Nano-biosensors Applied In Sensitive Detection Of Cancer Biomarkers

As one of the most common diseases in the world,cancer has become the focus of global health issues.In order to improve the controllability of cancer and reduce the mortality rate,early detection and accurate diagnosis are usually required.According to the existing research findings,some cancer biomarkers can often indicate the early detection of cancer,tumor classification and stage status,and even the risk assessment of cancer treatment process.In view of this,it is urgent to develop simple,efficient,sensitive and accurate methods for detecting cancer markers.Nano-biosensing technology,as a kind of biosensing technology based on functional nanomaterials,can not only complete the detection of small biological molecules,peptides,proteins,nucleic acids and other cancer biomarkers,but also can realize in living cells real-time monitoring of biomarkers and indicate the accurate dosing of lesions.Nanobiosensor overcomes the difficulty of detecting cancer markers in vivo and promotes personalized treatment of cancer.In this paper,several novel nano-biosensors have been developed by functionalizing a variety of nanomaterials and combining them with isothermal nucleic acid amplification methods to detect the content or activity of prostate-specific antigen（PSA）,intracellular micro RNA and different intracellular depurine/pyrimidine endonuclease（APE1）.At the same time,the imaging analysis of cancer biomarkers in living cells is realized,which provides a new example for the early diagnosis of cancer.The detailed research contents are described as follows:PSA is the preferred marker for diagnosis of prostate cancer.It is of great significance in the diagnosis,curative effect observation and prognosis monitoring of prostate cancer.At present,the clinical detection of the disease is mostly the traditional heterogeneous immune method,the detection procedures are complicated,time-consuming,and it is not conducive to the immediate diagnosis of the disease.Therefore,it is important to develop a simple and sensitive detection method for detection of PSA.In chapter 2,we developed Upconversion Nanoaurora,a novel homogeneous immunoassay approach for single-step,ultrasensitive detection of protein biomarkers.This approach relies on two designed antibody-modified upconversion nanoparticle（UCNP）probes,activator UCNPs enabling efficient generation of singlet oxygen（¹O₂）under near infrared excitation,and reporter UCNPs with luminescence caged by ¹O₂-cleavable quenchers.Specific detection is realized based on target mediated formation of a sandwiched complex between these two probes which allows efficient diffusion of ¹O₂ from the activator to uncage the reporter UCNPs.This approach is demonstrated using a tumor biomarker prostate specific antigen with an analogous detection format by detecting the luminescence intensity or a digital imaging format by counting the luminescent spots.The results reveal this approach affords a high signal-to-background ratio,a wide dynamic range and a very low detection limit,especially using the digital imaging format.This approach may provide an invaluable paradigm for developing highly accessible and ultrasensitive tools for diagnostics and biomarker discovery.DNA tetrahedron is a novel DNA nanostructure.As is known to all,DNA is a endogenous material,so the DNA-based nanostructure has extraordinary biocompatibility and biological stability.Since it was reported,DNA tetrahedron has been widely used in the research of drug delivery,biosensors and gene regulation in living cells.In chapter 3,we constructed a three-dimensional（3D）DNA nanoprobe called DNA“Nanoflare”（DNF）using DNA tetrahedron and Y-shaped DNA.DNF utilizes all the terminal of DNA tetrahedron and Y-shaped DNA,and can simultaneously deliver up to eight probes into cells.Compared with common DNA tetrahedron and Y-shaped DNA scaffolds,the loading capacity is largely improved.On the surface of the probe,there are two groups of double-stranded DNA fluorescent probes formed by hybridization of fluorescent strand（F strand）and quenched strand（Q strand）.DNF delivers the double-stranded DNA probes to the cells,where the target mi RNAs displace F-strand through strand displacement to restore the fluorescence of the system and form“nanoflare”in the cells.The multiplex“nanoflare”constructed by us can rapidly and sensitively perform simultaneous imaging analysis of multiple intracellular tumor-related mi RNAs,which has the potential for diagnosis of cancer and guidance of cancer treatment.The detection of cancer markers also includes some targets of low abundance,whose low expression in cells will lead to the development of cancer.For s uch targets,ordinary detection methods cannot detect them effectively in real time due to their insufficient sensitivity,so it is necessary to develop a detection method with high sensitivity.Hybridization chain reaction（HCR）is a simple and efficient nucleic acid isothermal signal amplification technology,which can improve the detection sensitivity.In chapter 4,we developed a well-defined 3D DNA nanostructure that,for the first time,can realize cross-linked hybridization chain reaction（C-HCR）in living cells for signal amplification and fluorescent imaging of multiplex mi RNAs.This 3D DNA nanostructure（TYH）is easily prepared by a simply annealing of customized single-stranded oligonucleotide strands to form a DNA tetrahedron carrying two hairpin probes at its each vertex via Y-shape DNA.The DNA nanostructure can efficiently deliver DNA probes into cells and specifically generate fluorescence signal by target-triggered hybridization chain reaction.This sensor offers efficient intracellular signal amplification and enables ultrasensitive fluorescence imaging of multiplexed mi RNAs.Due to the close proximity between hairpin probes,the prepared DNA nanostructure offers higher reaction efficiency than standard HCR,which shorten the reaction time of TYH-HCR and improve the detection sensitivity.The DNA nanostructure can provide a useful platform for low-abundance and multiplex biomarker simultaneous detection and imaging for cell biology and diagnostics.Apurinic/apyrimidinic endonuclease 1（APE1）plays a key role in the base excision repair（BER）pathway of DNA lesions to maintain the genome stability.It is also involved in regulating cellular responses to oxidative stress conditions.Abnormal expression/localization of APE1 has been found in tumor.Therefore,imaging of APE1 activity is critical for understanding the BER pathway in complicated intracellular circumstances as well as for screening the drug candidates for BER.In chapter 5,we report the development of a novel stochastic bipedal DNA walker that,for the first time,realizes direct intracellular base excision repair（BER）fluorescence activation imaging.In our design,the bipedal walker DNA was generated by BER-related human apurinic/apyrimidinic endonuclease 1（APE1）-mediated cleavage of DNA sequences at an abasic site in the intracellular environment,and it autonomously travelled on spherical nucleic acid（SNA）surfaces via catalyzed hairpin assembly（CHA）.Our nanomachine outperforms the conventional single leg-based DNA walker with an improved sensitivity,kinetics and walking steps.Moreover,in contrast to the single leg-based DNA walker,the bipedal DNA walker is capable of monitoring the fluorescence signal of reduced APE1 activity,thus indicating amplified intracellular imaging.This bipedal DNA-propelled DNA walker presents a simple and modular amplification mechanism for intracellular biomarkers of interest,providing an invaluable platform for low-abundance biomarker discovery leading to the accurate identification and effective treatment of cancers.