Research On Biocolorimetric Sensors Based On Functional Nucleic Acid Molecules And Metal-based Nanomaterials

In the field of analytical chemistry and sensors,colorimetry has a series of characteristics:simple,fast,low-cost,low requirements for equipment,visual detection,good accuracy,etc.It has obvious advantages in point-of-care detections and attracted wide attentions from researchers.With the development of various nanomaterials,sensing strategies,biotechnologies,instruments and equipments,colorimetric sensing has been rapidly developed.However,the targets of traditional colorimetric sensors are mostly concentrated in DNAs,metal ions,small molecules,etc.,and the sensitivity of them is relatively low.In addition,based on various natural enzymes and their mimics,such as nanozymes,DNAzymes,etc.,a large number of colorimetric sensors based on cascade catalytic reactions have been reported.However,these sensors still have some shortcomings.One of the problems of them is that they are often affected by inherent intermediate products in the samples,which can lead to obvious positive deviations in their detection results.Currently,nanomaterials used in colorimetric sensors can be mainly divided into two categories,one is plasmonic metal nanomaterials with localized surface plasmon resonance(LSPR),and the other is nanomaterials with enzyme-like activities.They are widely used in colorimetric sensing.In addition,as a kind of biological materials,DNA has many advantages such as low cost,editability and target recognition function,etc.The combination of DNA with nanomaterials can often endow biosensors with superior performance and specific functions.In view of the above-mentioned problems of low sensitivity and limited target range of colorimetric sensors as well as that the enzyme cascade-based colorimetric sensors are easily interfered by inherent intermediate products in samples,in this thesis,we focused on the development of colorimetric sensors with superior analytical performance through the combination of DNA and nanomaterials.The following two works have been carried out:1.A facile and sensitive colorimetric detection method was firstly reported for RNase A activity detection based on the target-regulated protection effect of chimeric DNA probes on the salt-induced aggregation of plasmonic gold nanoparticles.A chimeric DNA-RNA probe(r U)was designed as the substrate for RNase A,which containing a single uracil base as the recognition site and was much cheaper than RNA.Without the addition of RNase A,the r U probe would form stable duplex with the complementary DNA(Cr U)and the double-stranded DNA could not be adsorbed to the surface of Au NPs.On the contrary,when the target was added,the r U probe could be recognized by RNase A and hydrolyzed to generate two DNA fragments.The split DNA fragments could not hybridize with Cr U.Thus,the two fragments and the Cr U would both exist as single-strand and absorb on the surface of Au NPs rapidly due to the presence of exposed bases,which could keep the dispersion of Au NPs in high concentration of salt.By measuring the change of color and absorption spectra induced by plasmonic Au NPs aggregation,the activity of the RNase A could be quantitatively detected.In this sensing system,a single RNase A could efficiently identify and hydrolyze multiple substrate chains.Thus,high sensitivity could be obtained without additional signal amplification process.The constructed sensor had a wide linear range(1.5-1000 pg/?L)and the detection limit was 1.0 pg/?L(S/N=3).In addition,this colorimetric sensor for RNase A activity allowed the detection of targets by visual method and also possessed the advantages of low cost,simple operation,rapid response and low biological toxicity.This work developed a simple and highly sensitive colorimetric RNase A activity detection method for the first time,which also maintained the advantages of the Au NPs-Na Cl colorimetric system and might be beneficial for the development of point-of-care RNase A activity detection methods.2.In order to solve the problem that enzyme cascade-based sensors were easily interfered by inherent intermediate products,inspired by the dual-input response character of the“AND”logic gate,a novel colorimetric enzyme cascade sensor based on“AND”logic-controlled activation of peroxidase mimic catalysis system was reported for the first time,which had excellent performance against interference from inherent intermediate product.Considering the acid-degradable property of copper peroxide nanodots(CPs),it was combined with glucose oxidase(GOx)for developing a GOx-CPs cascade sensing platform.With the additive of glucose,the glucose was catalysed by GOx and generate gluconic acid(H⁺)and H₂O₂.Thus,the decrease of p H value would lead to CPs decomposition and cause the release of H₂O₂ and Cu²⁺.The released Cu²⁺would form G4-Cu²⁺with the G4 DNA and further work as peroxidase mimic,which could utilize H₂O₂ from both CPs decomposition and glucose oxidation for oxidizing the colorimetric substrate TMB.Thus,by measuring the absorption spectra of oxidized TMB,qualitative glucose detection could be realized.Since a novel“AND”logic which triggered by sole analyte for activating the peroxidase mimic sensing system was applied and the inherent intermediate product was only the sole input,the proposed GOx-CPs cascade sensing system might have more accurate detection signal than the traditional enzyme cascade catalytic system according to the“AND”logic.The experimental results exhibited that the proposed strategy possessed a wide linear range(20-1000?M)and a low detection limit(8.34?M)for glucose detection.More importantly,the system could effectively reduce the interference from inherently existed intermediate product(H₂O₂)for glucose detection.Compared with classical enzyme cascade detection method(glucose oxidase-horseradish peroxidase,GOx-HRP),this method had higher detection accuracy for glucose in samples containing H₂O₂,which exhibited the feasibility of applying the“AND”logic to reduce interference from inherent intermediate product.At the same time,the strategy also could be expanded.For example,other targets that can produce acid and H₂O₂ can be combined with CPs to develop sensors.In addition,the catalytic products of other enzymes or enzyme mimics can also be used as inputs to construct more logic-based colorimetric cascade sensors,which is beneficial for the application of enzyme mimics and logic gates in sensors.