Patent Application
20050014027
The invention relates to cross-linked bead polymers doped with superparamagnetic iron oxide, to a method for producing the pearl polymers, as well as to their use in nucleic acid diagnosis.
Recently, so-called genetic diagnosis have gained increased significance.
Genetic diagnosis has found its way into the diagnosis of human diseases (among others, detection of pathogens, detection of mutations in the genome, discovery of circulating tumor cells, and identification of risk factors for the predisposition for a disease). Veterinary medicine, environmental analysis, and food testing are also areas, however, that genetic diagnosis has recently found application. A further area of application is in studies by pathology/cytology institutions or in the context of forensic inquiries. In addition, however, genetic diagnosis is also being used in the context of quality control (e.g., studies of blood samples for freedom from pathogens), and the legislature is planning to create legislation for such tests in the future. Methods that are also used in genetic diagnosis (such as hybridization and amplification techniques, for example, PCR (polymerase chain reaction), bDNA (branched DNA assay), or NASBA (nucleic acid sequence based amplification technology) are also a part of the routine methods when conducting scientific studies.
Obtaining gene samples from biological material such as cells, blood, serum, or urine is an important partial step in genetic diagnosis.
EP 0 707 077 describes a method for isolating nucleic acids from biological material by using a soluble, weakly basic polymer. In this method, a precipitant product in an acidic pH range is produced out of a soluble, weakly basic polymer and the nucleic acid. The precipitant product is separated from the nonprecipitant components of the biological material and washed, and the nucleic acid from the precipitant product is released by adjusting to a basic pH value.
A disadvantage of the method in EP 0707 077 is that the manipulation, especially the separation and cleaning, of the precipitant product is difficult and takes a great deal of time. This method also cannot be performed, or can be performed only under difficult conditions, using automated analytic equipment.
U.S. Pat. No. 4,339,337 and U.S. Pat. No. 5,356,713 describe methods to manufacture magnetic beads of a vinyl aromatic polymer by using magnetic particles. However, these bead polymers do not contain any functional groups to link nucleic acids. In addition, the beads exhibit marked residual magnetism (remanence) whereby their dispersibility is made more difficult.
WO 8303920 describes a method for manufacturing magnetic polymer particles in which polymer particles are treated with solutions of iron salt, for example, whereby the iron is precipitated in the form of iron hydroxide. In this method, the precipitated iron compound is found both in the polymer particles and on the surface of the polymer particles. For a few applications, for example to amplify nucleic acids with TaqMan PCR, the iron compound on the surface can interfere.
U.S. Pat. No. 5,206,159 discloses a method to manufacture superparamagnetic polyacrylamide carriers. However, these carriers are not suited for removing nucleic acids.
A method to link DNA to magnetic microparticles is known from U.S. Pat. No. 5,705,628. The magnetic microparticles preferably have a particle size of 1 μm and possess a surface coated with carboxyl groups. In order to link the DNA to the particles, special salt concentrations must be used, and polyethylene glycol must be added in defined concentrations and with a specific molecular weight.
It has now been found that certain cross-linked bead polymers that have been doped with superparamagnetic iron oxide and that contain basic amino groups are extremely well suited for direct and automated isolation of nucleic acids.
The object of the invention is cross-linked bead polymers doped with superparamagnetic iron oxide and containing basic amino groups wherein the bead polymers contain copolymerized units of hydrophilic (meth)acrylate and amino (meth)acrylates.
The term (meth)acrylate means the derivatives of acrylic acid and methacrylic acid.
Hydrophilic (meth)acrylates are those whose homopolymers are more than 2.5% soluble in water at 25° C. Examples include 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, triethylene glycol monomethacrylate, tetraethylene glycol monomethacrylate, glycerol monomethacrylate, acrylamide, methacrylamide, and N,N-dimethyl acrylamide. Acrylamide is preferred.
Amino (meth)acrylates as defined in the present invention are derivatives of acrylic acid and methacrylic acid preferably with secondary and tertiary amino groups. The amino groups can also form a part of a cycloaliphatic or aromatic ring. Suitable amino (meth)acrylates include, for example N-(3-amino-propyl) methacrylamide, N-(3-imidazoyl-propyl) methacrylamide, N-(2-imidazoyl-ethyl) methacrylamide, N-(3-amino-propyl) acrylamide, N-(3-imidazoyl-propyl) acrylamide, N-2(2-imidazoyl-ethyl) acrylamide, N-1(1,1-dimethyl-3-imidazoyl-propyl) methacrylamide, N-(1,1-dimethyl-3-imidazoyl-propyl) acrylamide, N-(3-benzimidazoyl-propyl) methacrylamide, and (3-benzimidazoyl-propyl) acrylamide. Preferred amino (meth)acrylates are amino alkyl (meth)acrylates, such as N,N-dimethyl-aminoethyl-methacrylate, N,N-dimethyl-aminopropyl-methacrylate, N,N-dimethyl-aminoethyl-acrylate, and N-tert-butyl-aminopropyl-methacrylate. N,N-dimethyl-aminoethyl-methacrylate and N,N-dimethyl-aminopropyl-methacrylate are especially preferable. According to the invention, the amino groups may be present in the bead polymers either wholly or partially in protonated form, e.g., as hydrochlorides.
Potential crosslinkers include: ethylene glycol dimethacrylate, butanediol dimethacrylate, hexanediol dimethacrylate, pentaerytritol dimethacrylate, 1,2-glycerin-dimethacrylate, 1,3-glycerin-dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, trimethylol propane trimethacrylate, pentaerytritol trimethacrylate, pentaerytritol tetramethacrylate, ethylene glycol diacrylate, butanediol diacrylate, pentaerytriol diacrylate, 1,3-glycerin-diacrylate, triethylene glycol diacrylate, trimethylol propane triacrylate, pentaerytritol triacrylate, pentaerytritol tetraacrylate, allyl methacrylate, allyl acrylate, diethylene glycol divinyl ether, and methylene-N,N-bisacrylamide. Methylene-N,N′-bisacrylamide is preferred.
The amount of hydrophilic (meth)acrylate is 30 to 89% by weight, preferably 40 to 75% by weight, the amount of amino (meth)acrylate is 10 to 69% by weight, preferably 20 to 50% by weight, and the amount of crosslinker is 1 to 25% by weight, in each case based on the total amount of hydrophilic (meth)acrylate, amino (meth)acrylate, and crosslinkers.
According to the invention, the content of iron oxide in the superparamagnetic bead polymers is 2 to 80% by weight, preferably 4 to 50% by weight, most preferably 5 to 35% by weight, based on the weight of the unswollen bead polymers.
According to the invention, the bead polymers are superparamagnetic, which means that they possess low residual magnetization (remanence) and small coercive force. Their magnetic saturation is high, and they have strong attraction to an inhomogeneous magnetic field. After deactivating the magnetic field, they can be easily and completely dispersed in water or aqueous buffer solutions.
According to the invention, the particle size of the superparamagnetic bead polymers is 1 to 200 μm, preferably 5 to 100, and most preferably 10 to 50 μm. Microscopic image analysis is well suited for determining the average particle size (Ø) and the particle size distribution.
The ratio between the average of the volume distribution (Dv) and the average of the number distribution (Dz) is used as a measure for the breadth of the particle size distribution of the bead polymers. Narrow distributions of particle sizes as defined by the invention mean Dv/Dz≦2.5, preferably Dv/Dz≦2, and most preferably Dv/Dz≦1.5. According to the invention, it has been found that bead polymers with narrow distribution of particle sizes are especially good for isolating nucleic acids and provide results with particularly good reproducibility in amplification processes on the surface of the bead polymers.
According to the invention, the bead polymers are swellable in water. They have a swelling index of 1.25 to 8, preferably 2 to 6 (measured at 25° C.). The swelling index is defined as the quotient of the volume of the swollen bead polymers and the volume of the unswollen bead polymers.
To determine the swelling index experimentally, 10 ml of dried, sieved bead polymer are weighed into a 100 ml graduated cylinder. The quotient of the volume of the bed (V0) and the weighed (m) is the bulk volume Vbulk. The graduated cylinder is filled with water to 100 ml and allowed to stand for 10 to 20 hours at 25° C. During this time, it is shaken occasionally so that any air bubbles that occur can escape. The volume of the swollen bed is read and produces V1. The quotient of V1 and V0 is the swelling index.
A further object of the present invention is a method for manufacturing cross-linked bead polymers wherein a monomer mixture of hydrophilic (meth)acrylate, amino (meth)acrylate, a crosslinker, and, where appropriate, other monomer is polymerized to beads by inverse suspension polymerization and the latter are then doped with superparamagnetic iron oxide by an after-treatment with iron salt solution.
The term inverse suspension polymerization as defined by the invention is understood as a method in which the monomer mixture of hydrophilic (meth)acrylate, amino (meth)acrylate, a crosslinker, and, where appropriate, other monomer is activated with a free-radical former which is soluble in the monomer mixture and the activated monomer mixture is emulsified into droplets in a nonaqueous solvent by adding a dispersing agent and then the resulting droplets which have formed are cured by increasing the temperature.
The hydrophilic (meth)acrylate, amino (meth)acrylate, and the crosslinker correspond to the aforesaid compounds. The amino (meth)acrylate can beneficially be used, at least partly, in the form of ammonium, as a hydrochloride, for example. Other suitable monomers include N-vinyl pyrrolidone, vinyl imidazol, styrene, α-methylstyrene, chlormethyl-styrene, acrylonitrile, vinyl acetate, and maleic anhydride in amounts of up to about 25% by weight, based on the entire monomer mixture. It is beneficial to dilute the monomer mixture with water or water-alcohol mixtures. Suitable amounts of diluent are 10 to 200% by weight, for example, preferably 50 to 100% by weight based on the monomer mixture.
Azo compounds and peroxy compounds are suitable as free-radical initiators. When using water as a diluent, potassium peroxidisulfate and sodium peroxodisulfate are very suitable, also in combination with bisulfite or hydrogen sulfite. Further preferred free-radical initiators are the azo compounds such as 2,2′-azobis[2-(2-imidazoline-2-yl)propane] dihydrochloride and 2,2′-azobis(2-amidinopropane) dihydrochloride. The free-radical initiator is used in amounts of 0.02 to 2.5% by weight, preferably from 0.1 to 1% by weight, based on the total monomer mixture.
Hydrocarbons and halocarbons are well suited as nonaqueous solvents for the present invention, as well as low-viscosity silicon oils. The preference is for linear, branched, and cyclic aliphatic hydrocarbons, for example, hexane, heptane, n-octane, iso-octane, iso-dodecane, and cyclohexane. Mixtures of different hydrocarbons can also be used.
Oil-soluble polymers with a molecular weight of 2,000 to 1,000,000 are suitable as dispersing agents. The preference is for polymers containing copolymerized units from C6- to C22-alkyl(meth)acrylates and/or vinyl esters from C6- to C22-carboxylic acids, for example, polymers with copolymerized units of stearyl methacrylate, lauryl methacrylate, and vinyl stearate. Especially well suited are copolymers from C6- to C22-alkyl(meth)acrylates and vinyl esters from C6- to C22-carboxylic acids and hydrophilic monomers. The term hydrophilic monomers in this context means polymerizable olefinically unsaturated compounds that are wholly or partially soluble in water (more than 2.5% by weight at 20° C.). Examples include: acrylic acid and its alkali metal and ammonium salts, methacrylic acid and its alkali metal and ammonium salts, hydroxyethyl methacrylate, hydroxyethyl acrylate, diethylene glycol monoacrylate, diethylene glycol monomethacrylate, triethylene glycol monoacrylate, triethylene glycol monomethacrylate, tetraethylene glycol monoacrylate, tetraethylene glycol monomethacrylate, glycerol monoacrylate, aminoethyl methacrylate, N,N-dimethyl-aminoethyl-methacrylate, acrylamide, methacrylamide, vinyl pyrollidone and vinyl imidazol. The preference is for hydroxyethyl methacrylate, aminoethyl methacrylate, N,N-dimethyl-aminoethyl-methacrylate, acrylamide, methacrylamide, vinyl pyrrolidone, and vinyl imidazol.
Especially preferred dispersing agents are copolymers of
The quantity of the dispersing agent used is generally 0.1 to 8%, preferably 0.5 to 5%, by weight, based on the nonaqueous solvent.
The speed of stirring during polymerization is important for adjusting the particle size. According to the invention, the size of the obtained bead polymers decreases as the stirrer speed increases. The exact stirrer speed to adjust to a particular predetermined bead size strongly depends in the individual case on the reactor size, the reactor geometry, and the stirrer geometry. It has proved effective to determine the stirrer speed experimentally. For laboratory reactors with a reaction volume of 0.5 liters that are equipped with a gate stirrer, bead diameters of 10 to 25 μm are generally achieved at rotational speeds of 800 to 1,000 rpm when using copolymers of methacrylic acid-C13-ester and hydroxyethyl-methacrylate as a dispersing aid.
The polymerization temperature depends on the decomposition temperature of the initiator employed and the boiling temperature of the nonaqueous solvent. It is generally between 50 and 150° C., preferably between 55 and 100° C. The polymerization takes 0.5 to a few hours, for example, 10 hours.
After polymerization, the polymer can be isolated using usual methods, e.g., by filtering or decanting, and, if needed, dried after one or more washing steps. It is possible to fractionate the resulting bead polymer by physical methods in order to adjust to a narrower distribution of particle sizes. Suitable fractionation methods are, for example, sieving, sedimentation, and air classification.
The after treatment for doping with superparamagnetic iron oxide is done with mixtures of aqueous Fe2+ and Fe3+ salt solutions. The corresponding chlorides are very suitable. The molar ratio of Fe2+:Fe3+ should be 2:1 to 1:2. It is possible to start from iron salt solutions with a different Fe2+: Fe3+ ratio and to effect the optimal ratio of Fe2+: Fe3+ by using oxidizing or reducing agents. Suitable examples of oxidizing agents are peroxo and nitro compounds, and of reducing agents e.g., sodium bisulfite. The concentration of iron salt solutions is in general 10 to 50% by weight, preferably 20 to 40% by weight.
The iron salt solution is preferably brought into contact with a dried, anhydrous bead polymer. It is of particular benefit if the bead polymer swells by taking up the entire iron salt solution and if no excess iron salt solution remains in the interstices of the beads or on the surface of the beads.
The iron salts taken up by the swollen bead polymer are converted into the corresponding iron hydroxides by adding bases. Well suited for this are alkaline solutions of sodium hydroxide, sodium carbonate, or ammonia. Ammonia is preferred because an excess of it can be easily removed through evaporation. Ammonium salts formed are removed by thorough washing with water.
The iron hydroxide is converted into iron oxide (dehydrated) by heating the bead polymer. The heat treatment can be easily done in aqueous suspension at 65 to 100° C. Suitable times for heating are 0.5 to 5 hours. The conversion of the iron hydroxide into iron oxide is evident from a change in color from light brown to dark brown or black. Subsequently, the bead polymer can be separated and dried.
If desired, the dried bead polymers doped with superparamagnetic iron oxide can be processed one more time in the manner described above, whereby the content of superparamagnetic iron oxide is increased. In this way, it is possible to obtain iron oxide contents of more than 50% by weight.
The present invention further relates to a method for isolating nucleic acids from a sample, comprising the following steps:
The method according to the invention is suited to isolate and/or purify nucleic acids of different origins, for example from cells, tissue materials, blood, or pathogens. Prior to isolating the nucleic acids, the material to be studied is disrupted by using techniques known per se such as disruption via protease digestion, resulting in a sample suitable for subsequent steps A through C, a lysate. If needed, the lysis of the biological material takes places in an interim step after method step A). Other suitable disruption methods have been described in DE-A-4 333 805.
The sample is mixed with the bead polymer at a pH value of 7 or less, preferably in the range of 2 to 6, most preferably in the range of 2 to 3, at room temperature. The separation of the bead polymers is done using a magnetic field. The complex of nucleic acid and bead polymer obtained in this way can then be purified by washing with suitable buffers.
To release the bound nucleic acids from the complex, the pH of the complex is then adjusted to pH values above 7, preferably from 8 to 14, most preferably in the range of 12 to 14.
The bead polymers according to the invention provide higher adsorption and release rates than the soluble polymers disclosed in EP-A-0 707 077. Isolation can be performed more easily, i.e., with fewer steps and in shorter times. The purity of the isolated nucleic acids is higher, and in particular they contain fewer inhibiting byproducts so that amplification of the nucleic acids works especially well, for example through the so-called “PCR reaction” and “RT PCR.” The method according to the invention is also superior to the method described in EP-A-0 707 077 in relation to restriction enzyme digestion of the obtained nucleic acids.
The bead polymers according to the invention are also well suited to amplifying the adsorbed nucleic acids, for example through the so-called “TaqMan PCR reaction,” directly on the bead polymers (i.e., without step C).
Preparation of a Bead Polymer According to the Invention
1a) Preparation of a Dispersing Agent
In a 4 liter reaction vessel with a gate stirrer and gas feed and gas discharge hoses, a solution of 1,324 g cyclohexane, 511 g methacrylic acid-C13-ester, 57 g hydroxyethyl methacrylate, and 3.8 g dibenzoyl peroxide was heated under a nitrogen atmosphere to 78° C. over a period of 2 hours at 300 rpm, kept at this temperature for 10 hours, then heated to 90° C. and kept for another 1.5 hours at this temperature. Then, it was cooled to 25° C. 1,835 g of a 30.5% solution by weight of a dispersing agent was obtained. The Staudinger index, measured with an Ubbelohde viscosimeter at 25° C., was 72.6 ml/g.
1b) Preparation of a Cross-Linked Bead Polymer
In a 0.5 liter reaction vessel with a gate stirrer, reflux condenser, and thermosensor, 41.25 g of dispersing agent solution from 1a) and 240 g cyclohexane were introduced and stirred. 9.38 g N,N-dimethyl-aminoethyl-methacrylate was stirred with 13.3 g water and 5.89 g 37% strength hydrochloric acid for 5 minutes and neutralized with 0.6 g of 1N NaOH. This solution was then added to the reaction vessel. To this mixture were added 20.31 g acrylamide and 1.56 g methylene-N,N′-bisacrylamide, dissolved in 8 g methanol. 0.063 g potassium peroxide disulfate, dissolved in a mixture of 4.25 g water and 2 g methanol was added to the reaction mixture, which was then flushed at 450 rpm for 10 min with nitrogen gas. The stirring speed was increased to 1,000 rpm and the temperature raised to 60° C. over the course of 1 hour, and kept at this temperature for 10 hours. After cooling, the resultant polymer was removed by decanting from the reaction solution and purified three times each with cyclohexane, water, and methanol and dried at 40° C. in a vacuum oven. 14.7 g were obtained of dried bead polymer with an average particle size of 12 μm and a swelling index of 6 at 25° C. in water.
1c) Doping the Bead Polymer with Iron Oxide
In a stirring vessel with a magnetic stirrer and thermometer, 3.625 g iron (II) chloride tetrahydrate, 1.5 g iron (III) chloride (anhydrous), and 0.3 g sodium bisulfite were dissolved in 5.75 ml water.
5 g of dried bead polymer (1b) was introduced in a 100 ml three-necked flask and externally cooled with ice, and the iron salt solution described above was added. The resultant suspension was stirred for 35 minutes and externally heated with boiling water until the suspension had become a solid mixture. The bead polymer was stirred for 1 hour in another 500 ml flask with an alkaline solution of 67.5 ml water and 8.5 ml 26% ammonia solution (pH=9) and diluted with 250 ml water. After decanting the solution, this process was repeated several times.
The bead polymer treated with iron salt solution was mixed with 300 ml water and stirred for 30 minutes under a stream of air and then heated to 72° C. The pH value was set at 9 during the entire process by adding ammonia solution. 0.155 g potassium peroxodisulfate was then added to the solution and heated another 2.5 hours at 72° C. After decanting the liquid, the solid was washed five times with water while being treated with ultrasound. 5.1 g of black bead polymer with an iron content of 6% by weight, which is strongly attracted to an inhomogeneous magnetic field were obtained.
Preparation of a Bead Polymer According to the Invention
2a) Preparation of a Cross-Linked Bead Polymer
In a 0.5 l reaction vessel with a gate stirrer, reflux condenser, and thermosensor, 41 g dispersing agent solution from 1a) and 240 g cyclohexane were introduced and stirred. 9.38 g N,N-dimethyl-aminoethyl-methacrylate were stirred for 5 minutes with 13.3 g water and 6 g 37% hydrochloric acid. This solution was poured into the reaction vessel. To this mixture was added 18.75 g acrylamide and 3.13 g methylene-N,N′-bisacrylamide, dissolved in 8 g methanol. The reaction mixture was added with 0.313 g 2,2′-azobis(2-amidinopropane) dihydrochloride, dissolved in a mixture of 4.25 g water and 2 g methanol, and flushed at 450 rpm for 10 minutes with nitrogen gas. The temperature was raised within 1 hour at 800 rpm to 60° C. and the reaction was allowed to take place at this temperature for four hours. After cooling, the resultant polymer was separated from the reaction solution by decanting, and purified three times with cyclohexane, water, and methanol and dried at 40° C. in a vacuum oven. 15 g of dried bead polymer with an average particle size of 20 μm and a swelling index of 5 at 25° C. in water were obtained.
2b) Doping the Bead Polymer with Iron Oxide
5 g of the bead polymer from 2a) was doped with iron oxide as described under 1c). In doing so, the entire process was repeated twice. 5.4 g were obtained of black bead polymer with an iron content of 8.3% by weight, which is strongly attracted to an inhomogeneous magnetic field.
| Number | Date | Country | Kind |
|---|---|---|---|
| 100 33 583.7 | Jul 2000 | DE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP01/07326 | 6/27/2001 | WO |
