| Cancer is one of the three killers of health,resulting in a high death rate.The medical theoretical explanation of tumor is the gene mutation of normal cells.Many factors can cause tumor which threatens human lives,once it comes into being."Three-early dos"(early detection,early diagnosis and early treatment)is the most effective way to help reduce occurrence rate and death rate of malignant tumor,namely cancer.In the early stage,content of tumor marker is very low in patients’ body.The accurate and sensitive detection of tumor is the base of early diagnosis and treatment.The method of detecting molecule tools is thus of vital importance in the early cancer treatment stage,whice has many advantages over traditional ones that will cause trauma and time waste.Thrombin participates in many important physiological and pathological processes,including the activation of the tumorigenic potential and induction malignant cells in the normal cells.The emission level of nucleotide in human body reflects to some extent proliferation condition of cancer cell.Therefore,the the trace detection of thrombin and DNA is significant in the early diagnosis of cancer.A new approach was built to amplify the signal of SPR biosensing based on Nano-particles and Enzyme circulation,which combined the advantage of nanotechnology,specific binding theory and rolling-circle replication.The amplification was shown as a quantized numerical value by the transverter of SPR biosensor.First,we explored the feasibility of instruments and amplification methods,details are as follows:The avidin was immobilized on the chip.The specific binding of biotin-avidin was used to immobilize the biotin-modified capture DNA probes,the biological response of gold nanoparticles was used to show the signal amplification at a low target DNA concentration.A clear detection signal was obtained under the optimization of the amplification method.In the second part,a method with the rolling circle amplification and bio-barcode technology was built to amplify the signal for detection of thrombin.The thiol-modified antibody is immobilized on the chip by the gold-sulfur bond.The rolling circle amplification was achieved to form a long chain.The use of bio-barcode probe was combined to the rolling circle amplification products to obtained a large mass system,this system was combined with thrombin at last,as a result,different levels of thrombin was detected indirectly through the online detection of the entire system.The feasibility of this method has been verified.This detection method exhibits excellent specificity and sensitivity towards thrombin with a detection limit of 1.0 × 10’16M.the normalized SPR intensity was a good linear fit to the logarithm of thrombin in the range from 1.0 × 10-16 to 1.0× 10-11M.The third part is based on enzymatic cycling to amplify the signal with SPR biosensor to achieve the detection of target DNA.The thiol-modified hairpin DNA was immobilized on the gold substrate,with the presence of T4 ligase and Klenow polymerase,the partily complementary target DNA was combined with the hairpin DNA to form a structure whose middle section is double-stranded.With the DNA on the magnetic beads added at the presence of polymerase and the shear enzyme,the target DNA was dissociated for the next reaction.Bio-barcode which has a complementary DNA strand was injected into the SPR instrument online,amplifying the signal once more.The detection limit is 7.8 × 10-15 M(3σ).The linear ranged from 1.0 × 10-14M to 1 × 10-12 M. |
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