Fluorescence measurement An Avalanche Photo Diode (APD) is used to measure this fluorescence signal using a confocal microscope The signal was defined as the integrated area of the fluorescent spectral curve. The titration curve of hybridization of the DNA-GNPs with complementary strand T1 is measured in order to find the optimal condition of the efficience of quenching. Various volumes of synthesized DNA-GNPs are mixed with 2.5 μL of 100 nmol/L T1. Each of these mixtures are diluted to 50 μL with buffer A and then left for incubation at room temperature. the fluorescence signals are allowed to stay remain stable after equilibration for about 1 hour, they are then measured. For Detection of Cocaine 25 μL of DNA-GNPs, 2.5 μL of 100 nmol/L …show more content…
Step 2 : In the absence of cocaine, and upon hybridization, the fluorescence of TMR on T1 is quenched by the gold nanoparticle nearest to it. Step 3: The binding between aptamer and cocaine will now dissociate the duplex. Step 4 : After the above step the aptamer T1 will move away from GNPs, Step 5 : The fluorescence is restored In order to achieve a good quenching efficiency for the probe complex, a suitable and most appropriate concentration of DNA-GNPs has to be chosen. Hence the hybridization titration curve is measured with different volumes of DNA-GNPs which were mixed with the complementary T1.It is observed that the fluorescence signal decreases with the increase of the DNA-GNPs volume. a quenching efficiency of approximately 0.6 is obtained when DNA-GNPs volume was more than 25 μL. Therefore, we used 25 μL of DNA-GNPs per 50 μL mixture in the following experiments, and the final concentration of T1 was kept at 5 …show more content…
Varying concentrations of cocaine solution are incubated with a constant amount of DNA-GNP-DNA and T1 for about 30 min. Atlast the fluorescence intensity is measured . The recovery of fluorescence with increase of cocaine concentration is clearly observed. Qualitative and quantitative detection of cocaine in buffer A progressive Dilution strategy is applied to achieve the qualitative and quantitative detection of cocaine in buffer. Morphine is taken to be a negative control. Cocaine and morphine of concentration 100 μmol/L are taken and progressively diluted. It is diluted by the dilution factor of 2 with a buffer . And the fluorescence intensity is then measured at each and every step. In the presence of cocaine we can notice a slope on the progressive dilution curve. The presence of morphine in no way affects the rise or fall of the curve. Because of the specific binding seen between cocaine and the aptamer, a difference could clearly be seen between cocaine and morphine creating no confusion of any