Research On Novel Biosensing Methods Based On Nucleic Acid Amplification Technology And Novel Nanomaterials

Recently,biosensors have attracted increasing attention and greatly promoted the development of clinical diagnosis and drug screening in virture of their high sensitivity,good selectivity,short analysis time and low-cost.The development of nanomaterials has made an enormous impact on a wide range of fields,such as catalysis,computing,photonics,energy,biology,and medicine,which attract a tremendous interest in nanotechnology.Particularly,it has shown highly attractive potential to use nanotechnology in biological applications.The emerging field of nanobiotechnology holds the potential of revolutionizing biology and biomedical studies by employing new nanomaterial-based tools for investigative,diagnostic,and therapeutic techniques.In recent years,scientists have made great progress in developing different kinds of nanomaterials and nanofabrication techniques,greatly facilitating the advancement of nanobiotechnology.Functional nanomaterials enable many new opportunities for investigating complicated biological processes in virtue of their unique optical,magnetic,and mechanical properties,which are hard to study by traditional strategies,indicating exciting avenues in biological and biomedical fields.The combining of nanotechnology with biosensing technology has greatly promoted the rapid development of bioscience,nanoscience and biomedicine.This doctoral thesis developed a series of new biosensing methods by coupling nucleic acid probe with nanomaterials for sensitive,simple and low-cost detection of cholera toxin,microRNA profiling,hypochlorite and temperature,small molecule and imaging diagnosis and synergistic therapy for cancer.The detailed contents are described as follo ws:Cholera,a kind of acute infectious disease featured by watery diarrhea,remains a global threat especially in areas where access to safe drinking water and adequate sanitation cannot be guaranteed.The clinical manifestations of cholera are largely attributable to the actions of cholera toxin(CT)secreted by Vibrio cholera.Early detection is critical for the prevention of death and outbreaks caused by cholera.In chapter 2,We report the development of a novel plasmon coupling enhanced Raman scattering(PCERS)method,PCERS nanobeacon,for ultrasensitive,single-step,homogeneous detection of cholera toxin(CT).This method relies on our design of the plasmonic nanoparticles,which have a bilayer phospholipid coating with embedded Raman indicators and CT-binding ligands of monosialoganglioside(GM1).This design allows a facile synthesis of the plasmonic nanoparticle via two-step self-assembly without any specific modification or chemical immobilization.The realization of tethering GM1 on the surface imparts the plasmonic nanoparticles with high affinity,excellent specificity,and multivalence for interaction with CT.The unique lipid-based bilayer coated structure also affords excellent biocompatibility and stability for the plasmonic nanoparticles.The plasmonic nanoparticles are able to show substantial enhancement of the surface-enhanced Raman scattering(SERS)signals in a single-step interaction with CT,because of their assembly into aggregates in response to the CT-sandwiched interactions.The results reveal that the developed nanobeacon provides a simple but ultrasensitive sensor for rapid detection of CT with a large signal-tobackground ratio and excellent reproducibility in a wide dynamic range,implying its potential for point-of-care applications in preventive and diagnostic monitoring of cholera.MicroRNAs(miRNAs)are a class of small endogenous non-protein-coding RNA molecules with approximately 19-25 nucleotides,which play important regulatory roles in a wide range of biological processes.Extensive studies have revealed that many diseases,such as cancers,diabetes,and neurological disorders are directly associated with miRNA expression profiles.Mi RNA has been an emerging class of clinically important biomarkers for early diagnosis and targets for drug development.However,Mi RNA detection is still challenging because of their short lengths,sequence homology among family members,lability to degradation,and low abundance.Pursuit for sensitive and selective detection of miRNAs is still is highly demanding for biomedical research and clinical early diagnosis.In chapter 3,we developed a novel CHA coupled enzymatic amplification strategy,which can be realized in a single-step isothermal reaction,for highly sensitive detection of miRNA.When used for miRNA detection,the CHA-ERA assay was shown to give a favorable detection limit as low as 50 f M across a wide dynamic range up to 1 n M.Hence,the CHA-ERA indeed provided a novel platform for efficient nucleic acid amplification and miRN A expression analysis or related theranostic applications.Reactive oxygen species(ROS)have emerged as prevalent and important components of both physiological and pathological processes.ROS are important signaling molecules that are produced endogenous ly from oxygen and play key roles in regulating a wide range of physiological functions.Among various ROS,·OH,ClO~-,and ONOO-have strong oxidant properties so they are considered to be highly ROS(h ROS).It is reported that overaccumulation of h ROS cou ld induce oxidative stress and is associated with many pathological conditions.Thus it is useful to develop probe for visualizing h ROS for elucidating the biological roles.In the other hand,acatastatic temperature is tightly associated with various pathological cellular dysfunctions,which may lead to many diseases such as inflammation of tissues,and cancer.Therefore,it is extremely vital to explore novel strategies to effectively and accurately detect ClO~-and temperature instantly.In chapter 4,we investigated a new silica nanoparticle-based fluorescent sensor,which can be used as a dual-response ratiometric fluorescent sensor(DRFS)for the detecting ClO~-and temperature independently and effectively in aqueous solution.The sensor was fabricated via covalent binding technique,which a kind of fluorescent silica nanoparticle(FSNP,as hypochlorite sensing unit)and rhodamine B isothiocyanate(RBITC,as temperature sensing unit)was selected to form this detecting system.One of the fluorescent moieties is immune from external environment to be a stable reference signal,while the other one can be activated by external stimuli.For this detecting system,the fluorescence quenching process of the FSNP would be occurred by strong oxidants when the ClO~-was added in the system,while emission intensity from RBITC remains nearly unchanged,resulting in the ratiometric detection of ClO~-in water.In addition,the as-prepared DRFS was also used as an excellent ratiometric fluorescent sensor for the accurately monitoring of temperature in aqueous solution,dues to its wide responsive temperature range.Compared with previously reported fluorescence sensors,the as-prepared sensor is possessed of several appealing features,including facile preparation,low cost,short response time,good water solubility and low cytotoxicity.Detection of intracellular HCl O was conducted employing living RAW 264.7 macrophage cells.We expected that the project may offer a new thought for developing low-cost and dual-response ratiometric fluorescence biosensor for biological and environmental applications.ATP is an important substrate in living organisms.Being a major energy currency of the cell,it plays a critical role in the regulation of cellular metabolism and biochemical pathways in cell physiology.It has also been used as an indicator for cell viability and cell injury.Therefore,the determination of ATP is essential in biochemical study as well as clinic diagnosis.In chapter 5,we developed a novel split aptamer mediated proximity dependent hybridization mediated HCR amplification,called PHCR.Two aptamer fragment probes will get close in the present of target ATP,the terminals of which are further extended with two tail sequences to serve as initiator of HCR amplification,which can activate the fluorescence of hairpin probes.With the help of prepared semiconducting polymer dots,the probe can be delivered into cancer cells to realize detection of ATP in living cells.The proposed method is promising for the employment in many biological fields,such as the assay of DNA and protein,the delivery of gene and drug,and intracellular tracking,as well as in vivo monitoring,etc.Currently,cancer still remains one of the most fatal diseases,causing huge social and economic burden.The current therapeutic methodologies by single mode are rarely sufficient to overcome cancer.In chapter 6,we developed a tumor-targeted fluorescent imaging and combined therapy assay based on DNA-conjugated greplene oxide.A cancer cell-targeted aptamer and PH sensitive probe were assembled onto PEGylated greplene oxide,which can be released in response to the acid environment in cancer cells,with concomitant of activated fluorescence intensity and released of DOX in ds DNA.The released probe can be used to inhibit the activity of DNMT1 by covalently trapping as well.Moreover,the photothermal effect of GO also enable photothermal treatment.The results revealed that developed assay has good performance in combined therapy.Therefore,the developed nanotheranostics system might provide a promising platform for image-guided diagnosis and combined therapy of cancer.