Novel Fluorescence Nano Sensors For Sensitive And Visualized Detection Of Bladder Cancer

Background:Bladder cancer?BCa?,a very common aggressive malignancy,has become the9th most common cancer worldwide.It is well known that the earlier diagnosed and treated to BCa patients,the better outcomes they would get.Therefore,the early detection of BCa is particularly important.Currently,cystoscopy is still the gold standard method for patients with urinary symptoms.However,it will bring some discomfort to patients,due to cystoscopy is an invasive examination method.Furthermore,the cost is expensive,which limits the prevalence of cystoscopy and its application to the screening of healthy people.Compared with the traditional cystoscopy,the design and application of non-invasive diagnosis and monitoring methods in BCa patients have a wide range of social needs.As a highly specific tumor biomarker,telomerase has critical potential application value in early diagnosis and detection of cancer.Objective:?1?Based on the characteristics of telomerase,a ratio fluorescence nano-probe was designed and prepared into a test paper for the detection of telomerase activity in the urine of bladder cancer patients,which was applied in the early diagnosis of bladder cancer.?2?Based on the characteristics of telomerase,a novel nano-beacon?QD-BHQ2?was developed to visually monitor telomerase activity in vitro or in living cells.Methods:?1?The extraction of telomerase:telomerase was extracted from hela cells or fresh urine samples of bladder cancer patients,respectively.?2?Telomerase extension reaction:the extracted telomerase reacted with telomerase primers?TS?,dNTPs and telomerase reverse transcription buffer to form telomerase extension reaction products.?3?The Rox-DNA funtionalized quantum dots?CdZnTeS QDs?,a ratiometric fluorescent nano-probe,was carried out by one-pot hydrothermal method.?4?An HRP-mimicking hemin/G-quadruplex,which was formed with the help of telomerase activity,K⁺,and hemin,catalyzed H2O2 into H2O and O2.?5?The nano-beacon?QD-BHQ2?,which was composed of oligonucleotide stem ring,quantum dot fluorescence group and quenching group,was synthesized by using a simple method in aqueous solution,and the CdTe:Zn²⁺QDs were functionalized by a phosphorothioate modified hairpin BHQ2-DNA.?6?The G-quadruplex solution was added to the citrate buffer containing a certain dose of H2O2,then the appropriate amount of Rox-DNA modified CdZnTeS quantum dot solution was added,and the fluorescence spectrum of the fluorescent probe was recorded under the excitation wavelength of 340 nm.The activity of telomerase was detected indirectly according to the change of fluorescence intensity.?7?The preparation of bladder cancer early diagnosis test paper:filter paper was soaked in Rox-DNA modified CdZnTeS quantum dot solution and tris buffer solution,and then dried at room temperature.Subsequently,the color of test paper corresponding to different concentrations of telomerase solution was made into the reference standard of bladder cancer early diagnosis test paper to complete the preparation of diagnostic test paper.?8?The quantum dot nano-probe was added to the telomerase extension reaction product,and the fluorescence spectra were collected and observed under the excitation wavelength of 330nm.The detection of telomerase activity could be achieved indirectly according to the fluorescence intensity.?9?The living cells imaging technology in situ:TS was transfection into hela cells by lipofectamine 3000,and nano-beacon?QD-BHQ2?was incubated with hela cells.The confocal microscopy imaging system was used to observe fluorescence,under the excitation wavelength of 561 nm,according to the situation of fluorescence can be indirect detection of telomerase activity in living cells.Results:Combining the properties of telomerase,we developed a novel quantum dot ratiometric fluorescence nano-probe and a new quantum dot nano-beacon.Based on these two probes,we constructed two novel systems for high sensitivity and visual detection of telomerase.At the same time,based on the quantum dot ratiometric fluorescence nano-probe,a simple,universal,inexpensive and visual monitoring test paper for the early diagnosis of bladder cancer was developed.In addition,quantum dot nano-beacon?QD-BHQ2?can be applied to the situ imaging detection of telomerase activity in living cells,which is of great significance for the diagnosis and treatment of cancer.Conclusion:In this study,two novel nano-probes were developed,which led to the development of bladder cancer early diagnosis test paper for urine telomerase detection and living cell imaging technology for monitoring intracellular telomerase activity.The detection of telomerase activity has a broad application prospect and is expected to be formally applied to various early clinical diagnosis methods of cancer in the future.