| Research purposes: Blood is the most common and informative biological material in forensic cases.Analyzing blood samples can provide strong evidence for case detection.However,due to the large volume of analytical instruments,expensive equipment and complex sample preprocessing,it is difficult to achieve convenient blood sample analysis.In order to improve the efficiency of case detection and reduce the analysis time,it is urgent to establish an analysis method for blood samples instant and rapid detection.In recent years,fluorescent adaptor sensing technology has been widely used in disease diagnosis,drug delivery,detection of trace toxins and pathogenic bacteria due to its high sensitivity,strong specificity,fast analysis speed and low cost.Based on this,this study aims to establish a rapid detection method for forensic blood samples using fluorescent aptamer sensing technology,and evaluate its application value in forensic medicine practice.Research methods:(1)Rapid detection of 17β-estradiol to identify blood of female origin in forensic blood samples based on carbon quantum dot aptamer(CQDsAPT)nanoprobe.In this work,we designed a green synthetic fluorescent sensor method.Carbon quantum dots(CQDs)were synthesized by one-pot hydrothermal method using shaddock peel as raw material,and CQDsAPT nanoprobes were formed after modification by aptamers.Due to the adsorption between aptamer and CQDs,the fluorescence signal of the system will be quenched when the aptamer and CQDs form the probe complex of CQDs-APT.After adding the target 17β-estradiol,the aptamer will be unadsorbated from CQDs and the fluorescence of CQDs will recover.In addition,we compared the ability of traditional DNA typing technology and fluorescent aptamer sensing to distinguish the sex of blood samples in the presence of PCR inhibitors by simulating blood samples from complex vectors.(2)Simultaneous and rapid detection of mi R-199 a and mi R-499 in forensic blood samples by dual-emission fluorescence sensing probe based on CdTe quantum dots.In this work,we first synthesized CdTe fluorescent quantum dots capped with beta-cyclodextrin,and then performed simultaneous detection of acute myocardial infarction(AMI)related micro RNAs(mi R-199 a and mi R-499)on the basis of T7 Exo-assisted target recycling amplification.As the recognition part of the probe,aptamers can specifically recognize and bind target micro RNAs.In addition,we not only further evaluated the sensitivity and selectivity of the probe,but also applied the developed dual-emission probe to the detection of micro RNAs in blood samples to evaluate its effectiveness in actual detection.Research results:(1)Compared with traditional synthesis methods,carbon quantum dots synthesized from shaddock peel have advantages of simple operation,green synthesis and low cost.By combining fluorescent CQDs with DNA sequence that can specifically recognize 17β-estradiol,CQDs-APT nanoprobe is formed to form a fluorescent aptamer sensor,which can detect17β-estradiol in the dynamic concentration range of 0.025 ng/m L to 1ng/m L in 10 minutes.In addition,fluorescent aptamer sensor technology can accurately distinguish female blood samples with high 17β-estradiol content,showing excellent female sample discrimination ability for blood samples with a certain concentration of PCR inhibitors that make all gene loci lost and can not be determined by DNA typing technology.(2)The fluorescence probe constructed by CdTe quantum dots has strong fluorescence characteristics,and has two emission peaks of 565 nm and 644 nm at the excitation wavelength of 370 nm.Under optimal experimental conditions,a dual-emission fluorescent probe was constructed to simultaneously detect mi R-199 a and mi R-499 in the dynamic range from 0.1 p M to 100 n M in 30-50 minutes.The multi-group controlled experiment proved that there was almost no cross-reaction between multiple targets of the dual-emission probe,which verified the reliability of the probe for simultaneous determination of mi R-199 a and mi R-499.Moreover,the difference between the experimental detection value of the probe and the actual added value is small,and the recovery rate is 81.3%～109.6% in blood sample detection.Compared with traditional detection methods,the method of simultaneous detection of multiple micro RNAs using dual-emission fluorescent probes has higher sensitivity,simpler operation,and shorter detection time.Conculsion: In this study,a rapid detection method of forensic blood samples was successfully established based on fluorescence aptamer sensing technology.Compared with the traditional analysis methods,this method greatly improves the work efficiency of forensic workers,not only lays a foundation for the development of future instant and rapid detection methods,but also makes the study of complex forensic cases possible.There are 17 pictures,11 tables and 116. |
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