Tumor-Related DNA Detection And Gene Regulation Using Function Nucleic Acid Composites

DNA is a crucial molecule that carries genetic information and underpins species diversity.DNA has diverse applications in fields such as food testing,clinical diagnosis,and environmental monitoring.In the case of tumor growth and apoptosis,circulating tumor DNA(ct DNA)fragments are released into bodily fluids,including blood,lymphatic fluid,and tissue fluid.These fragments originate from various tumor cells,reflecting the heterogeneity of tumors.By analyzing ct DNA,diagnostic data can be obtained for tumor occurrence,development,and prognosis,guiding tumor treatment,and improving the quality of life and five-year survival rate of tumor patients.However,detecting target DNA from complex backgrounds is challenging due to the low DNA concentrations and the presence of bioactive substances with intricate molecular structures in bodily fluids.Therefore,analytical chemistry assays must continually improve their sensitivity and specificity to meet the growing demand for DNA detection,improving the accuracy of tumor DNA detection.Biosensors are important in the early diagnosis of tumors,as they can convert physical or chemical signals into optical or electrical signals,allowing for the detection of biomolecules.Optical detection techniques possess advantages such as simplicity,convenience,and sensitivity,making them widely applicable in biomolecule detection.Various optical detection methods,including fluorescence,surface-enhanced Raman spectroscopy,chemiluminescence,colorimetry,surface plasmon resonance,and electrochemiluminescence,have been designed for highsensitivity and wide detection range.To improve the detection performance of lowabundance analytes,signal amplification strategies have been explored,such as nucleic acid isothermal amplification.This technique amplifies detection signals under constant temperature,including strand displacement reaction,hybrid chain reaction,rolling circle amplification,loop-mediated isothermal amplification,and circular amplification of target DNA.The combination of nucleic acid isothermal amplification technology and optical detection technology has been widely applied in the field of biosensing.This thesis combines nucleic acid isothermal detection with fluorescent biosensors to detect ct DNA and explores the application of gene editing spherical nucleic acids for gene regulation.The specific studies in this thesis are outlined below.1)In the first study,we developed a highly sensitive detection method for ct DNA,which is commonly present in the blood of cancer patients.To achieve this,we developed a two-dimensional nanomaterials biosensor by combining DNA double-strand hybridization and surface adsorption/fluorescence quenching effects.Specifically,nitrobenzene-modified black phosphorus(NP-BPs)biosensors were synthesized through covalent reactions between BPs and NP.We found that BPs and NP synergistically adsorb single-stranded DNA probes,thereby improving the fluorescence quenching efficiency and sensitivity.Notably,we discovered that when the target ct DNA forms a double-stranded structure with a single-stranded DNA probe,NP-BPs lose the ability to quench the fluorescence of the DNA probe,resulting in high sensitivity of ct DNA detection at room temperature.Our findings provide a novel approach for the development of biosensors with high sensitivity and specificity for the detection of ct DNA,which could have important implications for the early diagnosis and treatment of cancer.2)In the second study,we aimed to develop a convenient and portable method for detecting ct DNA by combining isothermal detection with a paper-based biochip.A DNA strand displacement probe was designed to recognize the target ct DNA,and the sensitivity of the detection method was amplified through multiple rounds of amplification reaction using exonuclease III.The isothermal detection technology was integrated with paper-based biosensors,which are cost-effective,lightweight,easily portable,and suitable for single-use analysis.The paper-based biosensor offers a novel method for portable ct DNA detection and holds promise for early tumor diagnosis through ct DNA detection.3)In the third study,we propose a novel approach to gene editing using protein spherical nucleic acids(Pro SNAs)nanostructures for precise regulation of tumor cell genomes.We synthesized clustered regularly interspaced short palindromic repeats(CRISPR)associated protein 9(Cas9)SNA gene editing Pro SNAs,which can efficiently enter cells,escape from endosomes,and target the nucleus.With the help of single-stranded guided RNA,Cas9 Pro SNAs recognize specific DNA sequences and cleave DNA,enabling precise regulation of functional gene expression.Moreover,Cas9 Pro SNAs can simultaneously edit and regulate different DNA loci in the same cell,enabling precise gene regulation simultaneously.Our results demonstrate the potential of Pro SNAs nanostructures as a promising tool for precise tumor treatment.