Application Of Aptamer Functionalized Fluorescent Nanoprobe In Protein And Cell Sensing

The integration of signal reporter and molecular recognition is the basis for the target detection and biological labeling and imaging. Since the discovery of aptamer in 1990s, they have been developed as a new recognition molecule during the past thirty years. Their applications in various subjects have attracted extensive attention from researchers, such as chemistry, biomedicine, nanomaterials and physics. On the other hand, due to their excellent fluorescence properties, fluorescent nanomaterials such as quantum dots and metal nanoclusters as signal reporter have been widely used in biotechnology area, for example DNA detection, immunofluorescence detection and cell and animal biology.Combining the specific recognition ability of aptamer and excellent fluorescence property of fluorescent nanomaterials, we designed and constructed new types of protein and cell sensors. Additionally, we synthesized aptamer functionalized fluorescent nanoprobe for the cell-type specific imaging and the improvement of traditional western blot analysis. The major contents were described as follows.1. Aptamer-Quantum dots Conjugates-Based Competitive Electrochemical Cytosensor for the Detection of Tumor Cell A novel competitive electrochemical cytosensor was reported by using aptamer (Apt)-quantum dots (Qdots) conjugates as a platform for tumor cell recognition and detection. The complementary DNA (cDNA), aptamer and Qdots could be assembled to the gold electrode surface. When the target cells existed, they could compete with cDNA to bind with Apt-Qdots conjugates based on the specific recognition of aptamer to MUC1 protein overexpressed on the cell surface, which resulted in the denaturation of double-stranded DNA structure and the release of the Apt-Qdots conjugates from the electrode. Electrochemical stripping measurement was then employed to determine the Cd2+concentration in Qdots left at the electrode. The peak current was inversely proportional to the logarithmic value of cell concentration ranging from 1.0×102 to 1.0×106 cells mL-1 with a detection limit of 100 cells mL-1. Meanwhile, the recognition of aptamer to the target cells could be clearly observed through the strong fluorescence from Qdots. This is an example of the combination of aptamer and nanoparticles for the application of cell analysis, which is essential to cancer diagnosis and therapy.2. Aptamer-Quantum Dot Conjugate-Based Western Blot for Multiple Protein DetectionWe described an aptamer-Quantum dot (apt-Qdot) conjugate-based western blot for multiple proteins detection coupling the highly specific recognition capabilities of aptamers to the corresponding target and the tunable fluorescence imaging of Qdot to read out signal. Human a-thrombin, platelet derived growth factor (PDGF-BB) and single-stranded binding protein (SSB) were chosen as model proteins and were simultaneously detected from a single western blot with red, green and orange color. Furthermore, by the modification of Qdot with poly (ethylene glycol) (PEG) molecules and random sequence oligo blocking, the nonspecific binding of apt-Qdot conjugate was reduced obviously in complex matrix which greatly improved the specificity of the method. The target proteins could be detected even in 10-time diluted human serum with a high selectivity. This work showed the potential of apt-Qdot conjugate-based western blot in protein detection, especially in the more complex, protein-rich matrices and would open the possibility for uses in clinical laboratories.3. Binding-Induced Fluorescence Turn-on Assay Using Aptamer-Functionalized Silver Nanocluster DNA ProbesWe presented a binding-induced fluorescence turn-on assay for human α-thrombin detection. Key features of this assay include affinity binding-induced DNA hybridization and fluorescence enhancement of silver nanoclusters (Ag NCs) using guanine-rich DNA sequences.Two aptamers (Apt 15 and Apt29) were used and were modified by including additional sequence elements. A 12-n.t. sequence was used to link the first aptamer with a nanocluster nucleation sequence at the 5’end. The second aptamer was linked through a complementary sequence (12-n.t.) to a G-rich overhang at the 3’end. Binding of the two aptamer probes to the target protein initiates hybridization between the complementary linker sequences attached to each aptamer, and thereby bring the end of G-rich overhang to close proximity to Ag NCs, resulting in a significant fluorescence enhancement. This fluorescence assay is performed in a single tube, and it does not require washing or separation steps. The principle of the binding-induced DNA hybridization and fluorescence enhancement of Ag NCs can be extended to other homogenous assay applications.4. Homogeneous Competitive Fluorescent Assay for Protein Detection by Silver Nanoclusters-Aptamer AssemblyWe present here a label-free, homogeneous competitive fluorescent assay for protein detection based on the fluorescence enhancement of DNA stabilized Ag NCs by guanine-rich DNA sequences upon hybridization and the fluorescence quenching by competitive target binding. The effects of several important parameters on the specificity and sensitivity of the assay were examined, including hybridization base pairs, spacer, probe ratio and buffer conditions. Under the optimal conditions, with the use of human a-thrombin as a model, a 25 pM detection limit was obtained. This fluorescent assay is performed in a single tube, eliminating immobilization, separation, washing steps and quencher or enhancer of traditional assays. Only one aptamer was required as recognition element making this method potentially universal to a wider range of targets.5. One-pot synthesis of aptamer functionalized silver nanoclusters for cell-type specific imagingWe presented a strategy to synthesize AS 1411 functionalized Ag NCs with excellent fluorescence through a facile one-pot process. Confocal laser scanning microscopy and Z-axis scanning confirmed that the AS 1411 functionalized Ag NCs could be internalized into MCF-7 human breast cancer cells and was able to specifically stain nuclei with red color. To our surprise, 3-[4,5-Dimethylthiazol-z-yl]-2,5 diphenyltetrazolium bromide (MTT) assay demonstrated the Ag NCs were cyto-compatible and better inhibition effect than pure AS1411 to MCF-7 human breast cancer cells. Besides, a universal design of the oligonucleotide scaffold for the synthesis of Ag NCs was extended to other aptamers, i.e. Sgc8c and Mucin 1 aptamer. Due to the facile synthesis procedure and capability of specific target recognition, this fluorescent platform will potentially broaden the applications of Ag NCs in biosensing and biological imaging.