CRISPR/Cas Powered Electrochemiluminescence Biosensing

Hongzhang Xiang; Yingjie Yang; Wenjing Wang

doi:10.53941/nenp.2026.100004

Abstract

In the face of fast-changing global public health threats, there is an urgent demand for novel detection platforms that combine high sensitivity with robust selectivity in real samples. Electrochemiluminescence (ECL), as a powerful analytical tool, offers excellent sensitivity and low background for biosensing. However, its reliance on traditional recognition elements limits the specificity and further applications in trace-level target detection. The emergence of CRISPR/Cas systems, with their programmable recognition and powerful signal amplification via nonspecific trans-cleavage activity, presents a transformative opportunity. In this review, we focus on the development of CRISPR/Cas-powered ECL biosensing platforms for overcoming these challenges. We first introduce the fundamental mechanisms and main integration strategies that couple the precise nucleic acid recognition and collateral cleavage of CRISPR/Cas with the sensitive light emission of ECL transduction. Subsequently, we systematically highlight the broad applicability of these hybrid platforms for detecting diverse targets, including pathogenic nucleic acids and small molecules, with direct relevance to disease diagnostics, environmental monitoring, and food safety. Finally, we discuss the current challenges and future prospects of these platforms, emphasizing their potential as next-generation tools for field-deployable, ultra-sensitive analysis in public health security.

References

1.
Ji, T.; Liu, Z.; Wang, G.; et al. Detection of COVID-19: A review of the current literature and future perspectives. Biosens. Bioelectron. 2020, 166, 112455.
2.
Baker, R.E.; Mahmud, A.S.; Miller, I.F.; et al. Infectious disease in an era of global change. Nat. Rev. Microbiol. 2022, 20, 193–205.
3.
Bowsher, G.; McNamara, T.; Bernard, R.; et al. Veterinary intelligence: Integrating zoonotic threats into global health security. J. R. Soc. Med. 2021, 114, 545–548.
4.
Fuller, R.; Landrigan, P.J.; Balakrishnan, K.; et al. Pollution and health: A progress update. Lancet Planet. Health 2022, 6, E535–E547.
5.
Li, S.G.; Zhang, H.Y.; Zhu, M.; et al. Electrochemical Biosensors for Whole Blood Analysis: Recent Progress, Challenges, and Future Perspectives. Chem. Rev. 2023, 123, 7953–8039.
6.
Zeng, N.; Wu, Y.; Chen, W.; et al. Whole-cell microbial bioreporter for soil contaminants detection. Front. Bioeng. Biotechnol. 2021, 9, 622994.
7.
Yuan, G.L.; Hassan, M.M.; Yao, T.; et al. Plant-Based Biosensors for Detecting CRISPR-Mediated Genome Engineering. ACS Synth. Biol 2021, 10, 3600–3603.
8.
Wilkinson, J.L.; Boxall, A.B.A.; Kolpin, D.W.; et al. Pharmaceutical pollution of the world’s rivers. Proc. Natl. Acad. Sci. USA 2022, 119, e2113947119.
9.
Quesada-González, D.; Merkoçi, A. Nanomaterial-based devices for point-of-care diagnostic applications. Chem. Soc. Rev. 2018, 47, 4697–4709.
10.
Wang, F.; Xiang, L.; Leung, K.S.-Y.; et al. Emerging contaminants: A one health perspective. Innovation 2024, 5, 100612.
11.
Furst, A.L.; Francis, M.B. Impedance-based detection of bacteria. Chem. Rev. 2018, 119, 700–726.
12.
Li, Z.; Qin, W.; Liang, G. A mass-amplifying electrochemiluminescence film (MAEF) for the visual detection of dopamine in aqueous media. Nanoscale 2020, 12, 8828–8835.
13.
Liu, Z.; Qi, W.; Xu, G. Recent advances in electrochemiluminescence. Chem. Soc. Rev. 2015, 44, 3117–3142.
14.
Barhoum, A.; Altintas, Z.; Devi, K.S.; et al. Electrochemiluminescence biosensors for detection of cancer biomarkers in biofluids: Principles, opportunities, and challenges. Nano Today 2023, 50, 101874.
15.
Dinel, M.P.; Tartaggia, S.; Wallace, G.Q.; et al. The fundamentals of real-time surface plasmon resonance/electrogenerated chemiluminescence. Angew. Chem. Int. Ed. 2019, 58, 18202–18206.
16.
Du, F.X.; Chen, Y.Q.; Meng, C.D.; et al. Recent advances in electrochemiluminescence immunoassay based on multiple-signal strategy. Curr. Opin. Electrochem. 2021, 28, 100725.
17.
Han, D.; Yang, K.; Sun, S.G.; et al. Signal amplification strategies in electrochemiluminescence biosensors. Chem. Eng. J. 2023, 476, 146688.
18.
Zhang, Y.X.; Wu, Y.P.; Wu, Y.F.; et al. CRISPR-Cas systems: From gene scissors to programmable biosensors. Trends Anal. Chem. 2021, 137, 116210.
19.
Qi, Y.; Li, K.; Li, Y.X.; et al. CRISPR-based diagnostics: A potential tool to address the diagnostic challenges of tuberculosis comment. Pathogens 2022, 11, 1211.
20.
Broughton, J.P.; Deng, X.D.; Yu, G.X.; et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat. Biotechnol. 2020, 38, 870–874.
21.
Ackerman, C.M.; Myhrvold, C.; Thakku, S.G.; et al. Massively multiplexed nucleic acid detection with Cas13. Nature 2020, 582, 277–282.
22.
Jinek, M.; Chylinski, K.; Fonfara, I.; et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337, 816–821.
23.
Makarova, K.S.; Wolf, Y.I.; Iranzo, J.; et al. Evolutionary classification of CRISPR-Cas systems: A burst of class 2 and derived variants. Nat. Rev. Microbiol. 2020, 18, 67–83.
24.
Li, Y.; Li, S.Y.; Wang, J.; et al. CRISPR/Cas systems towards next-generation biosensing. Trends Biotechnol. 2019, 37, 730–743.
25.
Doudna, J.A.; Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science 2014, 346, 1258096.
26.
Chen, J.S.; Ma, E.B.; Harrington, L.B.; et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 2018, 360, 436–439.
27.
Gootenberg, J.S.; Abudayyeh, O.O.; Lee, J.W.; et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 2017, 356, 438–442.
28.
Dai, Y.F.; Somoza, R.A.; Wang, L.; et al. Exploring the trans-cleavage activity of CRISPR-Cas12a (cpf1) for the development of a universal electrochemical biosensor. Angew. Chem. Int. Ed. 2019, 58, 17399–17405.
29.
Karvelis, T.; Bigelyte, G.; Young, J.K.; et al. PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage. Nucleic Acids Res. 2020, 48, 5016–5023.
30.
Xiao, R.; Li, Z.; Wang, S.; et al. Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR–Cas12f nuclease. Nucleic Acids Res. 2021, 49, 4120–4128.
31.
Feng, W.; Newbigging, A.M.; Tao, J.; et al. CRISPR technology incorporating amplification strategies: Molecular assays for nucleic acids, proteins, and small molecules. Chem. Sci. 2021, 12, 4683–4698.
32.
Yu, L.Y.; Peng, Y.; Sheng, M.T.; et al. Sensitive and amplification-free electrochemiluminescence biosensor for HPV-16 detection based on CRISPR/Cas12a and DNA tetrahedron nanostructures. ACS Sens. 2023, 8, 2852–2858.
33.
Wang, Q.; Zhang, Z.H.; Zhang, L.; et al. Photoswitchable CRISPR/Cas12a-Amplified and Co3O4@Au nanoemitter based triple-amplified diagnostic electrochemiluminescence biosensor for detection of miRNA-141. ACS Appl. Mater. Interfaces 2022, 14, 32960–32969.
34.
Wang, Q.; Liu, Y.Q.; Yan, J.X.; et al. 3D DNA walker-assisted CRISPR/Cas12a trans-cleavage for ultrasensitive electrochemiluminescence detection of miRNA-141. Anal. Chem. 2021, 93, 13373–13381.
35.
Ge, H.R.; Wang, X.F.; Xu, J.F.; et al. A CRISPR/Cas12a-mediated dual-mode electrochemical biosensor for polymerase chain reaction-free detection of genetically modified soybean. Anal. Chem. 2021, 93, 14885–14891.
36.
Ling, L.M.; Yi, L.; Ling, Z.M.; et al. Y-shaped DNA nanostructures assembled-spherical nucleic acids as target converters to activate CRISPR-Cas12a enabling sensitive ECL biosensing. Biosens. Bioelectron. 2022, 214, 114512.
37.
Zhang, H.X.; Zhuang, T.T.; Wang, L.; et al. Efficient Au nanocluster@Ti3C2 heterostructure luminophore combined with Cas12a for electrochemiluminescence detection of miRNA. Sens. Actuators B Chem. 2022, 370, 132428.
38.
Hang, X.M.; Zhao, K.R.; Wang, H.Y.; et al. Exonuclease III-assisted CRISPR/Cas12a electrochemiluminescence biosensor for sub-femtomolar mercury ions determination. Sens. Actuators B Chem. 2022, 368, 132208.
39.
Zhang, Y.; Hu, C.; Yin, Y.; et al. CRISPR/Cas12a-responsive smart DNA hydrogel for sensitive electrochemiluminescence detection of the Huanglongbing outer membrane protein gene. Anal. Chem. 2024, 96, 11611–11618.
40.
Wang, H.; Hang, X.; Wang, H.; et al. Label/immobilization-free Cas12a-based electrochemiluminescence biosensor for sensitive DNA detection. Talanta 2024, 275, 126114.
41.
Zhang, Y.; Yang, W.-G.; Su, M.-L.; et al. A reagent-based label free electrochemiluminescence biosensor for ultrasensitive quantification of low-abundant chloramphenicol. Microchem. J. 2024, 198, 110124.
42.
Fauci, A.S.; Morens, D.M. The perpetual challenge of infectious diseases. N. Engl. J. Med. 2012, 366, 454–461.
43.
Sung, H.; Ferlay, J.; Siegel, R.L.; et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Ca-Cancer J Clin 2021, 71, 209–249.
44.
Kevadiya, B.D.; Machhi, J.; Herskovitz, J.; et al. Diagnostics for SARS-CoV-2 infections. Nat. Mater. 2021, 20, 593–605.
45.
Hauck, T.S.; Giri, S.; Gao, Y.; et al. Nanotechnology diagnostics for infectious diseases prevalent in developing countries. Adv. Drug Delivery Rev. 2010, 62, 438–448.
46.
Chen, M.; Shou, J.; Wu, P.; et al. Electrochemiluminescence biosensor for human papillomavirus DNA based on nanoparticle charge density regulation through a target activated CRISPR/Cas12a system. ACS Appl. Nano Mater. 2023, 6, 23488–23495.
47.
Luo, P.; Huang, X.; Luo, F.; et al. Low-background signal-on homogeneous electrochemiluminescence biosensor for Hepatitis B virus detection based on the regulation of the length of DNA modified on the nanoparticles by CRISPR/Cas12a and hybridization chain reaction. Anal. Chem. 2023, 95, 14127–14134.
48.
Luo, S.; Wu, J.; Zhong, M.; et al. An electrochemiluminescent imaging strategy based on CRISPR/Cas12a for ultrasensitive detection of nucleic acid. Anal. Chim. Acta 2024, 1324, 343040.
49.
Liu, P.F.; Zhao, K.R.; Liu, Z.J.; et al. Cas12a-based electrochemiluminescence biosensor for target amplification-free DNA detection. Biosens. Bioelectron. 2021, 176, 112954.
50.
Li, L.; Yu, S.; Wu, J.; et al. Regulation of target-activated CRISPR/Cas12a on surface binding of polymer dots for sensitive electrochemiluminescence DNA analysis. Anal. Chem. 2023, 95, 7396–7402.
51.
Wang, C.; Cui, L.; Wu, J.; et al. Electrochemiluminescence of hot exciton nanomaterial with boosted efficiency for visual bioanalysis. Nano Today 2024, 54, 102131.
52.
Zhao, K.-R.; Wang, L.; Liu, P.-F.; et al. A signal-switchable electrochemiluminescence biosensor based on the integration of spherical nucleic acid and CRISPR/Cas12a for multiplex detection of HIV/HPV DNAs. Sens. Actuators B Chem. 2021, 346, 130485.
53.
Wei, J.; Song, Z.; Cui, J.; et al. Entropy-driven assisted T7 RNA polymerase amplification-activated CRISPR/Cas13a activity for SARS-CoV-2 detection in human pharyngeal swabs and environment by an electrochemiluminescence biosensor. J. Hazard. Mater. 2023, 452, 131268.
54.
Yang, F.; Wang, W.; Zhang, M.; et al. CRISPR/Cas12a-mediated electrochemiluminescence platform for environmental and human serum SARS-CoV-2 RNA monitoring using a self-enhanced ruthenium complex linked to zeolitic imidazole framework-8. Environ. Sci. Nano 2022, 9, 3417–3426.
55.
Zhang, K.; Fan, Z.; Huang, Y.; et al. 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. Talanta 2022, 236, 122868.
56.
Zhang, K.; Fan, Z.; Ding, Y.; et al. A pH-engineering regenerative DNA tetrahedron ECL biosensor for the assay of SARS-CoV-2 RdRp gene based on CRISPR/Cas12a trans-activity. Chem. Eng. J. 2022, 429, 132472.
57.
Zhang, K.; Fan, Z.; Ding, Y.; et al. Exploring the entropy-driven amplification reaction and trans-cleavage activity of CRISPR-Cas12a for the development of an electrochemiluminescence biosensor for the detection of the SARS-CoV-2 RdRp gene in real samples and environmental surveillance. Environ. Sci. Nano 2022, 9, 162–172.
58.
Havelaar, A.H.; Kirk, M.D.; Torgerson, P.R.; et al. World health organization global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Med. 2015, 12, e1001923.
59.
Shen, J.; Zhang, D.; He, Y.; et al. Evolving CRISPR/Cas system for food safety monitoring across the food supply chain. Trends Anal. Chem. 2024, 181, 118050.
60.
Jangid, H.; Panchpuri, M.; Dutta, J.; et al. Nanoparticle-based detection of foodborne pathogens: Addressing matrix challenges, advances, and future perspectives in food safety. Food Chem X 2025, 29, 102696.
61.
Scallan, E.; Hoekstra, R.M.; Angulo, F.J.; et al. Foodborne illness acquired in the United States—Major pathogens. Emerg Infect Dis 2011, 17, 7.
62.
Zhang, X.; Wang, X.; Zhu, L.; et al. Target responsive-regulated CRISPR/Cas12a electrochemiluminescence sensing of salmonella typhimurium integrating ultrafine Pt NCs-anchored MXenes-boosted luminol/O2 system. Biosens. Bioelectron. 2025, 283, 117558.
63.
Wang, C.; Zhang, Y.; Liu, S.; et al. Allosteric probe-triggered isothermal amplification to activate CRISPR/Cas12a for sensitive electrochemiluminescence detection of Salmonella. Food Chem. 2023, 425, 136382.
64.
Liu, Y.; Wang, F.; Ge, S.; et al. Programmable T-Junction structure-assisted CRISPR/Cas12a electrochemiluminescence biosensor for detection of Sa-16s rDNA. ACS Appl. Mater. Interfaces 2022, 15, 617–625.
65.
Wu, L.; Zhou, T.; Huang, R. A universal CRISPR/Cas9-based electrochemiluminescence probe for sensitive and single-base-specific DNA detection. Sens. Actuators B Chem. 2022, 357, 131411.
66.
Mao, X.; Lu, Y.; Gao, Z.; et al. Modular microfluidic sensor integrating nucleic acid extraction, CRISPR/Cas13a, and electrochemiluminescence for multichannel RNA detection. Anal. Chem. 2025, 97, 5085–5092.
67.
Zheng, Z.; Qian, Z.; Huang, D.; et al. Ultrasensitive homogeneous electrochemiluminescence biosensor for N-Nitrosodimethylamine detection based on vertically-ordered mesoporous silica film-modified electrode and CRISPR/Cas12a-driven HRCA with triple signal amplification. Anal. Chem. 2025, 97, 5828–5835.
68.
Li, W.; Li, Y.; Zhao, L.-D.; et al. Triple-helix as a target converter for trace pesticide detection based on CRISPR/Cas12a-based ECL biosensor. Sens. Actuators B Chem. 2024, 409, 135599.
69.
Li, Y.; Yang, F.; Yuan, R.; et al. Electrochemiluminescence covalent organic framework coupling with CRISPR/Cas12a-mediated biosensor for pesticide residue detection. Food Chem. 2022, 389, 133049.
70.
Peng, X.; He, Y.; Zhao, J.; et al. CRISPR/Cas12a-mediated aptasensor based on tris-(8-hydroxyquinoline) aluminum microcrystals with crystallization-induced enhanced electrochemiluminescence for acetamiprid analysis. Anal. Chem. 2023, 95, 10068–10076.
71.
Cai, Q.; Wang, Y.; Jie, G.; et al. Nanoconfinement effect and nanozyme catalysis enhance ILu/HOF-14 electrochemiluminescence for biosensing. Anal. Chem. 2025, 97, 8592–8599.
72.
Shi, B.; Jia, Y.; Jia, D.; et al. Aggregation-Induced electrochemiluminescence of Ir(ppy)3-Functionalized ZIF-8 for microcystin-LR detection via the trans-cleavage activity of CRISPR-Cas12a. Anal. Chem. 2024, 96, 15050–15058.
73.
Hill, R.; Stentiford, G.D.; Walker, D.I.; et al. Realising a global one health disease surveillance approach: Insights from wastewater and beyond. Nat. Commun. 2024, 15, 5324.
74.
McSweeney, M.A.; Patterson, A.T.; Loeffler, K.; et al. A modular cell-free protein biosensor platform using split T7 RNA polymerase. Sci. Adv. 2025, 11, eado6280.
75.
Silva, S.R.; Katz, I.S.S.; Mori, E.; et al. Biotechnology advances: A perspective on the diagnosis and research of rabies virus. Biologicals 2013, 41, 217–223.
76.
Hu, C.; Xiang, H.; Yin, Y.; et al. Electrochemiluminescence resonance energy transfer biosensor for the human-associated clade of streptococcus suis based on prereduction-enhanced Yttrium MOFs. Anal. Chem. 2025, 97, 3153–3160.
77.
Liu, S.; Wang, C.; Wang, Z.; et al. Binding induced isothermal amplification reaction to activate CRISPR/Cas12a for amplified electrochemiluminescence detection of rabies viral RNA via DNA nanotweezer structure switching. Biosens. Bioelectron. 2022, 204, 114078.
78.
Wang, Y.; Shen, B.; Luo, N.; et al. Self-enhanced nanohydrogel electrochemiluminescence biosensor based on CRISPR/Cas12a and gold platinum nanoparticles modification for high-sensitivity detection of Burkholderia pseudomallei. Chem. Eng. J. 2024, 486, 150279.
79.
Lee, J.A.; Halbert, S.E.; Dawson, W.O.; et al. Asymptomatic spread of huanglongbing and implications for disease control. Proc. Natl. Acad. Sci. USA 2015, 112, 7605–7610.
80.
Hu, B.; Rao, M.J.; Deng, X.X.; et al. Molecular signatures between citrus and Candidatus Liberibacter asiaticus. PLoS Pathog. 2021, 17, e1010071.
81.
Gottwald, T.R. Current epidemiological understanding of citrus huanglongbing. Annu. Rev. Phytopathol. 2010, 48, 119–139.
82.
He, Y.; Zhang, Y.; Xiang, H.; et al. Magnetic bead-assisted one-pot RCA-activated CRISPR/Cas12a electrochemiluminescence biosensor for the detection of citrus Huanglongbing pathogen. Biosens. Bioelectron. 2025, 290, 117986.
83.
Yin, F.; Sun, Q.; Huang, X.Z.; et al. Recent progress in signal enhancement of nanomaterials-based electrochemiluminescence systems. Trends Anal. Chem. 2023, 169, 117376.

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