Study On Optical Sensors For Molecular Recognition Based On DNA And Fluorescent Carbon Dots

The research in the development of sensitive,selective,rapid,and cost-effective optical sensors for biomedical analysis,food safty,environmental monitoring,and the detection of terrorism agents is inspiring intensive efforts.In this dissertation,DNA biosensors combining nucleic acid amplification straretegies and magnetic separation technologies are bulit to realize the determination of trace target biomolecules.Moreover,DNA and carbon dots are utilized to tune the synthesis of metal nanoparticles for molecular recognition.We further explore the synthetic routes to preparing high-performing carbon dots.At the same time,combining the fluorescence and UV-vis spectra analysis technique,we explore the optical property and application of the carbon dots in aqueous solutions,and establish the optical sensors to detect the biomolecules,food additive,and environmental pollutants.The main contents of this dissertation are summarized as follows:1.Turn-on fluorescence detection of pyrophosphate anion based on DNA-attached cobalt oxyhydroxidePyrophosphate anion?PPi?is very important in biochemistry as it is involved in many bioenergetic and metabolic processes.Turn-on fluorescence detection of pyrophosphate anion was developed with DNA-attached cobalt oxyhydroxide?CoOOH?.Herein,CoOOH nanoflakes were fabricated by the oxidation and exfoliation method.The single-stranded DNA?ssDNA?could interact with CoOOH nanoflakes through electrostatic interaction.When carboxy-fluorescein?FAM?labelled ssDNA was added to CoOOH solution,it bound to CoOOH nanoflakes and resulted in quenched fluorescence.However,in the presence of PPi,FAM labelled ssDNA could be released because PPi showed stronger interaction during the competition with DNA for CoOOH nanoflakes.As a result,PPi detection was realised based on the gradually recovered fluorescence with increasing PPi concentration.This detection method was optimized and reached a low detection limit of 0.3?M.Interference from compounds that contain phosphate group and some other anions was minimal.The methodology showed great promise for the fluorescent PPi detection in diluted human serum samples.2.Sensitive mutant DNA biomarkers detection based on magnetic nanoparticles and nicking endonuclease assisted fluorescence signal amplificationBased on the nicking endonuclease?NEase?-assisted target recycling and magnetic nanoparticles?MNPs?separation process via the streptavidin-biotin system,we developed a new,signal amplified and ultrasensitive fluorescence biosensor for the detection of mutant human p53 gene.The target mutant DNA hybridizes with the loop portion of hairpin probe?HP?modified with biotin and a fluorescein isothiocyanate dye?FITC?at its 5'and 3'ends separately and forms nicking site for NEase,which cleaves the HP and releases the target DNA.The released target DNA again hybridizes with the intact HP and initiates the DNA recycling process with the assistance of NEase,leading to the cleavage of a large number of HPs and detachment of the biotin labeled part with the FITC tagged signal portion.Only these cleaved fragments corresponding to target DNAs could remain in solution and function as a signaling flare,while the biotin labeled sequences including intact hairpin probes could be trapped and removed by the streptavidin coated MNPs.The developed method exhibits the detection limit as low as198 fM and high discrimination efficiency toward single-base mismatched sequence.Therefore,the novel NEase-amplified magnetic nanoparticle assay has great potential for sensitive and accurate detection of trace amounts of DNA in clinical diagnosis and biomedical research.3.Enzyme-free fluorescent biosensor for the detection of DNA based on core-shell Fe₃O₄ polydopamine nanoparticles and hybridization chain reaction amplificationA novel,highly sensitive assay for quantitative determination of DNA is developed based on hybridization chain reaction?HCR?amplification and the separation via core-shell Fe₃O₄ polydopamine nanoparticles?Fe₃O₄@PDA NPs?.In this assay,two hairpin probes are designed,one of which is labeled with a 6-carboxyfluorescein?FAM?.Without target DNA,auxiliary hairpin probes are stable in solution.However,when target DNA is present,the HCR between the two hairpins is triggered.The HCR products have sticky ends of 24 nt,which are much longer than the length of sticky ends of auxiliary hairpins?6 nt?and make the adsorption much easier by Fe₃O₄@PDA NPs.With the addition of Fe₃O₄@PDA NPs,HCR products could be adsorbed because of the strong interaction between their sticky ends and Fe₃O₄@PDA NPs.As a result,supernatant of the solution with target DNA emits weak fluorescence after separation by magnet,which is much lower than that of the blank solution.The detection limit of the proposed method is as low as 0.05 nM.And the sensing method exhibits high selectivity for the determination between perfectly complementary sequence and target with single base-pair mismatch.Importantly,the application of the sensor for DNA detection in human serum shows that the proposed method works well for biological samples.4.Tuning gold nanoparticles growth via DNA and carbon dots for nucleic acid and protein detectionDNA and carbon dots were utilized to co-mediate the growth of gold nanoparticles?AuNPs?.The properties of grown AuNPs were discussed and applied to colorimetric biomolecule determination.When target biomolecules were absent,ssDNA was adsorbed on the surface of AuNP seeds.Branched AuNP solution of blue color was obtained after carbon dots reduction.In the presence of target DNA,it hybridized with the ssDNA and double-stranded DNA?dsDNA?were formed.As a result,a spherical AuNP solution of red color was achieved after growth as dsDNA would not be adsorbed by AuNP seeds.A similar situation was observed with thrombin.The complex of thrombin and its aptamer was formed,giving rise to spherical AuNPs after growth.The target biomolecule determination was realized by virtue of color change,consistent with the peak shift in UV-Vis absorption spectrum.The detection limits were determined to be 5 nM for target DNA and 1.8 nM for thrombin,respectively.The biomolecule sensors showed the requisite selectivity against possible interferents,and the assay for target DNA exhibited the potential for distinguishing single-base mismatch.These results served as the basis for constructing future biomolecule sensors using DNA and carbon dots co-mediated growth of AuNPs.5.Visible and fluorescent detection of melamine in raw milk with one-step synthesized silver nanoparticles using carbon dots as the reductant and stabilizerUsing carbon dots as the reductant and stabilizer,a visible and fluorescent method was developed for melamine detection in raw milk with one-step synthesized silver nanoparticles.In this work,carbon dots?C-dots?were applied to reduce silver ions and stabilize the nanoparticles,resulting in the formation of silver nanoparticles?AgNPs?.As a result,the inherent fluorescence emission of C-dots was significantly reduced after the formation of AgNPs.However,in the presence of melamine,silver ions could interact with the nitrogen atoms in amine and triazine groups of melamine.With 0-2?M melamine,aggregated AgNPs were found after the reduction by C-dots,resulting in color and absorbance changes.With further increase of melamine?2–20?M?,both formation and aggregation of AgNPs were inhibited,and the fluorescence was gradually increased.This optical platform was optimized for melamine detection and then was applied to detecting melamine in raw milk samples.The results for melamine assay based on visible and fluorescent method showed the requisite sensitivity with a low detection limit of 30 nM,as well as high selectivity.6.Adenosine-derived doped carbon dots:from an insight into effect of N/P co-doping on emission to highly sensitive picric acid sensingThe various synthetic routes of carbon dots?C-dots?feature a considerable step toward their potential use in chemical sensors and biotechnology.Herein,by coupling phosphorus and nitrogen element introduction,the adenosine-derived N/P co-doped C-dots with fluorescence enhancement were achieved.By separately employing adenosine,adenosine monophosphate,adenosine diphosphate,and adenosine-5`-triphosphate as precursors,the effect of N/P co-doping on the fluorescence emission is discussed in detail.The formed C-dots with adenosine monophosphate exhibited strong blue fluorescence with a high quantum yield of 33.81%.Then the C-dots were employed as a fluorescent probe and utilized to develop a fast,sensitive,and selective picric acid sensor.The fluorescence of C-dots can be quenched by picric acid immediately,giving rise to a picric acid determination down to 30 nM.The possible mechanism of fluorescence quenching was discussed,which was proved to be inner filter effect and static quenching.Moreover,this method has the potential to detect picric acid in environmental water samples.7.Using high-energy phosphate as energy-donor and nucleus growth-inhibitor to prepare carbon dots for hydrogen peroxide related biosensingHerein,by coupling pyrophosphate introduction with high-energy phosphate bond design,the fluorescence performance of C-dots is improved greatly.The introduction of pyrophosphate acts as the nucleus growth-inhibitor to protect C-dots from assembling and growing into large carbon particles.The high-energy phosphate bond is designed as an energy-donor to provide energy for synthesizing C-dots.The C-dots exhibit enhanced fluorescence with a high quantum yield of 24.8%.Then the C-dots are employed as the fluorescent probe to develop hydrogen peroxide?H₂O₂?-related biosensors.The mechanism is based on the fluorescence quenching of C-dots caused by the highly reactive·OH and Fe³⁺during the Fenton reaction.A wide linear range?0.5-100?M?and a low detection limit?0.195?M?are achieved for H₂O₂ detection.Moreover,the probe can be applied to H₂O₂-related biosensing in the presence of oxidase.As a proof-of-concept,a glucose sensor is developed with glucose oxidase.The presented glucose sensor exhibits good selectivity and high sensitivity with a detection limit of 0.254?M.Additionally,the practical application of the proposed biosensor has been confirmed by detecting glucose in human serum samples.