STAINED SOLS OF NON-METALLIC ELEMENTS OR COMPOUNDS, THEIR PREPARATION AND USE

Abstract: The invention relates to a process for the preparation of a sol of a metallic or non-metallic element or compound, such as SiO₂, sulfur, selenium, tellurium, and copper, wherein the sol is formed in a medium which contains at least one dye in solution to obtain a stained sol.

Description: The invention relates to a process for the preparation of a sol of a non-metallic element or compound. Such a process is known both from very old and from very recent publications. More than hundred years ago, Schulze, J. Pract. Chem. (2), 32, 390, 1885, described a preparation of selenium sols (i.e. dispersions of selenium particles in an aqueous liquid; the sols may also be referred to as hydrosols) by adding SO₂ to an aqueous solution of selenious acid (H₂SeO₃). In 1902, Gutbier, Z. Anorg. Chemie 32, 52, 1902, described a preparation of tellurium sols by treating an aqueous solution of telluric acid (H₆TeO₆) with hydrazine hydrate. Gutbier, Z. Anorg. Chemie 32, 106, 1902, also described a preparation of selenium sols by using hydrazine hydrate as reducing agent. Brintzinger, Koll. Zeitschr. 78, 22, 1937, disclosed a process for the preparation of selenium, tellurium, gold, palladium, platinum, silver, molybdenum and tungsten sols by reduction (for example of SeO₂) with ascorbic acid or isoascorbic acid as a reducing agent. US-A-2329147 and DE-A-756906 disclose stained sols of metals, especially of heavy and precious metals. More recent publications are concerned with a particularly interesting use of sols, i.e. for the labelling of a component of the reaction between a specifically-binding substance and a corresponding bindable substance, in particular use of a sol as a label for a component of the reaction between a specifically-binding substance and a corresponding bindable substance in a method for assaying, detecting or measuring the presence and/or quantity of a component of said reaction in a test sample. In EP-A-0 007 654 (Akzo N.V., published Feb. 6, 1980) the use and preparation of sols of a metal or metal compound, in particular gold and silver sols, is described. EP-A-0 032 270 (Akzo N.V., published July 22, 1981) discloses the use and preparation of sols of a hydrophobic dye or pigment. The use and preparation of sols of non-metallic elements, such as selenium, tellurium and sulfur is taught in EP-A-0 298 368 (Abbott Laboratories, publ. Jan. 11, 1989) and in Dutch patent application NL-A-87.02769 and the corresponding part of EP-A-0 321 008 (in the name of H.B.T. Holland Biotechnology B.V., publication June 21, 1989). The utility of a sol as a marker system for e.g. immuno-chemical reactions (for example in dipstick assays) generally requires a good visibility or, in more general terms, a good detectability of the sol particles. Some kinds of sols are not quite satisfactory in this respect because they are either not colored or only weakly colored. Examples of sols whose utility would be improved if they would have a more clearly detectable color are elemental sulfur sols, sols of the compound SiO₂, elemental selenium sols, and elemental copper sols. The present invention provides a solution of this problem with uncolored or weakly colored sols and, in addition, creates new possibilities by a process for the preparation of a sol of a non-metallic element or compound, which process is characterized in that the sol is formed in a medium which contains at least one dye in solution to obtain a stained sol. It should be understood that the words "a sol of a non-metallic element or compound" refer to a dispersion (preferably an aqueous dispersion, but alcoholic dispersions and dispersions in other organic liquids are not excluded) of particles substantially consisting of a non-metallic element or compound, without any intention to exclude that the sol particles are not fully homogeneous and, for example, contain a minor amount of the non-metallic element in the form of a chemical compound, i.e. the particles in a selenium sol may contain some selenium oxide, but most of the selenium will be present in the elemental form. Preferably at least 60%, more preferably at least 70% and most preferably substantially all of the selenium will be present in the elemental form. The same applies to sols of other elements, such as sulfur and, tellurium sols, while sol particles of a chemical compound, such as SiO₂ may contain minor amounts of other compounds or elements. Of course, it is inherent to the words "a sol of a non-metallic element or compound" that the element or compound concerned is an element or compound which can exist in the form of a sol. Chemical elements such as hydrogen, helium, oxygen, etc., and chemical compounds such as sulfur dioxide and nitrogen oxides do not satisfy this inherent requirement and are consequently not covered by the words "element" and "compound" as used herein. As far as non-metallic elements are concerned, the invention is particularly concerned with sols such as selenium, sulfur, tellurium, arsenic and phosphorus sols. Most preferably, the invention relates to a process for preparing a selenium or sulfur sol. As far as chemical compounds are concerned, the invention is especially concerned with inorganic compounds. The most preferred embodiment of the invention, however, is a process for the preparation of stained silicium dioxide sols. According to the invention, the process step in which the sol is formed is carried out in the presence of a dye which has been dissolved in the reaction medium. As the sols are usually prepared in aqueous media, the dye should preferably be soluble in a polar solvent such as water, alcohol, or acetone. It is preferred to use a dye carrying a nett negative charge when a sol is prepared which contains nett positively charged sol particles, and to use a dye carrying a nett positive charge when a sol is prepared which contains nett negatively charged sol particles. Other charge combinations, such as the addition of a negatively charged dye to a negatively charged sol, are not excluded, however. The use of a dye which carries the opposite charge compared to the sol particles to be formed is beneficial to obtain an irreversible and strong binding of the dye to the sol particles. Indeed, it was experimentally found impossible to remove the dye from the sol particles, either by washing or by dialysing. In the case of flocculation of the stained sols, the dye remains tightly bound to the sol particles and is flocculated therewith; no dye leaves the flocculated particles. Particular embodiments of the invention are characterized in that a dye, selected from the group consisting of fluorescent rhodamine isothiocyanate, Brilliant Cresyl Blue, Malachite green, and Oracet Blue (Chroma, Stuttgart, West Germany), is used. The advantages of stained sols obtainable by the process of the invention are numerous. First of all, the invention makes it possible to provide originally uncolored sols with very bright colors, thus creating or improvimg their utility as a marker system. For example, the invention makes it possible to provide SiO₂ sol particles, which are white by their own, with any desired color, such as red, blue, green, etc. In addition, the invention allows for the preparation and use of one kind of sol particle in different colors. When two or more differently stained SiO₂ sols having clearly distinctive colors are prepared and each is coupled to a different binding substance, e.g. different monoclonal antibodies, two or more agents can be recognized/detected on the same dipstick. Also, one of such differently stained sols could serve as a control of the correct reaction. Another advantage of the invention is the fact that fluorescent dyes can be used, since it is a known fact that the use of fluorescent dyes can lead to a higher assay sensitivity compared to the normal dyes. In view of these unexpected and highly advantageous properties of sols prepared by the process of the invention, the invention also extends to sols of a non-metallic element or compound, which are obtainable by the process of the invention. As is evident from the cited publications EP-A-0 007 654, EP-A-0 032 270, EP-A-0 298 368 and NL-A-87.02769, sols of non-metallic elements or compounds may be used for the labelling of a component of the reaction between a specifically-binding substance and a corresponding bindable substance. The invention extends to such a use, in particular to the use of a sol of a non-metallic element or compound, which is obtainable by the process according to the invention, as a label for a component of the reaction between a specifically-binding substance (e.g. protein, DNA, RNA, polysaccharide) and a corresponding bindable substance in a method for assaying, detecting or measuring the presence and/or quantity of a component of said reaction in a test sample. The invention will be explained in greater detail by means of the following examples of preferred embodiments thereof. It should be noted, however, that the examples are not intended to limit the scope of the invention but merely serve to illustrate the invention. The operations described hereunder were performed at room temperature, unless otherwise stated. Example 1 (a) Preparation of a SiO₂ sol stained with fluorescent rhodamine isothiocyanate 100 mg Rhodamine isothiocyanate (RITC, Sigma) was dissolved in 862 ml absolute ethanol (Merck). To this solution, 74 ml of a 25% aqueous NH₃ solution (Merck) was added, using a magnetic stirrer for mixing both solutions. Then 64 ml tetraethyl orthosilicate (Merck) was added quickly with vigorous stirring. The initial clear purplish/red solution becomes visibly opalescent after about 7 minutes. After 120 minutes, the sol was purified by dialysis (by means of an artificial kidney). During the dialysis, the pH of the sol was decreased from pH 10.0 to pH 6.5. The dialysis was terminated as soon as the dialysate no longer contained spectrophotometrically measurable quantities of RITC: (A 1 cm / 560 nm = 0). Thus, an opalescent, purple SiO₂ sol contained in an aqueous medium was formed. The average diameter of the spherical SiO₂ particles was 156 ± 8 nm. (b) Adsorption of a monoclonal mouse IgG to colloidal RITC/SiO₂ Prior to use, the sol was diluted to A(λ = 560 nm) = 1.0. The pH of 5 ml of the RITC/SiO₂ sol was adjusted to pH 7.0 by means of 0.1 M K₂CO₃. Mouse ascites (50 µl) was added under gentle stirring to said 5 ml neutralized RITC/SiO₂ sol. After an incubation of 15 minutes, the volume was made up with water to 15 ml. The formed RITC/SiO₂-IgG(ascites) conjugate was centrifuged at 1300xg (4°C) for 5 minutes. The supernatant was collected on a suction filter and the loose pellet containing the conjugate was re-suspended in 500 µl 5 mM NaCl solution. (c) Test procedure and results A concentration series of goat anti-mouse polyclonal IgG (1280 ng to 2.5 pg) was spotted onto nitrocellulose test strips (1) . As a negative control, a concentration series of β-galactosidase (1500 ng to 2.0 pg) was included. Cross-incubation (2) of the various strips was effected both with RITC/SiO₂-IgG(ascites) conjugate and with unprotected RITC/SiO₂ sol. The following results were obtained. Goat anti-mouse spots were specifically stained by the RITC/SiO₂-IgG(ascites) conjugate up to 40 ng. Goat anti-mouse spots incubated with unprotected RITC/SiO₂ sol did not show any specific staining. β-galactosidase spots did not exhibit any specific staining either with the RITC/SiO₂-IgG(ascites) conjugate or with unprotected RITC/SiO₂ sol. Note (1): the test strips were made of nitrocellulose paper having a pore diameter of 0.45 µm and being cut to a size of 1.2 cm x 5.5 cm. The spots applied to the test strips had a volume of 1 µl. After spotting the concentration series, the strips were dried at 37°C for 20 minutes. Immediately thereafter, the bare parts of the strips (i.e. the parts that were not covered) were blocked with a 3% Bovine Serum Albumine (BSA) solution in Phosphate Buffered Saline (PBS) at 37°C. After 20 minutes, the strips were dried at 37°C and were then ready for use. Note (2): the test strips were incubated in a 1:50 dilution of the probe in PBS + 1% BSA for at most 2 hours. During the incubation, the strips and incubation mixture were agitated on a shaker in polypropylene tubes. The sensitivity of a probe was determined by the lowest visually detectable concentrations of spotted (immobilized) protein in non-optimized systems. Example 2 Preparation of a SiO₂ sol stained blue with Brilliant Cresyl Blue 100 mg Brilliant Cresyl Blue (BCB, B.D.H.) was dissolved in 862 ml absolute ethanol (Merck). To this solution, 74 ml of a 25% aqueous NH₃ solution (Merck) was added, using a magnetic stirrer for mixing both solutions. Then 64 ml tetraethyl orthosilicate (Merck) was added quickly with vigorous stirring. The initial clear purple solution becomes visibly opalescent after about 7 minutes. After 120 minutes, the sol was purified by dialysis (by means of an artificial kidney). During the dialysis, the pH of the sol was decreased from pH 10.0 to pH 6.5 which resulted in a color change from purple to blue. The dialysis was terminated as soon as the dialysate no longer contained spectrophotometrically measurable quantities of BCB: (A 1 cm / 560 nm = 0). Thus, an opalescent, blue SiO₂ sol contained in an aqueous medium was formed. The average diameter of the spherical SiO₂ particles was about 150 nm. Example 3 Preparation of a SiO₂ sol stained green with Malachite Green 100 mg Malachite Green (MG, Sigma) was dissolved in 862 ml absolute ethanol (Merck). To this solution, 74 ml of a 25% aqueous NH₃ solution (Merck) was added, using a magnetic stirrer for mixing both solutions. Then 64 ml tetraethyl orthosilicate (Merck) was added quickly with vigorous stirring. The initial clear colorless solution became visibly opalescent after about 7 minutes. After 120 minutes, the sol was purified by dialysis (by means of an artificial kidney). During the dialysis, the pH of the sol was decreased from pH 10.0 to pH 6.5; as a result, the opalescent colorless sol turned dark blue-green. The dialysis was terminated as soon as the dialysate no longer contained spectrophotometrically measurable quantities of MG: (A 1 cm / 560 nm = 0). Thus, an opalescent, green SiO₂ sol contained in an aqueous medium was formed. The average diameter of the spherical SiO₂ particles was about 150 nm. Example 4 (a) Preparation of a selenium sol stained with Oracet Blue Oracet Blue (0.005 g) was dissolved in 1 ml ethanol. After 10 minutes, 9 ml demineralised water was added, followed by a filtration of the solution (solution A). A second stock solution was prepared by dissolving 0.005 g Oracet Blue in 1 ml acetone. After 10 min, 9 ml demi-water was added, followed by a filtration of the solution (solution B). SeO₂ was dissolved in demi-water to a concentration of 0.2 g/l, and NaBH₄ was dissolved in demineralised water to a concentration of 25 g/l. Several sols were prepared under vigorous stirring by adding 1 ml of the above NaBH₄ solution and different amounts of Oracet Blue and demineralised water to 100 ml of the above SeO₂ solution. Data on the sols prepared are given in Table I. After an incubation of 30 minutes, the sols were purified by means of dialysis. The sols did not change color upon dialysis. Moreover, addition of NaBH₄ to Oracet Blue alone did not result in a change of color, indicating that the color change from orange-red to brown of the selenium sol was due to the formation of a stained selenium sol. (b) Adsorption of IgG to colloidal stained Se-sols The pH of 50 ml sol was adjusted to pH 7.5 by addition of 0.01 M K₂CO₃. Then, 100 µl polyclonal rabbit antibodies (serum) directed against nortestosteron were added. Coupling took place at room temperature for 10 minutes. The mixture was then centrifuged at 4,600xg (4°C) for 10 minutes. The supernatant was removed and the pellet was suspended in 5 ml NaCl solution (1/20 th part of the original volume). The anti-nortestosteron IgG-stained selenium probe thus obtained was used for detection purposes. (c) Test procedure and results Different amounts of conjugated BSA-nortestosteron were spotted onto nitrocellulose strips. After drying and blocking the nitrocellulose with a 2% BSA solution, the nitrocellulose strip was incubated with the stained sol. After a few minutes, dark brown spots became visible. Stained sols which had been coated with irrelevant rabbit sera did not stain the spots. The same procedure with unstained Se-sols resulted in less visible orange-red spots.

Claims: 1. A process for the preparation of a sol of a non-metallic element or compound, characterized in that the sol is formed in a medium which contains at least one dye in solution to obtain a stained sol. 2. A process according to claim 1, characterized in that a dye is used, which is soluble in a polar solvent such as water, alcohol, or acetone. 3. A process according to claim 1 or 2, characterized in that a dye carrying a nett negative charge is used when a sol is prepared which contains nett positively charged sol particles, and that a dye carrying a nett positive charge is used when a sol is prepared which contains nett negatively charged sol particles. 4. A process according to claim 1, characterized in that a dye, selected from the group consisting of fluorescent rhodamine isothiocyanate, Brilliant Cresyl Blue, Malachite green, and Oracet Blue, is used. 5. A process according to any of claims 1 to 4, characterized in that a sol of the chemical compound SiO₂ is prepared. 6. A process according to any of claims 1 to 4, characterized in that a sol of the element selenium (Se) is prepared. 7. A process according to any of claims 1 to 4, characterized in that a sol of the element sulfur (S) is prepared. 8. A process according to any of claims 1 to 4, characterized in that a sol of the element tellurium (Te) is prepared. 9. A stained sol of a non-metallic element or compound, which is obtainable by the process of any of claims 1 to 8. 10. The use of a stained sol of a non-metallic element or compound, which is obtainable by the process of any of claims 1 to 8, for labeling a component of the reaction between a specifically-binding substance and a corresponding bindable substance. 11. The use of a stained sol of a non-metallic element or compound, which is obtainable by the process of any of claims 1 to 8, as a label for a component of the reaction between a specifically-binding substance and a corresponding bindable substance in a method for assaying, detecting or measuring the presence and/or quantity of a component of said reaction in a test sample.

Inventors: KEIZER, GERRIT DIRK, VAN DOORN, ALBERT WILLEM JACOB, VAN ES, REMCO MARIA, VAN GELDER, WILHELMUS MARTINUS JOZEF, WICHERS, JAN HERMAN
Applicants: AGROTECHNOLOGISCH ONDERZOEKSINSTITUUT (ATO), H.B.T. HOLLAND BIOTECHNOLOGY B.V.
Application year: 1989
Main classification: B01J13
IPC codes: C07H23, G01N33, B01J13