| Malignant tumors are serious public health concerns.In 2020,there were19.29 million new cancer cases worldwide,among which 4.57 million new cancers occurred in China,accounting for 23.7%of the world,and the number of new cases and deaths ranked first in the world.Lung cancer has emerged as one of the malignant tumors with the fastest growing morbidity and mortality.Early diagnosis and treatment are crucial for the prevention and treatment of lung cancer.It has been found that miRNA,as a non-coding smallRNA,is involved in many important life processes,controls many biological functions,such as cell differentiation and apoptosis,and has an important role in the development of many diseases,abnormally expressed in many tumor patients,which can be used as a biomarker for early tumor diagnosis.However,the detection of free miRNAs in circulating body fluids is difficult due to the complex in vivo environment,which is susceptible toRNase degradation,extremely unstable in body fluids and low in abundance.Exosomes,as nano-scale membranous microvesicles secreted by cells,contain a variety of biomarkers,including nucleic acid molecules,proteins,lipids,etc.,and are secreted in higher amounts in patients than in normal subjects.miRNA,as the content of exosomes,is protected fromRNAase degradation by the membranous lipid bilayer of exosomes.The abundance of miRNAs in exosomes is higher than that of free miRNAs in circulating body fluids,and the content of miRNAs in exosomes is also more stable.In recent years,the potential value of exosomal miRNAs as tumor markers is attracting widespread attention,and the study of exosomal miRNA detection methods based on biosensing technology is important for the clinical application of exosomal miRNAs,of which MicroRNA-21（miR-21）is associated with the proliferation and metastasis of lung cancer.Therefore,accurate and sensitive detection of miR-21 is of great value in the early diagnosis of lung cancer and monitoring the effect of lung cancer treatment.ClusteredRegularly Interspaced Short PalindromicRepeats-CRISPR associated（CRISPR-Cas）,a gene editing technology,has greatly contributed to the advance of life sciences since its introduction.The CRISPR-Cas system is widely found in bacteria and constitutes their acquired immune system to defend against foreign invading nucleic acid molecules.The CRISPR-Cas system itself helps bacteria and archaea destroy invading phage genetic material throughRNA-mediated interference strategies,and has been successfully implemented in vitro.It is widely used for nucleic and non-nucleic acid diagnostics.The system mainly consists of two parts,CRISPRRNA（crRNA）and Cas protein.crRNA can complement the target sequence and thus guide Cas protein for sequence-specific recognition and cleavage.CRISPR/Cas12a has highly specific target-activated cleavage activity,and the activated Cas protein can non-specifically cleave other nucleic acid fragments,a property that could be widely used for the detection of nucleic acid molecules,providing an important way to establish new methods for molecular diagnosis,but the limited sensitivity of this system,if used alone for the detection of nucleic acids,has prevented its further widespread use.Terminal deoxynucleotidyl transferase（TdT）,a non-template-dependent eukaryotic DNA polymerase,was first discovered in mammalian cells in 1960,and its biochemical properties and biological functions contributed to the development of diagnostic assays.The sequence composition of the extended polymer chain is closely related to the type of d NTP used in the reaction,so TdT can be used as a powerful tool to construct signal amplification probes.It acts as a multifunctional tool enzyme for quantifying targets in many studies,and is often applied in signal amplification strategies.If TdT is applied to biomarker detection,label-free signal amplification can be achieved by one-to-many conversion mode.In this study,a fluorescent biosensing system based on signal amplification strategy was constructed,combining TdT extension with CRISPR/Cas12a for the detection of miR-21 in lung cancer exosomes.Next,a signal amplification combined with CRISPR/Cas12a for the detection of miR-21 in exosomes was performed.This method can quantify the target miR-21,and the fluorescence signal amplification can significantly improve the specificity and sensitivity of the assay.The detection results showed good linearity in the concentration range of 0.5 p M～500 p M（R~2=0.9939）,and the detection limit of this method reach 161 f M,which can quantify the miRNA with low abundance.In addition,this method was successfully applied to the analysis of exosomal miR-21 from A549 cells,and detected exosomal miRNAs in the blood of lung cancer patients and healthy control individuals,respectively,showing good potential for clinical application,which is important for the application of exosomal miRNAs in the early clinical diagnosis of malignant tumors. |
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