Study On The Design Of Nucleic Acid Signal Amplification Strategies And Their Homogeneous Biosensing Applications

The development of methods which can be used for the highly sensitive and specific detection of protein markers and small antibiotic molecules in complex substrates is of great significance in the early disease diagnosis and food safety fields.Compared with traditional detection methods,the functional nucleic acids-based biosensors have attracted extensive attentions and researches in recent years.As biosensors possess the advantages like high sensitivity,simple operation,good selectivity and low sample consumption,they can be easily applied for the accurate and rapid detection of the target analytes in complex systems.In addition,due to the oligonucleotide properties of functional nucleic acids,various technologies such as isothermal nuclease-assisted signal amplification,catalytic nucleic acid signal amplification and enzyme-free nucleic acid assembly signal amplification can be conveniently combined with them to construct a large variety of strategies which can greatly improve the detection sensitivity of biosensors.Therefore,based on the requirements on the highly selective and accurate detection of trace bioanalytes such as protein markers and small antibiotic molecules on complex matrices,this thesis combines the special biological function of functional nucleic acids with various isothermal signal amplification technologies to carry out three reseaches on the design and construction of novel protein and antibiotic homogeneous biosensing strategies,which are listed as follows:1.Target biorecognition-triggered MNAzyme assembly for highly sensitive colorimetric biosensorsWhen the target biorecognition-triggered assembly of two Mg²⁺-dependentDNAzymes(MNAzymes)is employed for dually catalytic release of peroxidasemimicking G-quadruplex DNAzymes(G-DNAzymes),this work successfully develops a novel homogeneous colorimetric method for an ultrasensitive bioassay of platelet-derived growth factor-BB(PDGF-BB).The first MNAzyme assembly is realized through a highly specific aptamer biorecognition-driven proximity ligation reaction.Its catalytic cleavage toward the two designed hairpin substrates not only releases a large amount of G-DNAzymes for colorimetric signal transduction but also enables the spontaneous assembly of another MNAzyme for signal amplification.This leads to the successful detection of PDGF-BB in a wide linear range from 2.0 pg m L^-1 to 20 ng m L^-1 with a very low detection down to 0.088pg m L^-1.As the whole reactions including aptamer biorecognitions,DNA hybridizations,and catalytic cleavages of MNAzymes are conducted in a homogeneous solution,this method has very simple manipulations and also has high repeatability.In addition,the high specificity of the aptamer biorecognitiontriggered signal transduction decides the excellent selectivity of the method.This bioassay does not require an expensive instrument and nucleic acid labeling for signal readout or any nanomaterial,enzyme,or nuclease for signal amplification.Thus,it displays an extensive potential for clinical diagnostic applications.2.Enzyme-assisted target recycling for ZNAzyme release to drive the gold aggregation for homogeneous biosensingHerein we combine the exonuclease ?(Exo ?)-catalyzed release of a Zn²⁺-ligation DNAzyme(ZNAzyme)with the ZNAzyme-driven strand displacement reaction(SDR)to successfully develop a novel homogeneous colorimetric biosensing method for kanamycin(Kana)antibiotic detection.The base sequences of ZNAzyme the Kana-aptamer are first designed in a hairpin DNA.After its highly specific biorecognition toward target analytes,the enzymatic reaction of Exo ? will induce the target recycling and simultaneous release of the ZNAzyme-containing strand.Then,the ZNAzyme can catalyze the ligation of two oligonucleotides into a long DNA chain and thus cause a SDR and the aggregation of gold nanoparticles(Au NPs)labeled by two linker DNAs.Meanwhile,the ZNAzyme will release to participate in the catalytic cycling reaction again.Due to the aggregation of Au NPs for colorimetric signal transduction,and the Exo ? and SDR-assisted dual signal amplification,this method shows a wide linear range of five orders of magnitude and a very low detection limit down to 8.1 fg m L^-1.In addition,it also has excellent selectivity,repeatability and reliability,and well excludes many inherent disadvantages involving in conventional chromatographic and biosensing methods.Thus,this method shows a great potential for practical applications.3.Enzyme-assisted target recycling and G-DNAzyme dynamic assembly for homogeneous chemiluminescence biosensingBy combining the synergistically catalytic target recycling of Exo ? andMNAzyme with the dynamic assembly of G-DNAzyme by hybridization chain reaction(HCR),this work successfully develops a homogeneous chemiluminescence biosensing method for the highly sensitive detection of kanamycin.When the S1strand containing the functional sequence of the MNAzyme hybridizes with the Kana-aptamer strand of S2 to form a DNA duplex through the base complementation reaction,the catalytic activity of MNAzyme can be well inhibited.Once Kana exits,the specific biorecognition reaction between Kana and its aptamer can lead to the formation of the S2/Kana complex.Meanwhile,the released MNAzyme strand can be used to capture of its hairpin substrate DNA of H1 which is modified on streptavidin(SA)by the specific avidin-biotin interaction.In the presence of Mg²⁺,the MNAzyme can catalytize the cleavage of H1 into two segments of SA/L1 and L2.So the free L2can bind with S2/Kana to form the S2/Kana/L2 complex and then induce the enzymatic reaction of Exo ? for realizing the target recycling.The SA/H1 complex can be used to initiate the HCR between the two hairpin DNAs of H2 and H3 and thus produce a large number of G-DNAzyme structures neatly arranging on the two sides of the formed DNA duplexes.After combining hemin,these G-DNAzymes can be used to catalyze the chemiluminescence of luminol for realizing the signal transduction of the method.Under optimal conditions,the method has a wide linear range of 6 orders of magnitude with a detection limit low to 7.2 fg m L^-1.More interesting,the introduction of SA in this method not only increases the local concentration of L1,but also improves the molecule diffusion rate in homogeneous solution.So the reaction rate of HCR within a given time was efficiently improved to achieve the purpose of signal amplification.Therefore,this method has a great potential for practical applications.