Research On Biosensing Technology Based On Upconversion Nanoparticles And Other Novel Nanomaterials

Biosensing technology has attracted increasing attention and holds great potential in practical analysis and bionanomedicine because of many advantages such as good selectivity, short analysis time, low-cost, and live online analysis. Recently, nanomaterials have been widely used in many fields due to their intriguing properties, such as their quantum size effect, surface effect, small size effect and macroscopic quantum tunneling effect, and nanomaterials exhibit a series of unique properties in optic, mechanics, magnetics, and electrics. It has greatly promoted the rapid development of bioscience, nanoscience and biomedicine by combining nanotechnology with biosensing technology. This doctoral thesis developed a series of new biosensing methods for sensitive, simple and low-cost detection of phospholipase D(PLD), m RNA and enzyme activity which were based on upconversion nanoparticles(UCNPs), cobalt oxyhydroxide(Co OOH) nanoflakes and gold nanoclusters(Au NCs), and also realized the functions of biosensing, imaging diagnosis and synergistic therapy for cancer. The detailed contents are described as follows:In Chapter 2, PLD is a critical component of intracellular signal transduction and has been implicated in many important biological processes. It has been observed that there are abnormalities in PLD expression in many human cancers, and PLD is thus recognized as a potential diagnostic biomarker as well as a target for drug discovery. We developed a phospholipid-modified nanoprobe for ratiometric upconversion fluorescence(UCF) sensing and bioimaging of PLD activity. The nanoprobe could be synthesized by a facile one-step self-assembly of a phospholipid monolayer composed of poly(ethylene glycol) phospholipid and rhodamine B-labeled phospholipid on the surface of UCNPs. The fluorescence resonance energy transfer(FRET) process from the UCF emission at 540 nm of the UCNPs to the absorbance of the rhodamine B occured in the nanoprobe. The PLD-mediated hydrolysis of the phosphodiester bond made rhodamine B apart from the UCNP surface, leading to the inhibition of FRET. Using the unaffected UCF emission at 655 nm as an internal standard, the nanoprobe can be used for ratiometric UCF detection of PLD activity with high sensitivity and selectivity. The PLD activity in cell lysates was also determined by the nanoprobe, confirming that PLD activity in a breast cancer cell was at least 7-fold higher than in normal cell. Moreover, the nanoprobe has been successfully applied to monitoring PLD activity in living cells by UCF bioimaging. The results revealed that the nanoprobe provided a simple, sensitive, and robust platform for point-of-care diagnostics and drug screening in biomedical applications.In Chapter 3, ascorbic acid(AA), a potent antioxidant readily scavenging reactive species, is a crucial micronutrient involved in many biochemical processes. We developed a Co OOH-modified upconversion nanosystem for fluorescence sensing of AA activity in human plasma. The nanosystem consisted of UCNPs, which served as energy donors, and Co OOH nanoflakes formed on the surface of UCNPs, which acted as efficient energy acceptors. The FRET process from UCNPs to the absorbance of Co OOH nanoflakes occurred in the nanosystem. The AA-mediated specific redox reaction reduced Co OOH into Co2+, leading to the inhibition of FRET, and resulting in the recovery of upconversion emission spectra. The nanosystem can be used for sensing AA activity with sensitivity and selectivity. Moreover, due to the minimizing background interference provided by UCNPs, the nanosystem has been applied to monitoring AA levels in human plasma sample with satisfactory results. The proposed approach may potentially provide an analytical platform for research and clinical diagnosis of AA related diseases.In Chapter 4, multifunctional nanoplatform always integrates biosensing, imaging diagnosis, and therapeutic functions into a single nanocomposite, which have great important significance of biomedicine and cancer clinical diagnosis and therapy. Here, we developed a multifunctional polydopamine(PDA) modified upconversion nanosystem for intracellular m RNA detection and imaging-guided photodynamic and photothermal(PDT-PTT) synergistic therapy. The nanosysytem can be synthesized by loading hydrophobic UCNPs into silica shell in addition to positively charged hydrophilic methylene blue(MB) being introduced, and then modifying PDA shell through an in situ self-polymerization method, thus yielding core-shell-shell nanoconstruct UCNP@Si O2-MB@PDA. Because there was a significant difference in the adsorbent ability of PDA between single-stranded DNA(ss DNA) and double-stranded DNA(ds DNA), we could construct UCNP@Si O2-MB@PDA-hp DNA nanoprobe for sensing intracellular m RNA. In addition, the FRET process from the UCF emission at 655 nm of the UCNPs to the absorbance of the MB could be employed for PDT. Moreover, due to the strong near-infrared absorption and high photothermal conversion efficiency of PDA, the FRET process from the UCF emission at 808 nm of the UCNPs to the absorbance of the PDA could be employed for PTT. The results revealed that the developed multifunctional nanosystem could integrate biosensing, imaging diagnostics and synergistic therapy, which providing a new strategy for the design of novel multifunctional nanoconstructs for biomedical applications.In Chapter 5, phosphorylation of nucleic acids with 5’-OH termini catalyzed by polynucleotide kinase(PNK) is an inevitable process and has been implicated in many important cellular events. Here, we found for the first time that there was a significant difference in the adsorbent ability of Co OOH nanoflakes between ss DNA and ds DNA, which resulted in the fluorescent dye-labeled ds DNA still retaining strong fluorescence emission, while the fluorescence signal of ss DNA was significantly quenched by Co OOH nanoflakes. Based on this discovery, we developed a Co OOH nanoflake-based nanoprobe for the fluorescence sensing of T4 PNK activity and its inhibition by combining it with λ exonuclease cleavage reaction. In the presence of T4 PNK, dye-labeled ds DNA was phosphorylated and then cleaved by λ exonuclease to generate ss DNA, which could adsorb on the Co OOH nanoflakes and whose fluorescence was quenched by Co OOH nanoflakes. Due to the high quenching property of Co OOH nanoflakes as an efficient energy acceptor, a sensitive and selective sensing approach with satisfactory performance for T4 PNK sensing in a complex biological matrix has been successfully constructed and applied to the screening of inhibitors. The developed approach may potentially provide a new platform for further research, clinical diagnosis, and drug discovery of nucleotide kinase related diseases.In Chapter 6, 2,4,6-trinitrotoluen(TNT) is a well-known explosive with significant effects on the environment and human health. We developed a glutathione-capped gold nanoclusters(GSH-Au NCs) nanoprobe for sensitive fluorescence sensing of TNT in real environment sample. Because of the strong charge interaction between the electron-rich amino group of glutathione(donor) and the electron-deficient aromatic ring of TNT(acceptor), it could form Meisenheimer complex between GSH-Au NCs and TNT, and the fluorescence of GSH-Au NCs was quenched by FRET process and aggregation. A sensitive and selective sensing approach with satisfactory performance for TNT in real environment sample has been successfully constructed.