Gold Nanoparticles Quenching Fluorescence Probe For Glucose Detection And Cell Imaging Based On Fluorescence Resonance Energy Transfer (fret)

Semiconductor nanoparticles (NPs) or quantum dots (QDs) have unique photophysical properties such as size-controlled fluorescence, have high fluorescence quantum yields, and stability against photobleaching, which offer significant advantages as optical labels for biosensing.The integration of nanoparticles with biomaterials yields novel hybrid nanobiomaterials of synergetic properties and functions.The new type of fluorescent nanoprobe with its special quantum size effects and small dimension effects exhibits many different optical characteristics compared to the homogeneous single-molecule or large object and has been successfully applied in the detection of biological samples and cell imaging. Recently, biological assembly of nanosensor with protein-modified nanoparticles applied in biosensing and biodetection has atrracted extensive attention. For example, QDs have been used as a wonderful material in enzyme-based biological analyses and applications. Therefore, the design of elegant new assembled nanobiosensor for realizing the analysis and determination of bioactive molecules in vivo or in vitro has become a great challenge to scientific workers.Fluorescence resonance energy transfer (FRET) is one of the most powerful and widely used fluorescence technique available for probing structure and dynamics in media. The efficiency of FRET is dependent upon donor-acceptor proximity and spectral overlap. The most used donor is fluorescein due to its high quantum yield. The use of quenching acceptors is becoming increasingly popular in FRET systems, whether the acceptor partner is fluorescent or not.As a photoluminescent quencher, gold nanoparticles (AuNPs) can ultra-efficiently quench the molecular-excitation energy in chromophore-AuNP composites. AuNPs have been of great interest because of their high extinction coefficient and a broad absorption spectrum in a visible light that is overlapped with the emission wavelength of usual energy donors. In comparison with the organic quencher, AuNPs have unique structural and optical properties, low toxicity, well biocompatibility for new applications in biosensing and molecular engineering.Glucose, an important bioactive substance, plays a prominent role in the natural growth of cells. Its lack or excess can produce detrimental influence on cellular functions. Currently, there are many references reported cancer cells have increased rates of glucose metabolism relative to normal cells.The reason remains unclear, but that cancer cells metabolize glucose extensively is generally accepted.To date, many methods available for the glucose assays have been reported, among which,fluorescence-assay method based on a competitive binding reaction between glucose oxidase(GOD), Dextran and glucose, which has advantages in terms of good selectivity and nondestructive characteristics, has been extensively used for glucose detection. However, these methods were mostly confined to the toxicity, detection limit and could not determine glucose directly in biological samples, which restricted application in cellular signal transduction and cell imaging. Hence, a design of a selective and stable fluorescent probes for glucose detection in living cells is of special interest for biochemistry. We carried out two aspects of investigation:First, a simple and effective nanoprobe based on FRET for specific detection of glucose was designed. Dextran-FITC were used as the donor, while AuNPs modified with apo-glucose oxidase (apo-GOx) acting as a quencher. The detection mechanism is based on the switching off FRET through the high specific recognition of apo-GOx to glucose. Evidences available indicated that apo-GOx is highly specific to glucose and a higher affinity of apo-GOx for glucose over Dextran. In the absence of glucose the binding of AuNPs-apo-GOx and FITC-Dextran resulted in a high FRET efficiency. In the presence of glucose, FITC-Dextran of the nanobprobe is displaced by glucose which competes with Dextran on the binding sites of apo-GOx, resulting in the fluorescence recovery of the quenched FITC. The results show that the linear range of this method is 20nM ~ 0.2μM with the detection limit as low as 5 nM, and has excellent selectivity for glucose over other sugars and most biological species present in living cells. The nanoprobe was successfully applied in cellular imaging.Second, a new-typed nanoprobe with two gold nanoparticles of different sizes was designed for glucose detection.11-MUA modified gold nanodots (LAuND) were emplyed as donors and Dex-Au-NP as quencher. Based on the competitive combination between Dextran, glucose and apo-GOx, in the presence of glucose, Dextran is displaced by glucose which competes with Dextran on the binding sites of apo-GOx, resulting in the fluorescence recovery of quenched LAuND.