| Objective:Tumor associated DNA exists in the patient's blood,saliva,urine,and other secretions.Compared with the normal DNA,these DNA usually occur base mutations,and detection of these base mutations contribute to tumor screening and detection of tumor distant metastasis.Alpha thrombin is a kind of serine protease which has a dual function of blood coagulation and anticoagulation.Its abnormal expression in the body is directly related to the occurrence of thrombus diseases.However,current method for alpha thrombin operation detection has these problems,such as complex operation,expensive and low sensitivity.Based on associative toehold activation concatemer induced catalyzed hairpin assembly amplification,target DNA and human alpha thrombin were detected in this study.Feasibility,sensitivity and specificity of this method were analyzed.Methods:1.The formation of associative toehold activation concatemer: H1 can be opened in the presence of target molecule,the newly exposed sequence of H1 opens H2 to form a H1:H2 complex,this complex further opens H1 for next circulation.Thus,numerous HATA products can be produced.However,in the absence of target molecule,the two hairpins do not initially interact with each other.2.Catalyzed hairpin assembly reaction: The free and separated sequence of associative toehold activation concatemer can serve as the trigger of CHA.Because one target molecule can produce an associative toehold activation concatemer,which has numerous triggers for CHA,the first step of signal amplification can be realized.3.Mimic horseradish peroxidase reaction and further signal amplification: The production of CHA reaction,known as G-quadruplex DNAzyme,has the mimic horseradish peroxidase activity.It can dramatically enhance the catalytic ability of hemin,thus can realize the further signal amplification.4.Feasibility analysis: Native polyacrylamide gel electrophoresis and ultraviolet visible spectrophotometer(Uv-vis spectrophotometer)were used to characterize feasibility of this method.5.Experimental conditions optimization and performance evaluation: the complementary length of H1 with target DNA,H3 concentration,reaction time and temperature of CHA were optimized;Sensitivity and specificity of this biosensor were evaluated in detail.Results:1.Native polyacrylamide gel electrophoresis showed that the separated branch integrated DNA reaction and CHA were feasible.Uv-vis spectrophotometer showed that CHA were triggered by the strands on the associative toehold activation concatemer.2.Target molecules triggered signal amplification system,and a class of G-quadruplex DNAzyme were produced,which could catalytic substrate and the characteristic absorption peak was at 418 nm.The linear relationship between the absorption intensity and the log of target DNA concentrations was explored.3.Performance of colorimetric biosensor based on branch integrated DNA reaction and CHA: linear range for target DNA is 1.0 p M-75 n M;detection limit is 0.14 p M(S/N=3);linear range for human alpha-thrombin is 1.0 p M-2.5 n M;detection limit is 0.68 p M(S/N=3);It has high single-base mutation recognition ability.Conclusions:Based on associative toehold activation concatemer induced CHA,a novel colorimetric biosensor was developed for detecting target DNA and human alpha-thrombin.In this reaction system,the sensitivity of colorimetric reaction had been improved significantly.Besides,proximal mismatch identification showed high specificity and single-base mismatch recognition performance.Compared with traditional colorimetric experiment,this experiment had low detection limit,high specificity,easy operation,without rinse steps,etc.It has potential in clinical biomedical research,tumor markers detection,and application in forensic tests. |
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