Biochemical Analysis Based On Piezoelectric Electrochemistry And Chromatography With Electrochemical Detection

Developing new materials and in-situ real-time analytical methods with the help of nanobiotechnology for detecting bioactive molecules and monitoring cells can promot the development of biomedicine and other relevant fields to a large degree. In this dissertation, on the basis of the brief reviews of magnetic nanoparticles, quartz crystal microbalance (QCM) and high-performance liquid chromatography-electrochemical detection (HPLC-ECD) as well as their bioanalytic applications, we conducted a series of studies in cell analysis and detection of bioactive molecules by using QCM and HPLC-ECD, as summarized below.1. We used QCM,3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and fluorescence/optical microscopic inspections to investigate the cytotoxicity of paramagnetic selenium-ferroferric oxide (Se-Fe3O4) nanocomposites on human osteoblast-like MG-63cells. The Se-Fe3O4nanocomposites were synthesized through reduction of selenious acid by ascorbic acid in the presence of nanosized Fe3O4. We find that the Se-Fe3O4nanocomposites are able to induce cell death in a dose-dependent way, and an external magnetic field increases the cytotoxicity. The QCM technique is employed for the first time to monitor the toxicity of Se-Fe3O4on tumor cells in a real-time manner, and this simple, inexpensive and dynamic tool is promising in studying the magnetic drug-carrier systems for better understanding of the relevant magneto-chemotherapy processes and mechanisms.2. We prepared core-shell type magnetic silica nanospheres (Fe3O4@SiO2) as the drug carriers for the anticancer drug doxorubicin (DOX) and studied the cytotoxicity of doxorubicin-loaded magnetic silica composite (DOX-Fe3O4@SiO2) on human breast cancer cells (MCF-7) by dynamic QCM monitoring, cyclic voltammetry (CV) characterization, MTT assay and optical microscopic inspections. We find that the DOX-Fe3O4@SiO2are able to induce cell death in a dose-dependent way and exhibit higher cytotoxicity under external magnetic field in comparison with the free DOX, suggesting the potential of the Fe3O4@SiO2for targeting drug-delivery. The QCM sensor-based in situ/process monitoring in combination with ex situ/static analyses based on CV, MTT and optical microscopic inspections provide a reliable and informative experimental platform for investigating incubation of cells and interventions from exogenous substances.3. Hyaluronic acid coated magnetic nanoparticles (HA-MNPs) were synthesized for selectively collecting and detecting of leukemia cells (CCRF-CEM) in combination with QCM measurement. The limit of detection of8×103cells mL-1is obtained. HA-MNPs could be used to successfully extract cells from complex matrixes like human plasma samples based on the specific binding of HA with CD44receptor overexpressed on leukemia cell surfaces. The HA-MNPs-based magnetic separation combined with sensitive QCM measurement provide a simple, rapid and economical method for collecting and detecting leukemia cells, and this method may have great potential for wider applications in biomedical research and clinical diagnostics.4. A highly sensitive35MHz QCM was used to monitor in real time the adsorption of thrombin onto a gold electrode and the following binding process of solution hirudin to thrombin immobilized on the electrode. By fitting the experimental data, the average binding molar ratio of hirudin to adsorbed thrombin and the binding equilibrium constant are estimated to be1.13:1and8.75×105M-1, respectively, which are consistent with those obtained by surface plasmon resonance measurements. This method is characteristic of less reagent-consumption and high sensitivity for investigation of the target-drug interaction between biomacromolecule and small drug molecule, which is promising for wider applications in biomedical and biosensor fields.5. A new HPLC wall-jet/thin-layer amperometric electrochemical detector (ECD) was developed for simultaneous analysis of dopamine (DA) and homovanillic acid (HVA) at a glassy carbon electrode. Compared with conventional thin-layer mode and wall-jet mode, the present wall-jet/thin-layer ECD has the advantages of enhanced capture of electroactive DA and HVA on the working electrode to give enhanced responses and more convenient washing/refreshment of the working electrode surface. Under optimized conditions, the HPLC-ECD calibration curves show good linearity from0.01to100μM for DA and HVA, and the obtained limits of detection (LODs) of1.1nM for DA and0.7nM for HVA are lower than those obtained with an UV-vis detector and a commercial electrochemical detector. The balance among response-signal, signal-background and noise level is also discussed. In addition, a demethylation electrooxidation mechanism of HVA is suggested through potentiostatic bulk electrolysis, electrospray ionization-mass spectrometry, fluorescent spectrophotometry and CV studies.6. HPLC combined with a sensitive wall-jet/thin-layer amperometric electrochemical detector was developed here for simultaneous determination of guanine (G) and adenine (A), and the detection conditions were optimized. Under optimized conditions, the HPLC-ECD calibration curves show good linearity (R2>0.999) for G and A, and the obtained LODs of0.6nM for G and1.4nM for A are lower than those reported in most literatures. The method was tested on calf thymus double-strand DNA with satisfactory results. Simplicity, high sensitivity and good reproducibility of this HPLC-ECD may provide wider potential applications for flowing solution system studies in biomedical field.