Optical Detection Of Biomolecules In Tumor Cells Based On Nucleic Acid Probes

With the rapid development of the economy and the continuous progress of society,the level of healthcare has gradually improved.However,the fact of the continuously rising of the cancer incidence indicates that cancer is still one of the major threats to human health.It’s of great significance for the early prediction and later treatment of diseases by carrying out sensory detection of tumor-related biomolecules.Early diagnosis and treatment of sensory diseases can reduce the cost of treatment,release the pain of patients and greatly improve the cure rate.Biosensing technology is an emerging technology involving multiple disciplines,which has many advantages like convenience,high efficiency and specificity,real-time continuous detection,and wide application.Nucleic acid molecules,as indispensable macromolecular substances in the organism,are mainly responsible for the transmission of genetic information in the organism.Meanwhile,the nucleic acid molecules can participate in many important life activities such as protein synthesis and maintainance of the body’s normal immune function.Therefore,nucleic acid molecules can be used as an ideal biosensor recognition probe for the detection and analysis of biomolecules in tumor cells.Human telomerase is a special ribonucleoprotein reverse transcriptase that can maintain the continuous reproduction of cells by adding telomere repeat sequences at the end of telomeres.In normal human cells,the activity of telomerase is inhibited.While in most malignant tumor cells,telomerase is abnormally activated,resulting in the extension of the telomere repeat sequence,so that the tumor cells continue to survive.It has been reported that 85 ～ 90% of tumor tissues express high-level telomerase activity,indicating that telomerase has the potential to be a new biomarker for tumor diagnosis and a new target for tumor treatment.Therefore,the sensitive detection of telomerase activity in tumor cells is of great significance for the early diagnosis and prognostic treatment of cancer.In this paper,we designed a cascade amplification method based on nucleic acid probes with the application of molecular beacons MB1 and MB2 as signal reporter molecules,realizing the highly sensitive detection of telomerase in tumor cell extracts and fluorescence imaging of telomerase activity in living cells.In the presence of telomerase,the(TTAGGG)n telomere repeating sequence will add to the end of the TS primer and forms a telomere extension product(TEP).The TEP competes with the C-DNA/G-DNA duplex and replaces the G-DNA out of the duplex.The replaced G-DNA opens the stem-loop structure of nucleic acid probe MB1 by hybridization and recovers the fluorescence signal of MB1.Furthermore,the MB1 probe will hybridize with MB2 probe,which open the stem-loop structure of MB2 probe and ultimately enhance the fluorescence signal of the system.Additionally,the released G-DNA trigger the next strand displacement reaction.This strategy provides an enzyme-free and PCR-free amplificaction strategy,which can be applied to qualitatively and quantitatively detection of telomerase activity in tumor cells.This cascade amplification strategy based on biomolecular beacons effectively improves the sensitivity of the detection,and the limit of detection(LOD)is estimate to 20 He La cells.The in situ fluorescence imaging technology verified the practicability of this strategy for distinguishing tumor cells from normal cells.Besides,the reaserch of telomerase activity inhibitor detection indicates that this strategy has potential applications in the discovery and screening of anticancer drugs.Adenosine triphosphate(ATP)is a high-energy phosphate compound and commonly exist in various living cells,which plays an important role in regulating various metabolic processes and biochemical pathways in life.In this assay,we designed a nucleic acid probe based on ATP aptamer and its complementary strand.The water-soluble conjugated polymer brush(PPE-PLL)was used to amplify the fluorescence signal.By measuring the fluorescence resonance energy transfer between PPE-PLL and the fluorophores labeled at the end of the complementary strand,we realized the detection of ATP with high sensitivity and specificity.This sensing strategy can also be applied to detect ATP in complex systems.This optical detection method based on nucleic acid aptamer and water-soluble conjugated polymer has many advantages,such as simple operation procedures,fast,high efficiency,low cost and so on.It shows brilliant application prospects in ATP-related biological analysis.