades easily. Antigen detection uses the principle of specific binding between antigen and antibody, and has long been used to quickly diagnose diseases such as influenza, Legionnaires’ disease, streptococcal pharyngitis, and acquired immunodeficiency syndrome (AIDS). The rapid epidemic of the new coronavirus has prompted new antigen-based or other rapid testing methods. In areas where the risk of new coronavirus infection is high, fast and simple, antigen testing can effectively share the pressure of RT-PCR testing. There are at least four main structural proteins in the SARS-CoV-2, including spike proteins (S protein), small envelope proteins (E protein), membrane proteins (M protein), and nucleocapsid proteins (N protein), which can be used as biomarkers for viral diagnostics.^8–12 Moreover, virus copies present in saliva range from 9.9 × 10² to 1.2 × 10¹¹ copies per mL throughout the duration of the infection.¹³ Because of these low numbers, developing sensitive detection methods and kits of SARS CoV-2 protein in bodily fluids will break through the key technical bottleneck of clinical sample inactivation and virus degradation that lead to decreased detection sensitivity.

Surface plasmon resonance (SPR) technology possesses the advantages of fast response, not needing labeling of the analyte, real-time qualitative and quantitative analysis, and providing reactant molecular bonding information, thus exhibiting great potential in clinical applications.^14–18 However, the inability of traditional SPR sensing technology to detect trace targets in complex matrices limits its practical application. The growing use of nanomaterials in fabricating SPR sensor chips and response signal enhancers is promising for SPR sensing.^19–23 Among them, two-dimensional (2D) materials have become a hot spot in the biosensing field due to their unique dimensional characteristics.^24–28 2D nanomaterials provide a large specific surface area for loading foreign molecules, and the interaction between 2D nanomaterials and the foreign molecules causes changes in electrical response. These 2D nanomaterial-based sensing platforms exhibit fast response speeds, high sensitivity, and good selectivity.

In 2011, Gogotsi and his colleagues proposed a new family of 2D nanomaterials called MXenes,²⁹ attracting widespread interest among researchers. MXenes specifically refer to a kind of material system with the structural formula of M_n+1X_nTx (n = 1–3), and can be produced by the selective etching of A layers from M_n+1AX_n phases, where M is an early transition metal, A represents an A-group element, X is either carbon and/or nitrogen and T refers to the group/modifier on the surface of the 2D material.^30–32 During the etching process, A atoms are replaced by OH or F atoms, and the massive MAX phase becomes a loose structure similar to an accordion. The interaction between M_n+1X_n layers becomes weaker and easier to separate. Afterwards, ultrasonic treatment of the MAX phase treated with the etchant can easily achieve the stratification of MXene.^33–35 Unlike the challenge of modifying graphene, MXene materials allow for flexible chemical modification and show good compatibility with water, organic solvents and organisms. The unique properties of excellent electrical conductivity, hydrophilicity, large specific area, easy modification and high stability make MXene materials competitive among 2D materials in the field of biosensing.^36–38 Very little research has investigated the potential of MXene-based SPR sensing chip in SARS-CoV-2 antigen detection.

One of the SARS-CoV-2 structural proteins, the S protein coated at the outer shell of the virus, can be cleaved by the host cell furin-like proteases into two separate S1 and S2 subunits. The S1 subunit contains the RBD sequence and is surface exposed while S2 subunit is membrane bound (Fig. 1), and they are the major targets for SARS-CoV-2 therapy and detection.^39–41,52 Antigen detection is a good choice for testing viral loading in early stages of the COVID-19 disease. This work explored 2D MXene-based SPR sensing strategy for detecting SARS-CoV-2 spike S1 protein abbreviated as S1 protein. In this study, we developed a Ti₃C₂-MXene-based biosensing chip functionalized with anti-S1 protein (CAP) as a sensing platform. After S1 protein was captured, PDA–AgNPs nanohybrids conjugated with anti-S1 protein (DET) as signal amplifiers were flowed over the sensing chip interface to further enhance detection sensitivity by forming a sandwiched structure. Our SPR biosensor targeting S1 protein exhibited an ultralow limit of detection (LOD) of 12 fg mL⁻¹. More importantly, the selective detection of S1 protein in artificial saliva and human serum confirmed its potential for clinical applications.

Fig. 1 Schematic showing the detection procedure of the proposed biosensor.

2. Materials and methods

2.1 Materials and reagents

Mouse anti-SARS-COV-2 spike S1 protein (Catalog No. RM17568) abbreviated as Ab₁, mouse anti-SARS-COV-2 spike S1 protein (Catalog No. RM17569) abbreviated as Ab₂, and recombinant SARS-COV-2 spike S1 protein with its tag (Catalog No. RP01262) abbreviated as S1 protein were purchased from ABclonal. Bovine serum albumin (BSA) was purchased from Ding Guo Biotechnology Company. Ti₃AlC₂ MAX phase was purchased from Carbon-Ukraine Ltd. Dopamine (DA) and 3-mercaptopropinic acid (MPA) were purchased from Jk Chemical. Hydrogen tetrachloroauratehydrate (HAuCl₄·3H₂O) was purchased from Acros. Artificial saliva was purchased from Shenzhen Zhongwei Instrument Equipment Co., LTD. Human serum samples were provided by China–Japan Union Hospital of Jilin University. Sodium phosphate buffered saline (PBS, 0.01 mol L⁻¹, pH 7.4) was prepared before use. All water used in this assay was prepared through a Millipore purification device. Antibody and antigen solutions were subpackaged and stored at −20 °C, and other biological reagents were kept at 4 °C.

2.2 Synthesis of PDA–AgNPs nanohybrids

PDA nanospheres were synthesized according to the previous report with slight modifications.⁴² Briefly, 1.5 mL of ammonia solution (25–28%) was added to a mixture of ethanol (30 mL) and DI water (60 mL) under vigorous stirring at room temperature. Thirty minutes later, 6 mL of dopamine hydrochloride solution (0.05 g mL⁻¹) was added to the above mixture and stirred vigorously for 40 h. The dark brown supernatant was centrifuged with ethanol and DI water in sequence at 13 000 rpm for 10 min. The obtained PDA nanospheres were then dispersed in 25 mL of DI water.

To prepare PDA–AgNPs nanohybrids, 60 mL of AgNO₃ (25 mmol L⁻¹) solution was mixed with 12 mL of PDA nanosphere solution and stirred continuously for 30 min to promote Ag⁺ adsorption on the surface of PDA nanospheres. Then, 20 μL of NaBH₄ solution (0.15 mol L⁻¹) was added to the mixture and stirred for 1 h. The resulting product was washed with DI water by centrifugation at 13 000 rpm for 10 min. The obtained PDA–AgNPs nanohybrids were redispersed in 15 mL of PBS and stored at 4 °C for further use.

2.3 Preparation of PDA–AgNPs–Ab₂ nanoconjugates

PDA–AgNPs–Ab₂ nanoconjugates were prepared by adding 150 μg of Ab₂ into 1.5 mL of PDA–AgNPs nanohybrid solution prepared above under gentle stirring and kept at 4 °C for 12 h. Antibodies can be attached firmly to the surface of PDA nanospheres by Schiff-base reaction between amino residues of Ab₂ and quinone groups formed in PDA. The redundant binding sites of PDA–AgNPs nanohybrids were blocked by 10 mg mL⁻¹ BSA in PBS to eliminate nonspecific binding. Free BSA was removed by centrifugation, and the precipitate was redispersed in 1.5 mL of PBS containing 1% BSA. The obtained conjugates were stored at 4 °C prior to use.

2.4 Fabrication of the sensing platform

Ti₃C₂-MXene nanosheets were prepared by etching bulk Ti₃AlC₂ as we reported previously.⁴³ The bare gold film was ultrasonically cleaned in ethanol and dried with N₂ flow. The cleaned gold film was coated on a glass slide and then fixed on the bottom of a reactor to be used as a sensing chip. Ti₃C₂-MXene (500 μL, 0.8 mg mL⁻¹) solution was injected into the reactor and incubated for 3 h at room temperature to complete the assembly on the bare gold film. Next, free Ti₃C₂-MXene nanosheets in the reactor were gently washed with DI water, and PBS was injected to balance the baseline. Afterwards, Ab₁ solution (500 μL of 100 μg mL⁻¹) was injected into the reactor and immobilized on Ti₃C₂-MXene nanosheets. After 3 h, free Ab₁ in the reactor was removed by PBS, and then 10 mg mL⁻¹ BSA solution was injected and incubated for 20 min to block the non-specific binding sites on the sensing platform. Finally, PBS was injected to wash off free BSA in the reactor, and the prepared sensing platform was ready to capture S1 protein in samples.

2.5 SPR detection

In this work, S1 protein was used as the detection model to develop an SPR-based antigen detection method for COVID-19 diagnosis. Sample solutions were prepared by spiking PBS with S1 protein standard solutions at various concentration levels. SPR measurements were performed on an angle modulation SPR biosensor based on the conventional Kretschmann configuration, and the in situ SPR responses that originated from refractive index changes of the sensing interface were collected by a TR2005 spectrometer (RES-TEC Resonant Sensor Technology, Germany). For S1 protein detection, samples with different concentrations were flowed over the sensing platform and incubated for 30 min, respectively. The SPR chip surface was washed with PBS to remove S1 protein that was not captured, and then 8 mg mL⁻¹ PDA–AgNPs–Ab₂ nanoconjugates were injected into the reactor, forming the sandwiched immunocomplexes to enhance response signals. After incubation for 30 min, unreacted PDA–AgNPs–Ab₂ nanoconjugates were removed from the reactor through PBS washing. The obtained SPR responses were recorded as shifts of resonant angle before injecting S1 protein samples and after PBS rinsing of PDA–AgNPs–Ab₂ nanoconjugates in the sensing system. A schematic of the sensing procedure is illustrated in Fig. 1. The immunoassay was executed at normal pressure and room temperature, and all SPR measurements in this work were repeated three times to guarantee repeatability.

3. Results and discussion

3.1 Characterization

Ti₃C₂-MXene nanosheets were synthesized from parent MAX-phase ceramics with a series of procedures of delamination, disintegration, and probe sonication breakage. X-ray powder diffraction (XRD) was employed to identify the crystalline structures of the prepared Ti₃C₂-MXene nanosheets, and the diffraction peaks located at ∼6.72°, 13.7°, 38.8° agree well with the previous report⁴⁴ (Fig. 2a). X-ray photoelectron spectroscopy (XPS) measurement was used to evaluate the chemical status of the Ti₃C₂-MXene nanosheets. The survey spectrum demonstrates that the prepared Ti₃C₂-MXene is mainly composed of elements Ti, C, O and F (Fig. 2b), and O and F originated from the –OH and F terminal groups formed on the surface of theTi₃C₂-MXene nanosheets during etching.²⁹ The prepared Ti₃C₂-MXene nanosheets were thin, highly transparent, and wrinkled from the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images (Fig. 2c and d).

Fig. 2 Structural characterization of Ti₃C₂-MXene nanosheets. XRD spectrum, XPS spectrum, SEM image and TEM image (a–d).

The components of the PDA–AgNPs nanohybrids were measured through XPS analysis. Compared with the survey spectrum of PDA, the existence of an Ag peak demonstrated the successful introduction of Ag content, and the Ag 3d spectrum resolved into two spin–orbit components located at the binding energies of 374.25 eV and 368.15 eV is consistent with the Ag 3d_3/2 and 3d_5/2 peaks of metallic silver, respectively⁴⁵ (Fig. 3a and b). TEM was used to characterize the morphology of the prepared PDA nanospheres and PDA–AgNPs nanohybrids. As shown in Fig. 3c, PDA nanospheres exhibited a uniform and regular spherical morphology, and the average diameter was approximately 70 nm. By in situ reduction of AgNO₃ solution, PDA nanospheres were covered with dense AgNPs with an average diameter of 7 nm, and no isolated AgNPs were observed in the TEM image (Fig. 3d). Through Schiff-base reaction between the quinone groups in PDA and amino group in the antibody, Ab₂ can be conjugated with PDA–AgNP nanohybrids to prepare PDA–AgNPs–Ab₂ signal enhancers without using any further surface activation or treatment and maintain high reaction activity. The UV-Vis absorption spectrum of PDA–Ag nanohybrids showed the characteristic adsorption peak of AgNPs at 400 nm. After conjugating with Ab₂, the appearance of a typical protein absorbance peak at 285 nm indicates the successful binding of Ab₂, and peak position of AgNPs shifted from 400 nm to 410 nm due to a part of Ab₂ adsorbing on the surfaces of AgNPs via the interaction of AgNPs and amino groups of Ab₂ (Fig. S1†).

Fig. 3 XPS survey spectra of PDA and PDA–AgNPs nanohybrids (a), the high-resolution XPS spectrum of Ag 3d (b), and TEM image of PDA (c) and PDA–AgNPs nanohybrids (d).

3.2 Fabrication of sensing platform

Ti₃C₂-MXene nanosheets were assembled on a flat gold film via the strong metal–carbon coupling between MXene and the gold film for immobilizing antibody,⁴⁶ and the whole process of preparing the sensing platform was monitored by the change of the SPR spectrum. The assembly of Ti₃C₂-MXene nanosheets could be completed within 12 h, leading to a resonant angle shift of 0.64° (Fig. 4a). The SEM image of Ti₃C₂-MXene modified sensing chip showed a thin layer of Ti₃C₂-MXene nanosheets well distributed on the surface of gold film with satisfactory surface coverage (Fig. 4b). The immobilization of Ab₁ on the sensing chip could be completed within 60 min, and the resulting resonant angle shift was 0.6°. The stability test was performed by washing the prepared sensing chip with PBS, and the obtained blueshift of the resonant angle with 10 washing cycles was less than 8% of the initial resonant angle shift (Fig. 4c), which ensures the stability of the proposed Ti₃C₂-MXene-based sensing platform. Moreover, we evaluated the shelf life of this sensing chip which was kept at 4 °C in a dark area by measuring its detection activity at regular intervals. The prepared Ti₃C₂-MXene-based sensing chip still showed 86% of the initial activity after storing for 21 days, further confirming its good stability (Fig. 4d).

Fig. 4 The resonant angle changes before and after assembling Ti₃C₂-MXene nanosheets and the immobilization of Ab₁ (a), SEM image of Ti₃C₂-MXene nanosheets modified gold film (b), SPR spectra of the prepared sensing platform before and after 10 times washing with DI water (c), and stability measurement of the Ti₃C₂-MXene-based sensing platform (d). Error bar = ±S.D. and n = 3.

3.3 S1 protein detection

For the proposed strategy, the SPR response signals were effectively amplified by introducing PDA–AgNPs–Ab₂ nanoconjugates into the sensing system after S1 protein was captured on the sensing platform. Hence, the optimal dosage of PDA–AgNPs–Ab₂ nanoconjugates in this assay was investigated. PDA–AgNPs–Ab₂ nanoconjugates were prepared with different concentrations and injected into the reactor that had incubated 10⁻⁶ g mL⁻¹ S1 protein. The resonant angle exhibited a continuous redshift with the increase of the PDA–AgNPs–Ab₂ nanoconjugate concentration up to 8 mg mL⁻¹, and then the curve was level, which indicates that PDA–AgNPs–Ab₂ nanoconjugates captured by the sensing platform had reached saturation (Fig. 5a). Hence, 8 mg mL⁻¹ was selected to be the optimal concentration of PDA–AgNPs–Ab₂ solution in this assay. Furthermore, blank human serum samples were determined by the present method to estimate the nonspecific adsorption of the sensing platform towards PDA–AgNPs–Ab₂ nanoconjugates. The obtained resonant angle exhibited no obvious shift, confirming negligible nonspecific interaction between the prepared sensing chip and the signal amplifiers.

Fig. 5 Relationship between the resonant angle shifts and concentrations of PDA–AgNPs–Ab₂ nanoconjugates (a), relationship between SPR responses and S1 protein concentrations measured by the proposed biosensor (b), the corresponding SPR spectra of S1 protein in the concentration range of 10⁻¹³ to 10⁻⁶ g mL⁻¹ (c), and the resonant angle shifts for the detection of 10⁻⁶ g mL⁻¹ S1 protein by MPA modified sensing platform, Ti₃C₂-MXene modified sensing platform, Ti₃C₂-MXene modified sensing platform combined with Ab₂ signal enhancer, Ti₃C₂-MXene modified sensing platform combined with PDA–Ab₂ signal enhancer, and Ti₃C₂-MXene modified sensing platform combined with PDA–AgNPs–Ab₂ signal enhancer (d). Error bar = ±S.D. and n = 3.

3.4 Sensitivity of the immunoassay

Under the optimal experimental conditions, the obtained SPR response signals exhibited a linear increase with the logarithm of S1 protein concentration ranging from 10⁻¹³ to 10⁻⁶ g mL⁻¹ (Fig. 5b), and the linear relationship of the relative resonant angle change (Δθ) versus concentration of S1 protein (C) was described as Δθ = 0.6098 + 0.0459 lg C (R² = 0.9983). The obtained LOD was as low as 12 fg mL⁻¹, which was defined as a signal-to-noise ratio of 3 s (where s is the standard deviation of the blank solution, n = 10). Fig. 5c displays the SPR spectra of detecting S1 protein with a concentration of 10⁻¹³ to 10⁻⁶ g mL⁻¹ by the proposed biosensor. The high sensitivity achieved by the signal amplification strategy should be attributed to the following three aspects: (1) owing to hydrophilicity, a large specific surface area, high stability and good biocompatibility, the Ti₃C₂-MXene-modified sensing chip shows higher capacity of immobilizing antibodies and maintaining their biological activity compared with the conventional organic molecular layer, which effectively improves the detection sensitivity; (2) the formation of Ab₁/S1 protein/Ab₂–AgNPs–PDA sandwiched structure greatly increases the mass captured by the SPR sensing chip, amplifying the response signal by increasing the real part of the refractive index of the chip surface; (3) the intense electromagnetic coupling between the dense AgNPs located on PDA nanospheres and the underlying Au film leads to the increase of the imaginary part of the refractive index, enhancing the detection sensitivity. Our biosensor shows advantages of a better detection limit and detection range compared with those reported in the literatures (Table 1). Reproducibility of the proposed SPR biosensor was evaluated with the relative standard deviation (RSD) which was achieved by conducting independent testing of each sample in triplicate with different sensors. The obtained RSDs for detecting S1 protein were less than 5% (Fig. 5b), suggesting that the preparation procedure is highly reproducible.

Table 1 Comparison of different methods for detecting the SARS-CoV-2 virus

Method	Analyte	Sample	LOD	Detection range	Ref.
Magnetic particle spectroscopy	Spike protein	PBS	1.56 nM	3.13–400 nM	47
Photoelectrochemical	S protein	Pharynx swabs	72 ng mL^−1	0.5–8 μg mL^−1	48
Colorimetric	S protein	Influenza A virus	4.98 ng mL^−1	4.98–139 ng mL^−1	49
ELISA	S-RBD	Blood	20.6 fg mL^−1	0.34–1065 pg mL^−1	50
Electrochemical	Spike protein	Saliva	1 pg mL^−1	1–1000 pg mL^−1	51
LSPR	S1 subunit	Saliva	0.26 nM	1–100 nM	52
Electrochemical	S-RBD	Saliva	1.68 ng mL^−1	1–100 ng mL^−1	53
Colorimetric	S1 subunit	PBS	11 ng mL^−1	10–100 ng mL^−1	54
Chemiluminescence	S-RBD	Pseudovirus	0.1 ng mL^−1	0.2–100 ng mL^−1	55
SPR	S-RBD	Serum	37 nM	25–1000 nM	56
SPR	S1 subunit	Serum	12 fg mL^−1	0.0001–1000 ng mL^−1	This work

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We further performed control experiments of a traditional mercaptopropionic acid (MPA)-based sensing platform, Ti₃C₂-MXene-based sensing platform, Ti₃C₂-MXene-based sensing platform combined with Ab₂ signal amplifier, Ti₃C₂-MXene-based sensing platform combined with PDA–Ab₂ signal amplifier, and Ti₃C₂-MXene-based sensing platform combined with PDA–AgNPs–Ab₂ signal amplifiers for determining S1 protein analyte with a concentration of 10⁻⁶ g mL⁻¹ to reveal their roles in enhancing the detection sensitivity with the present sensing strategy (Fig. 5d). The Ti₃C₂-MXene-based sensing platform was demonstrated to be more sensitive than a traditional MPA-modified sensing chip because of the increased number of available binding sites for loading antibodies. The classic sandwich method was used to further improve the detection sensitivity, and we evaluated Ab₂, PDA–Ab₂ and PDA–AgNPs–Ab₂ as response signal amplifiers, respectively. PDA nanospheres offer a large surface area for loading Ab₂ and enlarge the mass arriving on the interface of the sensing chip, resulting in more effective signal amplification compared with Ab₂ signal amplifiers. AgNPs have been reported to be significant in enhancing SPR detection sensitivity and widely applied in SPR biosensing by virtue of the electromagnetic coupling between AgNPs and the gold chip to amplify SPR response signals. Electromagnetic coupling effect and considerable increase of mass captured on the sensing chip derived from the formation of dense AgNPs on PDA nanospheres results in the obvious signal amplification. The present dual signal amplification strategy achieves a ∼8.5-fold increase in response signal over that of an MPA-based sensing strategy for detecting 10⁻⁶ g mL⁻¹ S1 protein. The prepared sensing platform can be reused by impulse injection of massive PBS with a large amount of ions into the reactor to dissociate antigen from antibodies immobilized on the sensing chip. The reusability of the fabricated biosensor was investigated by measuring the SPR responses to 10⁻⁸ g mL⁻¹ S1 repeatedly, and 17% of its initial activity lost after six reuse cycles (Fig. S2†).

3.5 S1 protein detection in artificial saliva and serum samples

Evaluating the detection selectivity is critical due to the actual requirement of real sample analysis. The practicability of the S1 protein biosensor was tested in a complex matrix including artificial saliva and human serum to mimic the biological conditions. The utilized artificial saliva and human serum samples were diluted 10 times by PBS prior to the addition of S1 protein for reducing non-specific interference of other components. The resulting recoveries of S1 protein in artificial saliva ranged from 91% to 114% with RSDs of 3.8% to 6.2% (Table 2), indicating that the proposed biosensor could potentially be used to determine S1 protein content for clinical diagnosis and screening of COVID-19. In addition, the spiked serum samples also exhibited a satisfactory recovery of 92% to 120% with RSDs of 3.5% to 6.8% (Table S1†), further demonstrating good performance of the present sensing strategy in analysis of real biological samples.

Table 2 Determination of S1 protein concentration in artificial saliva samples using the proposed biosensor (n = 3)

Content of S1 protein (ng mL^−1)	Spiked (ng mL^−1)	Detected (ng mL^−1)	Recovery (%)	RSD (%)
None	100	98	98	5.3
None	10	9.1	91	6.0
None	1	0.96	96	4.5
None	0.1	0.105	105	5.7
None	0.01	0.0093	93	3.8
None	0.001	0.00114	114	6.2

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4. Conclusion

In summary, we present an SPR-based antigen detection method capable of detecting SARS-CoV-2 via spike S1 protein recognition. Our biosensor provides a sensitive, and selective means for determining S1 protein using a sandwiched amplification strategy based on a Ti₃C₂-MXene modified sensing platform and PDA–AgNPs–Ab₂ response signal amplifiers. The proposed SPR biosensor provides a wide detection range with a low detection limit of 12 fg mL⁻¹. Moreover, this method exhibited satisfactory precision in evaluating S1 protein levels in artificial saliva, indicating its potential application of early diagnosis and screening of COVID-19. We believe that our work provides a rapid and real-time response, and sensitive and selective sensing strategy which could be easily applied in diagnosing other viral variants and diseases just by changing the antibodies on the sensing platform and signal enhancers. In addition, this work might offer rewarding profits and act as a reference for extending applications of MXene-based biochips in clinical diagnosis.

Author contributions

Conceptualization, Q. W., P. R.; methodology, Q. W.; validation, Q. W., W. W.; formal analysis, Q. W., P. R.; investigation, Q. W.; data curation, Q. W., W. W.; writing – original draft preparation, Q. W.; writing – review and editing, Q. W., F. C. and P. R.; visualization, P. R.; supervision, P. R.; project administration, P. R.; funding acquisition, Q. W., F. C. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We greatly acknowledge the Natural Science Foundation of Jilin Province (YDZJ202101ZYTS024), the National Natural Science Foundation of China (31771093), and the Program for JLU Science and Technology Innovative Research Team (JLUSTIRT, 2019TD-36). Jim Hesson copyedited the manuscript (https://www.academicenglishsolutions.com/editing-service).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2an00426g

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