| Urine has many advantages as the ideal source of biomarkers,such as easily collection,less protein contained,and it directly reflects pathological changes of the kidneys.Thus,urinalysis is often used as a routine medical test for the screening of diseases such as cancers and urological diseases at the early stage.There are many methods for the detection of disease markers in urine,including chromatography,electrochemical methods,fluorescence spectroscopy and colourimetric methods.Among them,colourimetric methods have great potential in early screening of disease,for its advantages of sensitivity,easily operation and rapidity,and better accessibility to the grassroots.In this dissertation,the principle of target molecules detecting with nanomaterials was investigated.And it is achieved that specific detection of target molecules by using aptamer and other sensitive molecules to modify nanomaterials.Conbined with different portable colorimetric sensing systems,a variety of colorimetric sensors for detecting urinary disease markers were constructed.Which were an adenosine detection system based on aptamer and gold nanoparticles,a glucose detection system based on thiomalic acid-modified copper nanoclusters,a citric acid detection system sodium dodecyl sulfate modified silver nanoparticles,and a oxalic acid detection system bovine serum protein and dithiothreitol modified copper nanoclusters,and a simultaneous detection system based on combined enzymatic.This research was supported by the National Natural Science Foundation of China-Russian Fund for Basic Research and the Key Research and Development Project of Zhejiang Province.The main content and innovations of this dissertation are given as follows:1.A colorimetric sensor based on modified aptamer and Au NPs was proposed,combined with the bionic electronic eye,the portable and rapid detection of adenosine was achievedA colourimetric sensing method based on modified aptamers and gold nanoparticles(Au NPs)with electronic eye colourimetric system was developed for the detection of adenosine in urine,which is a colorectal cancer disease marker.The effect of the aptamer modifier on the detection was investigated,and the aptamer with biotin modification made the Au NPs more stable in high salt solution,showing a larger linear range of detection from 5.0 μM to 60.0 μM and a lower limit of detection of 0.17 μM with adenosine.The method showed good selectivity for adenosine.In addition,the adenosine assay enables the detection of adenosine in artificial urine.With a homemade electronic eye colorimetric system for portable and rapid detection of adenosine.Compared with other methods,this adenosine detection system has the advantages of suitable detection range,low cost,simple operation and rapid in situ detection,thus providing a possibility for pre-screening of colorectal cancer.2.A method based on thiomalic acid-modified copper nanoclusters as bionic enzymes is proposed for the rapid and quantitative detection of glucose in urineA colorimetric sensor design method based on thiomalic acid-modified copper nanoclusters(MSA-Cu NCs)as both a hydrogen peroxide mimetic enzyme and a chromogenic agent for the detection of glucose,a marker of nephrogenic diabetes,in urine was designed.The copper nanoclusters are aggregated by using hydrogen peroxide oxidation to break the S-Cu coordination bonds on MSA and Cu NCs,which enables the detection of hydrogen peroxide without the use of other chromogenic agents compared to other methods.By combining glucose oxidase,the detection of glucose in urine can be achieved in the range of 0.5-5 m M with a lower limit of detection of 0.12 m M.The advantage of this method is low cost operationally detectable and quantitatively detectable compared to existing commonly used urine glucose detection techniques,and therefore this assay offers another possibility for urine glucose detection.3.A sensor based on sodium dodecyl sulfate modified silver nanoparticles combined with a portable CD spectrometry i for the rapid detection of citric acid in urine was proposedA simple and sensitive colorimetric sensor based on the competitive binding of sodium dodecyl sulfate modified silver nanoparticles(SDS-Ag NPs)and citric acid to aluminium ions was developed for the detection of citric acid,a disease marker for urine stone.In this case SDS acts both as a modifier of Ag NPs to achieve specific binding to the specific molecules and as a stabiliser to avoid the use of citric acid in the synthesis of nanoparticles.After optimisation of the detection conditions the method has a good detection range and lower limit of detection in both the microplates reader and the home-made portable CD spectrometer.In addition,the selectivity of the method was verified,showing that several common substances in urine did not interfere with the detection of citric acid.Good recoveries were obtained in artificial urine samples.Thus,a colourimetric sensing method based on SDS-Ag NPs for the detection of citric acid shows good promise for application.4.A sensor based on copper nanoclusters modified with bovine serum proteins and dithiothreitol as bionic enzyme is proposed for the rapid detection of oxalic acid in urine.A sensor based on copper nanoclusters modified with bovine serum proteins and dithiothreitol(BSA-DTT-Cu NCs)as both the hydrogen peroxide-mimetic enzyme and the chromogenic agent was designed for the detection of oxalic acid,a urinary stone marker in urine.The Cu NCs were modified with BSA and DTT,which allowed the Cu NCs to remain stable for several months,and the catalytic effect of BSA-DTTCu NCs prepared from different batches was consistent for hydrogen peroxide.Compared to HRP,the Cu NCs are less sensitive to changes in p H and temperature and are more adaptable to environmental changes.By optimising the concentration of the chromogenic agent species,incubation time and temperature,the oxalic acid was detected in the range of 1-12 m M with a lower limit of detection of 0.32 m M.In addition,the method was validated in the detection of real samples,demonstrating its potential in practical assays.5.A method for simultaneous detection of oxalic acid and citric acid in urine was designed,with the integrated instrument for the pre-treatment and detection of urine samples,the method for rapid screening of urinary stone disease through urine was establishedA simultaneous assay for oxalic and citric acid in urine based on a bionic enzyme was designed.BSA-DTT-Cu NCs were used as bionic peroxidases to achieve noninterference between oxalic acid detection and citric acid detection.The detection range of oxalic acid was 1-12 m M with a detection limit of 0.18 m M,while the detection range of citric acid was 0.5-2.5 m M and detection limit was 0.11 m M.In addition,a multifunctional automatic analyser with automatic sampling,pre-processing and detection was developed to automate the extraction,enrichment and detection of urine samples.A method for rapid screening of urinary stone disease was established,with a diagnostic accuracy of 70% with simultaneous testing,which validates the potential of this sensing system for practical screening of urolithiasis. |
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