Paper Microfluidic Chip Electrophoresis - Direct Chemiluminescence Imaging Techniques Linked With Human Serum Proteins

In the past twenty years, there has been a great progress in microfluidic systems for chemical and biochemical analysis. In these systems, channels with tens to hundreds of micrometers wide are fabricated by micromachining techniques including etching and molding channels into PDMS, glass, silicone, or other plastics or polymers. However, there are several demerits for them. First of all, conventional process for creating microfluidic channels requires not only expensive equipments and materials, but also complex steps. Secondly, traditional microfluidic systems require well trained healthcare providers, who are rare and valuable resource, to illuminate the data. Thus, it is not convenient for the use in the developing countries and rural areas. Paper-based microfluidic devices and other paper-based detection devices have been developed in biological assays, medical diagnosis, drug development and monitoring the quality of environment recently. Paper can be patterned into hydrophilic channels separated by hydrophobic walls printed. Compared with conventional microfluidic systems, paper-based devices are very attractive for its portability, simplicity, cheapness and microliter sample volume requirement.The analysis of proteins in human serum is of great significance in biochemistry and medical applications because protein may often serve as the indicator for the (early) diagnosis of diseases. The most powerful impetus for the proteome analysis is the discovery of proteins that change in concentration in associations with a certain biological process or disease. Based on paper, quantitative colorimetric and immunostaining detections amongst others have been widely used in paper-based systems. However, the sensitivity of these detection techniques may still be improved.In recent years, a direct chemiluminescent (CL) imaging technique was introduced in the area of biochemistry research. The CL detection based on the reaction of hydrogen peroxide and 3-aminophthalic hydrazide (luminol) is in the presence of probe, providing outstanding analytical sensitivity. However, to our knowledge, major proteins in serum can not generate a CL signal directly, and only the proteins containing metal (ions) could produce CL emission. Therefore, several CL probes have been devoted to combine with the proteins to catalyze the CL reaction, extending the detection of range. However, strict conditions are still needed in these methods, such as pH range.Salbutamol is a synthesized and selectiveβ2-adrenoceptor agonist, which is widely used in the treatment of some chronic obstructive respiratory apparatus diseases. Varieties of methods for the detection of Salbutamol have been reported in recent years. However, there are still some disadvantages for the detection of Salbutamol by these methods. In present experiments, a novel method was applied for the detection of Salbutamol by direct chemiluminescent (CL) imaging after polyacrylamide gel electrophoresis (PAGE). Haptoglobin (Hp) was performed as a probe for the Salbutamol detection.The paper includes two parts, review and study.The first chapter is part of the overview section to describe the principle of polyacrylamide gel electrophoresis, capillary electrophoresis, microfluidic chip (electrophoresis) and paper-based micro devices, application and research. Focuses on the separation and detection of proteins and Salbutamol, we also introduced domestic and foreign protein testing technology, explained the background of this thesis, and to study the meaning, purpose and research content.The second chapter is research report.First, In this thesis, the development of chemiluminescence detection method for Salbutamol after electrophoresis was established, binding of drug with albumen in human serum and plasma concentration changing with time were discussed.Second, we fabricated paper-based microfluidic electrophoresis chip. Paper-based detection devices were used to separate, detect and quantify proteins in serum. Then we optimized the factors related to separation and detection. And we compared direct CL imaging with other three staining method, evaluated Sensitivity of this method. There are several merits for this method:(1) the procedure does not require expensive instruments and complex steps; (2) it requires low sample and reagent consumption and reduces the cost of analysis; (3) it provides a sensitive method.