Fabrication And Application Of DNA Biosensor Based On Magnetic Separation In Capillary

With the development of biotechnology,biosensors have been widely used in detection of food. Particularly, DNA biosensors are rapid, sensitive and stable. In this paper, we developed a new DNA biosensor based on magnetic separation in capillary with laser-induced fluorescence detection. Combined with the DNA sandwich hybridization and DNA aptamer technology, the biosensor has been successfully used for the qualitative and quantitative analysis of DNA, thrombin and Staphylococcus aureus(S. aureus).1. The DNA biosensor based on magnetic separation in capillary was fabricated. We assembled the device of biosensor by making use of the injection pump and laser induced-fluorescence detector. In this project, magnetic field and hydrodynamic drag force in capillary were applied to separate and the laser-induced fluorescence detector to realize the on-line detection. Two different sequences which were modified by magnetic nanoparticles and carboxy-fluorescein(FAM) respectively were used as the capture probe and the signal probe to detect target DNA. The ultraviolet–visible spectra(UV-vis), fluorescence spectra and particle size distribution were performed to characterize the combination of magnetic nanoparticles and capture probe. We also investigated the influence of bovine serum albumin. The parameters were optimized: the separation velocity was 30 ?L/min; the concentration of Bovine serum albumin was 0.8 mg/m L; the detection velocity was 150 ?L/min; the concentrations of magnetic nanoparticles and capture probe were 0.5 mg/mL and 50 nmol/L, respectively. A good linear response was obtained between the fluorescence peak area and the concentration of target DNA with a correlative coefficient of 0.994,and the limit of detection(LOD) was 2.7 fmol/L. Practically, the biosensor showed ideal specificity and stability.2. Thrombin, a typical representation of protein, was detected by the DNA biosensor based on magnetic separation in capillary. We used two aptamers of thrombin as probes for detection. The capture of thrombin by aptamer conjugated magnetic particles was demonstrated by coomassie brilliant blue. We further found that K+ and Mg2+ in buffer had a bad effect on the detection of thrombin. The incubation temperature, time and the concentration of signal probe were optimized. Under optimal conditions, the fluorescence peak area maintained a linear relationship with the concentration of thrombin with the detection limit of 9.7 nmol/L. The detection limit became 0.57 nmol/L when the reaction solution was diluted 10-folds. The biosensor has good specificity for the detection of thrombin, and the average recovery rate of thrombin from the diluted bovine serum samples was between 87.5% and 97.3%.3. S. aureus, a typical representation of pathogen, was detected by the DNA biosensor based on magnetic separation in capillary with 16 S rDNA and whole cell method, respectively. Fisrt, sequence comparison was used to select the specific DNA fragments from 16 S rDNA of S. aureus. And then we chose the capture probe and signal probe according to the DNA sandwich hybridization. The quantitative analysis of S. aureus was achieved via the detection of genome DNA which was extracted from S. aureus. The fluorescence peak area was linear with the concentration of S. aureus in the range from 57 to 560 CFU/mL, and the LOD was 42 CFU/m L. In the whole cell method, two aptamers of S. aureus were used as probes for detection. The transmission electron microscopy(TEM), fluorescence microscope and fluorescence spectra were performed to characterize the combination of S. aureus and aptamers. Under optimal conditions, the biosensor showed a wide dynamic range from 6.7 to 6.7×104 CFU/m L and the LOD was 3 CFU/m L. The proposed biosensor showed specificity on detection of S. aureus. We also performed detection on potable and tap water samples with satisfactory results.