|英文要約||Title: Technology Innovation Program for Small Business Innovation Research / Development of spectrometer for detection of endotoxin at a single molecule level (FY2011-FY2012) FY2011 Annual Report
(1) Endotoxin detection by other methods Endotoxin has been assayed by gelation of Limulus amebocyte lysate (LAL). For the sensitive detection, turbidity measurement and colorimetric analysis have been applied. Recently, some research groups reported that the early stage of LAL gelation is detectable by the laser scattering photometry. Another sensitive method has been introduced, that is a bioluminescence using firefly luciferase.(2) Advantage of the detection using SERS By comparing the sensitivity, and measuring time of the reported endotoxin assay methods, we confirmed advantage of the SERS technology. By that, we can overcome not only the the sensitivity but also the due time. In addition, endotoxin could be detected by a small quantity of LAL, less than 1/1000 scale of the conventional methods.(3) Data of SERS detection of endotoxin Silver-thiosulfate complex solution was mixed with aqueous LAL solution in equal volume (200 μL). The solution was dropped on a metal substrate with a diameter of 7 mm. After 3 minutes, the solution was removed and confirmed the silver nanocrystals were assembled on the metal plate. Aqueous solution of the standard endotoxin (500EU/L) was dropped on the SERS substrate. SERS spectra were measured by laser light excitation at 785 nm. At immediately after the dropping, SERS peak due to endotoxin appears at around 900 cm－1. Using a dilute LAL solution (1/1000 of the original solution), the peak was appeared at the same position. The reagent amount is dramatically low level of LAL for endotoxin assay, have been reported to date. By this SERS chip, we could evaluate 100EU/L endotoxin.For the quantitative analysis of the endotoxin, some endotoxin binding proteins, peptides were applied. Among them, lipopolysaccharide binding protein (LBP) indicated a new SERS peak due to endotoxin at around 1350 cm－1.(4) Market research for endotoxin detector We look into the commercial value of the endotoxin biosensors, the market which need endotoxin assay, the most convincing should be a blood dialysis. In Japan, the number of the patients is 290 thousands and shows an increase of 3 percent over every year. They are running dialysis 3 million times for a year in 4171 hospitals, etc. Thus, we confirmed our technology will meet the need of clients for the facile endotoxin detection.(5) Business plan Key point of the SERS technology is free from the contamination. The SERS chip could be installed into the glass capillary to eliminate the fungal or others that have positive effect on LAL. By strategic cost cutting, we are estimating a price of the SERS substrate at 500 yen per one sheet in 2013－2014 and that the price will drop about 40 % in 2015. Some patents on the basis of SERS substrate is under consideration.