Preparation Of Novel Optical Nucleic Acid-based Probes And Their Applications In Biochemical Analysis

The development of probes is one of the most important concerns in biochemicalanalysis. By virtue of its excellent properties, including flexibility in designing,convenience in signal acquisition, universality and abundance for targets detection,the optical nucleic acid-based probe has attracted most interest from researchers andbecome a type of significant detection tools for analytical applications. As seen fromthe structure composition of the optical nucleic acid-based probe, the signal reporteris the unit which can respond to the recognition events and transduce the targetimformation into detectable signal. Thus, it is essential to design and constructreasonable signal reporters. In addition, as seen from the history of the opticalnucleic acid-based probe, there is an increasing demand for sensitivity with advancesof technology. Therefore, the development of signal amplification technologies issignificative, which can amplify trace targets to detectable levels. Besides signalamplification, multiplex detection is another trend in modern analitical chemistry. Inthis thesis, we aim to synthetise novel signal reporters, construct new signalamplification and multiplex detection strategies, resulting in developing novel pticalnucleic acid probes for biochemical analysis. The main contents of this thesis aresummarized as follows.1. Poly(thymine)-templated formation of fluorescent copper nanoparticles andstudies on its fluorescence properties.In this work, poly(T) is screened as the specific ssDNA which can templated theformation of fluorescent copper nanoparticles (CuNPs), while other ssDNAs, such asrandom ssDNA, poly A, poly C, and poly G faile to serve as templates for fluorescentCuNPs. The size of CuNPs can be simply regulated by altering the length of poly T.There is a MegaStokes shifting (275nm) between its excitation and emissionwavelength (λex=340nm, λem=615nm). Positive correlations display between thefluorescence intensity of poly(T)-templated CuNPs and the length of poly(T), thepolymerization degree of poly(T), the concentration of poly(T) and the concentrationof copper ions. In addition, lower temperature, weak alkali and moderate ion strengthare favorable for the signal formation. Because of the excellent fluorescence of thepoly(T)-templated CuNPs, poly(T) can be used as a probe for further sensing.Simultaneously, selective metallization of ssDNA can be achieved using poly(T) as a template for CuNPs formation and other ssDNAs as linkers.2. Visual and portable strategy for copper(II) detection based on a striplikepoly(T)-caged and microwell-printed hydrogelWe here report a visual and portable strategy for Cu2+detection based onpoly(T)-templated fluorescent CuNPs an a strip-like hydrogel. Poly(T) was used asthe detection probe and caged in hydrogel. Uniform wells of microliter volume(microwells) are printed on the hydrogel’s surface. When Cu2+is injected into themicrowells, fluorescence of CuNPs can be observed under ultraviolet (UV)irradiation. The strategy integrates sample-injection, reaction and indication with fastsignal response, providing an add-and-read manner for visual and portable detectionof Cu2+, as well as a strip-like strategy. A detectable minimum concentration of20μM is achieved, and some practically applicable properties have been demonstrated.3. Poly(T)-templated fluorescent copper nanoparticles for ultrasensitivelabel-free nuclease assay and its inhibitors screeningWe report herein for the method to develop a novel ultrasensitive label-freemethod for the nuclease assay, and its inhibitors screening based on thepoly(T)-templated fluorescent CuNPs. In the presence of nuclease, poly T is digestedto mono-or oligonucleotide fragments with decrease of fluorescence. The proposedmethod is low-cost and simple in its operation without requirement for complexlabeling of probe DNA or sophisticated synthesis of the fluorescent compound. Thecapabilities for target detection from complex fluids and screening of nucleaseinhibitors are verified. A high sensitivity exhibites with a detectable minimumconcentration of5×107units μL1S1nuclease.4. Nucleic acid amplified detection based on dsDNA-templated fluorescentcopper nanoparticles and polymerase chain reactionThe dsDNA-templated fluorescent copper nanoparticles has been exploited as a“green” nano-dye for PCR-mediated amplified detection of nucleic acid. In thepresence of the corresponding target, polymerization is triggered, generating dsDNAwhich can efficiently template the formation of fluorescent CuNPs. The strategy hasmultifaceted excellences: simplicity, environment-friendly manner and gooddetection capabilities. Thus, this work will expand the way to apply thedsDNA-templated fluorescent CuNPs as a “green” nano-dye for signal transducing ina wider range of fields, such as aptamer-based detection of non-nucleic acid targets, molecular biology and biomedicine.5. Target-catalyzed dynamic assembly-based pyrene excimer switching forenzyme-free nucleic acid amplified detectionA new strategy for enzyme-free nucleic acid amplified detection has beenopened up by combining the signal-amplification capability of target-catalyzeddynamic assembly with the spatially sensitive fluorescent signal of thepyrene-excimer. In this strategy, three metastable pyrene-labeled hairpin DNAprobes were designed as assembly components. In the presence of the target, thedynamic assembly of branched junctions is circularly catalyzed and accompaniedwith the switching of the pyrene-excimer which emits at around488nm. Thus, thetarget DNA could be detected by this simple mix-and-detect amplification method,without expensive and perishable protein enzymes. A good detection capabilityexhibited with a detectable minimum target concentration of10pM.6. Engineering a unimolecular multifunctional DNA probe for analysis of Hg2+and Ag+We have designed a unimolecular multifunctional DNA probe (UMDP) that canrespond to two target ions (Hg2+and Ag+). The UMDP is a single stranded DNAlabled with fluorophore (FAM) and quencher moiety (DABCYL) at the5’ and3’termini respectively, and can form a molecular beacon structure in the presence ofHg2+or Ag+ions with the T–Hg2+–T or C–Ag+–C basepair built-in the stem skillfully.Therefore, when Hg2+ions or Ag+is present in the reaction solution, the UMDPhairpin structure is trigged and stabilized by target ions with concomitantfluorescence quenching. The two target ions can be simply distinguished using aC-rich DNA sequence. The UMDP probe shows excellent selectivity for target ions,with detection limis of0.67nM and0.95nM for Hg2+and Ag+, respectively. Thenovel strategy may provide a new pathway for multi-targets detection, molecularassembly, and molecular logic gates researches.7. Colorimetric multiplexed analysis of mercury and silver ions by using aunimolecular DNA probe and unmodified gold nanoparticlesA colorimetric assay for multiplexed analysis of Hg2+and Ag+ions isdemonstrated by using the unimolecular multifunctional DNA probe (UMDP) assensing element and unmodified gold nanoparticles (AuNPs) as color-reporting probe.The UMDP has a random coil structure that changes into two different hairpin-like structures with a T-Hg2+-T or C-Ag+-C basepairing built-in the stem upon bindingHg2+or Ag+ions, respectively. As a result, the conformation changes facilitate thesalt-induced AuNPs aggregation, leading red-to-blue color change with a red shift ofthe plasmon band in the UV-visible absorption spectrum. Both the color andabsorption changes of the system are selective for Hg2+and Ag+ions, with detectionlimis of250nM and500nM for Hg2+and Ag+, respectively. Moreover, the twotarget ions are simply distinguished by using EDTA. Then, the colorimetric responsesof the two target ions in spiked real samples are tested.