Immobilization of e-polylysine onto the probe surface for molecular adsorption type endotoxin detection system

摘要

Patients with renal failure become not able to expel the waste product, and they accumulate the toxic products for themselves. They therefore must use the hemodialysis to weed out the metabolic decomposition product. Hemodialysis for chronic renal failure is the most popular treatment method with artificial organs. However, hemodialysis patients must continue the treatment throughout their life, the results of long term extracorporeal dialysis, those patients develop the various complications and diseases, for example, dialysis amyloidosis etc. Dialysis amyloidosis is one of the refractory complications, and the prevention of this complication is important. Recently, endotoxin is thought to be the most likely cause of the complication. Endotoxin is one of the major cell wall components of gram-negative bacteria, and it forms the complex with proteins and lipopolysaccharide (LPS). It has various biological activities, e.g. attack of fever, when it gets mixed into human blood. In addition, it is known that large amount of endotoxin exists in living environment, and medicine is often contaminated with endotoxin. When contaminated dialyzing fluids are used to hemodialysis, above-mentioned dialysis amyloidosis is developed. Therefore, it is important that the detection and removal of endotoxin from dialyzing fluids. Until now, the measurement methods using Limulus Amebosyte Lysate (LAL) reagent were carried out as the tests for the presence of endotoxin. However, these methods include several different varieties of measurement techniques. The following are examples of them, gelatinization method, turbidimetric assay method, colorimetric assay method and fluoroscopic method. However, these techniques needed 30-60 minutes for the measurement. From these facts, they are not able to use as a "real-time endotoxin detector". The detection of endotoxin has needed to carry out immediately, for that reason, a new "real-time" detection method is desired. We focused attention to adsorption reaction between E-polylysine and endotoxin. e-polylysine has the structure of straight chain molecule composed by 25-30 residues made by lysine, and it is used as an antimicrobial agent, moreover, cellulose beads with immobilized e-polylysine is used as the barrier filter for endotoxin removal. Therefore, it is expected that the endotoxin be adsorbed to the immobilized e-polylysine onto the probe. As the result of this reaction, the mass of the probe is increased, and endotoxin can be detected by using of Quartz Crystal Microbalance (QCM). In our previous research, we have already acquired the proteins immobilization technique onto Au and Si surface. In this report, the proposal of molecular adsorption type endotoxin detection system, and the immobilization of E-polylysine onto the probe are described. We use X-ray Photoelectron Spectroscopy (XPS) to confirm the E-polylysine immobilization, and the adsorptive activity of immobilized e-polylysine is measured by XPS and AFM. The purpose of this study is to bring about the realization of "Real-time endotoxin detection system".