Sensitive Single Molecule Technology For Bioassay Based On Quantum Dots

In this paper the novel single particle detection techniques were developed based on the unique optical properties and blinking behaviors of fluorescent quantum dots(QDs) with fluorescence correlation spectroscopy(FCS) and total internal reflection fluorescence microscopy(TIRFM) using QDs as fluorescent probes.The main work is as follows: An aptamer-based single particle technique was developed for the sensitive and specific detection of thrombin in human serum based on fluorescence correlation spectroscopy(FCS) using quantum dots(QDs) as the fluorescent probes and aptamer as molecular recognition unit for thrombin. The measurement principle is that, the determined characteristic diffusion times of QDs by FCS are positively proportional to its sizes, and self-assembly of aptamer-modified QDs induced by thrombin lead to the linear increase of diffusion time with thrombin concentration. Firstly, we investigated the diffusion and blinking behaviors of QDs in a reaction by FCS and TIRFM imaging system, and the experimental results documented that the aptamer-modified QDs were bound together with “one-by-one” structure induced by aptamer-recognized thrombin. And then, the assay conditions were optimized in order to achieve the best sensitivity and specificity. Meanwhile, in the preparation of aptamer-modified QDs probes the brightness per particles(BPP) characterization was developed to assess the effects of different purification methods on their optical properties of QDs or its bioconjugates. Under the optimized condition, the linear concentration range of this method is from 5.0 nM to 500 n M thrombin, and the limit of detection is 2.6 nM. Finally, this method was successfully applied to homogeneous determination of thrombin in human serum samples. Furthermore, BPP characterization on QDs based on FCS were developed to evaluate the adverse effect of different purification methods on the fluorescence properties of QDs and its bioconjugates including size exclusion chromatography(SEC) and ultrafiltration separation. When QDs were used to label aptamer, it is essential to remove excess aptamer using purification procedure similar to the labeling procedures of fluorophores. However, it was found in their BPP characterizations that ultrafiltration purification method can result in the reduction of QD’s PL intensity and stability due to the possible surface deterioration, which is greatly different to SEC.The blinking is characteristic of individual QDs emitter. However, the blinking property disappears with self-aggregate of QDs. A novel single particle counting technique based on the blinking change was developed to detect the thrombin with TIRFM. In the bioassay, when the added thrombin react with the aptamer modified in the surface of QDs, the QDs gradually aggregate each other, which results in that blinking phenomena of QDs vanish and the ratio of non-blinking dots to total dots(α value) increase with concentration of thrombin. The α value change can reflect the content of thrombin in reaction solution. The effect of modification ratio of aptamer to QDs, concentration of fluorescent probes on the sensitivity of the method was investigated and optimized. And fluorescence lifetime and TEM characterization also confirmed the self-aggregate of QDs with thrombin. And under the optimal conditions a good linear relationship between α value and concentration of thrombin was constructed, the detection linear range is from 5 nM to 100 n M and the detection limit is 4.2 nM. This analysis method can be successfully applied in the detection of thrombin. We hope that the established method can extend to other detection of more target molecules.