| With the development of genome project and protein project, more sensitive, accurate, convenient and economic detection methods for biological macromolecules have been the focus of biology, medicine and chemistry. In recent years, signal amplification detection technologies based on electrochemical, optical, piezoelectric technologies and the use of various enzymes and nanoparticles, have realized highly sensitive detections of biological macromolecules. How to utilize the existing molecular biology techniques and tools to convert the information of biological molecules to detectable signals with high sensitivity and specificity, how to further expand the use of signal amplifying methods in molecular biology and biomedical application, and how to design novel signal conversion means and develop novel signal amplification detection technology platform to address the research process to life in the new issue, are still significant topics for researches in biomedical and analysis chemical area.In this thesis, a series of fluorescence signal amplifying detection technology platforms based on a variety of nucleic acid enzymes, nanoparticles and fluorescent nucleic acid probes have been developed for quantitative analysis of protein and DNA detection. The main researches included in this dissertation are presented as following:1. Ultrasensitive Fluorescence immunosensor for Detection of Protein Based on a Combination of the Magnetic Nanobeads and the assembly of fluorescent DNA nanotagsAlong with the efficient magnetic nonparticle amplifying strategy for sensitive detecting biomolecules, magnetic nanoparticle-based label sensors toward targets have been developed with its enrichment and magnetic separation properties. Sensor based on functionalized magnetic nonparticle has gained much attention as a new type tool of non-isotopic immunoassay labels. Magnetic particles act as a carrier and a bridge when functionalized magnetic particles are used to be as signal labels. On one hand, the significant signal intensity could be gained due to the application of amplification strategy. On the other hand, the background signals from labels nonspecific adsorption could be reduced due to the application of magnetic particles. In immunoassay, the application of the sensor based on magnetic nanoparticles can improve the sensitivity by reducing false positive signals with magnetic separation technology and increasing label signal intensity by its enrichment as a carrier.In this paper, aiming at the detection need of low abundant proteins, we report an ultrasensitive fluorescence immunoassay based on a novel and sensitive fluorescence biosensor by the combination of MNB and assembly of fluorescent DNA nanotags (SYBR Green I/BT-DNA conjugates) for detection of target protein. Highly amplified fluorescence signal and low background signal are achieved by using a double amplification (DNA amplification and the signal amplification feature of SYBR Green I/BT-DNA) and magnetic separation. In the high concentration, on logarithmic scales, the fluorescence intensity was found to exhibit a linear correlation to human IgG concentration from5.0×10-13mol L-1to8.0×10-15mol L-1with a linear correlation coefficient of0.995. In the low concentration, one protein brings one MNB onto the substrate. The linear relationship between the number of beads and human IgG concentration is in the range of50to3.0fM. The linear regression equation for human IgG in the range of50to3.0fM was determined to be y=13.135+7.684×1015x(R=0.996).2. Sensitive Detection of Proteins Using Assembled Cascade Fluorescent DNA Nanotags Based on Rolling Circle AmplificationSensitive proteins detection is critically important in basic discovery research and clinical practice because a few molecules of protein are sufficient to affect the biological functions of cells and trigger pathophysiological processes. To improve the sensitive, Great efforts have been made to develop some new protein detection techniques. Among them, fluorescence detection techniques combined with signal amplification are one of the most popular techniques. Specific nucleic acid sequences amplification is a basic technology of molecular biology. With the development of molecular biology and the application of the cross subject biochemistry and biophysics in the field, new nucleic acid amplification technologys have emerged. Among them, RCA (Rolling Circle Amplification) technology with high specificity, high sensitivity and simple operational gradually draw people's attention in recent years. And RCA is more and more used in basic research, clinical diagnosis analysis and the field of medicine. Compared with other nucleic acid amplification technology, RCA technology has the following advantages:high sensitivity, high specificity, diversity and high throughput, isothermal reaction and simple operation and so on.Aiming at the detection need of low abundant proteins and the problems existing in the amplification techniques by multiple fluorophores and nanoparticle probes, this work proposed a novel cascade fluorescence signal amplification strategy for ultrasensitive detection of protein, which based on the rolling circle amplification (RCA)-aided and assembled cascade fluorescent DNA nanotags as signal label and multiplex binding of the biotin-streptavidin system. The designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a3-decade concentration range from1.0pM to1.0fM with a limit of detection as low as0.9fM. It was demonstrated that the proposed strategy shows a broad dynamic range, ultra-high sensitivity, acceptable reproducibility, excellent specificity and a low matrix effect. Futhermore, the method does not require complicated conjugation chemistry and shows fast reaction kinetics and simply manipulating in assembly of fluorescent DNA nanotags. Compared with functionalized nanoparticles labels, it avoids the complicated and tedious synthesis process. The proposed strategy would become a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.3. A Versatile Platform for Ultrasensitive Detection of Protein:DNA enriching magnetic nanoparticles-Based Rolling Circle Amplification ImmunoassayIn fluorescence immunoassay, for the detection of trace amounts of biological analytes, a key factor of the detection and quantification of trace levels of protein analytes is to increase label fluorescence signal intensity to improve sensitivity. Great efforts have been made to develop some new protein detection techniques relying on labeling with different probes to improve sensitivity. In general, high-sensitivity assay labels involve the use of certain signal amplification strategies by which a single signal-reporting tag is able to incorporate numerous detectable elements. Typical high-sensitivity assay labels include enzymes or their polymeric forms as well as nanoparticles such as dye-doped nanoparticles, gold nano-particles and quantum dots. Notwithstanding the importance of these techniques, the signal amplification capacity of these assay labels is still limited.With the development of molecular biology and the application of the cross subject biochemistry and biophysics in the field, new nucleic acid amplification technologys have emerged. Among them, RCA (Rolling Circle Amplification) technology with high specificity, high sensitivity and simple operational gradually draw people's attention in recent years. And RCA is more and more used in basic research, clinical diagnosis analysis and the field of medicine. Compared with other nucleic acid amplification technology, RCA technology has the following advantages: high sensitivity, high specificity, diversity and high throughput, isothermal reaction and simple operation and so on.Along the direction of implementation of oligonucleotide-based immunoassay labels, to develop signal-reporting labels with maximized signal amplification and minimized nonspecific adsorption for highly sensitive proteins detection, we reported for the first time a DNA enriching magnetic nanoparticles and assembled fluorescent DNA nanotags based RCA immunoassay, magnetic nanoparticles-RCA immunoassay, as an alternative strategy. On logarithmic scales, the fluorescence intensity at the maximum emission wavelength was found to exhibit a linear correlation to human igG concentration over a6-decade concentration range from1pM to10aM with a linear correlation coefficient of0.997. The calculated limit of lower detection is8.3aM in a3s rule. The ultrahigh sensitivity and the broad dynamic range were attributed to the following reasons:(ⅰ) the primary amplification via functionalized magnetic nanoparticle detection probe attaching numerous primer DNAs for RCA, that is to say, a high ratio of primer DNAs are attached to an antigen;(ⅱ) the released numerous primer DNAs initiate a secondary RCA amplification;(ⅲ) a tertiary "assembled fluorescent DNA nanotags" amplification;(iv) a low nonspecific adsorption of functionalized magnetic nanoparticle detection probe by magnetic separation. This technique also enabled easy preparation of the detection probe, and isothermal end-point detection with simple instrumentation. It was demonstrated that the technique gave high specificity, low matrix effect and dose-response sensitivity. This technique holds great promise as an ultrasensitive, specific, powerful technology for protein detection in proteomics and clinical diagnostics.4. Sensitive and Homogeneous protein detection based on Exo I assisted separation-free and Nicking Enzyme assisted fluorescence signal amplificationAptamers, which are novel in vitro selected functional DNA or RNA structures from random-sequence nucleic acids libraries, possess high recognition ability to specific targets. Due to their inherent selectivity, affinity and their multifarious advantages over the traditional recognition elements, aptamers can rival antibodies for molecular recognition and detection. Compared with antibodies, the biggest advantage of aptamer-based methods is that oligonucleotides can be synthesized chemically with ease and extreme accuracy at low cost nowadays. Furthermore, they are thermally stable, reusable, and show good stability during long-term storage. At present, aptamers sensor for fluorescent detecting protein is mainly based on the change of fluorescence polarization degree or the fluorescence signal intensity after aptamer and the target protein binding. In this method, signal change is in the small areas, fluorescence background is bigger, linear range is not wide. It is difficult for low concentration of protein determination in some degree.Exonuclease I catalyzes the removal of nucleotides from single-stranded DNA in the3'to5'direction. The single-stranded DNA chains would be degraded to5'-terminal dinucleotides with the release of deoxyribonucleoside5'-monophosphates from the3'-termini of the single-stranded DNA chains. Nicking Enzyme can identify and cleave double-stranded nucleotides specific sites. In this work, we developed a sensitive and homogeneous protein detection based on Exo I assisted separation-free and Nicking Enzyme assisted fluorescence signal amplification. In this study, we firstly synthesize a protein detection probe containing a thrombin aptamer sequence and an ex-DNA recognition probe sequence. The ex-DNA was designed to be complementary to the MB-quenching fluorescence (MB). The MB probe is a short oligo-DNA, which carried the recognition sequences and cleavage site for the nicking endonuclease Nb.BbvcI, and was labeled with the fluorescent dye6-carboxyfluorescein (6-FAM) and its quencher DABCYL at the5'-and3'-ends, respectively. Nicking endonuclease is an enzyme that binds to its asymmetrical recognition sequence in double-stranded DNA and nicks only one specific strand of the duplex. When thrombin and its aptamer incubated together, the thrombin bound an aptamer and thereby was protected from degraded by exonuclease I. The protected protein detection probes hybridize with MB probe. Therefore, a fluorescence signal appears only when the MB probe was cleaved by Nb.BbvCI. After nicking, the hybrid MB probe became less stable, and the cleaved strand dissociated from the target, thus resulting in the complete disconnection of the fluorophore from the quencher. The released target strand could then hybridize to another MB probe and initiated the second cycle of cleavage. Finally each target strand could go through many cycles, resulting in cleavage of many probes. The detect limit was estimated to be PM. It was demonstrated that the proposed strategy shows an ultra-high sensitivity, acceptable reproducibility, excellent specificity and a low matrix effect. In addition, the strategy has promising application in the monitoring of many other proteins with high sensitivity and excellent specificity.5. A facile, label-free amplified DNA detection system based on rolling circle amplification strategy assisted quadruplex formationIn particular ionic strength and pH conditions, G-quadruplexes is comprised of single nucleotide with rich G basic groups through the counterpart of the Hoogsteen hydrogen bonding. N-methyl mesoporphyrin IX (NMM) is an anionic porphyrin characterized by a pronounced structural selectivity for G-quadruplexes. It is weakly fluorescent by itself, but exhibits a dramatic fluorescence enhancement upon binding to quadruplex DNA.Magnetic nanoparticle-based sensors have gained much attention with its enrichment and magnetic separation properties as a new type tool of assay sensor. RCA (Rolling Circle Amplification) technology with high specificity, high sensitivity and simple operational gradually draw people's attention in recent years. In a typical RCA process, DNA polymerase replicates the circular template hundreds to thousands of times. Therefore, the end products of an RCA reaction are extremely long ssDN A molecules. RCA has a wide range of applications in the biochemical analysis and clinical diagnosis due to its high sensitivity and specificity, isothermal operation and many other advantages.In order to design convenient, cheap, fast, easy operation, high sensitivity, good selectivity nucleotide detection method, in this work, we developed two facile, label-free amplified DNA detection systems.(1) A magnetic bead-based label-free fluorescent turn-on enzymatic amplification assay for DNA detection using RCA assistant ligand-responsive G-quadruplex formation. The developed assay exploits three amplifications (Exo III cleavage cycle amplification, RCA, a dramatic fluorescence enhancement of G-quadruplex/NMM). Here, the integration of a series of amplification techniques further improves the sensitivity. In addition, the developed assay realizes label-free DNA detection, which is important for retaining the high target binding activity.(2) In order to further simplified experimental steps, shorten the reaction time, avoid vulnerability to contamination, we developed a facile, label-free amplified DNA detection system based on a dumbbell probe-mediated rolling circle amplification assisted quadruplex formation for highly sensitive DNA detection.
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