Model Study Of Label-free Fluorescence Detection Of CA15-3 Based On G-tetramer/NMM Complex

Breast cancer is one of the main killers threatening the health of women,and there is currently no effective treatment.Therefore,it is extremely important to effectively improve the detection sensitivity of markers in the early diagnosis of breast cancer and preoperative examination.Compared with conventional detection methods,fluorescent aptamer biosensors have been widely studied due to their better sensitivity,specificity,reproducibility and simple operation.Among them,label-free fluorescence technology has been gradually developed because it can avoid the adverse effects of fluorescent labeling and is low-cost.In this study,the fluorescence produced by the specific binding of G-quadruplex and N-Methyl Mesoporphyrin IX(NMM)was used as the signal probe,and label-free fluorescent aptamer sensors ware designed to achieve the specific,sensitive and rapid detection of breast cancer antigen CA15-3 is as follows:(1)Firstly,a label-free fluorescence detection logic gate was constructed based on graphene oxide and G-quadruplex/NMM complex.And the structure of the designed hairpin DNA was predicted by NUPACK software,and its thermodynamics were evaluated stability.QGRS software was used to evaluate the ability of the circular guanine-rich sequence of hairpin DNA to form G-quadruplexes.Subsequently,fluorescence experiments were used to analyze the feasibility and optimize the experimental conditions.Under optimized conditions,the designed label-free fluorescent biosensor can detect CA15-3 in a linear range of 10-500 U·mL-1,and the detection limit is 10 U mL-1.Compared with the system containing the target CA15-3,adding the same amount of different types of protein only got low fluorescence intensity,indicating that the designed model has great specificity.Finally,the recovery rate test in the biological sample(diluted healthy human serum)confirmed the accuracy and repeatability of the results of this design scheme.(2)Designed a label-free fluorescent aptamer biosensor based on triple-helix DNA and G-quadruplex/NMM complex to detect CA15-3.The triple-helix DNA structure formed by DNA base complementary pairing and Hoogsteen hydrogen bonds can "lock"guanine-rich sequences.In the presence of the target CA15-3,it can partially bind to the aptamer to open the triple helix structure,releasing the guanine-rich sequence.In the presence of potassium ions,the guanine-rich sequence forms a G-quadruplex,and the NMM in the chimeric solution produces fluorescence.In the same way,fluorescence experiments are used to verify the feasibility and optimize a series of conditions.The measured linear range is 0.01-5 U·mL-1,and the detection limit is 0.01 U·mL-1.The accuracy and reproducibility of the design scheme was confirmed by the detection of diluted human serum samples and the calculation of the recovery rate.Replace CA15-3 with different proteins to obtain a smaller fluorescence difference with the blank control to verify the specificity of the designed label-free fluorescence detection proposal.Finally,compared with the detection limit and linear range of other CA15-3 methods,the designed proposals have better linear detection range and detection limit.By designing label-free fluorescent aptamer sensing models,simple,rapid,sensitive and specific detection of breast cancer antigen CA15-3 is realized,and it can be accurately tested in actual biological samples(diluted healthy human serum).The designed label-free fluorescence sensing models have the advantage of low-cost and time-saving compared with the existing methods,and at the same time provides a certain reference for functional nucleic acid in label-free fluorescence biosensing and disease detection.This study has certain significance for the exploration of new methods for simple and low-cost detection of specific markers in the early diagnosis,postoperative status,recurrence rate,and metastasis monitoring of breast cancer patients.