Multilayer Enzyme Assembly for the Development of a Novel Fiber Optic Biosensor
- PDF / 410,995 Bytes
- 6 Pages / 414.72 x 648 pts Page_size
- 111 Downloads / 153 Views
reaction. In a certain range, the decrease in the CL signal is proportional to the pesticide concentration. By monitoring the change of the initial velocity of the CL signal in the presence of the pesticide, one can determine the concentration of the pesticide. Due to encouraging results in experimental studies in the bulk solution, we have developed a fiber optic biosensor for pesticide detection based on the same detection mechanism. The advantages of using fibers in our biosensor include: the possiblity to do in-situ monitoring and, ease of fabrication of the sensor tip, and portability. In our fiber optic biosensor, the enzyme is immobilized on the tip of the fiber. The CL signal is collected and transmitted by the same fiber to a detector. There are two critical issues in developing our fibre optic biosensor: optimum fiber tip geometry and robust enzyme immobilization on the tip. To optimize the collection efficiency of our fiber optic biosensor, we have designed and fabricated tapered fiber tips with different geometries. Based on our experiments of the fiber tip tapering effects on the collection efficiency at different light source distributions( 2 ), a combination tapered tip configuration has been selected and used in our biosensor( 3 ,4 ). Robust immobilization of the enzyme on a fiber tip is a challenge. In the past, we have developed several techniques( 5 ,6 ) to immobilize proteins, which include covalent attachment and entrapment in a sol-gel matrix. This multilayer enzyme assembly technique has proven to be an effective and robust immobilization method for our fiber optic biosensor(3 ). The technique utilizes a bifunctional amino coupling reagent to crosslink different layers of enzyme. This method offers the flexibility of controlling the number of enzyme sites on a fiber surface. Multilayers of alkaline phosphatase have been characterized using ellipsometry and surface plasmon resonance. 125 Mat. Res. Soc. Symp. Proc. Vol. 414 01996 Materials Research Society
Chemiluminescence experiments have also been conducted to study the CL signal as a function of the number of layers immobilized.
0
0-0 ocI
-C
0r-0i-
alkaline phosphatase OPOCSPD
3
HPO4=
CSPOD
+
[
I
3
hv Fig. 1 Chemiluminescence reaction of CSPD® catalyzed by alkaline phosphatase. EXPERIMENT Materials The substrate solution - CSPD® was supplied as a part of the Southern-Light Chemiluminescent Detection Kit by Tropix Inc. (Bedford, MA). Diethylamine (DEA) was also a part of the kit for the preparation of assay buffer. Sapphire II, a CL amplifying reagent (referred to as enhancer), was also purchased from Tropix Inc. Magnesium chloride was purchased from Fisher Scientific (Fair Lawn, NJ). Paraoxon and alkaline phosphatase (1000 units/ml) were purchased from Sigma Chemicals Company (St. Louis, MO). The enzymes were dialyzed and purified before they were used. Bis(sulfosuccinimidyl) suberate (BS 3 ), a coupling agent, was purchased from Pierce Chemical Co. (Rockford, IL). Toluene and 3-aminopropyltriethoxysilane were supplied by Aldr
Data Loading...
