Doped sol-gel glasses for obtaining chemical interactions

DOPED SOL-GEL GLASSES FOR OBTAINING

CHEMICAL

זכוכיות סול-גל לוכדות לקבלת אינטראקציה כימית

The present invention relates to a method for obtaining an interaction between reagent/s in a solid support and diffusible solutes or components in an adjacent liquid or gas phase, wherein said reagent/s are trapped in sol-gel glass (hereinafter also referred to as doped sol-gel glass) which provides the solid support to the reagent/s.

The method according to the present invention can be applied to a variety of interactions between the doped sol gel glasses and reagent/s in an adjacent liquid or gas phase. The present invention can be useful in a myriad of applications: for quantitative and/or qualitative analyses, for extraction or separation of solutes from liquid solutions, and for many other applications. For example, the above method can be useful for detection of ions by chemical interaction between the ion/s in an aqueous phase and reagents trapped in the sol-gel glass, or vice versa, via characteristic color test reactions, or other routine detection methods. Another example is utilization of the above method for qualitative and/or quantitative analyses of pollutants.

The method can be applied as well for medical diagnostic purposes e.g. for detecting inorganic ions and/or small organic molecules in blood, urine and other body liquids. Another example, according to the present invention, is a chemical interaction between a substrate/antigen in the liquid phase and an enzyme/ antibody trapped in the sol-gel glass.

For centuries, inorganic glasses have been prepared by high temperature melting methods. This has imposed a major limitation upon the technological application of glasses: additives were restricted to thermally stable inorganic materials, while precluding the incorporation of labile organic molecules.

A recent major development in material science has been the preparation of inorganic (silica) glasses through the low temperature sol-gel synthesis (Brinker, C.J., Scherer, G.W., Sol-Gel Science, Academic Press, San Diego (1990)). An amorphous bond network of the glassy material is prepared by the room-temperature polymerization of suitable monomers, usually metal alkoxides, according to schemes such as:

nSi(OCH₃)₄ + mH₂0 -> (־-O-Si-O-)״ +

4nCH₃0H + (m-2n)H₂0 in which water is consumed by

H₂O + Si(OCH₃)₄ -> Si-OH + CH₃OH and released by

Si-OH -> Si-O-Si + H_aO.

By sol-gel glass one also means the product obtained by a polymerization of metal alkoxide mixtures which bear both hydrolyzable and nonhydrolyzable substituents.

The result of the plymerization is a transparent porous solid (xerogel) with surface areas of up to hundreds m²/gr and by narrow pores (0.5-500 nm).

The low-temperature glass synthesis allows doping inorganic (silica or other) glasses, with essentially any organic molecule. This possibility was used for trapping of e.g. photoactive molecules by adding the compound to the starting mixture at the onset of polymerization (Avnir, D., Levy D., Reisfeld, R., J. Phys. Chem. 88, 5956 (1984)), The compound remained permanently trapped, i.e. non-leachable system have been obtained. These doped sol-gel glasses have been used as photoactive materials, such as:

(a) Dye laser materials;

(b) Thin-film optical filters;

(c) Fluorescent solar collectors;

(d) Photochromic and phosphorescent glasses.

The interaction between the optical properties of trapped molecules and their environment was employed for monitoring the progress of the glass formation sequence: monomer -> oligomer -> sol -> gel -> xerogel. This allowed to scrutinize the evolution of such parameters as porosity, water content and degree of (cage) polarity (Kaufman, V.R., Avnir, D., Structural changes Along the

Sol-Gel-Xerogel Transitions, Langmuir 2, 717 (1986);

Kaufman, V.R., Avnir, D., Pines-Rojanski, D., Huppert,

D., Water Consumption During the Early Stages of the Sol-Gel Polymerization, J. Non-Cryst. Solids 99, 379 (1988)).

Sol-gel glasses demonstrate several technologically attractive properties:

(a) the ability to isolate a single doping molecule in an individual cage, even at high concentrations of additive, thus avoiding interfering side photophysical processes and interactions with impurities or photodecomposition products;

(b) thermal and photochemical stability as well as transparency in the U.V. range above 250 nm.

(c) lack of leaching of the trapped compound, simplicity of preparation, and easy technological manipulation allowing production in any desired geometry, including films.

Surprisingly, it was found that molecules trapped in sol gel glasses, may interact with diffusible solutes or components in an adjacent liquid or gas phase in the pore space. Said finding opened a new wide range of applications of doped sol-gel glasses as solid media for chemical interactions.

The present invention relates to a method for obtaining an interaction between one or more reagents in a solid support and diffusible solute/s or component/s in an adjacent liquid or gas phase, wherein said reagent/s are trapped in the sol-gel glass which serves as the solid support. Said reagent/s can be any organic compound, organometallic, or inorganic compound, or any biological material capable of being trapped in the solgel glass.

The diffusible solute/s or components can be any organic compound, stable organic radical, organometallic, or inorganic compound or biological material capable to interact with the trapped reagents.

The interaction between the reagent in the solid support and the diffusible solute in the liquid phase or a component in the gas phase can be a chemical interaction such as for analytical tests or chemical reactions. The method according to the present invention can be a specific color test reaction, N.M.R. or E.S.R. analysis, analysis via emission or absorption, luminescence, fluorescence, phosphorescence tests and/or electrochemical tests. The analytical reagent can be a pH indicator, redox reagents or an ion complexant etc.

The chemical interaction according to the present

ר invention can take place between anions or cations in a liquid or gas phase and reagents trapped in the sol gel glass or vice versa. For example the interaction may take place between metal ions and a specific reagents via a characteristic colour-test reaction, as in: (1) the determination of Fe*² cation with o-phenanthrolin, (2) the determination of Co*² with 1-nitroso-2naphtol, (3) the determination of Ni*² wherein the reagent is dimethylglyoxime, (4) the determination of S04²־ anion wherein the reagent is sodium rhodizonate and BaF2, or benzoinoxime, (5) the detection of H* is one of many examples for a pH sensors. The analytical test can be carried out by dipping the doped sol gel glass in the solution and observing the resulting color change.

The above method can be useful for the analysis of ore contents in soil, sea water, and rocks (e.g. uranium).

The sol gel glass according to the present invention can be in any shape suitable for the test. For example it can have the shape of rods, discs, cubes, sieves, powder, or thin films coating conventional glass plates or any other inert solid support. Thus, an electrochemical test according to said invention can be performed by preparing electrodes coated with doped sol gel glass layers. These electrodes may be used for clinical, analytical or industrial purpose, or as biosensors.

It should be emphasized that the method according to the invention can be useful for qualitative and for quantitative analysis.

The method according to the present invention can be applied to detection and analysis of pollutants in soil, in aquatic environments and characteristic water sources (including waste, industrial and municipal sources, lakes and swimming pools) in food, drugs, or in the air. The method may be applied to qualitative and/or quantitative analysis of pollutants. Said pollutants may be for example chlorides, nitrates, phosphates, herbicides, insecticides, inorganic ions and pollutants of organic origin. Detection devices according to this invention can be utilized as a prt of continuous monitoring systems.

The present invention can be utilized for extracting or separating molecular solutes from liquid solutions. The doped sol gel glasses can be used according to the present invention for all chromatographic purposes, including liquid, gas and thin layer chromatography. The extraction or separation is performed by passing the solution through columns made from appropriately doped sol gel material. The thin layer chromatography according to this invention can be ״־^,*’ ’ יי י *“.r־ x—r- . — t- ־ . ...

performed on conventional glass plates, paper or other inert solid support coated with doped sol-gel glass layers .

Medical diagnostic is another application of the present invention. For example, detection of inorganic ions, small organic molecules and other components in blood, urine and other body liquids. The invention can be applied also to the fractionation of body fluids.

The present invention relates, as well, to a method for preparation of bioactive materials (biocatalysts) by entrapment of enzymes in a forming sol-gel glass, which, following polycondensation of suitable monomers, serves as a solid matrix, bonding the enzyme and conveying to it mechanical, chemical and thermal stabilities.

The method, according to the present invention, can be applied to a variety of enzymes or enzyme systems, including co-immobilization of co-factors, organic and inorganic ligands, mono- and polyclonal antibodies, and their detection systems.

The method according to the present invention can be useful in a variety of applications, such as: (a) biochemical reactions and other bioconversions in organic and inorganic solvent solutions, (b) detection or qualitative determination of organic and inorganic molecules, which are substrates of the immobilized enzymes, or inhibitors, or modifiers of enzyme activity, (c) construction of bioelectronic detection devices, including construction of enzymes electrodes and biosensors.

Commercial applications of enzymes require successful immobilization. Immobilization allows reuse of an enzyme, protects it from harsh external conditions, from microbial contamination, and prolongs their useful lifetime. There are probably as many immobilization methods as there are enzymes. This proliferation of techniques reflects the complexity of the biological material and the variety of its uses. Simple inexpensive general techniques, resulting in stable and active enzyme catalyst are still in great demand (Kennedy, J. F. and White, C. A. in Handbook of Enzyme Biotechnology (Wiseman, A. ed.), Ellis Horwood Ltd, Chichester, pp. 147-207 (1985)).

An ideal enzyme catalyst should be bound to a mechanically and chemically stable, highly porous carrier. The bond linking the enzyme to the support is required to be stable under the catalyst application conditions to prevent leaching. The strong binding forces also have stabilizing effects on enzyme activity (Martinek, K, and Mozhaev, V. V. Adv. Enzymol. 57, 179, (1985)). The desired immobilization procedure should be simple, mild (non-denaturing) and generally applicable.

Enzymes covalently immobilized on controlled-pore glass beads offer an almost ideal solution to the problems of the support and of the binding force. However the preparation of catalyst by this immobilization technique is neither simple nor generally applicable. The beads are costly, require tedious chemical derivatization procedures, and lack in stability due to the continuous leaching of silica during prolonged usage (Kennedy, J. F. and White, C. A. in ״Handbook of Enzyme Biotechnology (Wiseman, A. ed.), Ellis Horwood Ltd, Chichester, pp. 380-420 (1985)).

The most generally applicable immobilization procedure is a simple entrapment of the enzyme in a forming gel of natural or synthetic polymers. The main shortcoming of this technique is the loss of the enzyme by leakage through a nonuniform net of polymer molecules. Rather weak interactions between the enzyme and the matrix result in a relatively nonrestricted diffusional movement of polypeptide chains. This can be of a benefit, whereas conformational transitions are required for successful catalysis. Otherwise, this diffusional freedom of motion can negatively affect immobilized enzyme stability.

Several properties of the sol-gel glasses make them especially attractive as possible enzyme catalyst supports: (a) the ability to entrap large amounts of additives; (b) the thermal and chemical stability of the matrix; (c) simplicity of preparation with no need of covalent modification; (d) easy technological manipulation and production in any desired geometry, including thin films.

Recently, aggregates of whole yeast cells trapped in thin layers of SiO₂ gels deposited on glass sheets were demonstrated to possess invertase activity. Thin films with cell-free invertase preparation were devoid of activity (Carturan, G., Campostrini, R., Dire, S., Scardi V. and de Alteriis, E. J. Mol. Cat., 57, L13, (1989)).

The present invention relates therefore also to a method for obtaining bioactive materials based on an enzyme molecules trapped within the porous structure of a sol-gel glass. The entrapment is achieved by the addition of a cell-free enzyme to a mixture of monomer or monomers at the onset of polycondensation. In addition to the enzyme and monomer(s), the mixture should contain additives ensuring (1) highly porous nature of the forming glass providing minimal diffusional limitations to the binding of the substrate at the catalytic site and to the removal of the product, (2) the stability of the enzyme during the polymerization and its tight binding preventing leaching of the enzyme.

Unexpectedly, we have found (1) that proteins can be trapped within the matrix of a forming sol-gel, (2) that several cell-free enzymes, belonging to various classes: hydrolases, oxidoreductases, lyases etc., can be effectively entrapped in such composite bioactive sol-gel glasses, while retaining high enzymatic activity, (3) that strong binding forces retain the enzyme in the matrix, thus producing a considerable stabilizing effect.

The sol-gel immobilized enzymes may be used as biosensors for hormonal tests or for any industrial purposes, including diagnostic and synthetic purposes. Said enzymes can be doped in sol gel glass layers coated on electrodes for probing any substrate. The enzymatic interaction according to the present invention can be applied also to radioactive tests and also for enzymatic column chromatography (crushed powder sol gel glasses may be used as support for enzymatic column chromatography).

The sol gel glass can be applied, according to the present invention, as active specific membranes allowing selective incorporation of the trapped molecules or ions or any other species.

The abovementioned applications are examples only and do not intend in any way to limit the scope of the invention.

The present invention relates also to the application of doped sol gel glasses according to this invention as well as for the preparation of sol gel glasses and doped sol gel glasses for such applications.

When prepared as thin film the width of the sol gel glass may be from molecular monolayers up to macroscopic layers. Said thin film can be part of multi-layered array of thin films. Said glasses may be supported on an electrode or optical support.

The unique transparency of sol gel glasses in the range above 250 nm, makes them highly applicable to quantitative spectrophotometric and spectrofluor!metric tests. Trapping of host molecules is relatively simple and does not require specific synthetic methods such as those associated with covalent linking of reagents to solid supports. Moreover, inherent properties of sol gel glasses such as high surface area, the wide range of available pore sizes and the thin film technology, make them highly attractive for potential applications as solid supports for a variety of reagents.

Examples

A. Preparation of doped sol-gel glasses

The polycondensation of alkoxysilans is associated with gelation of the sol, which after drying is densified by a mild heat treatment to form a glass. The properties of the final glass are determined by the chemical and physical conditions during the process of preparation. They depend upon the ratio metal (e.g. silane)/alcohol/water, the alkoxide pH, the presence of a catalyst, the temperature, the drying time and the amounts of organic additives, such as surface active agents.

Pore size and surface area are controlled by addition of acid or base (see Scherer and Brinker, 1990, cited above). Addition of NaF to the starting (TMOS) solution leads to an increase in average pore size.

1. A standard mixture for preparation of doped solgel glasses contained TMOS (5 ml) H20 (2.4 ml) and methanol (6. ml). The appropriate catalyst and the desired reagent were added in the required amounts (water or methanol solutions). Gels were formed within several minutes (base-catalyzed) or several hours (acid-catalyzed). Gelation was carried in a sol-gel glass was carried out with a starting solution composed of methanol (3 ml), TMOS (2.5 ml), 6 mM phosphate buffered saline (PBS, 0.25 ml) and Anti-IL-2R antibody (0.25 ml, from the kit). After stirring for 50 min. the sample was allowed to stand for 5 days at 27°C.

The resulting glass (4 mg, 0.4% of the total amount) was crushed and washed three times with 350 μΐ of the washing solution” from the kit (0.2 ml surfactant in 200 ml PBS). To this solution 100 μΐ the sample diluent (buffered serum protein) and 50 μΐ of the human IL-2R standard solution were added and mixed for 15 sec, followed by covering and incubating for 2 hours at 37°C. After three washings, 100ml peroxidase- conjugated AntiIL-2R antibody solution was added and incubated at 37°C for 2 hours. After three washings with the washing solution, 100 pg of o-phenylenediamine dissolved in a substrate diluent (buffered H2O2) were added and incubated for 20 min at room temperature. An absorbance 0D_49o= 0.93 was recorded, compared to 0D₄9o= 0.357 obtained with the above commercial kit , with a similar amount of Anti-IL-2R monoclonal antibody absorbed into polystyrene microtiter wells.

out at room temperature in glass bottles covered with aluminium foil. The gels were then transferred to an incubator and kept at 37-45°C. The samples were used after they reached a constant weight (about two weeks). The above procedure yields glass in any desired shape (rods, disks etcJL.—

2. An alternative technique of preparing sol-gel glasses is based on thin-layer coating of conventional glass supports. A characteristic procedure for the preparation of such thin layers began with a mixture containing methanolic or ethanolic solution of dopant (85 ml), TMOS (10 ml), Triton X-100 (3 g) and 0.03N HCL or 0.01N NaOH (3 ml). After mixing the solution was allowed to stand for 30 min at 25-35°C and was then used for coating. Coating was performed by dipping a glass plate into the solution, followed by drying for several minutes at room temperature. The resulting porous coating layer (0.25-0.5 pm).

B. Representative examples of reactivity of reagents trapped in sol-gel glasses

1. Tests were carried out by the immersion for 5 min of an appropriately doped sol-glass in aqueous solutions containing ions or molecules to be detected. Arrows denote transitions from the reagent-doped glass to the same glass after immersion in the tested solution. The doped glasses represent four classes of reactions: (a) a glasstrapped organic reagent with an inorganic cation to be determined in the solution; (b) same with inorganic anions; (c) a glass doped with an inorganic ion, testing a solution containing an organic molecule (reversal of a & b); (d) glass doped with a pH indicator.

Representative examples of color tests are shown in Table 1. Top (left to right): a) Glass doped with

1,1O-phenanthrolin after immersion in: b) 10⁵־M Fe²⁴־ solution, c) Fe²', d) 10²M Fe²⁺. Glass was prepared in the presence of the reagent (0.005%) and 5x10~³M NaOH as catalyst in the starting aqueous solution.

Middle: Doped glasses (top) and some glasses after immersion in solutions containing several ions:

a) Reagent for A1³* was quinalyzarin (1,2,5,8tetrahydroxyanthraquinone). To the starting solution (see above) 2 drops of a solution with the following composition were ?idded: 18 cc methanol containing

0.01 gr of the reagent, 2 cc pyridine and two drops of 0.1 N NH4OH. b) Reagent for Co²⁺ was nitroso naphthol. Starting solution contained 0.25% of the reagent and 3 drops of 0.1N HCI as catalyst, c)

Reagent for Ni²* was dimethylglyoxime (starting solution, contained 0.25% of the reagent and 3 drops of 0.1N NH40H as catalyst, d) Reagent for the

S04²־ anion was sodium rhodizonate & BaF₂.

Starting solution contained 0.25% sodium rhodizonate, 3,5x10 gr. BaF₂ and 3x10³־־N NaOH. Bottom left: Glass doped with Fe²* (top, yellow) after immersion in a solution containing ophenanthrolin (bottom, red). The glass, prepared in the presence of an acid catalyst (a few drops of concentrated HCL), was used before complete drying (container was capped after 3 days).

Bottom right: Two different reagents for the determination of Cu²* (top: glass with reagent, bottom: same after immersion in Cu²* solution). Left: Rubeanic acid as a reagent. Starting solution contained 0.15% of the reagent and 0.25% sodium tartaric in the presence of 0.01N NaOH. Right: Benzoinoxime (Cupron) as reagent. Starting solution contained 0.25% of the reagent with one drop of concentrated HCL. After 3 minutes 3 drops of concentrated NH4OH were added.

C. Representative examples of bioprocesses involving proteins.

1. Preparation of sol-gel immobilized enzymes.

Enzyme solutions (0.2 ml, 10 mg/ml) in non-buffered water, which may contain various additives were mixed in the cold (4°C) with either methanol or polyethylene glycol (PEG 400). The concentrations of additives (such as e.g. NaF, NaOH, HC1), the volumes of methanol and PEG 400 were as indicated in the examples below. Tetra-methoxy silane (TMOS, 1 ml) was then added. The tubes containing the reaction mixture were transferred to a shaking water bath at 8^qC. The bath was allowed to reach the room temperature during 2-3 h. In samples containing PEG 400, the polymerization was completed in about 3 h. All the liquid remaining on the top of sol-gel was then removed by suction. In methanol-containing mixtures gelation took place in about 4-5 h. The polymerized sol was allowed to dry for a week at 30°C.

2. Retention of protein by the sol-gel glass.

All the glasses prepared according to example C1 were ground to a size of about 60-100 mesh and packed in 2 ml-columns. The columns were eluted with

0.5 M NH4HCO3 (250 ml), followed by water (250 ml). This cycle was repeated twice. All washing solutions were collected, concentrated by freezedrying, and assayed for protein content and for respective enzyme activity. It was found that neither significant enzyme activity nor protein could be detected in the eluates.

3. Entrapment of trypsin in sol-gel glasses.

Trypsin (E.C. 3.4.21.4, from bovine pancreas, 11,000 U/mg) was supplied by RAD Chemicals, Rehovot, Israel. Trypsin entrapped in sol-gel was prepared as described in example C1. Assays were performed on the washed glasses at 25°C at pH 8 using N-benzoylL-arginine-4-nitroanilide (3.3 mM) as the substrate. The concentration of NaF in the enzyme solution and the addition of methanol or PEG 400 (ml per ml TMOS) as well as the enzymatic activity of the glass catalyst (expressed in per cent of trypsin activity initially added to the condensation mixture are shown in the following Table:

<td rowspan="3"> NaF (mM)</td><td colspan="4"> Trypsin Activity (per cent of initial)</td>

<td> MeOH (ml/ml TMOS)</td><td colspan="3"> PEG (ml/ml TMOS)</td>

<td> 0.6</td><td> 0.2</td><td> 0.4</td><td> 0.6</td>

<td> 0</td><td> 0.1</td><td> 11.4</td><td> 33.0</td><td> 33.6</td>

<td> 1</td><td> 0.6</td><td> 16.9</td><td> 24.7</td><td> 29.7</td>

<td> 10</td><td> 1.9</td><td> 10.0</td><td> 18.2</td><td> 23.6</td>

<td> 100</td><td> 2.8</td><td> 4.1</td><td> 21.6</td><td> 17.5</td>

4. Entrapment of acid phosphatase in sol-gel glasses.

Acid phosphatase (E.C. 3.1.3.2, from wheat germ,

0.45 u/mg) was purchased from Sigma. The acid phosphatase-containing sol-gel glasses were prepared as described in the example Cl . The assays were performed on the washed glasses at 25°C at pH 5.6 using p-nitrophenyl phosphate (6 mM) as the substrate. The activity yield, calculated in percents of enzyme activity used initially for the preparation of glasses, is shown in the following Table:

<td rowspan="3"> SOLVENT</td><td> NaF, mM</td>

<td> 1.0 3.0 10.0</td>

<td> ACID PHOSPHATASE YIELD, %</td>

<td> Methanol 0.6 ml/ml PEG 400, 0.2 ml/ml PEG 400, 0.4 ml/ml</td><td> 1.9 - 39.2 46.6 56.4 21.1 39.8 41.0 46.7</td>

5. Thermal stability of immobilized acid phosphatase in different sol-gel glasses.

The acid phosphatase-containing sol-gel glasses (example C4) were incubated at 70°C in citrate buffer (pH 5.6, 0.1 M) for various periods of time (up to 5 min). The activity of acid phosphatase was determined, as described in the legend to Table 1.

The half-life time was calculated assuming the 1st order inactivation kinetics. The half-life time of the soluble enzyme at the same conditions was below

0.1 min.

<td></td><td> NaF, mM</td>

<td> SOLVENT</td><td> 1.0 3.0 10.0</td>

<td></td><td> HALF-LIFE TIMES AT 70°C, min</td>

<td> Methanal 0.6 ml/ml</td><td> 3.9</td>

<td> PEG 400, 0.2 ml/ml</td><td> 3.3 3.3 12.0</td>

<td> PEG 400, 0.4 ml/ml</td><td> 3.2 3.1 3.5</td>

6. Entrapment of peroxidase in sol-gel glasses.

Peroxidase (E.C. 1.11.1.7, from horseradish, 200 U/mg) was obtained from Sigma. Sol-gels doped with peroxidase were prepared as shown in the example C1. All the glasses prepared with the addition of PEG 400 were active, although it was not possible to determine the extent of their activity quantitatively, since the dye formed by oxidation of several substrates was adsorbed strongly in the glass. Semi-guantitative comparison of the dye stain shortly after the addition of the assay mixture indicated improved activity yields at higher concentrations of PEG 400. In contrast to trypsin and catalase, sol-gel glasses made at elevated concentrations of NaF were more active. Glasses prepared in methanol-containing mixtures were devoid of peroxidase activity.

7. Entrapment of trypsin in sol-gel glasses.

Trypsin solution (1.0 ml, 2 mg/ml) in non-buffered water was mixed with either 20 mM NaF (0.1 ml) or the same volume of water, and with one of the following: (1) methanol, (2) polyethylene glycol solution (PEG 6000, 20% w/vol in water), or (3) glycerol solution (75% w/vol in water). The mixture was cooled to 4°C. Tetra-methoxy silane (TMOS, 1 ml) was then added. The tubes containing the reaction mixture were transferred to a shaking water bath and allowed to reach the room temperature. The polymerized sol was allowed to dry for a week at 30°C. The resulting glasses were treated as described (example C2). Trypsin activity of the trypsin-doped sol-gel glasses expressed as the yield of activity used for the preparation of the catalyst is presented in the following Table.

<td rowspan="2"> ADDITIVES</td><td colspan="2"> TRYPSIN ACTIVITY, % of initial</td>

<td> +NaF</td><td> -NaF</td>

<td colspan="3"> Methanol 17.2 26.8 PEG 6000, 51.3 46.1 Glycerol 82.0 38.1</td>

8. Entrapment of aspartase in sol-gel glasses.

Escherichia coli cells (ATCC 11303) were cultured as described (Chibata, I, Tosa, T., and Sato, T. Meth. Enzymol. 44, 739-746 (1976)). Saline-washed cells (4 g wet weight) were suspended in water (2ml) and disrupted by sonication. The homogenate was cleared by centrifugation (10,000xg, 30 min, 4°C) and used for the preparation of sol-gel glasses. The homogenate (0.5 ml) was mixed with NaF solution (0.2 ml) at the concentrations indicated in the Table below. Methanol (0.6 ml) or PEG 400 (0.2 ml) was then added followed by TMOS (1 ml). All the additions were made at 4°C. The resulting sol was kept overnight at room temperature and then washed with an excess of 50 mM phosphate buffer pH 7. Aspartase activity was measured and expressed in pmoles/min/g cells (wet weight). The results are presented in the Table. For comparison the polyacrylamide gel-entrapped whole cells from the same batch, prepared according to Chibata (1976), possessed aspartase activity of 133 pmoles/min/g cells .

<td rowspan="3"> NaF solution mM</td><td colspan="2"> Aspartase Activity, pmole/min/g cells</td>

<td> MeOH (ml/ml TMOS)</td><td> PEG (ml/ml TMOS)</td>

<td> 0.6</td><td> 0.2</td>

<td> 0</td><td> 105.0</td><td> 79.0</td>

<td> 1</td><td> 15.8</td><td> 127.7</td>

<td> 100</td><td> 64.5</td><td> 39.8</td>

9. Preparation of protein-doped glasses by NaOH catalyzed polycondensation. Immobilization of alkaline phosphatase.

A mixture of tetra-methoxy silane (TMOS, 5 ml), methanol (6 ml) and 1 mM NaOH in methanol (0.1 ml) was cooled to -20°C and mixed with an ice-cold solution (0.9 ml) of alkaline phosphatase (ALP, E.C.

3.1.3.1 from bovine intestinal mucosa, Type I--S, 8.5 U/mg, Sigma Chem. Co) containing 1.5 mg of the enzyme. A cloudy mixture was allowed to reach room temperature under stirring. The resulting viscous opaque material was kept for 10 days at 37°C. During this time the glass formation was completed and it reached a constant weight. The glass was ground and washed as described in the example C2. The enzyme activity was determined in NaOH-glycine buffer (40 mM, pH 9.5) at 25°C using p-nitrophenyl phosphate as the substrate. The yield of the alkaline phosphatase activity after immobilization was estimated at about 30%. The half-life time of the immobilized enzyme at 70°C (pH 9.0) was 4.7 min, as compared to 2.6 min for the soluble ALP at the same conditions.

10. Immobilization of chitinase

The glass trapped enzyme was prepared by adding 1.0 ml of the enzyme chitinase (EC 3.2.1.14, cloned from Serracia marcensens and expressed in E. coli, 200 units) in the phosphate buffer (10 mM, pH 6.3), to a solution obtained by stirring 3.0 ml methanol and

2.ml TMOS for 15 minutes.

11. Antibody reactions

Interleukin-2 receptor (IL-2R) is the protein that mediates the action of interleukin-2 (IL-2), an immune system growth hormone. Levels of soluble IL2R have been shown to be elevated in a number of pathological conditions, and may thus be of significant prognostic value. We have used an AntiIL-2R monoclonal antibody, trapped in a sol-gel glass, to determine IL- 2R, using a sandwich immuno assay test (cell-free Interleukin-2 Receptor CK 1020, 96 Test kit, T Cell Sciences, Inc., Cambridge, MA, USA). Trapping of Anti-IL-2R monoclonal antibody in a sol-gel glass was carried out with a starting solution composed of methanol (3 ml), TMOS (2.5 ml), 6 mM phosphate buffered saline (PBS, 0.25 ml) and Anti-IL-2R antibody (0.25 ml, from .the kit). After stirring for 50 min. the sample was allowed to stand for 5 days at 27°C.

The resulting glass (4 mg, 0.4% of the total amount) was crushed and washed three times with 350 μΐ of the washing solution from the kit (0.2 ml surfactant in 200 ml PBS). To this solution 100 μΐ the sample diluent (buffered serum protein) and 50 μΐ of the human IL-2R standard solution were added and mixed for 15 sec, followed by covering and incubating for 2 hours at 37°C. After three washings, 100ml peroxidase- conjugated AntiIL-2R antibody solution was added and incubated at 37°C for 2 Hours. After three washings with the washing solution, 100 pg of o-phenylenediamine dissolved in a substrate diluent (buffered H₃O₂) were added and incubated for 20 min at room temperature. An absorbance 0D_49o= 0.93 was recorded, compared to 0D_49Q= 0.357 obtained with the above commercial kit , with a similar amount of Anti-IL-2R monoclonal antibody absorbed into polystyrene microtiter wells.

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Sol-gel glass Xa _an effective suoDort 4

Uoande . ^P °^Γ ^*¹״^aily _active l+sands entrapped in this glass _a. , gxass at room temperature .״״».־־״, enteepp״, ״־ ־ o* their ene,»_{־tl־ ־ctl״lty Th־}._־־ associated with “ ““^־ יⁱ «״־־.91 9.1־1־• «־

־.־״.l.o_־־1״, _o״ ,״ _{lh־ phe״o>eno11 ־f}°* ^protein to the glass, entrapped molecules retains P-aaent stuay .

«mlno_Propyl_{criethoxyaUane} ״o״-spe״f_ic absorption _{of protein>}

״.».״״ _of. .₀₁,_{״־a M&lt; modlfl1־־}Potion o_f entrapped _{in tha} «re two problems d'or entrapping non-apeclfie absorption T־he second is that only a ״״.,״, a portion of the its biological activity. _in glass ^Wfls treated with provide a matrix which the

61Imitated

Secondly, the

Protein A method in order to increase were reactive.

. motilfl.d 9el-31.ee column end _u3ed ״»־ ,״״״׳. ׳“ .״* ”'” ־.tofrom 30x to35x ׳ρ_ΚΛ

־. ^Tha P^ur*ty of the IgG, PAGE, _was comparable .״ _״ .־״״!־״ . -״_hll ’ _h⁰ — ״״־ ״״.־־—« xiz ״. — ^{n 1a e4s1}*r and faster.

of the was to purify ¹”1״ ׳th . _{ws r־ngln9}as determined by $DS^

IgG

Immobilization of biologically relevant entities for diagnostic purposes has been explored by utilizing polymeric and oxide supports (3). wore recently, sol-gel glass has been investigated as a support for trapping of organic molecules in glass (4) and the preparation of porous sol-gel glass in which a variety of chemical reagents can be entrapped has been described (1)., In a subsequent paper (2), alkaline phosphatase and a number of other enzyme» were effectively entrapped in sol-gel glass with the retention of approximately 20« of their enzymatic activities. We report herein a method in which protein A is immobilized m a sol-gel glass column with the retention of 100« of its reactivity towards sheep IgG.

MATERIAL AND METHODS.

Insoluble protein A (100 mg of the insoluble mixture contains 0.44 mg of pure protein A) and commercial sheep IgG were purchased from Sigma (St. Louis) . Anti-rat visceral yolk sac (VYSi sheep serum (sheep antiserum) was prepared according to the method described by Brent et al. &lt;5). Binding buffer (pH 9,0) and elution buffer (pH 3.0) used for immunoglobulin purification were purchased from Bio-Rad (Richmond, CA).

Preparation of aol-qel glass, with and without protein A.

Non-derivatizec sol-gel glass was prepared according to the method described by Zusman et al. &lt;i) . A solution of tetramethoxysilane (TMOS, 1.0 ml), methanol (1.2 ml) and phosphate-buffared saline &lt;PBS, pH 7.4, 0.6 ml) was stirred at room temperature until gelation occurred, covered but not sealed, and maintained at room temperature for 14 days to complete polymerization.

Deriyatization of sol-gel glass. The solid non-derivatized sol-gel glass (300 to 500 mg) was derivatized by the addition of 2« (w/w) -T-aminopropyltriethoxyailane. dissolved in 0.5 ml PBS, according to the method adopted from Muramatsu et al. (6), originally used to modify piezoelectric crystals. After 2-3 days, the product was ground to a coarse powder with the aid of mortar ana pestle, loaded into a 10 ml syringe barrel containing a porous nylon membrane, and extensively washed, first with distilled water and then with several changes of PBS.

Sol-gel glass with protein A, Insoluble protein A (100 mg) was added to the TMOS-methanol-PBS mixture described above and was stirred on ice until gelation occurred. It was maintained at room temperature for 14 days to complete polymerization. The process of derivetization was performed as described above.

Sol-_Q-el glass., mixed with protein A, A portion of the ground polymerized glass &lt;350 mg) in 1 ml of methanol and 0.5 ml of PBS wea stirred with insoluble protein A &lt;50 mg). The amount of protein A retained on the sol-gel glass was calculated after first washing this glass extensively with a total of 250 ml of distilled water. The amount of protein A in the wash was determined spectrophotometrically (Response, Gilford, CIBA) by reading its absorbance versus a suitable control at 280 nm and 260 nm and calculation its concentration according to the method described by Peterson (7). The. amount of protein A retained on the sol-gel glass was calculated by subtracting the protein In the wash from the amount applied to the aol-gel glass.

' Retention and elution of immunoglobulin.

Before the glass columns were used in these experiments, they were thoroughly washed with binding and then with elute buffers. Sheep IgG (32 mg) was dissolved in binding buffer (8.5 ml) and was applied in 1.5 mi portions to each of five columns each containing 100 mg of the insoluble protein A in approximately 350 mg of sol-gel glass. After one hour, the columns were washed with several volumes of binding buffer to remove any non-absorbed IgG. Slution buffer (7 ml) was then applied to each column, the eluates pooled, and their protein content measured as described above.

In parallel, a sixth glass column, prepared by simply mixing 50 mg of insoluble protein A with derivatized sol-gel glass, was used for comparison utilizing the same procedure as described above.

Binding, capacity of _th.e._ar,otstin A gel-glass column fo.r sheeo IgG, Lyophilized sheep IgG (ISO mg) was dissolved in binding buffer (15 ml) and applied in equal portions to two SOl-gel glass • ί) π

־.. '1: .&lt;״׳:;. .: columns each containing 100 mg of insoluble protein A and also applied to a comparable protein A Affi-gel column (Econo-Pec, Bio-Ped). The procedures for the retention and elution of immunoglobulin were performed as described above for the sol-gel glass columns.

Purification of IqG from a sheep antiserum.

Lyophilized anti-rat VYS sheep serum &lt;200 mg) was dissolved in binding buffer &lt;2 ml) and applied to a column of gel-glass containing 50 mg of entrapped protein A. After one hour the column was washed with binding buffer until the eluate contained no detectable protein. Elution buffer was applied to the column and collected in 1 ml fractions. The first four fractions, which 1 contained moat of the protein, were pooled and the buffer exchanged for PBS according to the procedures described for the protein A Affi-gel column (Bio-Rad). Purification of IgG was performed using this column according to the standard protocol (Bio-Rad). Protein content was determined es described above.

Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine the protein profile of IgG obtained after purification by affinity chromatography using the two different columns. The 7.5m SDS-PAGE was performed according to standard procedures (Hoefer Sci. Ltd., San Francisco). Whole sheep antiserum was initially diluted to a concentration of 8 mg/ml with PBS: all other protein solutions were initially diluted with PBS to a protein concentration of 2.B mg/ml. The diluted solutions were mixed 1:1 (v/v) with the sample buffer

י - . ;. י

Approximately 70 pg ©£ protein in 50 pl of solution was applied to each well on a gel, except for the sheep antiserum, which waa 200 pg of protein per 50 pl of solution.

/ 27

RESULTS.

Sol-cel glass containing protein—A_»_ » Non-derivatized glass exhibited marked non-specific protein absorption: almost every protein in sheep sera was retained and could not be eluted from this glass column.

Non-specific protein absorption was eliminated by derivatization of sol-gel glass with a hydrophobic moiety, i.e., by the addition of 2X 1w/w> -r-aminopropyltriethoxyailane dissolved in 0.5 ml P8S.

Derivatized sol-gel glass alone, i.e prepared without protein A, did not bind measurable amounts of IgG.

Retention and elution of immunoglobulins _ in ._g_el-glass coluM£_ with entrapped or mixed protein A, Commercial sheep IgG dissolved in the binding buffer was applied to five gel-glass columns with entrapped protein A and to one column with mixed protein A. It was found that recovery of IgG in the elution buffer wes approximately 100« in columns with entrepped protein A.

Approximately ¾6¢¢ to 98« of the protein A simply mixed with derivatized gel-glass could oe eluted with water. Retained protein A simply mixed in the gel-glass column does not posses

<img file="IL93134A_D0001.tif" />

significant binding capacity: the amount of commercial sheep IgG eluted from this column was about 45¢.

Binding capacity for sheep_LsJiuu

The binding capacity of the gel-glass columns ranged between 4S to 52 mg XgG per 100 mg of insoluble protein A in sol-gel glass as compared to 41 mg of IgG which was obtained using the commercial protein A column. It was calculated that 1 mg of pure protein A binds 113 mg of commercial sheep IgG.

Purification of IgG from a sheep anti serum..

As shown in Fig. 6., the results of SDS-PAGE demonstrate that the IgG purified by affinity chromatography with protein A entrapped in the sol-gel glass is largely free from Other proteins present in the sheep antiserum. The yield of the IgG was IS!.3 mg from the 200 mg of lyopnilized antiserum per 50 mg of protein s contained An one eol-cffil column.

DISCUSSION.

There are many possible methoda for isolation IgG. end other proteins, from sera but the most common involves protein A (8>. snoraous efforts have been undertaken to simplify these procedures ao that they are practical for use in any laboratory. One ' direction taken is the search for better supports for biochemically active compounds which act as immunochemical sensors (9,10>. Various formulations of $ol־״gel glass have been intensively studied in this respect (4J. It was shown that sol gel glass prepared at room temperature can be used as a matrix for trapping chemically, active compounds <1,2>.

There are two problems concerning using of such sol-gel glasses for entrapping and their use for macromolecules binding ligands. One is the phenomenon of non-specific absorption of protein. The second la that only a portion of the entrapped molecules retains their biological activity (2>. Herein we have shown that a column prepared from derivatlzed sol-gel glass can be utilized for the immobilization of protein A and used for the purification of sheep IgG by affinity chromatography. More important, we have shown that treating the sol-gel glass With taminopropyltriethoxysilane eliminated non-specific absorption of protein and appeared to increase the' proportion of reactive molecules.

Protein A was entrapped in the modified gel-glass column and used to purify IgG from sheep sera by affinity chromatography with e yield ranging from SOX to 35*. The capacity of derivatized sol-gel glass with entrapped protein A was equal to commercially available protein A columns. The activity of entrapped molecules of protein A was greater (approximately 100*) than molecules of

Λ alkaline phosphatase entrapped in sol-gel glass (30%) (2).

Moreover, the method described herein is not less effective and more simple than those used by other authors (3>. It is likely that the method described in this article can be applied for the immobilization of monoclonal antibodies to be used for the isolation of specific antigens.

REFERENCES..

1. Zusman, R. , Rottman, C., Ottolenghi. M. and Avnir, D. &lt;1990) J. Ncn-Crystal. Solids, 122:107-109.

2. Braun, S.> Rappoport, S., Zusman, R;, Avnir, D. and Ottolenghi, M. <1990) Materials Letters,'10:1-5.

3. Wilchek, M. and Bayer, E.A. <1988> Anal. Biochem., 171:1-32.

4. Brinker, C.J. and Scherer, G.W. (1990) Sol-gel science. Academic Press, New York.

5. Brent, R.L., Johnson, A.J. and Jensen, M. &lt;1971) Teratology, 4:255-276,

6. Muramatsu, H_׳״ Dicks, J.M., Taaiya, E. and Karube I &lt;1987) Anal. Chem., 59:2760-2783.

7. Peterson, G.L. &lt;1983) Methods Enzymol., 91:95-119.

8. Hou, K.C., Zaniewski, R. end Roy, S. &lt;1991) Biotechnol. Appl. Biochem., 13:257-268.

9. Hearn, M.T.W. and Davies, J.R. (1990) J. Chromatography, 512:23-39.

10. Liang, S.P. and Laursen, R,A. &lt;1990) Anal. Biochem., 188:366-373.

Legend ζο figure.

s 6 A 7.55¢ SDS-page profile of proteins purified from commercial sheep IgG proteins were obtained sheep IgG from the end from _6nti-rat VYS sheep serum. The from: N, high molecular weight standard; commercial kit column; p, .sheep _{IgG fron} .9el-gU_aa column containing _{the R׳} rat VYS sheep serum &lt;200 pg/50 pl);

S, eheep IgG (70 pg/50 pl).

• 09 ί א if _Λ

EXAMF’L ES .

A. Preparation of doped sal-gel glasses exhibiting anion exchange properties.

In this section we exemplify the possibility to prepare anion exchangers using doped sol gel glasses. '

- Sol-gel glasses doped with aliquat 336 (technical. trioctylmethylammonium chloride) are shown to exhibit ion exchange properties and selective uptake of several ions over chlorides. Experimental details for the preparation of sol gel glasses: A solution of methanol, water and aliquat 336 was prepared and mixed with tetr amethoxy silane (TT1OS) in a 20 ml bottle. Typical weight ratios employed were (H_־D/methanol/TMOS/organic dopant) = (2.7/ 3.0/ 2.2/ 0.1-0.4). The bottles were covered with aluminum foil and were let to stand for several days. After the gel was formed tie glasses were transferred to a 40°C incubator for two weeks until no weight loss was detected. The glasses were then grinded prior to the selectivity tests.

Selectivity experiments: The glasses containing doped anion e> changer were immersed in a IN NaCl solution for several hours to remove traces of undesirable anions. The glasses were then washed thoroughly with distilled water and dried in a 50’C oven. Approximately one gram of the glass was immersed in 100 ml

Ο 9 ϊ54 ו !׳ II.

B. Thin Layer Chromatographic Detectors for separation and visualization of chemical species

In the fallowing we exemplify the usage of doped glasses for Thin Layer Chromatographic separation and spot visualization in one 5 tep.

Experimental Details:

Glass preparation: Glasses doped with 57. (weight) of 1,10-0phenanthroline or 57. l-nitroso-2-naphtho) were prepared,.

In a typical procedure 2.5 ml tetramethaxysilane (Petrarch), 3.0 ml methanol, 2.4 ml distilled water, 0,5 ml of 1 ml1׳ NaOH solution and 3.0 ml containing 1,10-0phenanthroline solution were mixed, allowed to gel and dried to a constant weight at 41°C for three days. ‘1 he dried glass contained 5.07. weight o-phenanthroline. An appropriate amount (Tables 1,2) □ f doped sol-gel glasses were crushed with 3.6 gr. of 70-230 mesh ASTM Kieselgel 60 (Merck) in a chinaware cup and sieved by a sieve shaker. The glass fraction smaller than 0.07 mm was used for the TLC plate preparation.

TLC plates prepara tian: TLC plates wer e prepared according to standard procedures (Ε. Stahl, Ihin Layer Chromatography, Springer-Verlag, Berlin, 2nd ed.) in ,־1 ו yp !.&lt; a I experiment a mixture of 0.2 gr. anhydrous CaSC),, 0.15 gr. Starch (Baker), 2.65 gr. crushed glasses were mixed with 10 ml distilled water. The slurry was spread on microscope slides (26x76 mm) forming 0.05 cm thick distilled water and allowed to stand for one day, after which stock solutions containing the counter ion (Br, S□/, N0_s־) were added.

The glasses were shaked gently on an Arig-Levi shaker for several hours until equilibrium was reached. The concentrations of the anions in the solution were analyzed by ion chromatography.

Results

Figures la-c demonstrate the binary exchange isotherms of S0₄, Br־ and NO/ versus Cl־ in glasses doped with Aliquat 336. Where C, and C_b, y, and y_h are the equilibrium concentrations of the counter ions a or b in the solution and in the resin phase respectively.

The following test confirmed that the exchange capacity of the doped glasses is due to entrapped ammonium groups rather than adsorption phenomena. Porous glasses prepared by the Sol-Gel process without any addition of organic dopant were immersed in a saturated solution of Aliquat 336. The glasses were then washed with one liter of distilled water according to the same procedure applied to the doped glasses prior to the selectivity tests. Exchange experiments with these glasses did not reveal any detectable exchange capacity, confirming that the immobilization of the ammonium salts in the polymerization process cannot be attributed to an adsorption mechanism only.

Iron Detection: TLC plates containing different percent of sol — gel glasses doped with 1,10-o-phenanthroline solution were developed in samples containing various concentrations of ferrous ammonium sulfate.

Cobalt/iron/copper detection: TLC plates of glasses doped with 0.0375Z l-nitroso-2-naphthol were exposed to several solutions containing various concentrations of cobaltous chloride, copper chloride and ferrous ammonium sulfate.

Results: Table 1 presents the lengths of stains developed after exposure of TLC Detection plates to various iron(II) samples as a function of the concentration of 1,10-o-phenanthroline in the qhromatographic media. The length of stain was shown to follow (regression coefficient, R = 0.983) the empirical equation!

L = 112.6 +22.369 Log ([Fe]) - 128.2 P where, L = length of stained section (mm) [Fe] = iron concentration in the sample (mole/liter)

P = o-phenanthroline weight percent in the TLC plate.

Table 2 depicts the lengths of the stained sections that were produced by exposure of l-nitroso-2-naphthol plate detectors to different concentrations of copper and cobalt ions. Since the 1nitroso-2-naphthol complexes with iron and cobalt exhibit different colors it is possible to separate the two compounds by a single TLC plate detector and to quantitate both concentrations with no need for further־ visualization technique. Thus, combining TLC separation ability, color visualization and quantitation into one step.

In a different experiment copper, cobalt and ferrous ions were separated using the same type of 1-nitroso *2-naphthol TLC plate. However, because the complexes of iron and copper exhibit similar shades of green it was necessary to distinguish between these two sections by further visualization with ammonia vapors.

TABLE 1

CALIBRATION EXPERIMENTS OF IRON(II) TLC PLATE DETECTORS

<td rowspan="2"> 7.0- Phenanthroline in glass</td><td rowspan="2"> 0.033</td><td colspan="3"> Length of Stain (mm)</td><td rowspan="2"> 0.099</td>

<td> 0.043</td><td> 0.066</td><td> 0.075</td>

<td> IRON Cone. (M) 0.00005</td><td> 13.5</td><td> 13.5</td><td> 9.5</td><td> 7.5</td><td> 5.0</td>

<td> 0.0001</td><td> LB.5</td><td> 1Θ.75</td><td> 15.0</td><td> 14.5</td><td> 12.0 ’</td>

<td> 0.00025</td><td> 26.0</td><td> 26.0</td><td> 21.5</td><td> 22.5</td><td> 19.0</td>

<td> 0.0005</td><td> 36.0</td><td> 36.0</td><td> 33.0</td><td> 27.5</td><td> 24.5</td>

<td> 0.001</td><td> 43.75</td><td> 42.5</td><td> 39.0</td><td> 38.0</td><td> 35.0</td>

TABLE

<td> LENGTHS OF</td><td> STAINS</td><td> PRODUCED BY</td><td> TLC PLATE</td><td> DETECTORS .</td>

<td> Analyzate:</td><td> Co(Il)</td><td> Cu(II)</td><td> Co(II)</td><td> Cu(II)</td>

<td></td><td colspan="2"> CONCENTRATIONS (mH)</td><td> Length</td><td> of Stains (mm)</td>

<td></td><td> 0.375</td><td> 5.0</td><td> 1.0</td><td> 10.0</td>

<td></td><td> 0.75</td><td> 5.0</td><td> 2.0</td><td> 9.0</td>

<td></td><td> 1.5</td><td> 5.0</td><td> 3.0</td><td> 9.0</td>

<td></td><td> 1.5</td><td> 10.0</td><td> 2.0</td><td> 16.0</td>

C. Masking chromatographic interferences by incorporation of doped sol-gel glass into the chromatographic media.

In the following example we demonstrate that the separation of amino acids containing High concentration of iron(II) can be improved by the addition of a few percent of 1,10-o-phenanthroline doped sol-gel glasses into the thin layer chromatographic media.

Experimental details!

Preparation of doped sol-gel glass: Preparation of sol-gel glasses doped with 1,10-o-phenanthroline was conducted according to the general procedure outlined in example B above. In a typical procedure 2.5 ml tetramethoxysilane (Petrarch), 3.0 ml methanol,

2.4 ml distilled water, 0.5 ml of 1 mM NaOH solution and 3.0 ml containing 1,10-o-phenanthroline solution were mixed, allowed to gel and dried to a constant weight at 41°C for three days. The dried glass contained 1.07. weight □-phenanthroline.

λ u

Preparation of TLC plates: The doped sol-gel glasses were crushed in a chinaware cup and sieved by a sieve shaker. The glass fraction smaller than 0.07 mm was used for the TLC plate preparation. 0.5 gr of doped sol gel glass and 3.6 gr. of 70-230 mesh ASTM Kieselgel 60 (Merck) were mixed with 0.6 gr. anhydrous CaS□0.3 ״ gr. starch and 9 ml distilled water. The slurry was spread on 5x20 cm glass plates. The TLC plates were than dried for 2 Hours in a S0°C oven forming solid 0.5 mm thick coatings.

Control plates, using the same ingredients without doped sol-gel glasses were prepared following the same procedure.

Amino—acid separation: One microliter aqueous solution containing freshly prepared 0.33 mg/ml L-histidine, 0.33 mg/ml D— phenylalanine, 0.33 mM ferrous ammonium sulfate and 0.067 gr/ml npropanol was applied to each plate. A mixture of 707. ethanol (957. pure) and 307. distilled water was used as eluent. Visualization of the spots was done by spraying 27. (weight) ninhydrin in ethanol solution followed by heating to 110°C.

Results:

Figure 2 depicts a comparison of the separation achieved by the two TLC plates. The R,s and the dimensions of the spots of the two amino acids in the TLC plate containing doped sol-gel glasses (plate A) were unaffected hy the 1>re^׳?nre of ferrous ions. The

I/! II.

spot size and the R, of the L-Histidine was considerably distorted in the presence of ferrous ions when the control TLC plate (without doped glass) was used (plate B) .

D. Tube Detectors for Gas Analysis;

In the following we demonstrate (Length of Stain) Tube Detectors containing methyl-orange doped sol-gel glasses for the determination of acidity and alkalinity in air.

Experimental details:

Glass Preparation; Sol-gel glasses doped with 0.057. methylorange indicator were prepared according to the procedure in example B.

Preparation of tube detectors: Glass capillar ies (0.7 mm in diameter and 10 cm length) were filled (up to 3/4 of their length) with Sol Gel glass pellets doped with the methyl orange reagent. The tubes were stoppered at both ends by 3 mm filter papers to hold the glass pellets in place.

Air Detection: Air was drawn by a vacuum pump through a solution containing either 257. ammonium hydroxide or 36 7. aqueous solution of hydrochloric acid and then through a methyl orange tube detector which has originally been in its acidic or basic form respectively.

?2 &lt;־'

Results:

Figure 3A depicts the color front (from yellow to orange) propagation when 0.03 ml/minute of air containing ammonium hydroxide was passed through pH tube detector. Figure 30 depicts i

a color front (from orange to yellow) propagation when 0.03 ml/minute of acidic air was passed through the detector. In both cases the front was Very sharp and its position could be accurately detected visually.

E. Tube Detectors for Liquid Phase Analysis

The strong entrapment of reagents in the doped sol-gel glasses makes the production of tube detectors for aqueous analysis possible. In the following we shall demonstrate tube detectors for quantitative determination of cobalt, iron and aluminum ions in aqueous solution.

Experimental details:

Glass preparation: The experiments were carried out using SolGel glasses prepared according to standard Sol-Gel procedures depicted above: In a typical procedure 5.0 ml tetramethoxysilane (Petrarch), 6.0 ml methanol solution containing the reagent, 1.2 ml distilled water and a 1 ml 1 mM NaOt-l solution (added for base catalysis) were mixed, allowed to gel and dried to constant weight at 41°C for three days. 0 typical reagpnt solution (e.g. the solution used for preparation of the iron glass detector in figure ll.

4) contained 70 mg 1,10-o-phenanthroline. After drying, the glasses were crushed in a chinaware cup and sieved by a sieve shaker. The fractions between 0.5 -0.4, 0.4 - 0.2, 0.2 - 0.1 mm and the glass pellets smaller than 0.1 mm were collected for further experiments.

Preparation of tube detectors! Glass capillaries (0.7 mm in diameter and 10 cm length) were filled (up to 3/4 of their length) with Sol Gel glass pellets doped with, the colorimetric reagents. The tubes were stoppered at both ends by 3 mm filter papers to hold the glass pellets in place.

Calibration curves: The following method was used in order to determine the response of tube detectors to different concentrations □f target analyzates. One end of the tube detector was immersed in approximately 2 mm deep sample solution. Capillarity forces drive the solution upward through the tube detectors. As the solution front advances up, a second color front marking the upper end of the stained portion of the Sol-Gel doped glasses ascends. The length of the stained portion of the glasses can be measured after the solution reaches the upper end of the capillary or after a certain amount of solution is passed through the glass column.

i'l ־ Ί

In some experiments the capillarity force was replaced by an external pumping device such as positive displacement pump or hydrostatic pressure.

Results;

Figure 4 depicts the response of disposable tube detectors containing glass pellets doped with 1,10-o-phenanthroline for iron(II) determination, l-nitroso-2-naphthol for cobalt(Il) and alizarin for aluminum(III) determination. The experiments depicted in figure 4 were carried out using glass capillaries filled with Sol-Gel pellets smaller than 0.1 mm and using capillarity force to drive the solution into the tube detectors. The color fronts (from white to red for iron, from yellow to brown for cobalt and from lilac to purple for aluminum) were sharp and could.be distinguished within accuracy greater than 1 mm. An almost linear dependence of the stain length on the concentration of the analyzates is exhibited.

F, Alkalinity and Acidity Tube Detectors

In the following we demonstrate that methyl orange doped sol-gel Tube Detectors can serve as alkalinity detectors and that the length of stain produced when water is sampled through the tube detectors represent alkalinity rather than pH measurement. Experimental

Tube Detector preparation: Tube detectors containing sol-gel glasses doped with 0.057. methyl orange were prepared according to the procedure specified in example E above.

The samples containing several combinations of sodium carbonate and sodium bicarbonate solutions were pumped into the capillaries at a constant rate using a home built positive displacement pump.

Results:

Table 3 depicts the length of stain produced by three sample solutions exhibiting the same pH (pH =9.7) and different alkalinities. The length of stain is almost proportional to the alkalinity (expressed in mg CaCO_s). Maintaining the same alkalinity (as in sample 3 and 4) and changing the pH did not change the length of stain response of the detectors.

Table 3

Response of Alkalinity Tube Detectors

<td> experiment number</td><td> Alkalinity (mg CaCO,)</td><td> pH</td><td> Sampled volume (ml)</td><td> Length of Stain (mm)</td>

<td> 1</td><td> 300</td><td> 9.7</td><td> 0.07</td><td> 2.7</td>

<td> 2</td><td> 1,500</td><td> 9.7</td><td> 0.07</td><td> 5.8</td>

<td> 3</td><td> 3,000</td><td> 9.7</td><td> 0.07</td><td> 9.7.</td>

<td> 4</td><td> 3,000</td><td> 11.0</td><td> 0.07</td><td> 10.0</td>

G. Fracture prevention!

We hereby demonstrate that doping sol-gel glasses with ionic surface active agents (e.g. quaternary ammonium ions) prevents cracking of doped sol-gel glass during drying and wetting of the glasses. Glasses doped with said dopants and with photometric reagents maintain their photometric activities.

Experimental Details:

Glass preparation! The experiments were carried out using Sol-Gel glasses prepared according to standard Sol-Gel procedures depicted above: In a typical procedure 5.0 ml tetramethoxysilane (Petrarch), 6.0 ml methanol and

2.4 ml water containing the reagent and cetylpyridinium bromide ar cetyltrimethyarnmonium bromide according to the concentrations depicted below (in some cases the reagents were dissolved in the methanol) and 1 ml 0.1 mM NaGH solution (added for base catalysis) were mixed, allowed to gel for three days and then dried to constant weight at 41°C for two weeks.

Results:

Figure 5 depicts several typical photometric detectors containing doped “surface active agents. The first upper section contains four examples of monolithic doped glasses before and after immersion in aqueous solution containing the analyzate (from left to right: Redox detector containing

Diphenylaminesulfonate barium salt (Img) and 5 mq cetylpyridinium bromide (CPB) before and after immersion in sodium hypochlorite solution; pH indicator containing phenolphthalein (2.5 mg) and 6.6 mg

?1 על cetyltrimethylammonium bromide (CTAB) before and after immersion in basic aqueous solution; iron Detector containing 1.2 mg o-phenanthroline and 5.0 mg CPB before and after immersion in a solution containing ferrous ammonium sulfate; nitrite indicator containing 4 mg 1-Naphthy lenediamine dihydrochloride, 10 mg sulphanilic acid and 6 mg C l AB). The lower row depicts few detectors (from left to rights copper detector containing 1 mg dithiooxamide and 6.25 mg CPB ; lead detector containing 1 mg galocyanine and

7.5 mg CPB ; pH detector containing 5 mg bromophenol and 6.6 mg CTAB; nickel detector containing 2 mg dimethyglyoxime and 6.25 mg CPB; aluminum detector containing 5 mg alizarin and 12.5 mg CPB)

In all cases the described procedure prevented cracking of the monolithic glasses even after few cycles of wetting and drying of the glasses.

0-/ י i

/ י ׳'

Figures

Figure 1! Selectivity isotherms of glass immobilized Aliquat 3368 ^v a.Br־/cr; b. NO_s־/Cr; c. S0//C1 ;(C_a, Y_a represent concentrations of the counter ion and C_eJ.Y_el represent the concentrations of chloride in the solution and glass phase respectively)

Figure 28 Amino acid separation in the presence of ferrous ionss A. Separation by a TLC plate that contains doped sol-gel glasses

B. Separation by a TLC plates without doped glasses

Figure 38 Length of stain development in alkalinity tube detectors: A. Transition from acidic form of methyl orange doped glasses to the basic form of the detector! B. Transition, from basic to acidic form

Figure 48 Response of several tube detectors to different concentrations of elements in water: a. Iron(II) detectors containing 1.77. 1.10-o-phenanthroline , pH = 4.0; Cobalt(II) detectors containing 0.037.1nitroso-2-naphthol, pH=6.0; C- Aluminum detectors containing 0.57. alizarin, pH =4

Figure 5s Monolithic glass detectors doped with ionic surface ' active agents to prevent cracking. Upper Section: detectors before and after immersion in solution containing the analyzates; lower rows Monolithic glass disks.