| Breast cancer has become the most common cancer among women in the world,with more than 2 million new cases and more than 600,000 deaths in 2021.Early diagnosis and treatment are the best ways to improve the survival rate of breast cancer patients.Liquid biopsy has the advantages of less trauma,real-time and convenience,showing its great potential in the early diagnosis of breast cancer.The actual effect of liquid biopsy is closely related to two factors: one is the detection bioassay used;the other is the choice of biomarkers.Electrochemical is a fast,highly sensitive,low-cost,and diverse detection mode analysis method,which has great advantages as a detection tool for liquid biopsy.The biomarkers of tumor liquid biopsy mainly include: circulating tumor cells,exosomes,nucleic acids,proteins,etc.Exosomes has become a popular biomarker for tumor liquid biopsy because of their relatively high content and stability,and the biological information it contains is closely related to the cells in the primary tumor.The miRNA-1246 in exosomes is protected via exosome membrane vesicles from being hydrolyzed by RNase in body fluids,so its content is stable.The miRNA-1246 in exosomes is closely related to the occurrence and development of breast cancer.In view of this,this paper designed a biosensor based on CRISPR/Cas12 a combined with magnetic nanoparticles for the detection of breast cancer derived exosomes and their miRNA-1246 on the electrochemical platform,and optimized the influencing factors to make the sensor have superior performance,so as to realize the quantitative detection of breast cancer exosomes and their miRNA-1246,and provide a fast,sensitive and exclusive detection method for the early diagnosis of breast cancer.1.CRISPR/Cas12 a combined with magnetic nanoparticles for electrochemical platform detection of breast cancer derived exosomes（1）An electrochemical biosensor based on CRISPR /Cas12 amagnetic nanoparticles aptamer was prepared and used to detect exosomes from breast cancer;The main influencing factors such as aptamer probe concentration,magnetic particle concentration,electrode modified probe concentration,Cas12 a concentration and Cas12 a enzyme digestion time were optimized to make the sensor have superior performance.（2）Exosomes from breast cancer cells（MCF-7 cells）were extracted by ultra separation technique and characterized by WB and TEM techniques.The results showed that exosomes were successfully isolated.（3）The sensor was used to detect the changes of electrochemical signals of target exosomes with different concentrations（Δ i）And the logarithm of exosome concentration showed a good linear correlation,and the linear range was 3 × 103～5 × 105 particles / ml,and the detection limit is as low as 280 particles / ml.And this method successfully distinguishes clinical breast cancer patients from normal patients.（4）The detection of exosomes in plasma samples from patients with breast cancer and healthy people shows that the biosensor has high sensitivity and superior specificity,which verifies the potential application value of the biosensor.2.Synergistic application of CRISPR /Cas12a-DSN-MNPs for electrochemical detection of miRNA-1246 in exosomes from breast cancer（1）CRISPR / Cas12a-DSN-MNPs synergistic electrochemical sensor was developed.The main influencing factors of detection,such as DSN enzyme digestion time,DSN enzyme concentration,magnetic particle concentration,biotin modified DNA probe concentration,Cas12 a concentration,Cas12 a enzyme digestion time and gold electrode modified probe concentration,were optimized to determine the best detection parameters.（2）The target mi R-1246 in the extracted total RNA of exosomes was detected,and the electrochemical signal changed（Δ i）There was a good linear relationship with the logarithm of mi R-1246 concentration,the linear range was 1f M-100 p M,and the detection limit was as low as 88 am.Complete pairing（miRNA-1246）,single base mismatch（SM）,double base mismatch（DM）,triple base mismatch（TM）and random sequence（miRNA21）were detected.The results showed that the electrochemical signal change of miRNA-1246 was significantly different from that of other substances,and the sensor had excellent specificity.In a word,we used CRISPR/Cas12 a and MNPs in the electrochemical detection platform.By designing two different strategies,we successfully detected exosomes from breast cancer and their miRNA-1246 under optimized test conditions.The two established methods were used to detect two different tumor markers,which can effectively compensate for the inaccuracy of tumor diagnosis results caused by only detecting one index. |
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