Quantitative Detection Of Single Molecules Within A Microfluidic Chip-based Single-cell Glucose

Glucose plays an essential role in all processes related to living cells. It providesthe necessary energy for living cells to maintain a normal operation of body. Undernormal circumstances, cytosolic glucose concentration maintains homeostasis.However, with the change of external environment, such as the presence of somestimulants, the amount of the glucose entering into the cell greatly decreases. Then itpromotes intracellular glucose metabolism, which induces oxidative stress and affectsthe signal transduction and gene expression. Therefore, not only the conventionalcontent detection of glucose is very important, but also the detection of lowconcentration of glucose has also important biological significance. However, veryfew studies have been reported about the latter.Currently intracellular glucose concentrations in cell population are researchedextensively. The acquired average results mask the cellular heterogeneity. Since thesize of a cell is too small, it’s difficult to achieve single-cell operation for traditionalanalysis methods. Microfluidic chip is widely used in single cell analysis due to itscharacteristics of miniaturization, integration and automation. The micro-andnano-scale can reduce the dilution of the cell contents, so it’s suitable forhigh-sensitivity detection. In addition, intracellular glucose imaging analysis usingfluorescent probes has realized visualization, but it can not analyse intracellularglucose quantitatively limited by the complicated synthesis or low sensitivity.This paper developed a new approach to analyse glucose quantitatively in asingle cell based on microfluidic chip and single molecule detection technology,which has potential value in the early diagnosis of metabolic disease. This workmainly includes the following two parts:In the chapter one, we gave a simple introduction about the biologicalsignificance of glucose detection. Then we summarized the current research status ofglucose sensors, the concept and composition of microfluidic analysis system, and itsadvantages in single cell analysis. In the chapter two, given that traditional bulk cellular analysis can not reflect thereal content of individual intracellular glucose and the method to detect lowconcentration of glucose is rare, we constructed a microfluidic chip-single moleculefluorescence imaging analysis system, which can achieve cell feeding, single cellcapture, fluorescence labeling and intracellular content detection. This system wascomposed of gravity-driven component, microfluidic chip and total internal reflectionfluorescence microscopy. Microwell was designed in the chip channel to capturesingle cell. The glucose was released from the cell after the introduction of lysis buffer.It was captured by the chip substrate and then labeled by quantum dots (QDs) withunique photophysical properties. The evanescent wave created by total internalreflection illuminated the sample, improving the signal to noise ratio greatly.Ultimately a quantitative analysis of glucose within a single cell was achieved.