POROUS SUBSTRATES WITH A HIGH CONCENTRATION OF AMINE GROUPS

Abstract: This invention relates to porous substrates with a high concentration of amines which are useful for transfer of substances from a gel or culture.

Description: POROUS SUBSTRATES WITH A HIGH CONCENTRATION OF AMINE GROUPS CROSS-REFERENCE TO RELATED APPLICATIONS This is a continuation-in-part of copending patent application Serial No. 07/304,737, filed on January 31, 1989. FIELD OF THE INVENTION This invention relates to porous substrates with a high concentration of amine groups and to the use of such substrates to transfer and immobilize nucleic acids, proteins, bacteria, and viruses from electrophoresis gels or culture media to a substrate. BACKGROUND OF THE INVENTION A number of materials and methods have been developed for transferring substances from a gel or culture medium to an immobilizing membrane. Ξ. M. Southern (J. Molecular Biology, Vol. 98, p. 503, 1975) has disclosed a method for transferring nucleic acids from electrophoresis gels to an adsorptive medium. In the Southern method, the membrane is placed between the gel and a few layers of paper towels. As buffer solution is forced through the gel and membrane and into the paper towels by capillary action, the nucleic acid sample is driven into and deposited on the membrane. This is referred to as transfer by blotting. Bittner et al. (Anal. Bioche ., Vol. 102, p. 459, 1980) disclosed a similar method in which an electrical potential is used drive the nucleic acid sample into the membrane. This is referred to as electrophoresis transfer or eiectroblctting. Adsorptive micropcrous membranes have also been ' seά to transfer bacteria and viruses from growth media. Several materials have been effectively used for use as transfer membranes, including nitrocellulose, nitrobenzyloxymethyi cellulose, aminobenzyioxymethyl cellulose, aminophenyl-thioether cellulose, diethyla inoethyl cellulose, and polyvinylidene fluoride. The cellulose-based materials are however brittle and frequently break up during the transfer process or the manipulations involved in subsequent analytical procedures. U. S. 4,455,370 broadly discloses the use of microporous adsorptive nylon films as transfer films. Methods for plasma-etching polyoiefins in the presence of ammonia are, of course, known in the art. ϋ. S. 4,340,479 discloses a process for preparing skinless hydrophilic alcohol-insoluble polyamide membranes comprising preparing a solution of an alcohol- insoluble polyamide resin in a polyamide resin solvent, inducing nucleation of the solution by controlled addition of a nonsolvent for the polyamide resin to the polyamide resin solution to obtain a visible precipitate of polyamide resin particles, thereby forming a casting solution; spreading the casting solution on a substrate to form a film on the substrate, contacting and diluting the film of casting solution with a nonsolvent for the polyamide resin, thereby forming a membrane, washing the membrane to remove solvent, and drying the resulting membrane. The reference teaches that although the starting polymers are available in a wide variety of grades, which vary appreciably with respect to molecular weight and in other characteristics, the formation of a hydrophilic membrane appears to be a function net of these characteristics, but of the chemical composition of the polymer. U. S. 3,876,738 discloses a process for the production of microporous films by casting or extruding a solution of a film-forming polymer in a sclvent system into a quenching bath which is comprised of a nonsolvent svste for the oolvmer. The colvmers emcioved are preferably nylon polymers. There is no discussion of • advantages which might accrue to the use of various molecular weight grades of nylon. SUMMARY OF THE INVENTION This invention corrects the problems associated with prior art membranes. This invention relates to a composition of matter, a polyamide substrate which has a high surface concentration of amine groups optionally supported by an inert support. This invention also relates to an improved method for the transfer of materials such as nucleic acids, proteins, bacteria, or viruses preferably from gel a or culture to a porous substrate, wherein the improvement comprises contacting the material with a polyamide substrate which has a high concentration of amine groups. This invention relates further to the transfer of nucleic acids or proteins from an electrophoresis gel to a polyamide substrate with a high concentration of amine ends by blotting or electroblotting. It also relates to transfer of nucleic acids, proteins, bacteria, or viruses from culture media (including plaques) by contacting the substance to be transferred in the medium with a polyamide substrate which has a high surface concentration of amine groups. DETAILED DESCRIPTION OF THE INVENTION This invention relates to a porous substrate which has a high surface concentration of amine groups. It is critical that the polyamide substrate have a high surface concentration of amine groups in order to bind nucleic acids or other biological materials. Preferably, the concentration of amine groups should be at least about 60 equivalents per 10 6 grams. Preferably, the surface area of the substrate should be at least about 1 m 2 /g, as measured by nitrogen adsorption. Suitable amine groups are characterized as -NRR', where R and R 1 are independently H, Ci-io branched, unbranched or cylic alkyl, Cς aryl or C7-- 12 aralkyl, where the alkyl, aryl or aralkyl groups may contain heteroatom substituents such as halogens or ether oxygens which do not interfere with binding of the biological samples to the porous substrate. Pore size of the substrates is expressed in terms of bacterial filtration ratings. For example, ability to quantitatively remove the organism Pseudomonas diminuta is accepted as defining a 0.2 μ bacterial rated filter, and ability to quantitatively remove the organism Serratia arcescens is acepted as defining a 0.45 μ bacterial rated filters. Materials with bacterial filtration ratings in the range of about 0.2 μ to 0.8 μ are preferred, although materials with larger or smaller ratings can be used. For transfer of DNA molecules smaller than about 1 kB and for RNA, the 0.2 μ rated materials are preferred. For transfer of DNA molecules larger than about 1 kB, the 0.45-0.8 μ materials are preferred. In contrast, pore size does not seem to be as -important in the transfer and retention of protein molecules, bacteria, or viruses. The polyamide substrate of this invention may be a homogeneous material, such as a polymeric film or membrane fabricated by techniques known in the art from polymers, or mixtures of polymers, which contain a high concentration of amine groups. A preferred substrate is a blend of low molecular weight poi amides (for high amine group concentration) and high molecular weight poiyamides (for strength) . Most preferably, the blend comprises about i to 90% by weight low molecular weight polyamide and about 10 to 99% by weight high molecular weight polyamide . Higher percentages of low MW material usually result in brittle blends. The concentration of amines in the low MW poiyamides should be :70 amine equivalents/10 δ g (i.e., 70 moles of amine groups per 10 6 g of polyamide) to impart a high surface concentration of amine groups to the substrate, but <1,500 amine equiv./10 6 g to maintain water-insolubility of the amine-rich portion of the polymer blend. The concentration of amine in the high MW poiyamides is <80 amine equilvanets/10 6 g. It will be appreciated by those skilled in the art that a compromise is necessary between the desire to have a substrate with a high surface concentration of amine groups and the necessity of incorporating a sufficient amount of high MW material to maintain structural integrity of the substrate. It may not, therefore, be possible to make a suitable substrate by using a large amount of high amine equivalent, low molecular weight polyamide. It has been found that for beneficial DNA adsorption, the final membrane has at least 60 amine ends/10^ g of polymer. 3ienάs of other low molecular weight, amine-ended polymers with compatible high molecular weight polymers should also give porous substrates with high surface concentrations of amine groups. High surface concentrations of amine groups can also be achieved by coating or impregnating an inert support with a polymer or mixture of polymers which contain a high concentration of amines. Suitable inert supports include, but are not limited to, cellulose, poiyolefins, poiyamides, polyesters, and fiberglass. The effectiveness of the substrate for transferring nucleic acids and other biological materials can be tested by procedures disclosed in U. S. 4,455,370, which cr csdures are herein incorporated bv references . In preparing composition according to the invention, it is preferable to use 0.074 ml material per ml of formic acid casting solution. Although material may not be necessary, its addition to the casting solution brings the polymer to the point of precipitation. Methanol is preferably used in the quench bath as a 1:1 solution of methanol:water. Any nonsolvent or nonsolvent mixture gives microporous structure. Glass is the preferred casting substrate, although any inert smooth surface allowing spreading of casting dope and easy release of membrane may be used. The compositions are preferably prepared at ambient temperature. EXAMPLES Comparative Example 1 Membrane Preparation High Amine 66 Nylon Membrane Can Not Be Made by the methods disclosed in U. S. 3,876,738 A 66 nylon sample was obtained with 80 equivalents of amine/10 6 g of polymer by means known in the art. A 16 g sample of the nylon was dissolved at ambient conditions in 63 mi of 96% formic acid and 9.0 ml of methanol. This solution was cast on glass as a 10 mil thick film and then quenched for 10 minutes in 1000 mi of either 1:3 or 3:1 methanol: ater. The film, still on the glass support, was removed from the bath and air dried. The film was too weak to be removed as a single piece either dry or after rewetting with water. A glass plate was sprayed with Teflon® Dry Lubricant before casting. The final films no longer adhered to the glass, but shrank and cracked to useless fragments on drying. Apparently, nylon samples with high concentrations of amine ends are too low in molecular weight to give ocrous substrates with adeσuate strenσth. Example Low Adsorption with Low Ends, Plain Membrane A 66 nylon sample (Zytel™ 42) was obtained with 20 equivalents of amine and 44 equivalents of carboxyl/10 6 g of polymer. A 72 g sample of this nylon was dissolved in 190 ml of formic acid. Then 14 ml of methyl alcohol were added and the solution tumbled again to homogeneity. This solution was cast 10 mil thick on glass, plunged 30 seconds later in 1000 ml of 1:1 methanol: ater, removed and drained after 3 minutes, air dried, stripped from the glass while wet with water, and dried again. In DNA adsorption experiments (in which a densitometer is used to quantify the adsorption of radioactive probe) this membrane averaged 1894 disintegrations per minute (DPM) . A duplicate membrane analysed for 24 amines/10 6 g of polymer. Example 2 Low Adsorption with Low Ends, Membrane on Reemay™ A 66 nylon sample (Zytel™ 42) was obtained with 20 equivalents of amine and 44 equivalents of carboxyl/10 6 g of polymer. A 72 g sample of this nylon was dissolved in 190 ml of formic acid. Then 14 ml of methyl alcohol were added and the solution tumbled again to homogeneity. This solution was cast 10 mil thick on a Reemay™ sheet mounted on a glass plate, plunged 30 seconds later in 1000 mi of 1:1 methanol:water, removed and drained after 3 minutes, air dried, stripped from the glass while wet with water, and dried again. In DNA adsorption experiments this membrane averaged 2547 disintegrations per minute (DPM) . Example 3 Intermediate Adsorption with Moderate Ends, Plain Membrane A S _ nylon sample (Zytel™ 42) was obtained with 20 equivalents of amine and 44 eσuivalents of carboxvi/10°c of polymer. Adipic acid was polymerized only to oligomer with hexamethyiene diamine affording low molecular weight nylon with <10 equivalents of carboxyl and 1170 equivalents of amine /10 6 g of polymer. Casting solution was prepared by dissolving 65 g of high molecular weight nylon, 7 g of oligomer, and later 14 ml of methyl alcohol in 190 ml of formic acid. This solution was cast 10 mil thick on glass, plunged 30 seconds later in 1000 ml of 1:1 methanol: ater, removed and drained after 3 minutes, air dried, stripped from the glass while wet with water, and dried again. In DNA adsorption experiments this membrane averaged 3015 disintegrations per minute (DPM) . A duplicate membrane analysed for 90 amines/10 6 g of polymer. Example 4 Intermediate Adsorption with Moderate Ends, Membrane on Reemay™ A 66 nylon sample (Zytel™ 42) was obtained with 20 equivalents of amine and 44 equivalents of carboxyi/10 6 g of polymer. Adipic acid was polymerized only to oligomer with hexamethyiene diamine affording low molecular weight nylon with <10 equivalents of carboxyl and 1170 equivalents of amine /10 6 g of polymer. Casting solution was prepared by dissolving 65 g of high molecular weight nylon, 7 g of oligomer, and later 14 ml of methyl alcohol in 190 ml of formic acid. This solution was cast 10 mil thick on a Reemay™ sheet spread en glass, plunged 30 seconds later in 1000 ml of 1:1 methanol: ater, removed and drained after 3 minutes, air dried, stripped from the glass- while wet with water, and dried again. In DNA adsorption experiments this membrane averaged 3520 disintegrations per minute (DPM) . Example 5 High Adsorption with High Ends, Plain Membrane A 66 nylon sample (Zytel™ 42) was obtained with 20 equivalents of amine and 44 equivalents of carboxyl/10°g 5 of polymer. Adipic acid was polymerized only to oligomer with hexamethyiene diamine affording low molecular weight nylon with <10 equivalents of carboxyl and 1170 equivalents of amine /10 6 g of polymer. Casting solution was prepared by dissolving 58 g of high 10 molecular weight nylon, 14 g of oligomer, and later 14 ml of methyl alcohol in 190 ml of formic acid. This solution was cast 10 mil thick on glass, plunged 30 seconds later in 1000 ml of 1:1 methanol:water, removed and drained after 3 minutes, air dried, stripped from 15 the glass while wet with water, and dried again. In DNA adsorption experiments this membrane averaged 3548 disintegrations per minute (DPM) . A duplicate membrane analysed for 180 ends/10 6 g of polymer. Exam le 6 20 High Adsorption with High Ends, Membrane on Re ay A 66 nylon sample (Zytel™ 42) was obtained with 20 equivalents of amine and 44 equivalents of carboxyl/10 δ g of polymer. Adipic acid was polymerized only to oligomer with hexamethyiene diamine affording low 25 molecular weight nylon with <10 equivalents of carboxyl and 1170 equivalents of amine /10 6 g of polymer. Casting solution was prepared by dissolving 58 g of high molecular weight nylon, 14 g of oligomer, and later 14 ml of methyl alcohol in 190 ml of formic acid. This 30 solution was cast 10 mil thick on a Remay™ sheet spread on glass, plunged 30 seconds later in 1000 ml of 1:1 methanol:water, removed and drained after 3 minutes, air dried, stripped from the glass while wet with water, and dried again. In DNA adsorption experiments this • ;<= membrane averaged 5129 disintegrations per minute (DPM) .

Claims: CLAIMS 1. A composition of matter comprising a polyamide substrate with a high concentration of amine groups. 2 . The composition of Claim 1 wherein the polyamide substrate is supported on an inert support. 3. The composition of Claim 1 wherein the concentration of amine groups is at least about 60 equivalents per 10 s grams of polymer. 4. The composition of Claim 1 wherein the surface area of the substrate is at least about lm /g as measured by nitrogen adsorption. 5. The composition of Claim 1 wherein the amine groups are characterized as NRR' where R and R' are independently H, Cι_ιo branched, unbranched or cylic alkyl, C 5 aryl or 07- 12 aralkyl, where the alkyl, aryl or aralkyl groups may contain heteroatom substitutes which do not interfere with binding of biological samples to the polyamide substrate. 6. A composition of matter comprising a polyamide substrate which is a blend of low moiecuiar weight poiyamides and high molecular weight poiyamides. 7. The composition of Claim 6 wherein the blend comprises from about 1 to about 90 weight percent of low molecular weight polyamide and from about 10 to about 99 weight percent of high moiecuiar weight polyamide. 8. The composition of Claim 6 wherein the concentration of amines in the low moiecuiar weiσht poiyamides is from 70 to 1500 equivalents per 10 6 grams of polymer and the concentration of amines in the high molecular weight poiyamides is less than 80 equivalents per 10 6 grams of polymer. 9. The composition of Claim 6 wherein the substrate is supported on an inert support. 10. The composition of Claim 6 wherein the concentration of amine groups is at least about 60 equivalents per 10 β grams of polymer. 11. The composition of Claim 6 wherein the surface area of the substrate is at least about lm 2 /g as measured by nitrogen adsorption. 12. The composition of Claim 6 wherein the amine groups are characterized as NRR' where R and R' are independently H, Cι_ιo branched, unbranched or cylic alkyl, C 5 aryl or C7- 12 aralkyl, where the alkyl, aryl or aralkyl groups may contain heteroatom substitutes which do not interfere with binding of biological samples to the polyamide substrate. 13. A method for transferring materials selected from nucleic acids, proteins, bacteria or viruses from electrophoresis gels or culture media which comprises contacting them with the polyamide substrate of Claim 1. 14. A method for transferring materials selected from nucleic acids, proteins, bacteria or viruses from electrophoresis gels or culture media which comprises contacting them with the polyamide substrate of Claim 2. 15. A method for adsorbing DNA which comprises contacting DNA containing material with the polyamide substrate of Claim 6.

Inventors: WHELAND ROBERT CLAYTON, WHELAND, ROBERT, CLAYTON
Applicants: DU PONT, E.I. DU PONT DE NEMOURS AND COMPANY
Application year: 1990
Main classification: C08L77
IPC codes: B01D57, B01D71, C08J9, C08L77, C12M1, C12N11, C12Q1, G01N27