glycerophosphorylcholine + H 2 OPLD! glycerol-3-phosphate + choline (1)
glycerol-3-phosphateG-3-PO!dihydroxyacetonephosphate + H 2 O 2 (2)
2H 2 O 2 + luminol + H 2 O 2 +OH-Co!
2 þ
hm+ 3-aminophthalate + N 2 +3H 2 O (3)
they used FIA-equipment with two mini-columns containing immobilized PLD and
immobilized glycerol-3-phosphate oxidase (G-3-PO). The H 2 O 2 produced [see
Equation (2)] reacts in a subsequent step with luminol at pH>7, and in the presence
of Co2+as catalyst under the production of chemiluminescence. The enzymes were
bound covalently to CPG (Sigma Chemicals Co.) functionalized with 3-aminopro-
pyltriethoxysilane via glutaraldehyde. The amount of enzyme bound was>90 %,
and the mini-columns could be used for 300 h during a 3-month period with inter-
mediate storage at 4 8 C and without deterioration of the enzymatic activity. Optimum
reaction conditions with respect to a maximum chemiluminescence yield were a
luminol and a Co2+concentration of 10-5M, a pH of 6 (maximum formation of
H 2 O 2 ) and a flow rate of 0.7 mL min–1. The detection limits for GP and GPC
are 5 10 –7M and 10–6M, respectively, and the sample throughput 40 per hour
for each analyte.
The development of a bi-enzymatic organic phase electrode by Campanella et al.
(1998a) is based on the poor solubility of lecithin and lecithin-containing products, as
it functions in organic solvents and allows the determination of phospholipids di-
rectly after solubilization. Again, the two enzymes PLD (Streptomyces chromofus-
cus) and choline oxidase were used in this biosensor immobilized toj-carrageenan.
This polysaccharide forms coils at higher temperatures. By cooling, the coils are
transferred to helices that aggregate to a gel (thermo-reversible gelation). There-
fore, the preparation of the immobilized enzymes is as follows: A solution of 0.2
gofj-carrageenan in 10 mL of water is first heated slightly and then poured
onto a Petri dish. After cooling and drying, a small disk is cut from the gel, and
25 lL of the enzyme solution (100 U of PLD and 500 U of choline oxidase) is
applied to its surface. A second disk is then pressed on the top of the first, in order
to obtain the enzyme membrane; this is then used in a gas diffusion electrode with
amperometric detection (Clark electrode). The best results with respect to both the
solubility of the substrate and the response and lifetime of the biosensor where found
when a water-saturated mixture of chloroform/hexane (50 % v/v) was applied. A
further increase in solubility of dietetic or pharmaceutical products containing phos-
pholipids, as well as in sensitivity, was observed in the presence of 1 % methanol.
However, in this case the life time of the electrode was reduced from 11 days to 3
days. The range of linearity was 2.1 mg l–1to 42.4 mg l–1(in the absence of metha-
nol) and 1.1 mg l–1to 66.1 mg l–1). The detection limits were 1.05 mg l–1and 0.55
mg l–1, respectively. In another paper, Campanella et al. (1998b) compared this
analytical device with a similar technique where the two enzymes were simply sand-
wiched between the gas-permeable membrane and the dialysis membrane after hy-
dration with a small amount of 0.1 M glycine buffer (pH 8.5). Both methods were
used to determine lecithin concentrations in several food samples. The electrode with
the membrane-entrapped enzymes had a response time of 10 min, which was twice
286 13 Preparation and Application of Immobilized Phospholipases
