The current major issue in improving detection sensitivity and selectivity is to design an electrochemical sensor that does not require PCR amplification for nucleic acid identification and measurement. Because of their great sensitivity, precision, and simplicity of downsizing, electrochemical biosensors have emerged as a research hotspot in the field of nucleic acid detection. The CRISPR/Cas12 system has emerged as a potent tool for nucleic acid detection due to its powerful cleavage activity and selectivity. Specific electrode changes combined with the CRISPR/Cas12 system can greatly improve the performance of electrochemical biosensors. In this study, the design concepts of electrochemical biosensors based on the CRISPR/Cas12 system and their application advancements in nucleic acid detection are discussed.
KeywordsCRISPRCas12Electrochemical Biosensor
Nadel, J., Athanasiadou, R., Lemetre, C., Wijetunga, N.A., Ó Broin, P., Sato, H., et al . (2015) RNA: DNA Hybrids in the Human Genome Have Distinctive Nucleotide Characteristics, Chromatin Composition, and Transcriptional Relationships. Epigenetics & Chromatin , 8, Article No. 46. https://doi.org/10.1186/s13072-015-0040-6
Vanova, V., Mitrevska, K., Milosavljevic, V., Hynek, D., Richtera, L. and Adam, V. (2021) Peptide-Based Electrochemical Biosensors Utilized for Protein Detection. Biosensors and Bioelectronics , 180, Article ID: 113087. https://doi.org/10.1016/j.bios.2021.113087
Zheng, C., Wang, K., Zheng, W., Cheng, Y., Li, T., Cao, B., et al . (2021) Rapid Developments in Lateral Flow Immunoassay for Nucleic Acid Detection. The Analyst , 146, 1514-1528. https://doi.org/10.1039/d0an02150d
Lockley, A.K. and Bardsley, R.G. (2000) DNA-Based Methods for Food Authentication. Trends in Food Science & Technology , 11, 67-77. https://doi.org/10.1016/s0924-2244(00)00049-2
Kesmen, Z., Gulluce, A., Sahin, F. and Yetim, H. (2009) Identification of Meat Species by Taq-Man-Based Real-Time PCR Assay. Meat Science , 82, 444-449. https://doi.org/10.1016/j.meatsci.2009.02.019
Hellberg, R.S.R. and Morrissey, M.T. (2011) Advances in DNA-Based Techniques for the Detection of Seafood Species Substitution on the Commercial Market. JALA : Journal of the Association for Laboratory Automation , 16, 308-321. https://doi.org/10.1016/j.jala.2010.07.004
Srisomwat, C., Yakoh, A., Chuaypen, N., Tangkijvanich, P., Vilaivan, T. and Chailapakul, O. (2020) Amplification-Free DNA Sensor for the One-Step Detection of the Hepatitis B Virus Using an Automated Paper-Based Lateral Flow Electrochemical Device. Analytical Chemistry , 93, 2879-2887. https://doi.org/10.1021/acs.analchem.0c04283
Lin, C., Hwang, D., Chiu, N., Weng, L., Liu, H., Mu, J., et al . (2020) Increased Detection of Viruses in Children with Respiratory Tract Infection Using PCR. International Journal of Environmental Research and Public Health , 17, Article 564. https://doi.org/10.3390/ijerph17020564
Lee, S.H., Yu, J., Hwang, G., Kim, S., Kim, H.S., Ye, S., et al . (2017) CUT-PCR: Crispr-Mediated, Ultrasensitive Detection of Target DNA Using PCR. Oncogene , 36, 6823-6829. https://doi.org/10.1038/onc.2017.281
Liu, H., Chang, S., Chen, S., Du, Y., Wang, H., Wang, C., et al . (2022) Highly Sensitive and Rapid Detection of SARS-CoV-2 via a Portable CRISPR-Cas13a-Based Lateral Flow Assay. Journal of Medical Virology , 94, 5858-5866. https://doi.org/10.1002/jmv.28096
Qiu, X., Xu, S., Liu, X., Han, L., Zhao, B., Che, Y., et al . (2022) A Crispr-Based Nucleic Acid Detection Platform (CRISPR-CPA): Application for Detection of Nocardia Farcinica. Journal of Applied Microbiology , 132, 3685-3693. https://doi.org/10.1111/jam.15424
Zhang, X., He, X., Zhang, Y., Chen, L., Pan, Z., Huang, Y., et al . (2023) A New Method for the Detection of Mycobacterium Tuberculosis Based on the CRISPR/Cas System. BMC Infectious Diseases , 23, Article No. 680. https://doi.org/10.1186/s12879-023-08656-4
Wang, Y., Liu, L., Liu, X., Wu, K., Zhu, X., Ma, L., et al . (2022) An Ultrasensitive PCR-Based CRISPR-Cas13a Method for the Detection of Helicobacter Pylori. Journal of Personalized Medicine , 12, Article 2082. https://doi.org/10.3390/jpm12122082
Kowalczyk, A. (2020) Trends and Perspectives in DNA Biosensors as Diagnostic Devices. Current Opinion in Electrochemistry , 23, 36-41. https://doi.org/10.1016/j.coelec.2020.03.003
Singh, A., Sharma, A., Ahmed, A., Sundramoorthy, A.K., Furukawa, H., Arya, S., et al . (2021) Recent Advances in Electrochemical Biosensors: Applications, Challenges, and Future Scope. Biosensors , 11, Article 336. https://doi.org/10.3390/bios11090336
Dai, Y., Somoza, R.A., Wang, L., Welter, J.F., Li, Y., Caplan, A.I., et al . (2019) Exploring the Trans‐cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor. Angewandte Chemie International Edition , 58, 17399-17405. https://doi.org/10.1002/anie.201910772
Thévenot, D.R., Toth, K., Durst, R.A. and Wilson, G.S. (2001) Electrochemical Biosensors: Recommended Definitions and Classification1international Union of Pure and Applied Chemistry: Physical Chemistry Division, Commission I.7 (biophysical Chemistry); Analytical Chemistry Division, Commission V.5 (electroanalytical Chemistry).1. Biosensors and Bioelectronics , 16, 121-131. https://doi.org/10.1016/s0956-5663(01)00115-4
Alhadrami, H.A. (2017) Biosensors: Classifications, Medical Applications, and Future Prospective. Biotechnology and Applied Biochemistry , 65, 497-508. https://doi.org/10.1002/bab.1621
Kim, J., Campbell, A.S., de Ávila, B.E. and Wang, J. (2019) Wearable Biosensors for Healthcare Monitoring. Nature Biotechnology , 37, 389-406. https://doi.org/10.1038/s41587-019-0045-y
Cesewski, E. and Johnson, B.N. (2020) Electrochemical Biosensors for Pathogen Detection. Biosensors and Bioelectronics , 159, Article ID: 112214. https://doi.org/10.1016/j.bios.2020.112214
Kabay, G., DeCastro, J., Altay, A., Smith, K., Lu, H., Capossela, A.M., et al . (2022) Emerging Biosensing Technologies for the Diagnostics of Viral Infectious Diseases. Advanced Materials , 34, Article ID: 2201085. https://doi.org/10.1002/adma.202201085
Zein, M.I.H.L., Hardianto, A., Irkham, I., Zakiyyah, S.N., Devi, M.J., Manan, N.S.A., et al . (2023) Recent Development of Electrochemical and Optical Aptasensors for Detection of Antibiotics in Food Monitoring Applications. Journal of Food Composition and Analysis , 124, Article ID: 105644. https://doi.org/10.1016/j.jfca.2023.105644
Magar, H.S., Hassan, R.Y.A. and Mulchandani, A. (2021) Electrochemical Impedance Spectroscopy (EIS): Principles, Construction, and Biosensing Applications. Sensors , 21, Article 6578. https://doi.org/10.3390/s21196578
Peng, Y., Pan, Y., Sun, Z., Li, J., Yi, Y., Yang, J., et al . (2021) An Electrochemical Biosensor for Sensitive Analysis of the SARS-CoV-2 RNA. Biosensors and Bioelectronics , 186, Article ID: 113309. https://doi.org/10.1016/j.bios.2021.113309
Faria, H.A.M. and Zucolotto, V. (2019) Label-Free Electrochemical DNA Biosensor for Zika Virus Identification. Biosensors and Bioelectronics , 131, 149-155. https://doi.org/10.1016/j.bios.2019.02.018
He, Y., Cheng, L., Yang, Y., Chen, P., Qiu, B., Guo, L., et al . (2020) Label-Free Homogeneous Electrochemical Biosensor for HPV DNA Based on Entropy-Driven Target Recycling and Hyperbranched Rolling Circle Amplification. Sensors and Actuators B : Chemical , 320, Article ID: 128407. https://doi.org/10.1016/j.snb.2020.128407
Liu, Y., Cao, Y., Wang, T., Dong, Q., Li, J. and Niu, C. (2019) Detection of 12 Common Food-Borne Bacterial Pathogens by TaqMan Real-Time PCR Using a Single Set of Reaction Conditions. Frontiers in Microbiology , 10, Article 222. https://doi.org/10.3389/fmicb.2019.00222
Abdulbari, H.A. and Basheer, E.A.M. (2017) Electrochemical Biosensors: Electrode Development, Materials, Design, and Fabrication. ChemBioEng Reviews , 4, 92-105. https://doi.org/10.1002/cben.201600009
Wang, C., Xia, K., Wang, H., Liang, X., Yin, Z. and Zhang, Y. (2018) Advanced Carbon for Flexible and Wearable Electronics. Advanced Materials , 31, Article ID: 1801072. https://doi.org/10.1002/adma.201801072
Lozano Untiveros, K., da Silva, E.G., de Abreu, F.C., da Silva-Júnior, E.F., de Araújo-Junior, J.X., Mendoça de Aquino, T., et al . (2019) An Electrochemical Biosensor Based on Hairpin-DNA Modified Gold Electrode for Detection of DNA Damage by a Hybrid Cancer Drug Intercalation. Biosensors and Bioelectronics , 133, 160-168. https://doi.org/10.1016/j.bios.2019.02.071
Khater, M., de la Escosura-Muñiz, A., Quesada-González, D. and Merkoçi, A. (2019) Electrochemical Detection of Plant Virus Using Gold Nanoparticle-Modified Electrodes. Analytica Chimica Acta , 1046, 123-131. https://doi.org/10.1016/j.aca.2018.09.031
Yu, H., Pu, Q., Weng, Z., Zhou, X., Li, J., Yang, Y., et al . (2021) DNAzyme Based Three-Way Junction Assay for Antibody-Free Detection of Locus-Specific N 6 -Methyladenosine Modifications. Biosensors and Bioelectronics , 194, Article ID: 113625. https://doi.org/10.1016/j.bios.2021.113625
Deng, L., zhou, S., Dong, J., Liu, Y., Huang, Z., Sun, H., et al . (2023) Crispr/cas12a and Primer-Assisted Rolling Circle Amplification Integrated Ultra-Sensitive Dual-Signal Sensing Platform for EGFR 19 Detection. Analytica Chimica Acta , 1279, Article ID: 341755. https://doi.org/10.1016/j.aca.2023.341755
Hoffman, A.S. and Hubbell, J.A. (2013) Surface-Immobilized Biomolecules. In: Ratner, B.D., et al ., Eds., Biomaterials Science , Elsevier, 339-349. https://doi.org/10.1016/b978-0-08-087780-8.00032-2
Zhang, D., Yan, Y., Que, H., Yang, T., Cheng, X., Ding, S., et al . (2020) CRISPR/Cas12a-Mediated Interfacial Cleaving of Hairpin DNA Reporter for Electrochemical Nucleic Acid Sensing. ACS Sensors , 5, 557-562. https://doi.org/10.1021/acssensors.9b02461
Lee, Y., Choi, J., Han, H., Park, S., Park, S.Y., Park, C., et al . (2021) Fabrication of Ultrasensitive Electrochemical Biosensor for Dengue Fever Viral RNA Based on Crispr/cpf1 Reaction. Sensors and Actuators B : Chemical , 326, Article ID: 128677. https://doi.org/10.1016/j.snb.2020.128677
Qing, M., Chen, S.L., Sun, Z., Fan, Y., Luo, H.Q. and Li, N.B. (2021) Universal and Programmable Rolling Circle Amplification-CRISPR/Cas12a-Mediated Immobilization-Free Electrochemical Biosensor. Analytical Chemistry , 93, 7499-7507. https://doi.org/10.1021/acs.analchem.1c00805
Globyte, V., Lee, S.H., Bae, T., Kim, J. and Joo, C. (2018) Crispr/Cas9 Searches for a Protospacer Adjacent Motif by Lateral Diffusion. The EMBO Journal , 38, e99466. https://doi.org/10.15252/embj.201899466
Uygun, Z.O., Yeniay, L. and Gi̇rgi̇n Sağın, F. (2020) CRISPR-dCas9 Powered Impedimetric Biosensor for Label-Free Detection of Circulating Tumor DNAs. Analytica Chimica Acta , 1121, 35-41. https://doi.org/10.1016/j.aca.2020.04.009
Ishino, Y., Shinagawa, H., Makino, K., Amemura, M. and Nakata, A. (1987) Nucleotide Sequence of the Iap Gene, Responsible for Alkaline Phosphatase Isozyme Conversion in Escherichia Coli, and Identification of the Gene Product. Journal of Bacteriology , 169, 5429-5433. https://doi.org/10.1128/jb.169.12.5429-5433.1987
Mohanraju, P., Makarova, K.S., Zetsche, B., Zhang, F., Koonin, E.V. and van der Oost, J. (2016) Diverse Evolutionary Roots and Mechanistic Variations of the CRISPR-Cas Systems. Science , 353, aad5147. https://doi.org/10.1126/science.aad5147
Chaudhuri, A., Halder, K. and Datta, A. (2022) Classification of CRISPR/Cas System and Its Application in Tomato Breeding. Theoretical and Applied Genetics , 135, 367-387. https://doi.org/10.1007/s00122-021-03984-y
Shi, Y., Fu, X., Yin, Y., Peng, F., Yin, X., Ke, G., et al . (2021) CRISPR-Cas12a System for Biosensing and Gene Regulation. Chemistry — An Asian Journal , 16, 857-867. https://doi.org/10.1002/asia.202100043
He, Q., Yu, D., Bao, M., Korensky, G., Chen, J., Shin, M., et al . (2020) High-Throughput and All-Solution Phase African Swine Fever Virus (ASFV) Detection Using Crispr-Cas12a and Fluorescence Based Point-Of-Care System. Biosensors and Bioelectronics , 154, Article ID: 112068. https://doi.org/10.1016/j.bios.2020.112068
Zakiyyah, S.N., Ibrahim, A.U., Babiker, M.S., Gaffar, S., Ozsoz, M., Zein, M.I.H.L., et al . (2022) Detection of Tropical Diseases Caused by Mosquitoes Using CRISPR-Based Biosensors. Tropical Medicine and Infectious Disease , 7, Article 309. https://doi.org/10.3390/tropicalmed7100309
Srivastava, S., Upadhyay, D.J. and Srivastava, A. (2020) Next-generation Molecular Diagnostics Development by CRISPR/Cas Tool: Rapid Detection and Surveillance of Viral Disease Outbreaks. Frontiers in Molecular Biosciences , 7, Article 582499. https://doi.org/10.3389/fmolb.2020.582499
Thompson, D. and Lei, Y. (2020) Mini Review: Recent Progress in RT-LAMP Enabled COVID-19 Detection. Sensors and Actuators Reports , 2, Article ID: 100017. https://doi.org/10.1016/j.snr.2020.100017
Patsali, P., Kleanthous, M. and Lederer, C.W. (2019) Disruptive Technology: CRISPR/Cas-Based Tools and Approaches. Molecular Diagnosis & Therapy , 23, 187-200. https://doi.org/10.1007/s40291-019-00391-4
Zhang, X. (2022) Development of Crispr-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry. Genes , 13, Article 2007. https://doi.org/10.3390/genes13112007
Liu, N., Liu, R. and Zhang, J. (2022) CRISPR-Cas12a-Mediated Label-Free Electrochemical Aptamer-Based Sensor for SARS-CoV-2 Antigen Detection. Bioelectroc hemistry , 146, Article ID: 108105. https://doi.org/10.1016/j.bioelechem.2022.108105
Chen, H., Li, Z., Chen, J., Yu, H., Zhou, W., Shen, F., et al . (2022) CRISPR/Cas12a-based Electrochemical Biosensor for Highly Sensitive Detection of cTnI. Bioelectrochemistry , 146, Article ID: 108167. https://doi.org/10.1016/j.bioelechem.2022.108167
Huang, L., Yuan, N., Guo, W., Zhang, Y. and Zhang, W. (2023) An Electrochemical Biosensor for the Highly Sensitive Detection of Staphylococcus aureus Based on SRCA-CRISPR/Cas12a. Talanta , 252, Article ID: 123821. https://doi.org/10.1016/j.talanta.2022.123821
Li, F., Ye, Q., Chen, M., Zhou, B., Zhang, J., Pang, R., et al . (2021) An Ultrasensitive CRISPR/Cas12a Based Electrochemical Biosensor for Listeria Monocytogenes Detection. Biosensors and Bioelectronics , 179, Article ID: 113073. https://doi.org/10.1016/j.bios.2021.113073
Cui, J., Luo, Q., Wei, C., Deng, X., Liang, H., Wei, J., et al . (2024) Electrochemical Biosensing for E . coli Detection Based on Triple Helix DNA Inhibition of CRISPR/Cas12a Cleavage Activity. Analytica Chimica Acta , 1285, Article ID: 342028. https://doi.org/10.1016/j.aca.2023.342028
He, Y., Jia, F., Sun, Y., Fang, W., Li, Y., Chen, J., et al . (2022) An Electrochemical Sensing Method Based on CRISPR/Cas12a System and Hairpin DNA Probe for Rapid and Sensitive Detection of Salmonella Typhimurium. Sensors and Actuators B : Chemical , 369, Article ID: 132301. https://doi.org/10.1016/j.snb.2022.132301
Wang, C., Zhang, Y., Liu, S., Yin, Y., Fan, G., Shen, Y., et al . (2023) Allosteric Probe-Triggered Isothermal Amplification to Activate CRISPR/Cas12a for Sensitive Electrochemiluminescence Detection of Salmonella. Food Chemistry , 425, Article ID: 136382. https://doi.org/10.1016/j.foodchem.2023.136382
Fu, X., Sun, J., Yu, B., Ye, Y., Sheng, L., Ji, J., et al . (2024) Investigating Enzyme Kinetics and Fluorescence Sensing Strategy of CRISPR/Cas12a for Foodborne Pathogenic Bacteria. Analytica Chimica Acta , 1290, Article ID: 342203. https://doi.org/10.1016/j.aca.2024.342203
Gu, X., Tang, Q., Kang, X., Ji, H., Shi, X., Shi, L., et al . (2024) A Portable Crispr-Cas12a Triggered Photothermal Biosensor for Sensitive and Visual Detection of Staphylococcus aureus and Listeria Monocytogenes. Talanta , 271, Article ID: 125678. https://doi.org/10.1016/j.talanta.2024.125678
Wu, J., Huang, Y., Ding, X., Kang, L., Wang, X., Li, D., et al . (2023) CPA-Cas12a-Based Lateral Flow Strip for Portable Assay of Methicillin-Resistant Staphylococcus aureus in Clinical Sample. Journal of Nanobiotechnology , 21, Article No. 234. https://doi.org/10.1186/s12951-023-02002-1
Cao, X., Chang, Y., Tao, C., Chen, S., Lin, Q., Ling, C., et al . (2023) Cas12a/Guide RNA-Based Platforms for Rapidly and Accurately Identifying Staphylococcus aureus and Methicillin-Resistant S . aureus . Microbiology Spectrum , 11, e04870-22. https://doi.org/10.1128/spectrum.04870-22
Li, Y., Shi, Z., Hu, A., Cui, J., Yang, K., Liu, Y., et al . (2022) Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus . Diagnostics , 12, Article 829. https://doi.org/10.3390/diagnostics12040829
Liu, Y., Liu, H., Yu, G., Sun, W., Aizaz, M., Yang, G., et al . (2023) One-Tube RPA-CRISPR Cas12a/Cas13a Rapid Detection of Methicillin-Resistant Staphylococcus aureus . Analytica Chimica Acta , 1278, Article ID: 341757. https://doi.org/10.1016/j.aca.2023.341757
Sun, X., Wang, Y., Zhang, L., Liu, S., Zhang, M., Wang, J., et al . (2020) CRISPR-Cas9 Triggered Two-Step Isothermal Amplification Method for E . coli O157: H7 Detection Based on a Metal-Organic Framework Platform. Analytical Chemistry , 92, 3032-3041. https://doi.org/10.1021/acs.analchem.9b04162
Zhu, L., Liang, Z., Xu, Y., Chen, Z., Wang, J. and Zhou, L. (2023) Ultrasensitive and Rapid Visual Detection of Escherichia Coli O157:H7 Based on RAA-CRISPR/Cas12a System. Biosensors , 13, Article 659. https://doi.org/10.3390/bios13060659
Mukama, O., Wu, J., Li, Z., Liang, Q., Yi, Z., Lu, X., et al . (2020) An Ultrasensitive and Specific Point-of-Care CRISPR/Cas12 Based Lateral Flow Biosensor for the Rapid Detection of Nucleic Acids. Biosensors and Bioelectronics , 159, Article ID: 112143. https://doi.org/10.1016/j.bios.2020.112143
Wang, Y., Ke, Y., Liu, W., Sun, Y. and Ding, X. (2020) A One-Pot Toolbox Based on Cas12a/crRNA Enables Rapid Foodborne Pathogen Detection at Attomolar Level. ACS Sensors , 5, 1427-1435. https://doi.org/10.1021/acssensors.0c00320
Xia, X., Ma, B., Zhang, T., Lu, Y., Khan, M.R., Hu, Y., et al . (2021) G-Quadruplex-probing CRISPR-Cas12 Assay for Label-Free Analysis of Foodborne Pathogens and Their Colonization in Vivo . ACS Sensors , 6, 3295-3302. https://doi.org/10.1021/acssensors.1c01061
Zhang, H., Yao, S., Sheng, R., Wang, J., Li, H., Fu, Y., et al . (2022) A Cascade Amplification Strategy for Ultrasensitive Salmonella Typhimurium Detection Based on DNA Walker Coupling with CRISPR-Cas12a. Journal of Colloid and Interface Science , 625, 257-263. https://doi.org/10.1016/j.jcis.2022.06.027