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Rapid determination of SARS-CoV-2 nucleocapsid proteins based on 2D/2D MXene/P–BiOCl/Ru(bpy)32+ heterojunction composites to enhance electrochemiluminescence performance

At the end of 2019, the novel coronavirus disease 2019 (COVID-19), a cluster of atypical pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been known as a highly contagious disease. Herein, we report the MXene/P–BiOCl/Ru(bpy)32+ heterojunction composite to construct an electrochemiluminescence (ECL) immunosensor for SARS-CoV-2 nucleocapsid protein (CoVNP) determination. Two-dimensional (2D) material ultrathin phosphorus-doped bismuth oxychloride (P–BiOCl) is exploited and first applied in ECL. 2D architectures MXene not only act as “soft substrate” to improve the properties of P–BiOCl, but also synergistically work with P–BiOCl. Owing to the inimitable set of bulk and interfacial properties, intrinsic high electrochemical conductivity, hydrophilicity and good biocompatible of 2D/2D MXene/P–BiOCl/Ru(bpy)32+, this as-exploited heterojunction composite is an efficient signal amplifier and co-reaction accelerator in the presence of tri-n-propylamine (TPA) as a coreactant. The proposed MXene/P–BiOCl/Ru(bpy)32+-TPA system exhibits a high and stable ECL signal and achieves ECL emission quenching for “signal on-off” recognition of CoVNP. Fascinatingly, the constructed ECL biosensor towards CoVNP allows a wide linear concentration range from 1 fg/mL to 10 ng/mL and a low limit of detection (LOD) of 0.49 fg/mL (S/N = 3). Furthermore, this presented strategy sheds light on designing a highly efficient ECL nanostructure through the combination of 2D MXene architectures with 2D semiconductor materials in the field of nanomedicine. This ECL biosensor can successfully detect CoVNP in human serum, which can promote the prosperity and development of diagnostic methods of SARS-CoV-2.

MXene architectures with 2D semiconductor materials in the field of nanomedicine. This ECL biosensor can successfully detect CoVNP in human serum, which can promote the prosperity and development of diagnostic methods of SARS-CoV-2.

Introduction
The novel coronavirus disease 2019 (COVID- 19), a cluster of atypical pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been known as a highly contagious disease. Owing to the rapid spread and multiple routes of transmission of SARS-CoV-2, it is a great challenge to guard against [1][2][3][4][5]. The clinical symptoms of COVID-19 disease include fever, cough, fatigue, shortness of breath, loss of smell and gastrointestinal symptoms, all these are similar to the common symptoms of influenza [6]. What's more, the asymptomatic patients and the ones during the incubation period are also infectious though they show none of mentioned symptoms, they can transmit the virus to the healthy persons [7]. Currently, the COVID-19 disease has posed great danger to the health of mankind and affected the global production and life order seriously, which is even far more destructive than 2003 SARS-CoV. Therefore, a rapid and effective strategy for diagnose of unprecedented COVID-19 at a large scale is of unparalleled significance [8]. At present, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) is regarded as the gold standard method for the diagnosis of COVID-19 [9]. Nevertheless, due to the expensive instruments, the complicated sample operation steps, the lack of the specialized laboratory and the trained personnel, the practical applications of this method is greatly restricted. In comparision, the coronavirus antigen testing is a promising candite to achieve the detection for SARS-CoV-2, which can overcome the above problems [10]. The nucleocapsid protein, as one of the major structural proteins encoded by SARS-CoV-2 genome, is abundantly expressed during infection [11,12], and is therefore regarded as a biomarker of SARS-CoV-2 in antigen testing [13]. Hence, a sensitive and selective method is critical for SARS-CoV-2 nucleocapsid protein (CoVNP).
Electrochemiluminescence (ECL), a technology combined chemiluminescence with electrochemistry, is induced via the electrochemical redox reaction of luminophores [14][15][16][17][18][19][20]. And ECL is considered to be a promising assay method for determination of CoVNP owing to its advantages of low background signals, high sensitivity, simple equipment and electrochemical controllability [21][22][23][24]. This advanced technology has been successfully applied in many researched fields including biomarkers detection [25], bio-imaging [26], point-of-care diagnosis [27], as well as DNA and proteins detection [28,29]. In particular, tris (2, 2 ′ -bipyridine) ruthenium (II) (Ru(bpy) 3 2+ ) and its derivatives have acted as ECL reagents on ECL sensors for long time because of their high ECL efficiency. Furthermore, tri-n-propylamine (TPA) is always utilized as the coreactant and reacts with Ru(bpy) 3 2+ to promote the ECL efficiency [30,31]. Nevertheless, in the pure Ru(bpy) 3 2+ /TPA system, Ru(bpy) 3 2+ is difficult to immobilize on the surface of electrode because of its good water solubility, which can not only cause Ru(bpy) 3 2+ to spill over and disperse freely in solution-phase, but also the consumption of the expensive Ru source [32]. Besides, to achieve good sensitivity, the high concentration of TPA is needed, which can lead to high background for the ECL system [33]. Taking these into consideration, the Ru (bpy) 3 2+ /TPA system is still not stable enough to achieve the excellent ECL performance for the ECL cycle. As a result, it remains urgently needed and extremely challenging to explore useful approach to attain a smooth channel for Ru(bpy) 3 [35,36].
Regrettably, the fast charge recombination rate and weak visible light response limit the application of BiOCl. Heteroatom doping is an effective way to improve the property of BiOCl, among various elements, phosphorus (P) is beneficial to the electron-hole separation, interfacial charge transfer and visible light absorption, resulted by its remarkable electron donating capability, applicable radius and variable valence [37]. However, we also find that the ultrathin P-doped BiOCl (P-BiOCl) has some limitations in ECL application on account of its own poor water dispersion and low conductivity. To address the aforementioned issues, it is imperative to develop a powerful supporting platform to anchor P-BiOCl nanosheets and promote the ECL signal intensity. MXene, a fast-growing family of 2D transitional metal carbides, nitrides and carbonitrides, has become a research hotspot since first reported by Gogotsi and his co-workers in 2011 [38]. Strikingly, MXene not only possesses well-defined layered architectures, muscular mechanical rigidity, superior stability, the active metal centers, but also high surface area. Owing to the outstanding physicochemical characteristics, MXene has exhibited satisfactory performance in various researched fields over the past decade, including supercapacitors [39,40], catalysis [41], ion batteries [42][43][44], solar cells [45], water purification [46], electromagnetic interference shielding [47], sensors [48,49] and so on. The most remarkable thing is that, research evidences show that the MXene based interfacial heterostructure materials are ideal candidates to fabricate effective ECL biosensors in recent years. For example, Yuan's group reports Ru-complex-grafted MXene nanosheets as an ECL indicator to fabricate ECL sensing platform for the tumor biomarker Mucin 1 (MUC1) [50]. Besides, Yang et al. prepare electroactive and catalytic 2D/2D Ti 3 C 2 T x /TiO 2 hybrids to accelerate the electro-oxidation of TPA and Ir(ppy) 2 (acac), the developed immunosensor shows high ECL efficiency in the determination of neuron-specific enolase (NSE) [51]. Inspired by the above innovations, we speculate that MXene is a good scaffold for P-BiOCl, and the formation of rich terminating surface groups (i.e., =O, -OH, and -F) on MXene after exfoliation can help the P-BiOCl easily attach on MXene [40,52,53].
Within this context, we design a 2D/2D MXene/P-BiOCl/Ru(bpy) 3 2+ heterojunction composite to construct a ECL biosensor platform for CoVNP. As displayed in Scheme 1, the synthesis and delamination of Ti 3 C 2 T x MXene is realized by in situ HF-generation and sonication exfoliation, then P-BiOCl and Ru(bpy) 3 2+ are anchored on MXene through solvothermal method and electrostatic interaction successively.

2+
hybrids are modified on the electrode to immobilize Anti-2019-nCoV-N McAb (Ab) and CoVNP. The ECL efficiency has been greatly promoted with MXene/P-BiOCl/Ru(bpy) 3 2+ /GCE in the presence of TPA as the coreactant for ECL "signal on". Concretely, the generation of the stronger ECL signal intensity is because MXene/P-BiOCl/Ru(bpy) 3 2+ can electrocatalyze the oxidation of TPA and greatly promoted the produce of intermediate TPA*, which also demonstrates that MXene/P-BiOCl/ Ru(bpy) 3 2+ is an efficient signal amplifier and coreaction accelerator.
Ultimately, the target CoVNP specifically binds to the MXene/P-BiOCl/ Ru(bpy) 3 2+ heterojunction composite, realizing the ECL intensity decrease of MXene/P-BiOCl/Ru(bpy) 3 2+ -TPA system for ECL "signal off". As expected, the developed ECL biosensor for CoVNP recognition possesses highly sensitive and selective ECL performance. Moreover, this sensor showcases excellent practical applicability in human serum. Notably, the combination of 2D MXene architectures and 2D semiconductor materials will guide for the design of high performance ECL biosensors, and it is valuable for the diagnosis and treatment of various diseases.

Preparation of Ti 3 C 2 T x MXene
Firstly, the multilayered Ti 3 C 2 T x (m-Ti 3 C 2 T x ) MXene is prepared via the selective etching of Al element from the raw material Ti 3 AlC 2 with in situ HF-generation. Specifically, 1.6 g of LiF is mixed with 20 mL of HCl (9 M) uniformly with constant stirring. Next, 1 g of Ti 3 AlC 2 is added to the above etching solution and the reaction proceeds at 40 • C for 24 h. The obtained product is centrifuged and washed several times with deionized water till the supernatant pH＞6. Then, the prepared m-Ti 3 C 2 T x MXene is dispersed in deionized water by ultrasonic treatment for 1 h under argon protection to avoid the oxidation of MXene. Finally, the single-layered Ti 3 C 2 T x (s-Ti 3 C 2 T x ) MXene is collected by centrifugation.

Preparation of MXene/P-BiOCl/Ru(bpy) 3 2+
The preparation of the 2D/2D MXene/P-BiOCl is performed via a solvothermal process according to a reported work with minor modifications [37]. Firstly, 5 mmol of Bi(NO 3 ) 3 ⋅5H 2 O is dissolved in 40 mL of ethylene glycol (C 2 H 6 O 2 ) with magnetically stirring to obtain transparent solution. Secondly, 5 mmol of KCl and 0.5 mmol of NaH 2-PO 2 ⋅H 2 O are dispersed in a beaker containing 10 mL of deionized water. Subsequently, the latter solution is added dropwise into the former one and kept continuous stirring for 1 h. Next, 1 mL of 5 mg/mL s-Ti 3 C 2 T x MXene dispersion is introduced into the above sufficiently mixed solution under stirring and kept for another 2 h. Then, transferring the resultant suspension into the 80 mL Teflon-lined autoclave and maintained at 180 • C for 24 h. After the solvothermal reaction, the resulting samples are centrifuged and washed several times with deionized water, ethanol and then dried at 60 • C. The product is named as MXene/P-BiOCl.

Fabrication of ECL biosensor
Before use, the glassy carbon electrode (GCE, 3 mm in diameter) is  To block the nonspecific site, 5 μL 1% the bovine serum albumin (BSA) is further coated on the electrode at 37 • C overnight for guaranteeing the specific interaction of Ab toward CoVNP. Finally, 5 μL of distinct concentrations of the target CoVNP is introduced and incubated at 37 • C for 60 min. What's noteworthy is that, every step is followed by phosphate buffer solution (PBS) washing to remove excess impurities. The as-prepared ECL biosensor MXene/P-BiOCl/Ru(bpy) 3 2+ /GCE is stored at 4 • C in a refrigerator for further use.

Electrochemistry and ECL measurements
Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements are tested in 5.

Characterization of MXene/P-BiOCl/Ru(bpy) 3 2+
The phase structure and morphology of the MXene/P-BiOCl/Ru (bpy) 3 2+ heterojunctions are first confirmed. The X-ray diffraction (XRD) patterns of Ti 3 AlC 2 , MXene, P-BiOCl, MXene/P-BiOCl and MXene/ P-BiOCl/Ru(bpy) 3 2+ are displayed in Fig. 1A, all these substances have good crystallinity. The diffraction peaks of Ti 3 AlC 2 can match with (PDF card 52-0875), and those of Ti 3 C 2 T x MXene after etching and exfoliation are in correspondence with the reported work [50]. The peaks of P-BiOCl shift slightly compared with those of BiOCl (PDF card 06-0249), resulted by the doping of P heteroatoms. Besides, it can be seen that the characteristic diffraction peaks are found in MXene/P-BiOCl and MXene/P-BiOCl/Ru(bpy) 3 2+ . The morphologies of the prepared nanomaterials are characterized by scanning electron microscopy (SEM). As exhibited in Fig. S1A, Ti 3 AlC 2 presents a tightly stacked morphology. Figs. S1B and S1C and Fig. 1B show that MXene after etching and exfoliation has larger interlayer spacing compared with Ti 3 AlC 2 . In Fig. S2, by shining a laser beam, a clear Tyndall Effect is observed in the s-Ti 3 C 2 T x MXene dispersion, in stark contrast to the deionized water, demonstrating the existence of a colloidal suspension. The SEM images of P-BiOCl indicate that it is a 2D sheet-like nanostructure with the stacked distribution, as depicted in Figs. S1D and S1E. Furthermore, when P-BiOCl nanosheets grow on the surface of MXene through solvothermal method, the self-assembled 2D/2D MXene/P-BiOCl heterojunction can be obtained (Fig. 1C, D, E). In addition, Fig. 1F confirms that Ru(bpy) 3 2+ has also anchored on the MXene/P-BiOCl by the electrostatic interaction.
To further investigate the microstructures of the composites, transmission electron microscope (TEM) and high-resolution TEM (HRTEM) are performed. The TEM image shown in Fig. 2A  . Bi 4f XPS spectrum is presented in Fig. 3B, the peaks at 163.9 and 158.6 eV are attributed to Bi 4f 5/2 and Bi 4f 7/2 , respectively. As shown in Fig. 3C, the peaks appearing at 284.5 and 280.8 eV refer to the binding energies of Ru 3d 3/2 and Ru 3d 5/2 , respectively.

Conductivity-enhanced ECL investigation of MXene/P-BiOCl/Ru (bpy) 3 2+
CV and the Nyquist plots recorded by EIS is adopted to validate the interfacial natures and conductivities of various materials in 5. . We also compare the property of the GCE and Au electrode, the corresponding ECL intensity-potential curves in Fig. S5 reveal that the GCE is superior to the Au electrode.

Electrochemical and ECL performance of the sensing platform
To characterize the fabrication procedure of the biosensor step by step, techniques including CV, EIS and ECL are conducted. As described in Fig. 4A, the largest redox peak presents on the bare GCE (curve a), whereas the redox peak current decreases dramatically when the GCE is modified by the binder Nafion (curve b), which can contribute to the poor conductivity of the binder. After the nanocomposites MXene/ P-BiOCl/Ru(bpy) 3 2+ decorating on the electrode (curve c), the current obviously increases due to its brilliant conductivity. Subsequently, it is readily see that the electrochemical signals continuously reduce with Ab (curve d), BSA (curve e) and CoVNP (curve f) stepwise incubating on MXene/P-BiOCl/Ru(bpy) 3 2+ /GCE, because of the non-conductivities of biomacromolecules [17].
Additionally, the corresponding EIS characterizations of the assembled biosensors are exhibited in Fig. 4B. The R et value of MXene/ P-BiOCl/Ru(bpy) 3 2+ /GCE (curve b) is much smaller than that of the bare GCE (curve a), forcefully demonstrating MXene/P-BiOCl/Ru(bpy) 3

2+
can improve the electron transfer. Then, along with the successive incubation of Ab (curve c), BSA (curve d) and CoVNP (curve e), the semicircle diameter increases gradually, because the electronically inert feature of biomacromolecules can block the electron transfer. The ECL responses of the biosensor construction process are also recorded. As can be seen from Fig. 4C, both of the bare GCE (curve a) and the Nafion/GCE (curve b) show extremely weak ECL signals. As  and CoVNP (curve f), the ECL signal gradually decreases. ECL intensitypotential curves in Fig. 4D show the same trend with Fig. 4C. All the experimental results of CV, EIS and ECL indicate that the proposed ECL biosensor has been successfully developed.

ECL mechanism exploration
The constructed ECL biosensor based on MXene/P-BiOCl/Ru(bpy) 3 2+ is employed as the specific recognition platform throughout the whole experiment, and the coreactant TPA is introduced to enhance the ECL intensity. Initially, when the prepared MXene/P-BiOCl/Ru(bpy) 3 2+ ink is decorated onto the surface of GCE, in turn, a strong initial ECL signal intensity is generated in the presence of TPA as the coreactant for ECL "signal on". Hereafter, CoVNP binds to the specific site of Ab, ultimately, when achieving the attachment of the plentiful target CoVNP on the electrode, electron transfer is blocked with remarkable ECL quenching for "signal off". The working mechanism of "signal on-off" is explicated through the electron transfer procedure as displayed in Scheme 2.

Optimization of experimental conditions
In order to obtain the satisfactory detection results, some main parameters that play vital roles for the detection efficiency are optimized, including pH value of PBS, incubation temperature, incubation time and concentration of Ab. As protein is highly sensitive to the pH of its surrounding environment, we investigate the compatibility of the biosensor to various pH conditions (ranging from 6.0 to 8.0). In Fig. S7A, the ECL intensity shows the best result as pH is 7.4, so the subsequent experiments are carried out at pH 7.4. As displayed in Fig. S7B, when the incubation temperature changes from 18 • C to 45 • C, the ECL signal gradually attenuates and tends to be stable until 37 • C. In view of 37 • C is close to the normal body temperature, so we do not select too high temperature as the optimal incubation temperature for the subsequent experiments. Therefore, 37 • C is considered to be as the best incubation temperature. Meanwhile, the effect of the incubation time for CoVNP should not be overlooked. Fig. S7C shows the ECL intensity decreases with the increasing of incubation time and reaches a plateau at 60 min, implying 60 min is the perfect time in our ECL strategy. Furthermore, the ECL intensity of the biosensor with the concentration of COVID-19 Ab is also explored, Fig. S7D shows that 20 μg/mL of Ab is optimal for the ECL performance of the developed biosensor.

ECL response of the biosensor towards CoVNP
To assess the analytical performance of the prepared ECL biosensor, different concentrations of CoVNP are determined under the optimal experimental condition. As displayed in Fig. 5A, the ECL response decreases gradually with the increment of the concentration of CoVNP enhancing from 1 fg/mL to 10 ng/mL. To our satisfaction, the ECL signal intensity presents a perfect linear relationship with the logarithm of the In addition, as listed in Table S1, as-constructed biosensor exhibits comparable or superior performance in determination for COVID-19 disease compared with other recently reported bioassay method. The excellent performance of the proposed detection method can be ascribed to that the heterojunctions MXene/P-BiOCl/Ru(bpy) 3 2+ can not only act as excellent accelerators of TPA, but also synergistically quench the ECL intensity.

Specificity, reproducibility and stability of the ECL biosensor
Specificity, reproducibility and stability are critical indicators for evaluating the properties of the established ECL biosensor. The specificity test is studied via employing different interferences including carcino-embryonic antigen (CEA), beta-human chorionic gonadotropin (HCG), neuron specific enolase (NSE) as well as prostate specific antigen (PSA) at the concentration of 10 ng/mL. As depicted in Fig. 6A, the ECL intensities of the mentioned interferences exhibit no significant differences to the blank sample. Besides, negligible differences of ECL responses are presented when detecting the mixture solution (10 ng/mL CEA, 10 ng/mL HCG, 10 ng/mL NSE, 10 ng/mL PSA and 1 ng/mL CoVNP) and the target 1 ng/mL CoVNP alone. The results substantiate that the biosensor can specifically recognize CoVNP. To investigate the reproducibility of the sensor, the fabricated electrode is adopted to detect five parallel samples incubated with 10 fg/mL CoVNP. As shown in Fig. 6B, the ECL intensities display little variation with the relative  standard deviation (RSD) of 2.64%, revealing excellent reproducibility of the fabricated sensor. Moreover, the operational stability tests are implemented by recording the ECL signals of different concentrations of CoVNP under 20 cycles consecutive scanning. And the results are presented in Fig. 6C and D, the ECL intensities show no significant change, the RSD are 3.37% for 100 pg/mL CoVNP and 1.49% for 10 ng/mL CoVNP respectively, which confirms that the ECL biosensor is endowed with superior stability.

Application in serum sample analysis
With the purpose of further validating the feasibility of the developed ECL biosensor in clinical application, the recovery studies are performed in human serum for CoVNP detection with the standard addition method. As displayed in Table S2, the recoveries of the ECL sensor are in the range of 93.0%-99.5%, and the RSD is within 5%. It means that our proposed MXene/P-BiOCl/Ru(bpy) 3 2+ /GCE ECL biosensor platform has satisfactory reliability and practical applicability.

Conclusions
In conclusion, the 2D/2D MXene/P-BiOCl nanocomposite is a desired candidate to provide a smooth channel for Ru(bpy) 3 2+ and TPA in ECL reaction and promote the ECL performance for the ECL cycle. Besides, MXene/P-BiOCl/Ru(bpy) 3 2+ can electrocatalyze the oxidation of TPA and greatly promoted the produce of intermediate TPA*, highlighting that MXene/P-BiOCl/Ru(bpy) 3 2+ is an efficient signal amplifier and co-reaction accelerator. Based on the MXene/P-BiOCl/Ru(bpy) 3 2+ -TPA system, the developed "signal on-off" ECL biosensor performs the reliable recognition for CoVNP, obtaining a wide linear range from 1 fg/ mL to 10 ng/mL and a low LOD of 0.49 fg/mL (S/N = 3). In addition, the combination of 2D MXene architectures with 2D semiconductor materials has potential to construct high-performance ECL sensing platforms. This work presents a new perspective for the applications of ECL in the nanomedicine field and clinical practice, preventing various diseases and guarding the health of mankind.

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.

Data availability
The data that has been used is confidential.