key: cord-0697444-ez8qg885 authors: Zhang, Kai; Fan, Zhenqiang; Huang, Yue; Ding, Yuedi; Xie, Minhao title: A strategy combining 3D-DNA Walker and CRISPR-Cas12a trans-cleavage activity applied to MXene based electrochemiluminescent sensor for SARS-CoV-2 RdRp gene detection date: 2021-09-10 journal: Talanta DOI: 10.1016/j.talanta.2021.122868 sha: fc6b726bc432ae7a99a79e01ee350da2d42883e6 doc_id: 697444 cord_uid: ez8qg885 Early diagnosis and timely management of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are the keys to preventing the spread of the epidemic and controlling new infection clues. Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. In this work, we first proposed the idea of activating CRISPR-Cas12a activity using double-stranded DNA amplified by a three-dimensional (3D) DNA walker. We applied it to the design of an electrochemiluminescent (ECL) biosensor to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. We first activated the cleavage activity of CRISPR-Cas12a by amplifying the target DNA into a segment of double-stranded DNA through the amplification effect of a 3D DNA walker. At the same time, we designed an MXene based ECL material: PEI-Ru@Ti(3)C(2)@AuNPs, and constructed an ECL biosensor to detect the RdRp gene based on this ECL material as a framework. Activated CRISPR-Cas12a cleaves the single-stranded DNA on the surface of this sensor and causes the ferrocene modified at one end of the DNA to move away from the electrode surface, increasing the ECL signal. The extent of the change in electrochemiluminescence reflects the concentration of the gene to be measured. Using this system, we detected the SARS-CoV-2 RdRp gene with a detection limit of 12.8 aM. This strategy contributes to the rapid and convenient detection of SARS-CoV-2-associated nucleic acids and promotes the clinical application of ECL biosensors based on CRISPR-Cas12a and novel composite materials. tract have yet to be clarified. SARS-CoV-2 infected patients usually present with pneumonia-like symptoms (fever, dry cough, and dyspnea) and gastrointestinal symptoms such as diarrhea, followed by severe acute respiratory infections. [2] Some cases may have acute respiratory distress with severe respiratory symptoms, complications, and even death. At present, there is no specific treatment for SARS-CoV-2 infected patients. Early diagnosis and timely management are the keys to preventing the spread of the epidemic and controlling new infection clues. [3] Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. Reverse transcription-polymerase chain reaction (RT-PCR) to detect viral nucleic acid is considered the gold standard for diagnosing SARS-CoV-2. [4] However, this method has higher requirements for experimental conditions, facilities, personnel, many influencing factors, operation steps, and long detection time. [1] More importantly, although the specificity of nucleic acid detection is high, due to various reasons, the false-negative rate is high, which limits the detection of nucleic acid. There is no doubt that the COVID-19's rapid and accurate identification can significantly help control the emerging pandemic. Biological protein machinery is ubiquitous in biological systems and performs various physiological functions, including mechanical drive, intracellular transport, and signal transduction [5] . Inspired by these biological machines, researchers have tried to create various artificial molecular machines and motors that can use controlled molecularlevel motion to perform specific tasks. Relying on the predictability, specificity, and versatility of Watson-Crick base coordination theory, researchers have designed and J o u r n a l P r e -p r o o f synthesized various mechanical devices made of DNA molecules, including DNA Walker, DNA tweezers, DNA motors, and DNA robots. [6] DNA Walker, which can be precisely controlled on the micron or nano DNA tracks, has shown great potential in biosensor analysis. [7] At present, the emergence of three-dimensional (3D) DNA orbits has brought new perspectives to DNA walkers' research. [8, 9] Compared with the restricted one-dimensional DNA track or two-dimensional DNA origami such as the DNA Walker track, the 3D DNA track composed of micro-or nano-particles has a larger specific surface area. [6] So the three-dimensional DNA track has a more vital ability to concentrate and expand. For example, Ellington and his colleagues first developed a 3D DNA Walker on the surface of particles using the principle of catalytic hairpin selfassembly (CHA). [10] In this way, similar DNA Walkers based on CHA principles have appeared one after another. Besides, Yin and his colleagues reasonably designed an entropy-driven 3D DNA walking sensor. [11] Different from the DNA Walker based on the CHA principle, the entropy-driven DNA Walker uses a single-stranded linear DNA molecular sequence, thereby avoiding the use of complex pseudoknots, the secondary structure of the kiss ring, or high background signals in the DNA molecules. A simple DNA hybridization reaction activates the enzyme-free three-dimensional DNA Walker. Various signal amplification methods for protein detection have been proposed. Compared with previous biosensors constructed based on isothermal amplification strategy, [12, 13] this 3D-DNA Walker can be based on a highly localized entropydriven reaction, and the chain substitution reaction triggered by the walking arm walking will be strictly limited to the Au particle surface, and no chain substitution reaction will occur across the particles. Thus the highly localized motion of a single molecule walking arm on the surface of a single nanoparticle will generate enough nucleic acid duplexes to allow a more complete DNA walker amplification reaction on the surface of that nanoparticle. As we all know, the polymerase chain reaction (PCR) is one of the most widely used amplification techniques and is very important for biological research. Instead, it is limited by labor-intensive work, precise temperature control, and potential differences between batches. In contrast, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a relatively new nucleic acid detection method. Since the application of the modified CRISPR/Cas9 system for gene editing of mammalian genomes, the CRISPR/Cas system has taken center stage in biotechnology. [2] A large number of researchers have worked to develop a variety of nucleic acid amplification systems by exploiting the side-cleavage (Trans) activity of CRISPR/Cas nucleases such as CRISPR/Cas12a, CRISPR/Cas12b, and CRISPR/Cas13a. [14] This technology is now considered an innovative approach for next-generation diagnostics and has been identified as having the potential to significantly impact the development of biosensors by providing a faster and more accurate ultra-sensitive nucleic acid detection method. [15] The CRISPR system consists of a guide RNA (gRNA) and a Cas12a protein, and the gRNA is able to direct Cas to recognize and cleave RNA or DNA molecules with specific sequences. [16, 17] Among them, Cas12a protein will continue to cut non-target DNA indiscriminately when it explicitly cuts the target DNA. Using this feature, DNA signal molecules with signal markers are added to the whole system, which will cause a change in signal once the DNA with signal is cut, and this technology combined with isothermal amplification technology can detect the nucleic acid to be detected with high specificity and sensitivity. [16] Electrochemiluminescence (ECL) technology has been active in the research of disease marker analysis. ECL not only has the advantages of ultra-sensitive detection and wide range of detection by traditional electrochemical methods, but also has the to meet the development needs of clinical treatment. [3] However, traditional electrochemiluminescence-based biosensors are not perfect and need new breakthroughs due to the disadvantages of electrode interface potential resistance that affects the binding efficiency of enzyme molecules, complex interface environment that increases the probability of non-specific adsorption, easy diffusion of luminescent reagents in homogeneous solutions, poor luminescence stability, inability to modulate ECL spectra and reversible modulation, etc. [16, 22] Therefore, this solution is not perfect and needs new breakthroughs. The continuous discovery of graphene derivatives and graphene-like 2D nanomaterials provides new development opportunities to develop solid-phase material-based luminescent electrochemical biosensors. [23] The reason is that 2D nanomaterials have the following advantageous properties: a) The electronic properties are significantly enhanced because the electrons are confined within the 2D planar structure, which makes 2D nanomaterials ideal electron transport carriers for optoelectronic sensors. b) The strong covalent bonds within the lamellae and the thickness of the atomic layers make 2D nanomaterials exhibit excellent mechanical strength, flexibility and light transmission. Among them, Ti3C2Tx (MXene), as a new 2D lamellar material, possesses high intrinsic photoelectric conversion effect, while the abundant functional groups on its surface enable efficient modification and good homogeneous dispersion, which makes it highly promising as an ECL material for the construction of various ECL sensors. [16] Considering that the amplification product of DNA track-based DNA Walker contains DNA double-stranded, we pioneered the combination of DNA walker and CRISPR/Cas12a amplification by using the amplification product double-stranded DNA as a linkage, in order to achieve highly sensitive detection of novel coronaviruses. This double-stranded DNA can further activate CRISPR/Cas 12a, which can indiscriminately cut the single-stranded DNA on the surface of the electrode, thus causing a change in the ECL signal, which can reflect the concentration of RdRp gene to be tested. Using this detection strategy, gene detection at 12.8 aM can be achieved. Therefore, the present method has potential application in the clinical screening of SARS-CoV-2 gene. Gold chloride trihydrate (HAuCl4·3H2O), sodium citrate, sodium borodeuteride (Table S1 ) were synthesized by Genscript Bio-technology Co. Ltd. (Nanjing, China) The PEI-Ru@Ti3C2@AuNPs-DNA7 probe was successfully synthesized with slight modification by referring to our previous literature. [16] As shown in Scheme 1, The system we designed contains two parts: a 3D nano-machine and a CRISPR/Cas12a-based nucleic acid amplification ECL sensor. In the first part， our nano-machine mainly contains a DNA-modified AuNP (DNA-AuNP) a walking leg (DNA5), and a fuel (DNA6). The DNA-AuNP is a conjugate with a three-stranded substrate complex (DNA1/DNA2/DNA3) and affinity ligand (DNA4) on a single AuNP. The detailed sequence information for the nano-machine is shown in Table S1 . DNA1 was modified with a thiol at 5' end to modified on the AuNP surface and hybridized with part of DNA2, which co-hybridizes to DNA3. This forms a sandwich structure with a toehold at the 5' end of DNA2. The recognition sequences for target DNA (RdRp gene) are designed to embed in DNA4 and DNA5, respectively. The binding target DNA and DNA5 to DNA4 bring DNA5 into proximity to the AuNP surface, leading to tethered to the AuNP to form a walkable leg with the capability to perform highly effective intramolecular hybridization. The entropy-driven catalytic reaction occurs as follows: DNA5 interacts with DNA2 via toehold and displaces DNA3 from We first performed the feasibility analysis of this system (Figure 1(I) ). When no nucleic acid to be tested was present in the system, the system showed an extremely low ECL signal (curve a) because it could not trigger the amplification system to produce DNA2/DNA6 duplex, so it could not further activate the cleavage activity of CRISPR-Cas 12a. When 1000 aM of the nucleic acid to be tested was added to the system, a large amount of DNA2/DNA6 duplex was generated and successfully activated the cleavage activity of CRISPR-Cas 12a through the amplification effect of the DNA walker on the surface of the gold electrode, which in turn led to an increase in the ECL signal (curve b). When no gold nanoparticle-modified DNA walker was present in the J o u r n a l P r e -p r o o f system, the same low ECL signal was generated because the DNA walker reaction could not be triggered and no raw DNA2/DNA6 duplex could be produced to trigger the cleavage activity of CRISPR-Cas 12a (curve c). When CRISPR-Cas 12a was not added to the system, although the nucleic acid to be tested was able to trigger the DNA walker reaction and produce the DNA2/DNA6 duplex, CRISPR-Cas 12a was not present in the system and therefore did not cleave DNA7 on the electrode surface, and thus, only a low ECL signal was obtained (curve d). All these data corroborate the mechanism of this experiment and prove the feasibility of our designed system. The PEI-Ru@Ti3C2@AuNPs composites were characterized by EDX (Energy dispersive X-ray spectroscopy) mapping as shown in Figure 1 (II). The ECL enhancement mechanism of the ternary system (Ru(bpy)3 2+ /PEI/Ti3C2) can be summarized as follows: (1) the co-reactants are modified PEI modified on MXene; (2) PEI is directly oxidized to PEI + under applied voltage and then directly deprotonated to PEI• radicals (PEI•); (3) PEI• reduced Ti3C2/Ru(bpy)3 2+ to form Ti3C2/Ru(bpy)3 2+ , which in turn reacts with PEI• + in the oxidized state to form Ti3C2/Ru(bpy)3 2+ *. Eventually, Ti3C2/Ru(bpy)3 2+ * is finally converted to Ru(bpy)3 2+ and the ECL light signal is generated. PEI − − → PEI + PEI · + − H + → PEI · PEI · +Ti 3 C 2 /Ru(bpy) 3 2+ → Ti 3 C 2 /Ru(bpy) 3 + + product PEI · + + Ti 3 C 2 /Ru(bpy) 3 + → Ti 3 C 2 /Ru(bpy) 3 2+ * + product Ti 3 C 2 /Ru(bpy) 3 2+ * → Ti 3 C 2 /Ru(bpy) 3 2+ + hν Electrochemical impedance spectroscopy (EIS), an effective method to probe the interfacial properties of electrode surface modifications, was also used to characterize the interfacial changes during the preparation of DNA walker and CRISPR-Cas12a based biosensors, and the results are shown in Figure 2A . The impedance plot of the bare gold electrode shows that the native impedance semicircle diameter is less than 200 (curve a), which indicates that the bare gold electrode has excellent conductivity and low impedance. When PEI-Ru@Ti3C2@AuNPs were modified on the electrode surface and became a uniform film, the electrode impedance increased significantly (curve b), which is mainly due to the dense PEI-Ru@Ti3C2@AuNPs film hindering the electron transfer, thus leading to a more significant impedance of the sensor. When DNA7 was assembled on the electrode surface through Au-S bonding, the impedance of the biosensor increased significantly (curve c). This indicates that DNA7 further hinders the electron transfer. When MCH was modified on the AuNP surface, the impedance value increased further (curve d), indicating that MCH sealed the electrode J o u r n a l P r e -p r o o f surface even further. When the prepared biosensor was incubated with the DNA2/DNA6 duplex activated CRISPR Cas12a/gRNA complex, the impedance value of the biosensor decreased significantly (curve e), indicating that DNA7 was cleaved by the CRISPR Cas12a/gRNA complex, resulting in an increased electron transfer rate. Therefore, this data suggests the successful assembly of the target sensor. Since DNA5 is the key to the DNA walker reaction, we optimized the length and Under the optimized conditions, we used the ECL biosensor system to detect the SARS-COV-2 RdRp gene. Figure cleaves the DNA7 on the electrode surface, causing an increase in ECL signal. Figure 3B insert also depicts an excellent linear relationship between the change in ECL signal intensity and the concentration of the RdRP gene, i.e., ECL=3.84163×C(RdRp)+314.72, R 2 =0.9952. The detection limit was 12.8 aM. In addition, we also did a comparison data between the gene to be tested and the previously reported SARS-COV-2 gene (Table 1) . We can see that our detection system has a more comprehensive linear range and lower detection limit compared to conventional sensors. DNA walker-based biosensors for RdRp gene detection. Therefore, next, we tested various nucleic acids to assess the specificity and stability of our constructed biosensing system ( Figure 4A ). We selected a blank solution (no target DNA), SARS-COV RdRp gene (COV, 1 fM), ORF1ab-COVID (OC, 1 fM) DNA1 with random mutation sites (M1, 1 fM), and DNA2 with random mutation sites (M2, 1 fM) as controls. As shown in Figure 4A , there was no significant difference in the electrochemiluminescence J o u r n a l P r e -p r o o f signal after using the blank solution, SARS-COV RdRp gene, DNA1 with random mutation sites and DNA2 with random mutation sites acting on this assay system. In contrast, the electrochemiluminescence signal had a greater degree of variation after acting the SARS-CoV-2 RdRp gene (1 fM) on the CRISPR-Cas 12a-based assay system. The reason for this experimental phenomenon may be that DNA containing the gene mutation site cannot promote the 3D DNA walker reaction on the surface of gold nanoparticles, and therefore does not activate the shearing activity of CRISPR-Cas12a. The results show that our constructed CRISPR-12a-based and 3D DNA walker has good selectivity for the RdRp gene of SARS-COV-2. Stability is also an important performance indicator for the success of CRISPR-Cas12a and 3D DNA walker-based biosensor construction. We measured the stability of the electrochemiluminescence signal, and the results are shown in Figure 4B . It can be seen from the figure that the electrochemiluminescence signal of this biosensor has good stability (RSD=4.21%) despite performing twenty scans three times. Thus, this indicates that the constructed CRISPR-Cas12a and 3D DNA walker-based biosensor is expected to be used for the detection of SARS-COV-2 RdRp gene. according to the standard addition method. As described in Table S2 , the experimental recoveries ranged from 98.97% to 102.51%, and the RSD values ranged from 3.2% to 4.57%. It indicates that the novel ECL biosensor has a good signal response for RdRp and can be applied to detect RdRp gene in real samples. Unlike most 1D or 2D DNA nanomachines, our 3D DNA walker is built on a 3D DNA This work was supported by the National Natural Science Foundation of China (21705061), the Jiangsu Provincial Key Medical Discipline (Laboratory) (ZDXKA2016017), and the Innovation Capacity Development Plan of Jiangsu Province (BM2018023) Zhou Detection of COVID-19: A review of the current literature and future perspectives Guan CRISPR-based detection of SARS-CoV-2: A review from sample to result Zhang Entropy-driven amplified electrochemiluminescence biosensor for RdRp gene of SARS-CoV-2 detection with selfassembled DNA tetrahedron scaffolds Szunerits Preanalytical Issues and Cycle Threshold Values in SARS-CoV-2 Real-Time RT-PCR Testing: Should Test Results Include These? Emerging bioanalytical applications of DNA walkers Enzyme-Powered Three-Dimensional DNA Nanomachine for DNA Walking Research progress of DNA walker and its recent applications in biosensor TrAC Jiang A catalytic assembled enzyme-free three-dimensional DNA walker and its sensing application A microRNA-initiated DNAzyme motor operating in living cells Ellington A stochastic DNA walker that traverses a microparticle surface Ye Rational Engineering of a Dynamic, Entropy-Driven DNA Nanomachine for Intracellular MicroRNA Imaging Angew Wang Ultrasensitive detection of hERG potassium channel in single-cell with photocleavable and entropy-driven reactions by using an electrochemical biosensor Xie Ultrasensitive fluorescence detection of transcription factors based on kisscomplex formation and the T7 RNA polymerase amplification method Le CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules Xing CRISPR/Cas13a Powered Portable Electrochemiluminescence Chip for Ultrasensitive and Specific MiRNA Detection Tang CRISPR-Cas12a-driven MXene-PEDOT:PSS piezoresistive wireless biosensor Nano Energy Huang Dual-Wavelength Electrochemiluminescence Ratiometric Biosensor for NF-κB p50 Detection with Dimethylthiodiaminoterephthalate Fluorophore and Self-Assembled DNA Yuan Perylene Derivative/Luminol Nanocomposite as a Strong Electrochemiluminescence Emitter for Construction of an Electrogenerated Chemiluminescence Peptide-Based Biosensor for the Determination of Prostate Chai Electrochemiluminescence Peptide-Based Biosensor with Hetero-Nanostructures as Coreaction Accelerator for the Xie Entropy-driven electrochemiluminescence ultra-sensitive detection strategy of NF-κB p50 as the regulator of cytokine storm Feng A signal-on photoelectrochemical aptasensor for chloramphenicol assay based on 3D selfsupporting AgI/Ag/BiOI Z-scheme heterojunction arrays Drosten Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Eurosurveillance Poon Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia Pan Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles ACS Nano Homogeneous circle-to-circle amplification for real-time optomagnetic detection of SARS-CoV-2 RdRp coding sequence Wang Dual-Functional Plasmonic Photothermal Biosensors for Highly Accurate Severe Acute Respiratory Syndrome Coronavirus 2 Detection ACS Nano Ultrasensitive supersandwich-type electrochemical sensor for SARS-CoV-2 from the infected COVID-19 patients using a smartphone Sens Cui A reversetranscription recombinase-aided amplification assay for the rapid detection of N gene of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) Virology 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.