key: cord-0708279-ir5pg4wn authors: Gao, Yakun; Han, Yingkuan; Wang, Chao; Qiang, Le; Gao, Jianwei; Wang, Yanhao; Liu, Hong; Yu Zhang; Han, Lin title: Rapid and sensitive triple-mode detection of causative SARS-CoV-2 virus specific genes through interaction between genes and nanoparticles date: 2021-02-17 journal: Anal Chim Acta DOI: 10.1016/j.aca.2021.338330 sha: 0399ac92f20bf9b568a70fdeb9409bfd8c5768c4 doc_id: 708279 cord_uid: ir5pg4wn The recent outbreak of coronavirus disease 2019 (COVID-19) is highly infectious, which threatens human health and has received increasing attention. So far, there is no specific drug or vaccine for COVID-19. Therefore, it is urgent to establish a rapid and sensitive early diagnosis platform, which is of great significance for physical separation of infected persons after rapid diagnosis. Here, we propose a colorimetric/SERS/fluorescence triple-mode biosensor based on AuNPs for the fast selective detection of viral RNA in 40 minutes. AuNPs with average size of 17 nm were synthesized, and colorimetric, surface enhanced Raman scattering (SERS), and fluorescence signals of sensors are simultaneously detected based on their basic aggregation property and affinity energy to different bio-molecules. The sensor achieves a limit detection of femtomole level in all triple modes, which is 160 fM in absorbance mode, 259 fM in fluorescence mode, and 395 fM in SERS mode. The triple-mode signals of the sensor are verified with each other to make the experimental results more accurate, and the capacity to recognize single-base mismatch in each working mode minimizes the false negative/positive reading of SARS-CoV-2. The proposed sensing platform provides a new way for the fast, sensitive, and selective detection of COVID-19 and other diseases. A causative coronavirus, since then named severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), outbroke in Wuhan, Hubei Province, China, in December 2019 [1, 2] . The World Health Organization (WHO) officially named 2019 novel coronavirus disease [3, 4] , with estimated incubation period of about 2 to 10 days [5] . The new pneumonia caused by SARS-CoV-2 virus is highly infectious disease, and the number of infections worldwide is currently increasing. Early diagnosis of COVID-19 is very important, not only to improve the survival rate of patients, but also to hold back the infection in time. As a result, it is urgent to develop a rapid, sensitive and accurate diagnosis methods in order to effectively identify these early infects. Current testing approaches for SARS-CoV-2 detecting can be divided into two categories, serological and nucleic acid. Serological assay is mainly to detect antibodies produced by individuals as result of exposure to the virus or antigenic proteins in infected individuals [6] . The determination of SARS-CoV-2 exposure relies largely on the detection of either IgM or IgG antibodies, for instance, using enzyme-linked immunosorbent assay (ELISA) [7] or field-effect transistors (FET) based biosensing platform [8] . However, the production of IgG and IgM antibodies in patients, termed seroconversion, usually occurs 5 -10 days after the initial symptoms appear [9] , therefore, nucleic acid-based testing is currently the common-used tool for early detection of SARS-CoV-2 infection. For RNA virus infections, RT-PCR [10, 11] is routinely used approach for COVID-19 diagnosis, LAMP [12] and metagenomics J o u r n a l P r e -p r o o f sequencing [13, 14] are also newly developed methods to diagnose Recently, in order to detect biomolecules with high sensitivity, a number of novel sensing approaches have been developed by using colorimetry [15, 16] , fluorescence, SERS [17, 18] , electrochemical [19, 20] , and surface plasmon resonance (SPR) [21, 22] . Each platform has its specific advantages, but it is still challenging to solve the problem of false negative detection, which is extremely important in epidemic prevention and control. Therefore, the method based on multiple signal output provides a powerful choice that can greatly reduce false negative signals and improves the accuracy of detection. For example, the combination of colorimetry and fluorescence have achieved highly sensitive detection of alkaline phosphatase [23] , SO 2 derivatives [24] ; the fluorescence-SERS dual-signal platform has accomplished selective detection of microRNA [25] , hydroxyl radicals [26] and K + [27] . The colorimetrical and SERS dual-signal sensors have achieved precise detection of mercury ion [28] and the electrochemical-photoelectrochemical dual-mode sensing platform has accomplished the selective detection of hydrogen sulfide [29] . However, most multiple platforms are confined in dual-mode sensing, and there are still much room to fabricate the multi-mode sensing platform to meet high-demand clinical sensing. Here, we develop a triple-mode biosensor based on AuNPs to detect specific RNA in SARS-CoV-2 virus including colorimetric, SERS, and fluorescence. As shown in The extraction and purification of viral RNA is not necessary for the proposed biosensor. Comparing with PCR based detection, the proposed biosensor has very simple detection process. DNA probe conjugated reaction solution can be stored in the reaction chamber and get ready for detection because of its long-term stability, the operator only needs to load detection sample and then separate the supernatant from aggregated AuNPs through centrifugation after addition of SSC buffer. The fluorescence intensity is tested by photoluminescence system and the absorption spectrum is tested by the automatic and portable microplate reader, while the Raman spectrum is recorded by a Micro Raman spectrometer. The proposed detection approach could detect 96 or 384 samples simultaneously using a 96 or 384 microplate. Among the SARS-CoV-2 genomic regions, it is discovered that three regions had conserved sequences: the RNA-dependent RNA polymerase gene (RdRp) in the open reading frame ORF1ab region, the envelope (E) genes and the nucleocapsid (N) protein genes [30] . Both the RdRP and E genes had high analytical sensitivity for detection [6] , therefore, we selected specific fragments of ORF1ab and E genes [31] in the sequence of Wuhan-Hu-1 strain (GenBank accession number MN908947) for virus recognition based on the specific gene segment. The AuNPs were synthesized by the reduction of HAuCl 4 using trisodium citrate [32] . The detailed procedure for RNA detection is as the following. First, DNA probes The size of AuNPs plays important role in the proposed biosensors, which determines their aggregation and the capacity to bind DNA probes [33] . AuNPs were In order to achieve high-performance detection, experimental parameters are The influence of reaction time between DNA and RNA is also investigated. The absorbance measurements are carried out according to the procedure described above. Selectivity is one of the most important parameters in RNA sensing. One and two To evaluate the sensitivity of the triple-mode sensor, samples containing different concentrations of target RNA are detected under the optimized experimental conditions. As shown in Figure 5a , the characteristic absorption peak of AuNPs at 520 nm gradually decreases, the absorption peak at 690 nm gradually increases, and the color gradually becomes blue from red with the increase of RNA concentration. In addition, a red shift (570 → 620 nm) in representative fluorescence peak is present with the increase of RNA concentration, and the fluorescence peak intensity is greatly increased, as in [42, 43] , where S b1 is the standard deviation of blank value, and S is the sensitivity at low concentration, that is, the slope of the standard working curve within the range of low concentration. After the calculation, the limit detection of absorbance is 0.58 pM, the limit detection of Raman is 2.17 pM, and the limit detection of fluorescence is 1.11 pM. AuNPs containing DNA probes were tested for stability within ten days. In order to simulate real sample detection, four target RNA segments with different concentrations are spiked in TE buffer as listed in Table 2 , and P-ORF1 probe is utilized in the reaction system. The concentration of target RNA varies from 10 nM to 0.1 nM, while the concentration of non-specific RNA-ORF1, E1, and E2 is kept at 1 nM. Figure 7a shows the absorbance spectrum of RNA detected in mixed samples. In the detection system with multiple-site probes, it is more sensitive because of higher hybridization opportunity at multiple detection sites. Four-site probes are utilized with a concentration of 0.0125 μM for each probe. In order to simulate real sample detection, four target RNA segments with different concentrations are spiked in TE buffer as listed in Table 3 , and their concentrations vary from 1 pM to 0.01 pM. The experimental results show that the detection sensitivity of the four-site probes system is higher than that of single-site probe system, and the detection signal is enhanced to 4 times as that of single-site system. Figure 9 shows that the detection limit of the triple-mode biosensor with four-site probes is 160 fM in absorbance mode, 259 fM in fluorescence mode, and 395 fM in SERS mode. We believe that the sensitivity of the sensor platform could be improved further by designing more specific probes since the RNA length is more than 20 thousand bases. The triple biosensor based on colorimetry, fluorescence, and SERS is capable of detecting SARS-CoV-2 viral RNA with high selectivity and low detection limit. The main parameters of different methods are listed in Table 4 . The developed triple-mode biosensor provides a few advantages for screening COVID-19 disease. The most important merit of the triple-mode biosensor is the detection accuracy through inter-proof results between three working modes, which reduces the false negative/positive reading of SARS-CoV-2 and avoids the spreading of the COVID-19. Another advantage of the triple-mode biosensor is its rapid process, which only needs 40 min for the whole detection process. J o u r n a l P r e -p r o o f randomly into short segments, but the short segments are suitable for our detection method even though partial targets may get broken. It is worth to note that the developed triple-mode biosensor can be used to detect any genes by using corresponding specific gene probes. Despite the several distinct merits of the developed triple-mode biosensor, there are still some limitations associated with current sensing system. The major limitation is the sensitivity. The acceptable detection range (160 fM -1 nM) of the current triple-mode biosensor is comparable to most developed existing gene biosensor, which may be suitable for the screening of COVID-19 patient with severe infection. But for patients with mild infection, the performance of our triple-mode biosensor needs to be improved by incorporating gene amplification processing. Recently, Yan [49] and Xing [50] reported the signal amplification strategy by using fluorescence molecular amplification technology, resulting in the ultrasensitive gene detection at the level of ~aM. We believe that the proposed triple-mode biosensor could reach much lower detection limit by incorporating the fluorescence signal amplification strategy. In summary, we fabricated a facile genes sensing platform based on AuNPs for triple-mode RNA biosensing, which is achieved by integrating colorimetric, SERS All the authors declare no conflicts of interest with this work. 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