| Metabolite plays an important role in biological systems. The basic unit of living creatures is cell. So analysis of cells metabolite can help us to understand the mechanism of biological systems under physiological and pathological stimuli responses and cell communications. Dissection of regulation and signaling mechanisms by metabolite analysis would pave the road for new windows for clinic diagnosis and treatment. Due to the complex matrix of the bio-sample, the content of disease related biomarkers are often in very low concentration. The method for their assay should be highly sensitive and selective. Conventional methods for cellular metabolite analysis are often tedious, needing long analyze time, showing low efficiency and costly. Some biomarkers can’t even be detected due to insufficient sensitivity. Microfluidic chip technology has been rapid developing in the current cell research due to high throughput and not requiring large sample volume preparation. In order to mimic in vivo microenvironment, developed microfluidic device was applied to reconstruct biological microenvironment and used for cell co-culture. Then a series of cell-based researches were carried out on chip for metabolite detection. Because of high specificity and affinity, aptamer is widely used in capture of a variety of proteins, small molecules or even cells. Functional nucleic acid (FNA) including aptamer and g-quadruplex sequences can be amplified by rolling circle amplification (RCA) and polymerase chain reaction (PCR), which enables great improvement of sensitivity. This article has studied the disease-related cell metabolites and their association with molecular mechanisms based on microfluidic platform and FNA. The four main works were as following:1) In order to realize the portable assay of thrombin, a convenient and high-throughput colorimetric assay was developed on an aptamer and microchip based device. For ultrasensitive detection of thrombin, aptamer was assembled with g-quadruplex, and then rolling circle amplification was applied to produce the multiplex G-quadruplex unit. G-quadruplex DNAzyme can catalyze the H2O2-mediated oxidation of 2,2’-azinobis (3-ethylbenzothiozoline)-6-sulfonic acid, which enhanced the visible light absorbtion at certain wavelength and changed the solution color. The application of RCA technology and G-quadruplex DNAzyme enabled a great improvement of the sensitivity. Finally, the developed method was successfully applied to detect thrombin from human plasma. The established method may be applied for other biomarkers assay, which implicated great potential in clinical diagnosis and medical investigation.2) A simple and rapid method for multiplex protein assay based on tunable aptamer and microchip electrophoresis has been developed. The mass-to-charge ratio of protein-DNA complex was depended on the charge of DNA, which was determined by the length of single-stranded DNA. For PDGF-BB, VEGF165 and thrombin assay, three tunable aptamers were designed respectively to modulate the target proteins’ electric quantity thus adjusting the electrophoretic mobility of proteins and allowing the protein molecules to be effectively separated by microchip electrophoresis. DNA of lowest molecule weight was used as internal standard to realize qualitative analysis. A non-specific DNA was applied as another internal standard to achieve the quantitative assay and to reduce interference. Finally, the proposed method was successfully applied to analyze cell secretions of PDGF-BB and VEGF165 and thrombin. This method could be applied for other cell metabolites analysis with slight alternation of the condition, especially for biomarkers assay, which might reveal the tumor development, and be helpful for drug screening and clinic diagnosis.3) An integrated microfluidic device for cell co-culture under different concentrations of oxygen and on-line qualitative and semi-quantitative analysis of secreted protein VEGF165 was developed based on functional nucleic acid schematics. The different oxygen gradient was produced by two-layer microfluidic device and continuous nitrogen flow supply. This microfluidic platform allowed investigation of distances effect on cell-to-cell communication. Besides, under 5%,15% and 20% oxygen gradient conditions, cellular morphology and proliferation as well as apoptosis were studied. Furthermore, reactive oxygen species and the VEGF 165 related genes such as HIF-1α and VEGF 165 genes were also investigated, which provided intensive study for cell-cell interaction. The results would provide new perspectives for the diagnosis and medical treatment of cervical cancer.4) A simple microfluidic device was designed to mix VEGF 165, functional nucleic acid and luminescence probe rapidly, and then applied to kinetic study of aptamer and protein. The device contained three functional parts. The application of a short U shape channel to every inlet can reduce the deviation of pressure and flow rate.40μm wide channels enabled rapid sample mixing from milliseconds to minutes. The long U shape channel in the downstream can then ensure complete sample mixing, and place observation sites for kinetic study. Different transient states in the protein-nucleic acid reaction could be monitored. Using a highly selective luminescent switch-on probe, the kinetic study between VEGF 165 and FNA was carried out in the microfluidic device.Finally, the developed method was used to detect VEGF165 from CaSki cell secretion under paclitaxel culture condition. The results would be beneficial to studies on cervical cancer biology and anti-cancer drug screening.5) Cell metabolite analysis have been extensively studied to understand the development of diseases, like cancer, and to investigate the effects of drugs. However, it is still challenging to analyze macromolecular and small molecule products from cells simultaneously. In this work, we have successfully developed a platform for cell co-culturing and determination of proteins and drug at the same time by Chip-Mass combined with fluorescence microscopy. Target protein from complex matrix is captured by specific aptamer which is grafted onto microchannel in advance, and after the second aptamer being coupled to protein, rolling circle amplification (RCA) is performed for high-sensitivity fluorescence detection. The usage of different aptamers promises the multi-protein determination in one assay. After protein determination, samples are desalted through solid phase extraction by Bond Elut Plexa cartridges in microchip (MPE), followed by ESI-Q-TOF MS analysis. This proposal is simple in set-up and operation, with the capability for rapid and accurate detection of biomarkers as well as drug metabolites, which has potential to the clininc diagnose and drug screening. |
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