The Applications Of Palladium Nanoparticles As A Novel Energy Acceptor In Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer ?FRET?, which is a highly sensitive and homogeneous assay technique, has been broadly used in studying the structure and function of biomacromolecules and quantitative detection of biomolecules. Organic dyes, fluorescent protein, quantum dots ?QDs?, upconversion nanoparticles ?UCNPs? and the recently developed carbon nanomaterials are used as fluorescence donor in the FRET system. While fluorescence acceptor mainly refers to organic dyes, carbon nanomaterials and noble metal nanoparticles such as gold and silver. In the early stage organic dyes were usually used as fluorescence acceptor. However, their low fluorescence quenching efficiency and necessary labeling process are unfavourable for the analytical performance. In recent years, carbon nanomaterials or gold and silver nanoparticles have also been found to exhibit excellent fluorescence quenching ability. Carbon nanomaterials such as carbon nanospheres, nanotubes, graphene and so on, have been widely used as fluorescence acceptor in FRET biosensors for sensitive detection of biomolecules and ions. But the introduction of surfactants or polar groups in order to improve their dispersion in water and the obvious nonspecific absorption make the analytical performance not so satisfactory. Therefore, it is very necessary to develop new energy acceptor for FRET biosensors with better analytical performance. Noble metal nanoparticles including gold and silver which have high molar extinction efficiency and relatively broad surface plasmon resonance peak, can act as effective fluorescence quenching materials towards various kinds of fluorescence donors. Considering the similar physicochemical characteristics of noble metal, we infer palladium nanoparticles ?PdNPs? also have excellent fluorescence quenching ability and can be used to construct FRET biosensors accordingly. So we investigate the fluorescence quenching ability of PdNPs towards organic dyes, graphitic-phase C₃N₄ ?g-C3N4? nanosheets and UCNPs which is hardly to be quenched. Meanwhile, we compared the fluorescence quenching ability of this novel energy acceptor with the widely used fluorescence acceptor-graphene.1. Considering the size-dependent plasmonic light absorption abilities of PdNPs, we synthesized larger PdNPs exerting strong light absorption which covers nearly the whole ultraviolet-visible range and then investigated its fluorescence quenching ability towards organic dyes. Based on the coordination interaction between PdNPs and bioprobes, such as single-stranded DNA ?ssDNA? and polypeptide molecules, organic dyes and PdNPs were brought into close proximity and the fluorescence of organic dyes was effectively quenched by PdNPs with a quenching efficiency of 97%. What is more, there were nearly no non-specific quenching caused by PdNPs, that is, the undesired quenching of fluorophores not bound to probes. After the introduction of the target ssDNA or protease into the FRET system, the specific affinity interaction between probes and target greatly weakened the coordination interaction between probes and PdNPs, and the FRET process was inhibited accompanied by the fluorescence recovery of organic dyes. The ultrasensitive detection of ssDNA and protease were both realized by this way. The linear detection range of target ssDNA is from 0.003 nM to 0.1 nM by this sensor, the sensitivity of which was greatly enhanced compared to simple mix-and-detect strategy without enzyme amplification.2. g-C₃N₄ nanosheets has recently been found to be a kind of carbon nanomaterial with high fluorescence quantum yield. In the above chapter, we have learned that PdNPs is capable of interacting with heteroatom through coordination, so g-C₃N₄ nanosheets with nitrogen-containing founctional groups on the surface have the potential to act as stabilizer for PdNPs. Thereafter we successfully synthesized well-dispersed PdNPs loaded on g-C₃N₄ nanosheets and found that the fluorescence of g-C₃N₄ nanosheets was greatly quenched by PdNPs. Considering the stable Pd-S bond formed between thiol-containing molecules and Pd, we infer that the addition of L-cysteine ?L-cys? into the nanocomposite would enlarge the distance between g-C₃N₄ nanosheet and PdNPs, which would result in the fluorescence recovery of g-C₃N₄ nanosheet. As we expected, the phenomenon happened and the quantitative detection of L-cys was realized accordingly with the linear range from 1 to 500 nM.3. UCNPs are a kind of inorganic luminescent nanomaterials which are hardly to be quenched. As the occurrence of FRET needs the distance between fluorescence donor and acceptor in the range from 1 to 10 nm, usually luminescent lanthanide ions only located on the surface of UCNPs can be effectively quenched by energy acceptors. In order to investigate the fluorescence quenching ability of PdNPs towards UCNPs, we synthesized PdNPs stabilized by 11-mercaptoundecanoic acid ?MUDA-PdNPs? with broad absorption in the visible range. The standard EDC/NHS conjugation technique was applied to label CEA aptamer onto the surface of hexanedioic acid ?HDA? modified UCNPs ?UCNPs-CEA aptamer?. Later MUDA-PdNPs were mixed with UCNPs-CEA aptamer. By use of the coordination between nucleic acid base and Pd, UCNPs were brought in close proximity to PdNPs, which resulted in the fluorescence quenching of UCNPs by PdNPs with a quenching efficiency of 85% or more. After adding CEA into the UCNPs-aptamer-PdNPs FRET system, CEA aptamer preferentially bound with CEA accompanied by its conformational change. It greatly weakened the coordination between CEA aptamer and PdNPs, which resulted in the fluorescence recovery of UCNPs, and the ultrasensitive detection of CEA was realized accordingly. In the concentration range from 2 pg/mL to 100 pg/mL, the fluorescence recovery was linear related to the concentration of CEA in buffer solution and the limit of detection was 0.8 pg/mL. The ultrasensitive detection of CEA in diluted serum was also realized.4. In order to compare the fluorescence quenching ability of PdNPs with graphene, being broadly adopted as energy acceptor, we developed a biosensor for kanamycin on the basis of FRET between UCNPs and graphene. Firstly, kanamycin aptamer was conjugated to HDA modified UCNPs. And then they were incubated with graphene. The ?-? stacking interaction between aptamer and graphene brought UCNPs and graphene in close proximity, resulting in the fluorescence quenching of UCNPs. After addition of kanamycin into the FRET system, kanamycin aptamer is more inclined to interact with kanamycin accompanied by the conformational change. In this way the ?-? stacking interaction between aptamer and graphene was weakened, and UNCPs were released from the surface of graphene, which resulted in the fluorescence recovery of UCNPs. And the detection of kanamycin in buffer and diluted serum was realized, accordingly.5. When graphene was used as fluorescence acceptor in the FRET system, the existence of obvious non-specific quenching phenomenon restricted the signal-to-noise ratio and sensitivity. While PdNPs, as a new kind of energy acceptor, with high fluorescence quenching ability and negligible nonspecific fluorescence quenching phenomenon, can be broadly used in FRET biosensors in the future.