Two-Photon Excitation Fluorescence Resonance Energy Transfer And Its Bio-analytical Applications

Fluorescence resonance energy transfer (FRET) is considered as a sensitive and reliable "ruler" over distances of 10-100 A and has broad applications in studying interactions of biological macromolecules, in immunoassay, and so on. However, in the application of conventional one-photon excitation (OPE)-FRET, where the energy donor is excited with ultraviolet or visible light, autofluorescence or scattering light always arises from biomolecules upon excitation of the energy donor. In addition, the energy acceptor is often coexcited with the energy donor because of the overlap of their excitation spectra. Therefore, new energy donor-acceptor pairs are desired to overcome the problem. Recently, two-photon excitation (TPE) has attracted a lot of attention in biological applications. In such a nonlinear optical process, two low-energy photons are simultaneously absorbed to reach the excited state. TP excitable materials can be excited in IR region to give emission in the visible region. Under TP excitation with IR light, the autofluorescence of biomolecules and the scattered excitation light could be eliminated, and the coexcitation of energy acceptor with energy donor could be avoided. Besides, the IR light causes much less photobleaching off the focal plane and less photodamage to biological samples. Therefore, TP excitable fluorophores would be a promising alternative as the FRET donor. TPE-FRET based bioassays would be free of interference from autofluorescence of biomolecules and the scattered excitation light in real biological systems. Owing to the advantages of TPE-FRET in complicated biological sample matrix and the originality of such idea, in this dissertation, several TPE-FRET models had been constructed with TP excitable small organic molecules and QDs as donors to demonstrate the abilities and advantages of TPE-FRET in complicated biological sample matrix.1. A FRET model using two-photon excitable small organic molecule trans-4-(N-2-hydroxyethyl-N-ethyl amino)-4’-(dimethyl amino) stilbene (DMAHAS) as energy donor has been constructed and tried in an assay for avidin. In the FRET model, biotin was conjugated to the FRET donor, and avidin was labeled with a dark quencher DABS-C1. Binding of DABS-C1 labeled avidin to biotinylated DMAHAS resulted in the quenching of fluorescence emission of the donor, based on which a competitive assay for free avidin was established. With using such donors that are excited in IR region, it is capable of overcoming some primary shortcomings of conventional one-photon FRET methods, especially in bioassays, such as the interference from background fluorescence or scattering light. The results of this work suggest that two-photon excitable small molecules could be a promising energy donor.2. A two-photon excitable small organic molecule 2-((E)-4-(dimethylamino) styryl)-5-((E)-4-aminostyrul) terephthalonitrile (abbreviated as TP-NH2) with large two-photon absorption cross section and competitive fluorescence quantum yield was prepared. Using the TP-NH2 molecule as an energy donor, a TPE-FRET based homogeneous immunoassay method was proposed. The donor and the acceptor (DABS-C1) were labeled to bovine serum albumin (BSA) separately, and anti-BSA protein was determined by employing an antibody bridging assay scheme. Rabbit anti-BSA serum containing other biomolecules was intentionally used as the sample to introduce interference. A parallel assay was performed using the traditional one-photon excitation FRET model, which failed to carry out quantitative determination due to the serious background luminescence arising from those biomolecules in the sample. The TPE-FRET model showed its strong ability to overcome the problem of autofluorescence and provided satisfying analytical performance. Quite good sensitivity and wide linear range (0.05-2.5 nM) for anti-BSA protein was obtained. The results of this work suggest that TPE-FRET could be a promising technique for homogeneous assays excluding separation steps, especially in complicated biological sample matrixes.3. The newly emerging TPE-FRET technique was extended to the determination of oligonucleotide. A new TPE molecule with favorable two photon action cross section was synthesized [2-(2,5-bis(4-(dimethylamino)styryl)-1H-pyrrol-1-yl)acetic acid, abbreviated as TP-COOH], with the tagged reactive carboxyl group allowing facile conjugation with streptavidin (SA). Employing the TP-COOH molecule as energy donor and black hole quencher 1 (BHQ-1) as acceptor, a TPE-FRET based homogeneous competitive hybridization model was constructed via a biotin-streptavidin bridge. Through the hybridization between a biotinylated single-stranded DNA (ssDNA) and a BHQ-1-linked ssDNA, and the subsequent capture of the as-formed hybrid by TP-COOH labeled SA, the donor fluorescence was quenched due to the FRET between TP-COOH and BHQ-1. Upon the competition between a target ssDNA and the quencher-linked ssDNA toward the biotinylated oligonucleotide, the donor fluorescence was recovered in a target-dependent manner. Good linearity was obtained with the target oligonucleotide ranging from 0.08 to 1.52μM. The method was applied to spiked serum and urine samples with satisfying recoveries obtained. The results of this work verified the applicability of TPE-FRET technique in hybridization assay and confirmed the advantages of TPE-FRET in complicated matrix.4. FRET performance driven by a two-photon excitation process has been limited by the photophysical properties of organic dyes and fluorescent proteins. Considering the large two-photon action cross-section of quantum dots (QDs) and the high specificity of hairpin shaped molecular beacons (MB), a new MB driven by two-photon excitation was developed with QDs as donor,6-carboxy-X-rhodamine (ROX) as acceptor via a biotin-streptavidin bridge. The TPE-MB can successfully recognize the complementary target DNA, non-complementary target DNA, and discriminate single mismatched target DNA in assay buffer. Furthermore, the method was applied to spiked serum with satisfying recognition of complementary target DNA with no interference from background fluorescence or scattering light. The results of this work suggest that the TPE-MB can be applied to quantitative detection of complementary target DNA in biological matrix and fluorescence in situ hybridization.