| Objectives:Prostate cancer(PCa)is the second most common cancer among men worldwide.In recent years,the incidence of prostate cancer in China is also increasing year by year.Prostate cancer is often treated with androgen-blocking therapy in the early stages,but often turns into hormone-independent prostate cancer after 2-5 years of treatment.Hormone-independent prostate cancer is not sensitive to the cytotoxic effects of low-dose chemotherapy or radiotherapy,while high-dose chemoradiotherapy can cause significant side effects.Therefore,it is still of great significance to seek new non-toxic and effective anti-tumor drugs to induce the death of prostate cancer cells.Traditional Chinese medicine is the treasure of Chinese traditional culture with a long history and has made great contributions to the healthy reproduction of the Chinese nation.A large number of clinical studies have confirmed that traditional Chinese medicine not only has the characteristics of lower toxicity,but also plays an important role in inhibiting tumor proliferation and metastasis,preventing complications,improving quality of life and reducing treatment side effects.At present,the research on the anti-tumor effect of traditional Chinese medicine mainly focuses on screening the effective components of traditional Chinese medicine and elaborating its regulation effect on tumor-related genes,proteins and other biomolecules.Prostate-specific antigen(PSA)is an important tumor marker for prostate cancer.It is used not only for early diagnosis and clinical staging of prostate cancer,but also for efficacy observation and follow-up.Various active ingredients of traditional Chinese medicine have been proved to down-regulate the expression of PSA gene and protein in androgen-dependent and androgen-independent pathways by blocking the gene expression of androgen-receptor in prostate cancer cells,thus inhibiting the occurrence and development of prostate cancer.In addition,microRNA(miRNA)is a kind of endogenous non-coding small RNA,whose expression profile varies according to tumor type,stage and efficacy,and is a potential tumor marker for the diagnosis,classification and efficacy observation of prostate cancer.The detection of the above biomarkers can not only meet the needs of early clinical diagnosis and curative effect observation,but also be used as a means of drug action mechanism research to assist the development of new drugs and drug screening.Therefore,it is of great significance to establish the detection technology of tumor markers for screening the effective components of traditional Chinese medicine.In the 1920s,people began to use modern scientific research methods to study the mechanism of action of traditional Chinese medicine.In recent years,a large number of innovative and interdisciplinary technologies have been applied to the experimental research of traditional Chinese medicine,playing a role in expanding the vision of traditional Chinese medicine and promoting the development of traditional Chinese medicine.Biosensor technology is an important new force.As a new technology developed by the intersection of life science and information science,biosensor technology has the advantages of high sensitivity,high precision,high throughput,real-time monitoring,rapid detection,small structure and economic use.Biosensors are composed of biomolecular sensing units and signal transducers.The sensing units can selectively identify the objects to be measured,form complexes,and turn the physical and chemical effects into electrical signals proportional to the concentration of the objects to be measured,so as to detect the amount of objects to be measured.Nanomaterials with the size of between 1-100 nm,have some special physical and chemical properties,such as large specific surface area,high surface energy,superparamagnetism,etc.Due to the above significant advantages,various new nanomaterials have been applied to the sensing units of biosensors to improve the detection performance of biosensors.Among them,fluorescent biosensor has attracted researchers’extensive attention because of its advantages such as simple operation,quick and sensitive,and good biocompatibility.In this report,we fabricate two fluorescent biological sensing platforms including graphene oxide and poly-dopamine magnetic nanoparticles(MNPs@PDA),respectively.The former platform was used to detect prostate cancer tumor markers PSA.The latter one was used to detect miRNAs related to prostate cancer,and was thereafter applied in monitoring the expression of miRNAs regulated by active ingredients of Chinese traditional medicine.Methods and results:The main content of this project includes the following 3 parts:Part Ⅰ:Fabrication of fluorescent nanobiosensor based on HCR signal amplification strategy and its application in detecting PSAThe gelred-embedded nucleic acid aptamer was constructed as the fluorescence donor,and graphene oxide(GO)was the fluorescence energy resonance energy transfer(FRET)biosensor of the fluorescence receptor.The fluorescent biosensor with amplification of hybridization chain reaction(HCR)was applied for the detection of PSA.First,the aptamer probe with stem-ring structure was synthesized.The sequence mainly consists of two parts:one is the recognition sequence that can specifically bind to PSA,and the other is the trigger sequence of hybrid chain reaction(HCR)of hairpin structure.The tested substance was added to the reaction system mixture,and then fluorescence intensity was performed.If PSA was added,the aptamer probe recognition sequence was combined with it.Then the hairpin structure was opened and the trigger sequence was exposed,triggering the hybridization chain reaction and forming the long chain product of double helix.GelRed,as a fluorescent nucleic acid dye,binds to long-chain products to produce a strong fluorescent signal.Graphene oxide(GO)has a weak affinity for the long chain products of the double helix,resulting in a strong fluorescence signal.However,without PSA added,HCR could not be triggered,and the hairpin chain was adsorbed by GO,resulting in fluorescence quenching and weak fluorescence signal due to fluorescence energy resonance energy transfer.The system with target PSA had strong fluorescence signal.In the case of the system without the target PSA,the fluorescence signal is weak.Then,conditions were optimized from the four aspects of pH,GO concentration,HCR reaction time and temperature.The optimal conditions were at pH 7.4,GO concentration 15 μg/mL,HCR reaction time 60 minutes and HCR reaction temperature 37℃.Under the optimized conditions,the fluorescence intensity of PSA at different concentrations was determined,and it was found that within the range of 100 pg/mL～200 ng/mL,the logarithm of PSA concentration showed a linear relationship with the fluorescence signal of the system,and the detection limit was as low as 10 pg/mL.In addition,we investigated the selectivity of the biosensor.After adding CD63,BSA,CEA,AFP,HCG,D-D dimer and random ssDNA into the reaction system,the fluorescence signal intensity produced by the biosensor was very weak compared with that of PSA,indicating that the biosensor had a high selectivity to PSA.Finally,PSA was added to 10%of all kinds of body fluid(human serum,saliva and urine),and the detected fluorescence signal was significantly different from the blank control,indicating the applicability of this platform to biological samples.Part Ⅱ:A fluorescent nanobiosensor based on double-chain specific nuclease amplification strategy and polydopamine magnetic nanoparticles for detection of miRNAFluorescence energy resonance energy transfer(FRET)biosensor based on the dual-chain specific nuclease(DSN)amplification and magnetic absorption enhanced polydopamine magnetic nanoparticles(MNPs@PDA)was constructed for the detection of miR-141.First,dopamine-coated magnetic nanoparticles were prepared by adding dopamine hydrochloride and magnetic nanoparticles into alkaline buffer.The synthetic MNPs@PDA was characterized by Raman spectrometer,infrared spectrometer and transmission electron microscope.The well-characterized MNPs@PDA after ultrasound was dispersed in water for backup.In the absence of target miRNA,fluorescein labeled DNA probes were adsorbed on the surface of MNPs@PDA due to π-π accumulation,leading to fluorescence quenching.In the presence of the target miRNA,the probe and miRNA were complementarily hybridized to form a DNA-RNA hybrid double helix with desorption from the MNPs@PDA surface.Because DSN can selectively digest DNA in DNA-RNA hybridization chains,labeled probes are enzymatically cleaved into small pieces while the target miRNA is released.The probe fragment has a weak affinity with MNPs@PDA due to its short length,so the fluorescence signal is restored.The released target miRNA is again hybridized with the next probe,and the above process circulates,resulting in geometric amplification of the fluorescence signal.In addition,due to the presence of internal filter effect(IFE),the quenching agent dispersed in the solution will reduce the fluorescence intensity.Therefore,MNPs@PDA dispersion in solution attenuates the fluorescence signal.We magnetically attracted MNPs@PDA to the bottom of the color plate with the fluorescence enhanced.Within a certain period of time,the more target miRNAs,the more probe fragments generated by enzyme digestion,and the stronger fluorescence signal,confirming that the experiment can quantitatively determine low abundance of target miRNAs.Then,conditions were optimized from four aspects of MNPs@PDA concentration,DSN enzyme digestion temperature,DSN enzyme concentration and enzyme reaction time,and the optimal conditions were set at MNPs@PDA of 10 μg/mL,temperature 50℃,DSN concentration 0.2 U/mL and reaction time 90 min.Under optimized conditions,the fluorescence intensity of miR-141 at different concentrations was determined.It was found that within the range of 5 pmol/L～5 nmol/L,the logarithm of miR-141 concentration showed a linear relationship with the fluorescence signal of the system,and the detection limit was as low as 0.42 pmol/L,which was an order of magnitude lower than the detection limit of 3.8 pmol/L without magnetic absorption.In addition,we investigated the selectivity of the biosensor.The non-complementary nucleic acid sequences miR-21,RS ssDNA,SM RNA and miR-141 were added to the reaction system for detection,and the target miR-141 was successfully identified,indicating the good selectivity of the biosensor.Finally,we directly detected the total RNA extracted from prostate cancer PC-3 cell line and normal RWPE-1 cell line by this sensor,and found that the expression of miR-141 from the cancer cell line was up-regulated,indicating the applicability of this platform to biological samples.Part III:Fluorescent nanosensor for monitoring regulation of active ingredients of traditional Chinese medicine on miRNA related to prostate cancerBased on the work in the second chapter,we monitor miR-141 expression in prostate cancer PC-3 cell lines before and after TCM applied by fluorescence biosensor based DSN signal amplification.We selected four kinds of single TCM,including curcumin,Ginsenoside Rh2,quercetin and honokiol.First,CCK-8 method and flow cytometry were used to determine the PC-3 cell activity after the treatment of four traditional Chinese medicine of different concentrations.The results showed that curcumin,ginsenoside Rh2,quercetin and honokiol inhibited the proliferation of prostate cancer cells in a dose-time dependent manner and induced apoptosis in a dose-dependent manner.Western blotting was used to detect the expression of apoptosis-related proteins in PC-3 cells after the treatment of traditional Chinese medicine,and the expression of pro-apoptotic proteins increased while the expression of anti-apoptotic proteins decreased.The results showed that the four active ingredients of traditional Chinese medicine had anti-prostate cancer effect,which could inhibit the proliferation of cancer cells and induce the apoptosis of cancer cells.Then,the PC-3 cells were cultured with four active ingredients of TCM,respectively.The total RNA of the cells in the treatment group and the blank control group were extracted with the kit,and the extracts of the cells in each group were detected with the aboved fluorescence sensor.Compared with the control group,the fluorescence signals of ginsenoside Rh2 and quercetin groups showed no significant difference,while the fluorescence signals of curcumin and honokiol group were significantly enhanced and increased with the increase of concentration.The results indicate that curcumin and honokiol promoted the expression of miR-141 in a concentration-dependent manner.At the same time,these results were further confirmed by RT-qPCR.The above results showed that the four active ingredients of traditional Chinese medicine had significant inhibition on cancer cells,and the survival rate of cell lines decreased and the apoptosis rate increased.And curcumin and honokiol up-regulate the expression of miR-141 in prostate cancer cells.Conclusion:1.GO fluorescent biosensor based HCR was constructed for PSA detection with high sensitivity and specificity.2.Fluorescent biosensor based on the amplification of DSN and polydopamine magnetic nanoparticles was constructed to detect miRNA with high sensitivity and specificity.3.Fluorescence platform was used to evaluate the expression of miR-141 in prostate cancer cells before and after drug treatement,providing a research basis and technical means for the discussion of drug target mechanism and drug screening. |
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