Dried tetramethoxysilane sol-gel containing a leachable reagent

Details Dried tetramethoxysilane sol-gel containing a leachable reagent Dried tetramethoxysilane sol-gel containing a leachable reagent

1998-11-16

2001-05-15

Naff, David M. (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving oxidoreductase

C435S004000, C435S174000, C435S176000, C435S182000

Reexamination Certificate

active

06232090

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to sol-gels and their use in water quality assays.
BACKGROUND OF THE INVENTION
Water courses may be contaminated with substances such as sewage, heavy metals, pesticides containing organic residues etc. Such substances act as free radical scavengers. They can therefore be assayed using a light-producing free radical reaction; the light emission is reduced or inhibited to a degree proportional to the amount of contaminant present, when compared to a distilled water control.
One such test, available under the trade name Aquanox, from Randox Laboratories Limited, involves a free radical reaction between a hydrogen acceptor (oxidant) and a hydrogen donor (luminol) in the presence of an enhancer. This reaction is catalysed by horseradish peroxidase (HRP) and results in light emission at a constant rate.
At present, in order for the Aquanox assay to be performed, a vial of freeze-dried signal reagent (containing luminol, enhancer and oxidant) is reconstituted with 5 ml borate buffer (pH 8.5), and then 100 &mgr;l of this solution, 20 &mgr;l enzyme reagent plus 1 ml water sample are added to a disposable cuvette. The reaction is started by the addition of the enzyme reagent. The separate additions of signal reagent, enzyme and sample to the cuvette have proved difficult for some users when undertaken in the field, especially to less technically skilled personnel.
Sol-gels are known. The sol-gel process involves the mixing of metal alkoxides, e.g. TMOS, i.e. tetramethoxysilane, in solution with water and a catalyst, at room temperature. In this process, a complex series of hydrolysis and polymerisation reactions occurs. The initially fluid solution becomes viscous as polymerisation proceeds, until a gel is formed. The gel is then dried, during which process liquid is expelled, causing substantial volume shrinkage of the gel, leaving a dry porous solid. The pore networks formed in dried gels do not scatter visible radiation and are therefore optically transparent.
Molecules added in the sol-gel process become entrapped in the growing covalent network. For example, Piechota and Sueverkruep, Acta. Pharm. Technol. 34:27s (1988), discloses sol-gels of unspecified composition, containing fluorescein. The marker was released at an essentially constant rate over 5-8 hours, and the rate varied with increasing phosphate content.
Further, EP-A-0439318 discloses a reagent trapped in a sol-gel glass, with the intention that a desired reaction with an analyte should occur within the pores of the network. The Examples disclose TMOS:solvent ratios of approx. 2:3 for sol-gel shapes and 1:8 for thin films.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that, by increasing the TMOS:solvent ratio, sol-gels can be produced that satisfactorily retain a reactant therein, but which can readily be leached out in the presence of water. This is particularly suitable when it is desired to supply reactants to a user in a mixture, but physically separate, until use.
According to one aspect of the present invention, a sol-gel containing a reactant is obtainable by reaction of water with, per part thereof, at least 2 parts of a metal alkoxide such as TMOS, and drying the resultant gel.
According to a second aspect of the invention, for a combination of first and second reactants that give a signal when mixed in the presence of an analyte in a liquid sample, the reactants are separately contained in sol-gels that release the reactants in the presence of the liquid.
DESCRIPTION OF THE INVENTION
By virtue of the invention, the reactants that are required for use in, say, the Aquanox system can be supplied in dry/solid form, e.g. as a dry mixture of any desired shape, films, pellets or powder. For the purpose of this specification, the term “reactant” is used to describe one or more components. If two or more components are contained in one sol-gel, they should not be mutually reactive.
The reactants that may be used in the present invention are not limited. Examples include organic and inorganic ligands, antibodies, enzymes, oxidising and reducing agents, reaction enhancers, signal-generators and labels. Similarly, the components of the sol-gels are not limited, although TMOS is preferred. The preferred alkoxide:water ratio (by volume) is 2.5:1 to 5:1.
The invention will now be described by way of example only with reference to the components used in the Aquanox system. Thus, the first reactant comprises at least luminol as hydrogen donor (reductant) and the second reactant comprises sodium perborate as hydrogen acceptor (oxidant). The first reactant may additionally comprise horseradish peroxidase, and either reactant may additionally comprise enhancer (p-iodophenol), or either or each of these additional components may be contained in further sol-gels. For use in an Aquanox cuvette, these four components may be provided in dried pellet form; no interaction occurs until the water sample is added. However, once water is added, the gels are such that sufficient of the components quickly leach out of their respective sol-gel pellets, and react together, free in solution.
The following Examples illustrate the invention, or are for the purposes of comparison. More specifically, Examples 1 and 2 (TMOS:water ratio is 5:1 or 5:2) are illustrative; Examples 3 to 5 (TMOS:water ratio is 3:2, 1:1 or 1:2) are comparative.
In the Examples, TMOS is tetramethoxysilane/tetramethyl orthosilicate. Citrate phosphate buffer, pH 6.0, comprises the acid and Na
2
HPO
4
. Borate buffer, pH 8.5, comprises sodium tetraborate, N-methylisothiazolone and 2-chloroacetamide.
Preparation of Sol-Gels
1. Sol-gel pellets of total volume 200 &mgr;l were prepared in the wells of Nunc clear microtitre plates, by the following method:
(i) HRP-pellets—into each well was pipetted 100 &mgr;l pH 6.0 citrate phosphate buffer which contained the following:
(a) 1, 1.5 or 2 mg/ml HRP only
(b) 1, 1.5 or 2 mg/ml HRP plus 1% polyvinyl alcohol (9000-10,000 mw) or 1% polysucrose (400,000 mw).
(ii) Luminol/p-iodophenol pellets—into each well was pipetted 100 &mgr;l 8.0 borate buffer which contained 12 mM luminol and 1.2 mM p-iodophenol.
(iii) Sodium perborate pellets—into each well was pipetted 100 &mgr;l pH 8.0 borate buffer which contained 50 mM or 100 mM sodium perborate.
2. Into each well was pipetted 100 &mgr;l TMOS sol containing, per ml, 30 &mgr;l 40 mM HCl and one of the following ratios of TMOS:H
2
O—5:1, 5:2, 3:2, 1:1 and 1:2. The TMOS sols were prepared in brown glass vials and the reagents were mixed by vortexing for approximately 30 seconds, until the solution was clear and the initial two layers had disappeared. After vortexing, the vials were cooled to room temperature before the sol was added to the wells.
Once the 100 &mgr;l TMOS sol was added to the wells, the concentration of reagents was therefore half the value initially present in the well, i.e. 0.5, 0.75 or 1 mg/ml HRP or 6 mM luminol/0.6 mM iodophenol or 25 and 50 mM sodium perborate.
3. After gelling occurred (within minutes), the HRP-containing gels were dried at ambient room temperature by placing the microtitre plates in a desiccator containing silica gel, in the dark, until no further weight loss was recorded. Those gels containing the signal reagent components were dried in vacuo in the desiccator, at ambient room temperature, in the dark.
Assay of Sol-Gels
The control Aquanox assay contained 1 ml deionised water, 100 &mgr;l reconstituted signal reagent and 20 &mgr;l enzyme reagent. The sol-gel pellets were assayed as follows:
(i) 1×HRP pellet+1 ml deionised water+100 &mgr;l reconstituted signal reagent.
(ii) 1×HRP pellet+1×luminol/iodophenol pellet+1×sodium perborate pellet+1 ml deionised water.
(iii) 1×luminol/iodophenol pellet+1×sodium perborate pellet+20 &mgr;l enzyme reagent.
(iv) The wash-water from the above pellets+the corresponding liquid enzyme or reconstituted signal reagent. To obtain this wash-water, 1 ml of deionised water was added

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