Studies On The Precision Spectroscopy Of Biological Coenzymes Using Novel Silver Nano-Materials

Biological coenzymes participate in many intracellular redox processes, including energy metabolism, mitochondrial function, gene expression, calcium homeostasis, cell division, cell apoptosis, and carcinogenesis. Due to the important roles that biological coenzymes play in redox processes, it is significant to further study the sensitive sensors that monitor them. Thus far, various efficient and reproducible conventional methods, such as high performance liquid chromatography (HPLC), gas chromatography coupled to mass spectrometry (GC-MS), capillary electrochromatography (CE), and enzymatic cycling assay, have been developed to monitor the level of biological coenzymes. Although those biochemical methods have high detection sensitivity, they have drawbacks:high cost, high time-consumption, tedious and destructive sample preparation, all of which may hamper the original properties of coenzymes. especially for the coenzyme molecules in biological activities.Recently, metallic nano-materials (such as, gold and silver) have attracted considerable attention because of their excellentoptical, electromagnetic, mechanical, and electrochemical catalyst properties. Therefore, the noble metal nano-materials have been widely used in the development of biosensors. In this thesis, we prepared novel silver nanoparticles/nanoclusters and studied the precision spectral characteristics between biological coenzymes and silver nano-materials via surface enhanced Raman scattering and fluorescence spectroscopy. Moreover, we further discussed the interaction mechanism between novel silver nano-materials and biological coenzymes in theoretical and experimental investigation, and some new suggestions and proposals were confirmed. The details are as follows:1. In the first part, we calculated the theoretical Raman spectra of aromatic molecules (FAD, NAD+/NADH, sodium fluorescein/fluorescein) by density functional theory (DFT). The calculation provided reasonable evidence for studying SERS spectra of aromatic molecules. We prepared PSSS-templated Ag NPs by reducing silver nitrate in one step. The named PSSS-templated Ag NPs are excellent SERS substrates, which can be used as a sensor to sensitively detect polycyclic aromatic coenzymes (FAD, NAD+/NADH) via π-π interactions. Compared with Creighton silver nanospheres, the PSSS-templated Ag NPs SERS substrates have many advantages, such as good stability, weak Raman background, and simple preparation. In addition, the PSSS-templated Ag NPs SERS substrates have good universality, which can solve problems that arise from sensitive detection of polycyclic aromatic molecules (FAD, NAD+/NADH, sodium fluorescein/fluorescein). The detection level for NAD+/NADH and sodium fluorescein/fluorescein can be as low as 5 nM or 10 pM, respectively.2. In the second part, FAD was chosen as a SERS probe to further study that π-π interactions and Van der Waals force interactions produced some influence in the signals of SERS. We synthesized two different SERS substrates: 2-phenylethanethiol modified Ag NPs andl-pentanethiol modified Ag NPs. When the polycyclic aromatic coenzyme FAD worked as a probe molecule, the interaction mechanism between the two thiols modified SERS substrates is π-π interaction and Van der Waals force interaction, respectively. The obtained results showed that the SERS spectra of FAD enhanced by π-π interactions are obviously superior to the SERS spectra enhanced via Van der Waals force interactions. In addition, the high SNR (signal-to-noise-ratio) of SERS spectra suggest that the π-π interaction mechanism is a little stronger than the Van der Waals force. However, due to strong Raman background from 2-phenylethanethiol modified SERS substrates, it can be concluded that 2-phenylethanethiol modified Ag NPs are not ideal SERS substrates to detect FAD by π-π interaction mechanism. In comparison with the 2-phenylethanethiol modified Ag NPs, PSSS-templated Ag NPs have obvious superiority.3. In the third part, we studied the spontaneous dimer phenomenon induced by π-π interactions between the biological molecule FAD and PSSS-templated Ag NPs in ultralow detection. Due to two special moieties of the FAD molecule, two Ag NPs were bridged by the flexible FAD molecule, and formed a dimer. The appearance of this dimer phenomenon can provide ideal enhancement sites (called hotspots), and those hotspots guarantee the strong SERS signal of FAD, located in the junction of dimer. The topological structure of silver dimmer was studied by atomic force microscopy (AFM) imaging. Then, we calculated the electromagnetic enhancement factor of the dimer by discrete dipole approximation (DDA). The morphology of the dimer mimics the experimental results imaged by AFM imaging. The simulation results show that the dimer can provide an enhancement factor of about 109.4. In the fourth part, we systematically studied the SERS spectra of single cyclic aromatic molecules based on PSSS-templated Ag NPs SERS substrates. The results showed that, due to n-n interactions, PSSS-templated Ag NPs SERS substrates can efficiently enhance and amplify the SERS signals of polycyclic aromatic molecules (such as FAD, NAD+/NADH, sodium fluorescein/fluorescein, Trp-Trp dipeptide), but it is very difficult to detect the SERS signals of single cyclic aromatic molecules (L-tryptophan, benzene, methylbenzene). Therefore, we proposed that π-π interactions can attenuate the Raman intensity of interacting aromatic molecules. For single cyclic aromatic molecules, due to the attenuation of the π-π interactions mechanism, the SERS signals of single cyclic aromatic molecules are hardly detected. However, for polycyclic aromatic molecules, the Raman signals of one adsorbed moiety were attenuated by the π-π interactions mechanism, and the obtained SERS signals were from other neighbored moieties. Finally, we confirmed and verified the proposal by theoretical simulation of a benzene dimer (C6H6-C6F6).5. Due to the similar structure between NAD+and NADH, the SERS spectra of NAD+ and NADH are almost identical, so it is difficult to distinguish NAD+and NADH by SERS spectra. In order to solve this problem, we synthesized and prepared fluorescent silver nanoclusters modified by the thiol molecule cysteamine. The cysteamine modified silver nanoclusters have an initial fluorescence emission located at 550 nm. The NAD+ molecules act as etching ligands to the cysteamine modified silver nanoclusters, resulting in another fluorescence emission, located at about 395 nm. We can efficiently label the NAD+ levels and NAD+/NADH ratios by measuring the dual emission intensity ratio (I395/I550).