Construction Of A Dual-mode Aptasensor Based On Branched Hybridization Chain Reaction And The Application In ATP Detections

Objective Adenosine-triphosphate（ATP）,as an unstable high-energy compound,is a direct source of energy in the body and plays a crucial role in many biological processes.In this study,a dual-mode aptasensor for ATP based on fluorescence and inductively coupled plasma-mass spectrometry（ICP-MS）detection was constructed with good sensitivity and detection accuracy,by combining quantum dots and branched hybridization chain reaction.The constructed sensor was used to quantitatively detect the ATP content in the blood of colorectal cancer patients and compared it with normal blood.Methods First,the primary amino group modified by the aptamer-containing oligonucleotide sequence was amidated and coupled with the activated carboxyl magnetic beads（MBs）,and the RNA content at 260 nm was measured using a UV spectrophotometer.Then,the four biotin-modified sequences that have been denatured at high temperature to form a hairpin structure are added to the magnetic bead aptamer solution at an appropriate ion concentration and subjected to branched hybridization chain reaction（b HCR）to form MBs-b HCR complexes were characterized using agarose gel electrophoresis.Finally,streptavidin-labeled quantum dots（QDs-SA）as fluorescent and elemental probes bind to the complex through a specific reaction between biotin-streptavidin to form the MBs-b HCR-QDs biosensor.The aptasensor and QDs-SA were characterized using Transmission Electron Microscopy（TEM）and fluorescence microscopy,respectively.To obtain the optimal experimental conditions,each condition in the experiment was optimized.First,the base arm length of the Ta sequence and the H2 sequence was optimized.The reaction time and temperature,the influencing factors of QDs,the ATP reaction time,and the digestion conditions of ICP-MS were optimized.Subsequently,under the optimal experimental conditions,the analytical performance of the sensor was examined by adding different concentrations of ATP and compared with the case where only linear HCR was introduced.At the same time,the specific of the sensor was analyzed by the specificity experiments of small molecules similar to ATP,and the ATP standard was added to the complex matrix samples to analyze the anti-interference ability of the sensor.Finally,the tissue and peripheral blood samples of colorectal adenocarcinoma patients were collected,and the ATP content was detected,and compared with the peripheral blood of normal people.Results According to the results of TEM and fluorescence microscopy,it is concluded that the MBs-b HCR-QDs biosensor has been constructed.After optimizing the experimental conditions,the optimal sequence is H2;the optimal concentration of Ta sequence is 7.2nmol·L^-1and Poly The optimal T arm length is 10 T bases;the optimal time for the amidation reaction between MBs and Ta is 8 h;the optimal concentration ratio of Ta:H1:H2:H3:H4 in b HCR is 2:5:5:10,The optimum time of hybridization reaction is 2h,and the optimum temperature is 37°C;the optimum concentration of QDs is 5nmol·L^-1,and the optimum time is 1h;the optimum time of ATP reaction is 2h,and the optimum time of ICP-MS is 2h.The digestion solution was 1 mol·L^-1HNO₃solution for 30 minutes.The analysis performance of the sensor shows that when b HCR is used as signal amplification when the ATP concentration is in the range of 0.01 nmol·L^-1-1.5 nmol·L^-1,the linear relationship obtained by fluorescence detection is y=1227*x+226.9,the detection limit is 8.89 pmol·L^-1;the linear relationship obtained by ICP-MS detection is y=3433996*x+127151,and the detection limit is 0.026 pmol·L^-1.While using one-dimensional linear HCR as signal amplification,when the ATP concentration is 10 nmol·L^-1-200 nmol·L^-1,the linear relationship obtained by fluorescence detection is y=4.278*x+191.9,and the detection limit is 2.549 nmol·L^-1;the linear relationship obtained by ICP-MS detection was y=12409*x+325063,and the detection limit was 0.0072 nmol·L^-1.At the same time,in the specificity verification,it can be seen that the enhancement of the fluorescence signal after adding ATP is significantly different from the other three solutions and the blank solution,and the ATP recovery rate in the real serum samples is distributed between 95-106%.Finally,the peripheral blood of CRC patients was tested for ATP,and compared with the peripheral blood of normal people,it was concluded that the ATP content of tumor patients was significantly higher than that of normal.Conclusion In this study,the specific binding of the aptamer to ATP will cause a change in conformation.Magnetic beads are added to bind the DNA sequence containing the aptamer for adsorption and separation,and the signal is amplified by branched hybridization chain reaction to complete the exponential amplification of the signal.Finally,the quantum dots as fluorescent and element-labeled probes were combined with the DNA polymers through the biotin-streptavidin reaction,and the combination of fluorescence and ICP-MS detection confirmed each other.Successfully constructed a simple and rapid reaction,no enzymes involved,high sensitivity and specificity based on b HCR and QDs labeling dual-mode ATP detection system.