key: cord-0702582-95go74k3
authors: Chen, Winston Yenyu; Lin, Hang; Barui, Amit Kumar; Gomez, Ana Maria Ulloa; Wendt, Michael K.; Stanciu, Lia A.
title: DNA-Functionalized Ti(3)C(2)T(x) MXenes for Selective and Rapid Detection of SARS-CoV-2 Nucleocapsid Gene
date: 2021-12-30
journal: ACS Appl Nano Mater
DOI: 10.1021/acsanm.1c03520
sha: 85393abe6693832b2253f8f8d3596bddffef6618
doc_id: 702582
cord_uid: 95go74k3

[Image: see text] Coronavirus disease 2019 (COVID-19) is an emerging human infectious disease caused by severe acute respiratory syndrome 2 (SARS-CoV-2, initially called novel coronavirus 2019-nCoV) virus. Thus, an accurate and specific diagnosis of COVID-19 is urgently needed for effective point-of-care detection and disease management. The reported promise of two-dimensional (2D) transition-metal carbides (Ti(3)C(2)T(x) MXene) for biosensing owing to a very high surface area, high electrical conductivity, and hydrophilicity informed their selection for inclusion in functional electrodes for SARS-CoV-2 detection. Here, we demonstrate a new and facile functionalization strategy for Ti(3)C(2)T(x) with probe DNA molecules through noncovalent adsorption, which eliminates expensive labeling steps and achieves sequence-specific recognition. The 2D Ti(3)C(2)T(x) functionalized with complementary DNA probes shows a sensitive and selective detection of nucleocapsid (N) gene from SARS-CoV-2 through nucleic acid hybridization and chemoresistive transduction. The fabricated sensors are able to detect the SARS-CoV-2 N gene with sensitive and rapid response, a detection limit below 10(5) copies/mL in saliva, and high specificity when tested against SARS-CoV-1 and MERS. We hypothesize that the MXenes’ interlayer spacing can serve as molecular sieving channels for hosting organic molecules and ions, which is a key advantage to their use in biomolecular sensing.

Coronavirus disease 2019 (COVID- 19) pandemic was first detected in December 2019 in Wuhan, China and has caused a global outbreak and a serious public health concern. The novel coronavirus was named severe acute respiratory syndrome 2 (SARS-CoV-2, initially named 2019-nCoV), the pathogen causing COVID-19, which causes respiratory and intestinal illness in both humans and animals. As of mid-October 2021, the rapid spread of SARS-CoV-2 has impacted more than 200 countries, infecting more than 240 million people with over 4 million confirmed deaths. 1 COVID-19/SARS-CoV-2 is still devastating people and countries around the world and seems to be difficult to tackle at this moment. There have been other recurrent outbreaks from emerging coronaviruses, such as severe acute respiratory syndrome (SARS-CoV-1) in 2003 and Middle East respiratory syndrome (MERS-CoV) in 2012. Thus, there is a critical need for the development of a rapid, inexpensive, and reliable identification method toward novel viruses that can greatly facilitate public health response to emerging viral threats.

Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is one of the most widely used laboratory methods for the detection of SARS-CoV-2 using samples from respiratory secretions. 2 The targets for RT-PCR molecular assays developed by various countries are genetically similar. The national RT-PCR protocols among various countries typically target the nucleocapsid (N) gene of SARS-CoV-2 due to its highly conserved nature and few nucleotide changes over time. 3 RT-PCR is highly sensitive and specific for detecting viral RNA by amplification of specific sequence regions. 4 However, molecular diagnosis using RT-PCR presents some drawbacks, including expensive fluorescent tags, long processing time, tedious sample preparation, need for laboratory facilities, and manpower issues, limiting testing capacity during large-scale outbreaks. Thus, there is still an urgent need to develop rapid and accurate diagnostic technologies for the detection of SARS-CoV-2.

Biosensors are promising candidates for providing an alternative and reliable solution to clinical diagnosis, real-time detection, and on-site monitoring. 5 Compared with the RT-PCR technique, electronic-based biosensors present the advantages of low cost, high speed, small size, and label-free detection, leading to the possibility of widespread biomedical applications. A recent report used thiolated-DNA-functionalized gold nanoparticles to detect SARS-CoV-2 sequences based on the laser-induced plasmonic photothermal reaction. 6 The field-effect transistor based on graphene conjugated with SARS-CoV-2 spike antibody was used to detect the SARS-CoV-2 spike protein via the antibody−antigen reaction. 7 Carbon nanotubes were functionalized with a host protein for the detection of SARS-CoV-2 spike protein based on an optical sensing approach. 8 In another report, SARS-CoV-2 viral particles were detected via electrochemical methods through the same antibody binding of spike protein. 9 Notably, detection of pathogens, including viruses, by recording changes in the surface electrical properties of electrodes upon DNA hybridization, interfacing with twodimensional (2D) materials has recently received extensive attention. 10 This sensing modality offers a specificity that is as high as that of RT-PCR, but without the use of fluorescent probes or laboratory-scale signal-transducing instruments, which significantly lower the detection cost, along with the advantage of higher-field deployability and faster potential turnaround to results. 11 Owing to their large surface area and unique electrical/optical properties, 2D materials, such as graphene, molybdenum disulfide (MoS 2 ), and tungsten disulfide (WS 2 ), 12,13 offer highly sensitive sensing channels for the development of novel biosensors. Although lateral flow assays (LFA) have been demonstrated for the detection of pathogens, they suffer from a poor analytical sensitivity in comparison to electronic-based biosensors. 4 MXenes, a new family of multifunctional 2D transition-metal carbides/nitrides with various intriguing properties, have been developed by Gogotsi and co-workers. 14−16 They are typically prepared by selectively etching away intermediate A layers from MAX (M n+1 AX n ) phases, which have a general formula of M n+1 X n T x , where M stands for an early transition metal, A is a A group element, X is carbon and/or nitrogen, and T x represents surface functional groups such as −O, −OH, and/ or −F. MXenes have a unique combination of properties, including metallic conductivity, surface hydrophilicity, large chemically active surface, high specific surface, and good mechanical properties and thus are promising for numerous applications including energy storage, 17 electromagnetic shielding, 18 water purification, 19 catalysis, 20 and chemical sensors. 21 However, MXenes have rarely been employed in biosensing applications. It has been recently reported that MXenes are also biocompatible and hold a great potential in biomedical applications, 22, 23 such as drug delivery and biomolecule adsorption using their abundant surface anchoring sites. 24−26 Another 2-D material, graphene, by comparison, has been extensively used in biosensors. Such graphene-based biosensors typically show low sensitivity and small changes in electrical conductance owing to the zero-band-gap electronic structure of graphene. 7, 11, 27 Although yet another 2D material class, transition-metal dichalcogenides (TMDCs) have a larger band gap and exhibit a higher sensitivity for biosensing, the high electrical noise level limits their practical use, even when labbased performance is adequate. 12, 28 In contrast, MXene-based biosensors have advantages of ultrahigh electrical conductivity, hydrophilic surface, offering not only a high signal-to-noise ratio for chemiresistive biosensors but also the ability to precisely tailor their properties for biomolecule sensing through surface modification. Thus, the MXenes are interesting candidates to investigate the development of high-performance biosensors. The functionalization strategy for 2D materials (e.g., graphene, MoS 2 , and Ti 3 C 2 T x ) is mainly based on noncovalent interactions and simple adsorption of biomolecules, 13, 29, 30 including π−π stacking, hydrogen bonding, and van der Waals interactions. 31 Herein, we report on the development of single-stranded DNA functionalization of Ti 3 C 2 T x MXene (ssDNA/Ti 3 C 2 T x ) and its novel use in a chemoresistive biosensing platform for the highly sensitive and fast detection of SARS-CoV-2 N gene. The 2D Ti 3 C 2 T x surfaces were used for the first time in such a biosensing platform and were functionalized by SARS-CoV-2 N gene-specific nucleic acid probes through noncovalent adsorption, which enables sequence-specific detection via hybridization with target SARS-CoV-2 genes. Targeting the N gene rather than the more often targeted protein S is another novel feature of this work along with the unique and simple, compared to other reports, chemoresistive mechanism. 7, 32 The hybridization of SARS-CoV-2 N gene with complementary DNA probes leads to the detachment of hybridized double-stranded molecules from the Ti 3 C 2 T x surface, resulting in an increase in the conductivity of the Ti 3 C 2 T x channel. Importantly, ssDNA/Ti 3 C 2 T x biosensors detect SARS-CoV-2 N gene with a very low limit of detections of below 10 5 copies/mL in saliva and a fast response. The capability of ssDNA/Ti 3 C 2 T x biosensors developed here to distinguish the SARS-CoV-2 N gene from those of SARS-CoV-1 and MERS-CoV is presented, thus providing the feasibility of ■ RESULTS AND DISCUSSION Figure 1 shows the preparation of ssDNA/Ti 3 C 2 T x biosensors for SARS-CoV-2 detection. First, the Ti 3 C 2 T x MXene was synthesized by the selective etching of Al layers from their Ti 3 AlC 2 MAX phase in a HCl−LiF premixed solution and further delaminated in water by ultrasonication. The Ti 3 C 2 T x dispersions were then spray-coated onto gold interdigitated electrodes. The detailed procedure for device fabrication is given in the Experimental Section. For the selective detection of SARS-CoV-2 N gene, Ti 3 C 2 T x films were functionalized with DNA probes through noncovalent binding based on weak interactions involving the phosphate backbone and/or nucleobases. 30, 33 The detection mechanism of SARS-CoV-2 N gene is based on sequence-specific hybridization. Specifically, when the ssDNA probe hybridizes with the target SARS-CoV-2 N gene (complementary sequences), the interaction between the formed double-strained DNA and layered Ti 3 C 2 T x is weakened, resulting in desorption of hybridized compounds off the MXene surface and thus increasing channel conductance. Moreover, only the specific bases of DNA can hybridize with their complementary sequences, which enhances the selectivity of the sensing platform. 13 Figure 2a ,b shows the scanning electron microscopy (SEM) images of Ti 3 AlC 2 and as-etched Ti 3 C 2 T x , respectively, indicating a successful transition from a bulk Ti 3 AlC 2 to an accordion-like Ti 3 C 2 T x MXene with interlayer spacing that can serve as molecular sieving channels for hosting organic molecules and ions. 15 The increased interlayer spacing of Ti 3 C 2 T x MXene is induced by the selective etching, which in turn facilitates molecular transport and creates extra active sites for the electrochemical reaction. Previous studies have proposed a similar intercalation effect for different metal cations, organic molecules, and polymers, resulting in improved electrochemical performance of MXenes. 34 0.98 nm for the (002) plane of Ti 3 C 2 T x , consistent with X-ray diffraction (XRD) analysis presented later. Moreover, the highangle annular dark-field scanning transmission electron microscopy (HAADF-STEM) imaging, along with the associated energy-dispersive X-ray elemental mapping, presented in Figure 2e , shows a uniform distribution of Ti, C, and O elements in Ti 3 C 2 T x nanoflakes. The XRD patterns of Ti 3 AlC 2 and as-etched Ti 3 C 2 T x are displayed in Figure 2f , further confirming the formation of Ti 3 C 2 T x MXene. After the selective etching of Ti 3 AlC 2 , an intense peak at 39.1°, corresponding to the (104) plane of Ti 3 AlC 2 , is greatly weakened, accompanied by a downshift of the (002) peak from 9.5 to 8.9°, indicating the removal of Al layers and a successful formation of the Ti 3 C 2 T x MXene. 38 X-ray photoelectron spectroscopy (XPS) was used to investigate the element composition and bonding configuration of ssDNA-functionalized Ti 3 C 2 T x (see Figure 3 , as well as Figure S1 , Supporting Information). The survey spectra and atomic concentrations (Table S1, Supporting Information) of elements for both samples indicate that ssDNA functionalized has increased nitrogen concentration, which is a key element in nucleic acids' sequence. The high-resolution spectra of Ti 2p, C 1s, and N 1s (Figure 3) , taken from ssDNA/Ti 3 C 2 T x films, also suggest the presence of DNA probes on Ti 3 C 2 T x MXene after surface functionalization. The high-resolution Ti 2p spectrum (Figure 3a ) was fitted with four doublets (Ti 2p 3/2 and Ti 2p 1/2 ) with an area ratio of 2:1 and a doublet separation of 5.7 eV, characteristics of Ti 3 C 2 T x . 39 The Ti 2p spectrum was deconvoluted into four peaks centered at 454.6, 455.6, 456.6, and 458.6 eV, corresponding to Ti−C, Ti 2+ , Ti 3+ , and Ti−O, respectively. 40 The C 1s spectrum in Figure 3b is fitted with four peaks centered at 281.6, 284.6, 286.1, and 288.5 eV, corresponding to C−Ti, C−C, CH x /CO, and COO, respectively. 41 The N 1s peak is a reliable indication that DNA is adsorbed on the surface of Ti 3 C 2 T x , as nitrogen is exclusively found in the nitrogen-containing base pairs of DNA. 42 The N 1s spectrum in Figure 3c can be identified with the binding energy value of 398.6 eV, demonstrating the successful adsorption of DNA onto the Ti 3 C 2 T x MXene surface. Figures 4 and S2 in the Supporting Information show the detection mechanism of SARS-CoV-2 N gene using a Ti 3 C 2 T xbased biosensing device functionalized with probe DNAs. When complementary SARS-CoV-2 N genes are introduced, the desorption of ssDNA probes out of the MXene surface induced by the hybridization process results in an increase in the conductance of the sensing channel. Due to the sequencerecognition property, noncomplementary genes will not be hybridized with ssDNA probes on the Ti 3 C 2 T x surface, which means that the sensing platform has sequence specificity. SARS-CoV-2 genomes encode four structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N). 43 Among them, a virus surface S protein mediates SARS-CoV-2 entry into human cells, and thus it is considered as a major target for both vaccine design and rapid diagnosis. 44 However, the SARS-CoV-2 S gene shares 76% similarity of amino acid sequence to the SARS-CoV S gene, and nonsynonymous mutations develop in the S protein when the SARS-CoV-2 pandemic progress. 45−47 By contrast, SARS-CoV-2 N gene is genetically stable and allows for few gene mutations over time. 48 Owing to the sequence specificity of the sensing platform, it can be easily modified to address present and future emerging mutations in the virus' N gene, which can be targeted by designing new probe sequences. The N protein of many coronaviruses is also highly immunogenic and expressed abundantly during infection. 49 Thus, the N gene of SARS-CoV-2 should be a promising target candidate for rapid diagnosis of SARS-CoV-2.

US Centers for Disease Control and Prevention (CDC) published two specific sequences of probes, 2019-nCoV_N-F and 2019-nCoV_N-R, for the purposes of identifying SARS-CoV-2 N gene. We integrated these recognition probes with 2D MXenes for the development of COVID-19 sensors. To validate the presence of the ssDNA probes on the Ti 3 C 2 T x surface, the current−voltage (IV) curves of the fabricated Ti 3 C 2 T x before and after probe DNA functionalization were recorded. As shown in Figure 5a , the ssDNA functionalization results in an increase in electrical resistance of the hosting Ti 3 C 2 T x , indicating the successful introduction of ssDNA probes. With the loading of DNA probes, the electrical resistance of the sensors increases because of the metallic property of Ti 3 C 2 T x MXene. 28 To evaluate the performance of ssDNA/Ti 3 C 2 T x sensors for the detection of SARS-CoV-2 N gene, we performed the real-time measurements of ssDNA/ Ti 3 C 2 T x sensors in a buffer solution with target SARS-CoV-2 N gene, using pristine Ti 3 C 2 T x sensors as a control ( Figure  5b ). ssDNA/Ti 3 C 2 T x sensors produce a rapid spike in relative conductance (−ΔI/I 0 ) upon adsorption of target DNA (SARS-CoV-2 N gene) before settling to steady-state values. The spike in ΔI/I 0 indicates a transient perturbation in electronic states at the sensor surface caused by structural reorganization upon DNA hybridization. These findings indicate that only ssDNAfunctionalized Ti 3 C 2 T x is sensitive to SARS-CoV-2 N gene over the concentration range from 10 5 to 10 9 copies/mL. The responses of ssDNA/Ti 3 C 2 T x sensors to concentration variations of SARS-CoV-2 N gene are displayed in Figure 5c , showing a nearly linear response to SARS-CoV-2 N gene over a wide range of concentrations, indicating a lower limit of detection and good reproducibility of sensing response. Notably, ssDNA-functionalized Ti 3 C 2 T x not only exhibits a high sensitivity to the target SARS-CoV-2 N gene but also provides an excellent detection limit, which further highlights the advantages of using ssDNA/Ti 3 C 2 T x chemiresistive sensors for SARS-CoV-2 viral nucleic acid quantitative detection. It is important to note that this detection limit is equal to the one offered by conventional laboratory-based RT-PCR detection. 50 Moreover, diagnosis of COVID-19 using a saliva sample eliminates the need for nasopharyngeal swabs, which are invasive and prone to a shortage. The viral load of virus stays a longer period in saliva compared to nasopharyngeal swab, 51 which may benefit the detection of viral nucleic acid. The saliva sample has a more complex composition that could affect the sensor performance. Therefore, it is worth evaluating the dynamic response of ssDNA/Ti 3 C 2 T x sensors to SARS-CoV-2 N gene in saliva. The real-time sensing results presented in Figure 5d ,e indicate that ssDNA/Ti 3 C 2 T x sensors maintain a good sensitivity, even when tested in saliva, down to a concentration of 10 5 copies/mL. This study provides the feasibility of detecting SARS-CoV-2 genes using 2D MXene functionalized with ssDNA recognition probes as a promising pathway for rapid detection of SARS-CoV-2 gene. To further investigate the theoretical limit of detection (LOD) of ssDNA/ Ti 3 C 2 T x sensors, the LODs for SARS-CoV-2 N genes in both buffer and saliva were calculated with a signal-to-noise ratio (SNR) of 3. 52 The relationship between the sensor response and the concentrations in buffer and saliva with a linear fitting model is shown in Figure S3 , Supporting Information. The ssDNA/Ti 3 C 2 T x sensors exhibit a similar slope and response, with a LOD of ∼10 3 copies/mL in both buffer and saliva, indicating that this platform has a superior sensitivity toward SARS-CoV-2 N genes. These estimations prove themselves to be accurate as they not only sufficiently model the ACS Applied Nano Materials www.acsanm.org Article experimental data of both in buffer and saliva but also reflect low variation in these different media.

We also validated the selectivity of ssDNA/Ti 3 C 2 T x sensors by preparing and testing oligonucleotide sequences from the closely related N genes of SARS-CoV-2, SARS-CoV-1, and MERS-CoV, all having a concentration of 10 5 copies/mL. As demonstrated in Figure 5f , the ssDNA/Ti 3 C 2 T x sensor shows almost no response to SARS-CoV-1 and MERS-CoV N genes, confirming that this biosensing platform possesses sequencespecific recognition for SARS-CoV-2 N gene. The selectivity results also suggest that SARS-CoV-2 N probes are highly specific for SARS-CoV-2 N gene, which is consistent with the agarose gel electrophoresis experiments demonstrating the specificity of SARS-CoV-2 N probes for detecting the N gene of SARS-CoV-2 ( Figure S4, Supporting Information) .

The detection of clinically relevant targets is essential and important for practical diagnostic development. Thus, the detection of N gene from heat-inactivated SARS-CoV-2 using ssDNA/Ti 3 C 2 T x sensors was performed ( Figure 6a ). As SARS-CoV-2 isolation and propagation require a Biosafety Level 3 (BSL-3) laboratory, the noninfectious SARS-CoV-2 was prepared by heating the virus to 65°C for 30 min and thus was safe to use under BSL-1 conditions. 53 The N gene target was confirmed by one-step qRT-PCR and agarose gel electrophoresis from heat-inactivated SARS-CoV-2 ( Figure  S5 , Supporting Information). The SARS-CoV-2 N gene sequence contains a single band with a length of 72 base pair (bp), consistent with previous PCR results. 53 The realtime sensing of the ssDNA/Ti 3 C 2 T x sensor under various concentrations of N gene from heat-inactivated SARS-CoV-2 is shown in Figure 6b . The sensor exhibits an evident current response increase with increasing the concentration of target gene. Figure 6c shows the relationship between the response values and target concentrations. The ssDNA/Ti 3 C 2 T x sensor is ultrasensitive to the SARS-CoV-2 N gene with (i) a low detection limit of less than 10 5 copies/mL and (ii) a nearly linear response over a wide range of concentrations from 10 5 to 10 9 copies/mL, which is suitable for detecting viral loads from the majority of reported patients at the onset of symptoms. 54−56 The sensor response shows slightly lower sensitivity compared to the sensor response in buffer and saliva, caused by a minimized device-to-device variation. The detectable viral load strongly depends on the days after symptom onset, and it has been reported that SARS-CoV-2 could be most reliably detected around symptom onset or a few days thereafter. 57, 58 The low detection limit and linear response toward SARS-CoV-2 N gene suggest that MXene-based biosensors fabricated here have the ability to detect low viral load.

Compared with other 2D materials (e.g., graphene and MoS 2 ), Ti 3 C 2 T x core materials present the advantages of unusually high electrical conductivity, functionalized, and hydrophilic surface, providing not only a high signal-to-noise ratio for chemiresistive biosensors but also the ability to precisely tailor their properties for biomolecule sensing through surface modification. Because of the low or even the zero-band-gap electronic structure of graphene, graphenebased biosensors typically show small changes in electrical conductance, limiting their response and sensitivity. 7, 11, 27 On the other hand, transition-metal dichalcogenides are larger band gap materials and exhibit a higher response but suffer from the intrinsically high noise level. 12, 28 Owing to a highly conductive Ti 3 C 2 core with abundant hydrophilic surface functionalities, MXene-based materials are an attractive candidate for the development of high-performance biosensing devices.

In conjunction with the recent studies of SARS-CoV-2 detection technologies (Table S2 , Supporting Information), the ssDNA/Ti 3 C 2 T x biosensing platform reported herein is rather straightforward to use. The use of self-collected saliva samples is less invasive, can also minimize health care worker exposure, and decrease the need for sample collection devices, such as swabs. The nucleic acid electrochemical biosensing proposed here is effective and reliable, which can reduce false positive or negative results. Targeting the N gene ensures the specific detection of SARS-CoV-2 owing to its low rate of mutation compared to the multiple mutations of S genes observed in the three new SARS-CoV-2 variants when COVID-19 pandemic progress. Furthermore, the biosensing technology examined here, which did not use fluorescent probes and laboratory-scale instruments, provides an insight for the preparation of SARS-CoV-2 biosensors with potential for low-cost manufacturing and mass production.

A DNA primer-functionalized Ti 3 C 2 T x biosensor was successfully developed for a highly sensitive, selective, and rapid detection of the N gene of SARS-CoV-2. The sensor response increases with the concentration of the added target DNA. A clear differentiable response to the N gene of SARS-CoV-2 with ssDNA/Ti 3 C 2 T x sensors can be observed at a ACS Applied Nano Materials www.acsanm.org Article concentration as low as 10 5 copies/mL in artificial saliva, which matches the current detection limits of conventional qPCR testing. The ssDNA/Ti 3 C 2 T x biosensor also demonstrates the same performance toward detecting the SARS-CoV-2 N gene in saliva samples. The cross-reactivity of the sensor was confirmed against SARS-CoV-1 and MERS-CoV. The detection mechanism of N gene from SARS-CoV-2 for DNA-functionalized Ti 3 C 2 T x is proposed as being dominated by the hybridization of highly selective DNA probes on Ti 3 C 2 T x surfaces with SARS-CoV-2 N gene. Under the current COVID-19 outbreak, this study shows the feasibility of developing a real time and highly reliable diagnosis devices for clinical tests based on DNA-functionalized Ti 3 C 2 T x MXenes.

■ EXPERIMENTAL SECTION Materials. Ti 3 AlC 2 (2 g, particle size <40 μm) was purchased from Carbon-Ukraine. Conductive gold ink (UTDAu25) was purchased from UT Dots (Champaign, IL). The plasmid controls of the viruses contain a complete nucleocapsid gene from 2019-nCoV, SARS, and MERS viruses, which were synthesized from Integrated DNA Technologies (IDT, Skokie, IL). The selected oligonucleotide probes were approved by the US Centers for Disease Control and Prevention (CDC) and given in Table S3 , 59 Supporting Information: nCOV-N Forward Probe, 5′-GACCCCAAAATCAGCGAAAT-3′, nCOV-N Reverse Probe, and 5′-TCTGGTTACTGCCAGTTGAATCTG-3′. The artificial saliva was purchased from Pickering Laboratories (Mountain View, CA). Heat-inactivated SARS-CoV-2 (strain: 2019-nCoV/USA-WA1/2020, ATCC VR-1986HK) was purchased from American Type Culture Collection (ATCC, VA). All other chemical reagents were of analytical reagent grade and used without further purification. Ultrapure water (>18.3 MΩ) was used throughout the experiment.

Preparation of Ti 3 C 2 T x MXene. Ti 3 C 2 T x MXene were prepared on the basis of the procedure reported by Gogotsi et al. 38, 60 Ti 3 AlC 2 powder was selectively etched to remove Al layers in a premixed acid solution of 9M HCl (20 mL) and LiF (3.2 g) and stirred at 200 rpm for 24 h at room temperature. The mixture was washed through several centrifugation cycles with ultrapure water until pH value of the supernatant reached approximately 6. The resulting Ti 3 C 2 T x sediment was collected and rewashed with ultrapure water by vacuum filtration using the PVDF membrane with 0.22 μm pore size, subsequently dried in vacuum at 80°C for 24 h. To obtain delaminated Ti 3 C 2 T x dispersion, 100 mg of Ti 3 C 2 T x multilayer powder was sonicated in 20 mL of ultrapure water with an ultrasonic bath (Branson, CPX2800H) for 1 h. The bath temperature was controlled at 4°C to prevent restacking of nanosheets caused by the thermal energy released during sonication. The delaminated Ti 3 C 2 T x nanoflakes were collected for fabricating SAR-CoV-2 biosensors.

Fabrication of ssDNA/Ti 3 C 2 T x Sensors. First, nanogold ink was printed by a commercial inkjet printer (Dimatix DMP-2850, Fujifilm) on a polyimide substrate containing six pairs of gold interdigitated electrodes with a total active electrode area of 8 mm × 8 mm. Then, the Ti 3 C 2 T x solution (5 mg/mL) was sprayed onto interdigitated electrodes using an airbrush (G-233, Master Airbrush) for 10 s. The spray conditions were achieved with an operating pressure of 80 psi, a 0.5 mm of nozzle size, an operating distance of 30 cm between the spray nozzle and substrate, and a steady moving speed of 10 cm/s in all directions. The stock solution of probe DNA solution (10 μM) was thawed on ice and desired concentration of the DNA ligand was prepared. The Ti 3 C 2 T x films were then functionalized by casting a 20 μL drop of mixed probe DNA solution (5 μM) and immediately dried under vacuum. The resistance values of the sensor were recorded (Table S4 , Supporting Information). No significant changes for 1 μM of DNA loading are due to the insufficient DNA loading on the sensor surface. Both 5 and 10 μM of DNA loadings have similar resistance value, suggesting that 5 μM of DNA loading is enough for SARS-CoV-2 detection. All DNA solutions used in this study were quantified on the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).

Characterization and Real-Time Sensing of SARS-CoV-2 Viral Sequences. The surface morphology and microstructure of the Ti 3 C 2 T x MXene were examined by scanning electron microscopy (SEM; S-4800, Hitachi), transmission electron microscopy (TEM; Talos 200X, FEI), high-angle annular dark-field scanning electron microscopy (HAADF-STEM), and X-ray diffractometry (XRD; X'Pert Pro, Panalytical) operated at 45 kV and 40 mA using Cu Kα radiation. X-ray photoelectron spectroscopy (XPS; PHI 5000 Versaprobe, ULVAC-PHI) was conducted to investigate chemical components and bonding structures. The N gene sequences were detected by 3% agarose gel electrophoresis at 120 V for 60 min. The DNA agarose gel was imaged by the Bio-Rad imaging system. The electrical performance was measured using a source measure unit (Keithley 2400). The sensor response is defined using the following formula: ΔI/I 0 = (I−I 0 )/I 0 , where I and I 0 represent the real-time current and the initial current of the sensors, respectively.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsanm.1c03520. Figure S1 , XPS survey spectra for Ti 3 C 2 T x and ssDNA/ Ti 3 C 2 T x ; Table S1 , atomic concentrations of elements; Figure S2 , working mechanism of the biosensor; Figure  S3 , linear relationship between response and concentration in buffer and saliva; Figure S4 , agarose gel electrophoresis of SARS-CoV-2 N gene using SARS-CoV-N probes; Figure S5 , agarose gel electrophoresis of N gene from heat-inactivated SARS-CoV-2; Table S2 , summary of recent studies for SARS-CoV-2 detection; Table S3 , selected sequences for SARS-CoV-2 detection; and Table S4 , resistance measurements of biosensors after different concentrations of DNA loading (PDF)

COVID-19 Situation Report; World Health Organization

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Diagnosing COVID-19: The Disease and Tools for Detection

How Nanophotonic Label-Free Biosensors Can Contribute to Rapid and Massive Diagnostics of Respiratory Virus Infections: COVID-19 Case

Dual-Functional Plasmonic Photothermal Biosensors for Highly Accurate Severe Acute Respiratory Syndrome Coronavirus 2 Detection

Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor

Rapid SARS-CoV-2 Spike Protein Detection by Carbon Nanotube-Based Near-Infrared Nanosensors

Detection of SARS-CoV-2 Viral Particles Using Direct, Reagent-Free Electrochemical Sensing

Human virus detection with graphene-based materials

Graphene/MoS(2) heterostructures for ultrasensitive detection of DNA hybridisation

Ultrasensitive Monolayer MoS2 Field-Effect Transistor Based DNA Sensors for Screening of Down Syndrome

Comparison of MoS2, WS2, and Graphene Oxide for DNA Adsorption and Sensing

Two-dimensional nanocrystals produced by exfoliation of Ti 3 AlC 2

2D Metal Carbides and Nitrides (MXenes) for Energy Storage

Rational Design of Two-Dimensional Transition Metal Carbide/Nitride (MXene) Hybrids and Nanocomposites for Catalytic Energy Storage and Conversion

Applications of 2D MXenes in Energy Conversion and Storage Systems

Titanium Carbide and Protein Electrodes for Stimuli-Responsive Electromagnetic Shielding

Water Treatment and Environmental Remediation Applications of Two-Dimensional Metal Carbides (MXenes). Mater. Today

Surface and Heterointerface Engineering of 2D MXenes and Their Nanocomposites: Insights into Electro-and Photocatalysis

The Rise of MXenes

Universal Ti3C2 MXenes Based Self-Standard Ratiometric Fluorescence Resonance Energy Transfer Platform for Highly Sensitive Detection of Exosomes

MXene Coupled with CRISPR-Cas12a for Analysis of Endotoxin and Bacteria

Insights into 2D MXenes for versatile biomedical applications: current advances and challenges ahead

DNA Detection with Single-Layer Ti3C2 MXene Nanopore

Real-time reliable determination of binding kinetics of DNA hybridization using a multi-channel graphene biosensor

Metallic Ti 3 C 2 T x MXene Gas Sensors with Ultrahigh Signal-to-Noise Ratio

A Graphene Nanoprobe for Rapid, Sensitive, and Multicolor Fluorescent DNA Analysis

Interaction of single-and double-stranded DNA with multilayer MXene by fluorescence spectroscopy and molecular dynamics simulations

Progress and biomedical applications of MXenes

Rapid and Sensitive Detection of anti-SARS-CoV-2 IgG, Using Lanthanide-Doped Nanoparticles-Based Lateral Flow Immunoassay

A label-free electrochemical biosensor for highly sensitive detection of gliotoxin based on DNA nanostructure/MXene nanocomplexes

MXene as a Charge Storage Host

MXene Molecular Sieving Membranes for Highly Efficient Gas Separation

Recent advances in 2D MXenes: preparation, intercalation and applications in flexible devices

Effect of Glycine Functionalization of 2D Titanium Carbide (MXene) on Charge Storage

Guidelines for Synthesis and Processing of Two-Dimensional Titanium Carbide (Ti 3 C 2 T x MXene)

Hybrids of Two-Dimensional Ti 3 C 2 and TiO 2 Exposing {001} Facets toward Enhanced Photocatalytic Activity

Transparent Conductive Two-Dimensional Titanium Carbide Epitaxial Thin Films

Template-Free 3D Titanium Carbide (Ti 3 C 2 T x ) MXene Particles Crumpled by Capillary Forces

Characterization of DNA Probes Immobilized on Gold Surfaces

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. The Lancet

Cell entry mechanisms of SARS-CoV-2

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

The Nucleocapsid Protein of SARS-CoV-2: a Target for Vaccine Development

The SARS coronavirus: a postgenomic era

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle

The Limit of Detection Matters: The Case for Benchmarking Severe Acute Respiratory Syndrome Coronavirus 2 Testing

Comparison of Saliva and Nasopharyngeal Swab Nucleic Acid Amplification Testing for Detection of SARS-CoV-2: A Systematic Review and Meta-analysis

Two-Dimensional Vanadium Carbide MXene for Gas Sensors with Ultrahigh Sensitivity toward Nonpolar Gases

Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR

Opportunities and Challenges for Biosensors and Nanoscale Analytical Tools for Pandemics: COVID-19

Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens

Viral Load Kinetics of MERS Coronavirus Infection

SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients

SARS-CoV-2 detection, viral load and infectivity over the course of an infection

-Novel Coronavirus (2019-nCoV) Real-time rRT-PCR Primers and Probes

Surface Functionalization of Ti 3 C 2 T x MXene with Highly Reliable Superhydrophobic Protection for Volatile Organic Compounds Sensing

ACS Applied Nano Materials www.acsanm.org Article