Multiplexed detection of SARS-CoV-2 based on upconversion luminescence nanoprobe/MXene biosensing platform for COVID-19 point-of-care diagnostics.

Multiplexed detection is essential in biomedical sciences since it is more efficient and accurate than single-analyte detection. For an accurate early diagnosis of COVID-19, a multiplexed detection strategy is required to avoid false negatives with the existing gold standard assay. Nb2CTx nanosheets were found to efficiently quench the fluorescence emission of lanthanide-doped upconversion luminescence nanoparticles at wavelengths ranging from visible to near-infrared spectrum. Using this broad-spectrum quencher, we developed a label-free FRET-based biosensor for rapid and accurate detection of SARS-CoV-2 RNA. To target ORF and N genes, two types of oligo-modified lanthanide-doped upconversion nanoparticles can be used simultaneously to identify-two sites in one assay via upconversion fluorescence enhancement intensity measurement with detection limits of 15 pM and 914 pM, respectively. Moreover, with multisite cross-validation, this multiplexed and sensitive biosensor is capable of simultaneous and multicolor analysis of two gene fragments of SARS-CoV-2 Omicron variant within minutes in a single homogeneous solution, which significantly improves the detection efficiency. The diagnosis result via our assay is consistent with the PCR result, demonstrating its application in the rapid and accurate screening of multiple genes of SARS-CoV-2 and other infectious diseases.

Introduction

The outbreak of novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible to humans and in some cases may even be fatal, which has triggered a global public health crisis [1]. Lung computed tomography (CT) scanning of patients based on specific imaging features is one of diagnostic method of COVID-19, but the accuracy of 76.4 % and untimely diagnosis limit its further implement [2]. Further, antibody serology assays can only detect antibodies within 1–2 weeks after disease onset, making early diagnosis difficult [3]. Rapid antigen tests have shown promise as means of obtaining results in a shorter period of time and requiring less sample preparation. However, its sensitivity is criticized by the majority of users, resulting in challenges for early and accurate diagnosis [4]. Current nucleic acid amplification testing (NAAT) such as reverse transcription-polymerase chain reaction (RT-PCR) is often used to detect the nucleic acids extracted from the SARS-CoV-2 specimens owing to optimized sensitivity, specificity, and ease of quantitative analysis [5]. However, the testing process is hindered by the long turnaround times, expensive laboratory equipment and trained personnel. As a gold standard, qPCR is plagued by the problem of false negatives. According to the new Coronavirus pneumonia diagnosis and treatment plan of the National Health Commission of China, up to 20–67 % of patients test positive only after multiple repeated tests [6], [7].

In order to diagnose SARS-CoV-2 accurately and rapidly, it is highly anticipated to develop point-of-care multiplexed detection strategies for the cross-validation of multiple characteristic-related biomarkers. The U.S. Center for Disease Control and Prevention (CDC) provides examples of gene selection and multi-component testing methods [8]. For each patient’s sample, three qPCR reactions were performed sequentially, in accordance with the guidelines set forth by the World Health Organization (WHO) [9]. Additionally, a variety of biosensors have been developed for multi-component detection, including electrochemical biosensors [10], colorimetric biosensors [11] and loop-mediated isothermal amplification (LAMP) using clustered regularly interspaced short palindromic repeats (CRISPER) [12]. Despite their success, the results of these biosensors were hampered by the undesired aggregation [13] and lengthy turnaround time [14]. Therefore, it became urgently necessary to develop rapid, sensitive, and specific diagnostic tools for multiplexed detection of the virus and its mutated forms.

Förster resonance energy transfer (FRET) has been widely used in biomedical and clinical applications to access molecular scale information [15]. Such a phenomenon occurs when the emission spectrum of the donor fluorophore overlaps with the absorption spectrum of the acceptor fluorophore between two proximal fluorophores [16]. According to E1, the efficiency of this transfer depends on the distance between the two fluorophores. Inverse sixth power dependence makes FRET measurements extremely sensitive to even small changes in distance. Based on the intrinsic properties, various FRET-based biosensors have been designed for virus detection [17]. Among these assays, the bio-receptors interact with the biomarkers of interest and induce a change in the luminescence intensity. Hence, the chemical and physical properties of the transducer/fluorophore are vital in the detection. Most organic dyes are subject to photobleaching, whereas inorganic quantum dots are prone to blinking. A further limitation is that their excitation wavelengths are typically in the UV–vis range, which severely limits the sensing sensitivity due to fluorescent background derived from biomolecules, as well as internal filtering effects due to absorption by other species [18], [19]. Fortunately, these disadvantages might be mitigated by utilizing lanthanide (Ln3+)-based upconversion nanoparticles (UCNPs) as fluorescence donors. UCNPs can absorb two or more low-energy photons and produce luminescence at a short wavelength than the excitation wavelength. The inner 4f-4f orbital electronic transitions, which are protected by the full 5s and 5p orbitals, are responsible for the upconversion fluorescence. Besides, the long-lived emission of Ln3+-based probes would improve lifetime measuring accuracy and allows for time-resolved detection or even multiplexed sensing by using various Ln3+ combinations. Because of their excellent chemical and photochemical stability, large anti-Stokes shift, low toxicity, lack of photobleaching, blinking and autofluorescence, UCNPs is well suited for biosensing detection.

Niobium carbide (Nb2C) MXene is a new emerging 2D material and its unique physical and chemical properties of Nb2CTx are very versatile and termination-dependent, making it desirable for various application including electrochemical biosensors [20], energy storage [21] and theranostics [22]. Its large surface area facilitates multiple-donor–single-acceptor configurations and broad absorption endows it high luminescence quenching ability, which makes it favorable for developing the FRET-based biosensor applicable to multiplexed detection [23], [24].

Considering these merits, here we develop a novel, simple and label-free FRET based biosensor for multiplexed detection of SARS-CoV-2 RNA. The test is relied on the Förster distance changes between the donors of oligo-modified different emission UCNPs and the acceptor of Nb2CTx, in which simultaneous detections of ORF1ab gene and N gene were performed, showing excellent selectivity and sensitivity. We adopted this simple “mix and detect” strategy for multiplex detection based on the following three considerations: (i) the analysis of two genes in one tube may increase the detection sensitivity, resulting in accumulation of detection signals, (ii) analyzing two genes simultaneously may improve detection efficiency because one positive signal of the two genes indicates SARS-CoV-2 infection, (iii) screening of COVID-19 can be more effective with the rapidity and simplicity of the detection workflow. Furthermore, with the capability of multisite cross-validation, the biosensor had been evaluated using SARS-CoV-2 variant (B.1.1.529/Omicron) clinical samples, which successfully distinguished the ORF and N gene. Positive or negative result of viral gene testing can be obtained within 5 min, which is fully in agreement with the PCR results. Thus, our simple and label-free multiplexed detection strategy enables on-site rapid and sensitive detection of multiple gene simultaneously and it can also be extended to the bio-detections of other nucleic acid and biomarkers.

Materials and methods

Materials

Nb2AlC (>99 %) was purchased from Laizhou Kai Ceramic material Co., LTD. Lanthanide (Ln) acetates (99.9 %), including thulium (Tm), erbium (Er), gadolinium (Gd), and ytterbium (Yb) were dissolved in D.I. water to form 0.2 M aqueous solution. 1-octadecene (1-ODE, 90 %, technical grade), Oleic acid (OA, 90 %, technical grade), cyclohexane (ACS grade, 99 %), ethanol (99.9 %), sodium hydroxide (NaOH, ACS grade, 97 %), methanol (99.9 %), concentrated hydrochloric acid (HCl, 37 %), phosphate buffered saline (PBS, Bio Performance, pH = 7.4) were purchased from Sigma-Aldrich. Ultrapure water (molecular biology grade) is used in all experiments involving DNA oligonucleotides (oligos). DNA oligos were purchased from Integrated DNA Technologies, Inc. All these chemicals were used as received without further purification.

Synthesis of few layers Nb2CTx MXene

Nb2CTx MXene is typically synthesized by selectively etching Nb2AlC with 5 M LiF/6 M HCl solution for 48 h at room temperature. Upon completion of the etching process (complete removal of Al elements), it is necessary to wash the product three times to remove the residual acid and salt. The multi-layered Nb2CTx MXene can be collected when the pH of the solution reaches 6. In the delamination step, TMAOH was used as an intercalant. 12 mL 25 % TMAOH was reacted with multi-layered MXene (20 mg) for 12 h. After the reaction, the suspension was washed and collected by centrifugation to obtain TMA-intercalated Nb2CTx. The sediment was dispersed in 2 mL distilled water, and the multi-layered MXene was further delaminated for 10 min by sonication, and then the solution was centrifuged to collect the few-layered Nb2CTx. To alleviate its oxidation, the obtained MXene was dried in vacuum.

Synthesis of blue-emitting NaGdF4:Yb/Tm UCNPs

In a typical coprecipitation synthesis, 0.008 mmol of thulium (III) acetate hydrate, 0.072 mmol of ytterbium (III) acetate, 0.32 mmol of gadolinium (III) acetate hydrate 4 mL OA and 6 mL ODE were added to a 50 mL three-necked flask under magnetic stirring at room temperature. The mixture was heated to 100 ℃ for 30 min to remove the water followed by 150 ℃ for 1 h to form the lanthanide-oleate coordination complexes. After that, removed the heating mantle and cooled the mixture to room temperature under magnetic stirring. To trigger the nucleation and growth of the NaGdF4 nanocrystals, 1 mmol NaOH-methanol stock solution and 1.32 mmol NH4F-methanol stock solution were then mixed by vortexing for 10 s to prevent NaF generated from sticking to the wall of the centrifuge tube and immediately injected it into the reaction mixture. Next, the temperature was raised to 50 ℃ and keep it for 1 h under magnetic stirring to promote the crystal growth. Connect the flask to a dual vacuum/gas manifold and degas for 10 min, and then raise the temperature to 290 ℃, and keep it for 1.5 h under the protection of argon. After the synthesis was finished, let the reaction mixture to cool to ambient temperature while stirring. The crude UCNPs was transferred into a 15 mL centrifuge tube, rinse the flask with 5 mL ethanol, and combine it into the centrifuge tube. The product was centrifuged at 6000 r.p.m. (∼3500 g) at room temperature for 3 min, and then purified using cyclohexane and ethanol. Finally, the UCNPs were dispersed in 4 mL fresh cyclohexane for further use.

Synthesis of red emitting-NaErF4:Tm UCNPs

0.398 mmol of erbium (III) acetate hydrate, 0.002 mmol of thulium (III) acetate, 4 mL OA, and 6 mL ODE were introduced to a 50 mL three-necked flask with magnetic stirring at room temperature. The mixture was heated to 100 °C for 30 min to remove water before being heated to 150 °C for 1 h to create the lanthanide-oleate coordination complexes. The heating mantle was then removed, and the fluid was cooled to room temperature using magnetic stirring. To initiate the nucleation and growth of the NaErF4 nanocrystals, 1 mmol NaOH-methanol stock solution and 1.32 mmol NH4F-methanol stock solution were vortexed for 10 s to prevent the NaF produced from clinging to the wall of the centrifuge tube and then introduced into the reaction mixture. The temperature was then increased to 50 °C and maintained for 1 h under magnetic stirring to promote crystal formation. Connect the flask to a dual vacuum/gas manifold and degas for 10 min before raising the temperature to 290 °C and keeping it there for 1.5 h under argon protection. After the synthesis was finished, the liquid was progressively cooled to room temperature while being stirred. The crude UCNPs were put into a 15 mL centrifuge tube, and the flask was rinsed with 5 mL ethanol before being combined into the centrifuge tube. The product was centrifuged at 6000 r.p.m. (3500g) for 3 mins at room temperature before being purified with cyclohexane and ethanol. Finally, the UCNPs were dispersed in 4 mL of fresh cyclohexane for further use.

Synthesis of ligand-free UCNPs

The UCNPs dissolved in cyclohexane were centrifuged and redispersed in dilute hydrochloric acid at pH ∼ 4. Then, the mixture was treated with sonication for 30 mins, followed by centrifugation at 14,000 rpm for 30 min and discard the supernatant. Next, repeat these washing steps until the brown color disappears. Then, wash it with deionized water to remove the excess acid. Finally, the obtained ligand-free UCNPs were dispersed in deionized water.

UCNPs modification with poly C10-complementary oligo

The ligand-free UCNPs (NaGdF4:Yb/Tm; NaErF4:Tm) were dispersed in 600 µL D.I. water, and the suspended UCNPs mixture was slowly added to an aqueous solution (300 µL) containing different types of C10-complementary DNA under the sonication, followed by the addition of 20 µL of 10 × TB-Na+ buffer (890 mM tris base, 890 mM bonic acid, 1 M NaCl, pH ∼ 8) to the mixture per hour for a total volume of 100 µL, making the nanocomposites dispersed in 1 × TB-Na+buffer. After incubation for overnight, the solution was purified with a 0.22 µm filter to remove large aggregates. Finally, free DNA in the supernatant were discarded by centrifugation to obtain DNA-modified UCNPs. The prepared DNA-modified UCNPs can be characterized by UV–vis spectroscopy.

FRET efficiency calculation

Nonradiative energy transfers are dependent on spectral overlap and intermolecular distance according to Theodor Forster’s quantitative theory. According to Forster, energy transfer rate is calculated as an integral of spectral overlap. The simplified equation for FRET efficiency is as follows:when the transfer efficiency is 50 %, a fluorescent distance between donors and acceptors is defined as R0. Where R is the distance between donors and acceptors. There is an inverse relationship between energy transfer efficiency E and distance between donors and acceptors. The transfer rate is about to reach its maximum (

PL response for nucleic detection

To verify the DNA probe detection, 1 × 10−9 M of each P1 and P2 modified probe were hybridized with different concentration of T1 and T2 (target DNA or SARS-CoV-2 RNA) in 494 µL of phosphate buffer (0.1 M, pH 7.4) respectively and followed by adding the Nb2CTx MXene aqueous with the final concentration of 200 µg mL−1. Fluorescence spectra were monitored by the FLS920P Edinburgh Instrument at room temperature. From UCL spectra, the fluorescence intensity enhancement efficiency (ηx) can be calculated by the E2.

Virus and cell line

A strain of B.1.1.529/Omicron (GeneBank: OM212472) has been isolated from the respiratory tract of COVID-19 patients at The University of Hong Kong. All experiments involving live SARS-CoV-2 were performed in accordance with approved operating procedures. A DMEM medium containing 10 % FBS, 2 mM l-glutamine, and 100 U/mL/mL penicillin was used to culture Vero-E6-TMPRSS2 cells. The cells were incubated at 37 °C in a CO2 atmosphere at 5 %. The sequences of all variants of this virus were determined through nanopore sequencing after virus cultures were obtained. A commercial RT-PCR kit (Novel Coronavirus (2019-nCoV) Dual Probes RT-PCR Kit; Beyotime, China) and a detection system (Applied Biosystems QuantStudio 5 Real-Time PCR System, Thermo Fisher Scientific, USA) were used to amplify extracted RNA for clinical diagnosis. RT-PCR results with a Ct value, 35 were considered positive.

Result and discussion

Multiplexed detection strategy

A simple, label-free and sensitive MXene-integrated biosensor was designed for multiplexed detection of SARS-CoV-2 RNA sequences. The design strategy is shown in Fig. 1 . During the process, different emitting UCNPs labeled with complementary oligonucleotides, serve as fluorescent probes, were fixed on MXene nanosheets due to noncovalent binding. Nb2CTx nanosheets quenched the fluorescence of UCNPs under the effect of FRET. The complementary oligo-labeled UCNPs hybridized with the RNA target to form double-stranded structure. Thus, nucleobases are buried between the densely negatively charged helical phosphate backbones which has strong repulsion with negatively charged Nb2CTx MXene, resulting in different emitting oligo-modified UCNPs desorbed from MXene and fluorescence signals turn on. It is worth mentioning that the long RNA sequences are prone to undergo unwanted aggregation and entanglement. Dual fluorescence probes were employed to indirectly increase the hybridization site of the target, thus enhancing the sensitivity and improving the detection efficiency via multisite cross-validation.

Fig. 1

The schematic illustration of the rapid multiplexed detection of SARS-CoV-2 RNA with enhanced sensitivity and detection efficiency based on a dual-upconversion emission/MXene integrated nanoplatform.

Synthesis and characterization of few-layer Nb2CTx and blue/red-emitting UCNPs

We synthesized high-quality 2D few-layer Nb2CTx NSs using a facile and scalable two-step method involving acid etching and intercalation. Currently, a top-down approach is widely employed for fabricating MXene by selective etching from their MAX phases. Owing to high chemical activity of the metallic Nb-C bonds, Nb2CTx MXene can be obtained at room temperature by eliminating weaker Nb-Al bonds from Nb2AlC powder in an aqueous 50 % hydrofluoric acid (HF). In Fig. S1, the (0 0 2) peak of Nb2C MXene is shifted to the lower 2θ position, which signals expanded interlayer spacing, demonstrating the successful synthesis of Nb2CTx nanosheets (NSs). To obtain the few-layer nanosheets, tetrabutylammonium hydroxide (TBAOH) was used to exfoliate 2D layered Nb2CTx. Protons (H+) exchange with bulky tetrabutylammonium ions (TBA+) which results in the material swelling and delamination. Fig. 2 A illustrates typical 2D morphology of the exfoliated Nb2C NSs, with transparent features evidence of ultrathin properties of the MXenes. As shown in Fig. 2B, selected area electron diffraction (SAED) pattern demonstrates the hexagonal symmetry structure of Nb2C NSs. The blue-emitting NaGdF4:Yb, Tm is well dispersed in the cyclohexane and displays exceptional crystallinity with a lattice spacing of 5.1 nm, which is indexed to the (1 0 0) plane of a hexagonal NaGdF4 crystal, as demonstrated in high resolution transmission electron microscopy (HR-TEM) images (Fig. 2D). Fig. 2E illustrates the mono-dispersibility of oleate-capped red emitting UCNPs (NaErF4:Tm) in cyclohexane, which were spherical in form and averaged 48 nm in size. The NaErF4:Tm UCNPs exhibited outstanding crystallinity with a lattice spacing of 5.4 Å, corresponding to the (1 0 0) plane of a hexagonal NaGdF4 crystal (Fig. S2). Considering its good biocompatibility, large surface area, abundance of terminations (—O—/OH—) and broad absorption across UV–vis-NIR spectrum, Nb2CTx is well suited for an outstanding substrate as well as a fluorescence quencher in a FRET-based biosensing system, allowing multiple-donor–single-acceptor configurations to be realized in a multiplexed detection. Fig. S3 demonstrates the absorption spectrum of different concentration of Nb2CTx and the absorption enhanced as the increment of the concentration of MXene. Additionally, as shown in Fig. 2F the blue and red emissions of two probes are well overlapped with the absorption of Nb2CTx NSs, and the quenching efficiency was calculated to be 80.82 % and 69.29 % (Figs. S4 and S5), offering great potential for fluorescence recovery and providing great possibility for ultrasensitive multiplexed detection.

Fig. 2

(A). TEM of few-layer Nb2CTx MXene. (B). SAED pattern of few-layer Nb2CTx nanosheets; (C). TEM and (D). HR-TEM images of NaGdF4:Yb,Tm. (E). TEM image of NaErF4,Tm UCNPs. (F). UV–vis absorption of Nb2CTx overlaps the blue-emitting and red-emitting UCNPs. The inset images in (C) and (E) are blue and red emission of NaGdF4:Yb,Tm and NaErF4,Tm UCNPs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Ultrasensitive detection of short oligonucleotide fragments

Based on aforementioned results and well-established fact of DNA-MXene interaction, we designed an MXene-integrated biosensor for multiplexed real-time SARS-CoV-2 RNA fragments detection. The fluorescent signal of UCNPs is turned on and increases with the concentration of targets. The spectral overlap between the blue/red emitting UCNPs and the absorption properties of Nb2CTx indicates the FRET efficiency of this integrated biosensor. Fig. 3 A shows the upconversion spectra of short ORF1ab-target detection using the FRET-based integrated assay. When the UCNPs-P1 contact with the Nb2CTx MXene, the emission intensity drops owing to the FRET effect between UCNP and Nb2CTx MXene. However, with increment of the amount of target, the intensity of UCNPs at 481 nm enhanced accordingly. The oligo modified UCNPs were specifically bound with the complementary oligo target via hybridization, resulting in weakened noncovalent binding ability. Therefore, the double strands structure will be detached from the MXene, which in turn will result in a longer Förster distance, thus increasing the luminescence intensity of UCNPs. The limit of detection (LOD) was determined to be 12 pM for ORF detection using the control signal plus three times standard derivation (Fig. 3 B). The upconversion spectra of N-target detection utilizing the MXene-based integrated assay are shown in Fig. 3 C. A similar phenomenon also occurs between UCNP-P2 and Nb2CTx, the intensity of UCNPs at 660 nm enhanced with increment concentration of N-target oligo, the UCNPs-probe specifically bound with the short gene via hybridization, resulting in weakened noncovalent binding ability. As the concentration of N target oligonucleotide increased, the recovered fluorescence was enhanced accordingly. As a result, the LOD was calculated to be 95 pM. (Fig. 3 D).

Fig. 3

(A and B) UC emission spectra of the Nb2CTx/blue-emitting UCNP assay in the presence of different concentrations of short ORF1ab target. (C and D) UC emission spectra of the Nb2CTx/red-emitting UCNP assay in the presence of different concentrations of short N-gene target. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Multiplexed simultaneous detection of long DNA fragments

We have successfully demonstrated the ultrasensitive detection of short ORF1ab and N oligo fragments using our UCNPs/MXene integrated biosensors. However, the clinical viral samples containing long gene sequences undergo unwanted aggregation and entanglement, increasing the steric hindrance, thus resulting in low sensitivity of the proposed biosensor (Fig. 4 A). To compensate for this risk of reduced sensitivity and enhance the detection efficiency, we developed a label-free approach for multiplexed upconversion fluorescence detection of long DNA fragment. The long DNA sequences are shown in Table S1. As shown in Fig. 4 B and 4C, two oligo modified probes with different upconversion emission can be mixed and quantified in one single assay. It is observed that blue and red emission intensities increase simultaneously with the presence of long DNA fragments. Calibration curves were formed in the simultaneous detection protocol using luminescence intensity enhancement as a function of the varying concentrations of long DNA targets (ORF1ab and N gene), enabling rapid and sensitive detection of ORF1ab and N gene (Fig. S6). The LOD were determined to be 15 pM and 914 pM for long simulated ORF1ab and N gene, which is slightly higher than that for short oligo detection owing to unwanted aggregation and entanglement of long gene sequence. To evaluate the analytical specificity of our proposed assay, a cross-reaction analysis was executed. The obtained results in Fig. 4 D and 4E show that the assay is capable of distinguishing SARS-CoV-2, SARS-CoV and bat-SL-CoVZC45. The upconversion intensity enhancement at 481 nm were 18.44 % for SARS-CoV, 30.54 % for bat-SL-CoVZC45 and 82.81 % for SARS-CoV-2, respectively. The test shows excellent specificity, thus it can be used for evaluating clinical samples.

Fig. 4

(A) The scheme of detection of long simulated target using dual emission UCNPs/MXene integrated biosensor. Photoluminescence spectra at different emission bands of the proposed biosensor for simultaneous multiplexed detection of (B) ORF-1ab and (C) N-target. (D) PL intensity recovery comparison for RNA-targeted regions among three highly homologous coronavirus. (E) The specificity analysis of the using dual emission UCNPs /MXene integrated biosensor.

Detection of SARS-CoV-2 Omicron (B.1.1529) variant samples

To demonstrate the readiness of the dual emission UCNPs/MXene integrated biosensor for rapid multiplexed detection of COVID-19, we applied it to the clinic diagnosis test. A positive control was conducted by using RT-PCR to detect the ORF gene of viral samples with defined quantities before testing clinic samples. Virus-free kit buffer and lysis buffer were used as negative controls. The cycle threshold (Ct) values of kit buffer and lysis buffer by RT-PCR were 34.989 ± 1.651 and 36.155 ± 2.556, whereas for viral samples with quantities of 0.5 μL, and 1 μL, each Ct values were 25.181 ± 0.381 and 22.955 ± 2.315, respectively (Fig. 5 A). Based on clinical reports of diagnosing COVID-19 patients with ORF genes, the Ct values of positive RT-PCR results are less than 35. Having determined the Ct values of the specific quantities of viral samples, we then mixed each viral sample with a mixture containing Nb2CTx and oligo modified UCNPs. It is encouraging to note that the fluorescence intensities at 481 and 658 nm would have recovered after 5 min of hybridization, indicating the presence of the ORF gene and N gene of SARS-CoV-2 (Fig. 5 B and C). Fig. 5 D illustrates the fluorescence enhancement at 481 nm and 658 nm of 20.04 %, 20.02 % when 0.5 μL of virus sample was added. For the 1 μL virus sample, the fluorescence enhancements were 24.18 % (481 nm) and 17.62 % (658 nm), respectively. A significant increase in intensity of the viral sample groups compared with the control group is associated with the presence of target RNA that activates the “Turn-on” mechanism of our assay. These results demonstrate that our assay realize multiplexed detection of SARS-CoV-2 ORF and N gene and enables high-sensitivity screening of positive and negative samples through PL spectrum. As a result, the diagnosis result via our assay in good agreement with the RT-PCR results, indicating that with a significantly shorter turnaround time of 5 min, our assay is capable of reliably screening the SARS-CoV-2 Omicron variants (B.1.1.529). This effective and rapid method thus provides an efficient alternative to commercial RT-PCR. The fast sample-to-answer time, simplicity of workflow, high sensitivity and multisite cross-validation of this test make it a possible replacement for the current rapid antigen tests at multiple point-of-care scenarios.

Fig. 5

(A) RT-qPCR results of ORF-gene in different volume viral samples (different amount viral samples serve as positive control groups, while the kit buffer and lysis buffer without virus and serves as negative control groups). (B) Blue emission and (C) red emission of UCNPs/MXene integrated biosensor when adding viral sample (0.5 µL and 1 µL of SARS-CoV-2 Omicron/B.1.1529 stock solution extracted from cells, sample to answer time: 5 min). (D) Peak intensities enhancement of the Nb2CTx/UCNP integrated biosensor after probing different quantities of viral samples, corresponding to (B) and (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Conclusion

Herein, we developed label-free multiplexed RNA analysis strategy based on the weakening of the FRET effect between different emission UCNPs and broad-spectrum quencher Nb2CTx to simultaneously detect two specific RNA gene sites of SARS-CoV-2. By measuring the upconversion fluorescence enhancement intensity, rapid and sensitive detection of ORF and N gene can be achieved in one single assay with limits of 15 pM and 914 pM, respectively. Additionally, this multiplexed and sensitive biosensor can be used to analyze two RNA fragments of SARS-CoV-2 Omicron variant for COVID-19 point-of-care diagnostics. The results obtained with the test system were in agreement with those tested by PCR, showing its great potential for rapid detection of multiple genes related to SARS-CoV-2 and other infectious diseases.

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.