SE531879C2 - Scintillation fibers made by electrospinning - Google Patents

Description

531 SWE aromatic compounds. The consequence will be that no scintillation signal will be generated.

The principle of proximity scintillation is found in US Patent 4,568,649 and in FCT application WO 91/08489, in which a support body used for proximity scintillation in radioimmunoassays is described. The support body is constructed of scintillation material, to which biological recognition components such as antigens, antibodies, etc. are bound. The biological recognition components are capable of selectively binding a target analyte. Several patents and patent applications (US 4271139; US 4568649; EP 1007971 A1) relate to the implementation of the SPA technique by using different scintillation materials and test formats. In all of the above examples, the selective recognition components are exclusively derived from biological macromolecules such as antibodies, membrane receptors, enzymes, lecithins, etc.

Molecular imprinted polymers (MIPs) are synthetic materials that have pre-designed molecular recognition properties. MIPs can be prepared using template-directed polymerization reactions in which the template forms stable complexes with functional monomers through crosslinking reactions. After the polymerization, the template can be removed from the crosslinked polymemetry, leaving well-defined cavities which are capable of reattaching the original template as well as its structural analogues. Under the names "synthetic enzymes" and "synthetic antibodies", U.S. Patent S110,833 describes the use of molecular imprinting as a general method for creating MIPs.

FCT application WO 94/1 1403 describes a method for producing molecularly shaped polymers as artificial antibodies and methods for using these artificial antibodies in therapeutic and diagnostic applications. Analogously to heterogeneous immunoassays, the amount of bound, radioisotope-labeled analytes was quantified after separation of unbound fractions by a centrifugation or filtration step. The separation step is often time consuming and difficult to automate, which makes it difficult to obtain a high throughput when a large number of samples are to be handled.

FCT application WO 02/068958 describes methods for preparing molecularly embossed polymers suitable for testing proximity scintillation. The embossed polymers contain at least one type of organic scintillator which is covalently bonded in the polymer. The immediate proximity between the molecular recognition sites and the bound scintillator makes it possible to use the material in non-separation samples for different analytes. As described in the document, an optimally embossed scintillation polymer should contain at least one of each of the following components: (1) molecular recognition sites, (2) an aromatic compound, and (3) an organic scintillator.

The requirement for bonding between the aromatic compound and the organic scintillator in molecularly embossed polymers can be obtained by copolymerizing styrene-like monomers and scintillator monomers or by chemical bonding of these components to prefabricated embossed particles. Swedish patent application 0502041-7 describes a simple electrospraying method for producing composites containing molecularly embossed particles. By applying a high voltage to a polymer solution emitted from a spinneret, electrospun continuous fibers are obtained. When the polymer solution contains molecularly embossed particles, these can be easily encapsulated in the polymeric fibers, giving composite fiber materials suitable for affinity separation.

Detailed Description of the Invention The scintillators described in this invention are prepared by electrospinning a polymer solution containing at least one of each of the following components: (1) aromatic compound, (2) scintillator, and (3) molecularly embossed particles. Figure 1 schematically shows the basics of an electrospinning experiment. Figure 2 shows the principle of signal conversion in scintillation beams containing molecularly imprinted particles.

The aromatic compound can either be of low molecular weight or be part of the polymer used in the electrospinning. When the aromatic compound is of low molecular weight, it can be added directly to a solution suitable for electrospinning, regardless of the type of polymer. An example of a non-aromatic polymer is polymethyl methacrylate (PMMA).

When the polymer used for electrospinning contains aromatic moieties, it is not necessary to add additional low molecular weight aromatic compounds to the spinning solution.

Polymers containing aromatic moieties are, for example, polystyrene (PS) and polyvinyltoluene (PVT).

The scintillator used in this invention may be either an inorganic or an organic sointillator. An organic scintillator should have low water solubility. Two examples of inorganic scintillators are yttrium silicate (YSi) and yttrium oxide (YOX). Some representative organic scintillators covered by this invention have any of the core structures shown in Figure 3. An example of organic scintillators is the LIO-diphenylanthracene (Figure 3).

It is possible to use polymers containing covalently bonded organic scintillators for electrospinning. In such cases, no additional scintillator needs to be added to the spinning solution.

The molecularly embossed particles can be obtained by precipitation polymerization, emulsion polymerization, mineral emulsion polymerization, fluorine emulsion polymerization, suspension polymerization, especially by fragmentation of polymer particles by mechanical grinding. The particle size of the molecular embossed particles used in this invention is between 5 nanometers and 10 micrometers. The molecularly embossed particles may be organic or inorganic particles or composite particles which are obtained from organic and inorganic monomers or precursors.

Example E> _ra__mp _ <= ._ l_l: Preparation of Molecular Embossed Nanoparticles Molecular embossed nanoparticles are synthesized using the precipitation polymerization method described in the literature (Yoshimatsu et al., Anal. Chim.

Acta 2007, 584, 112-121). In summary, the template molecule, (R, S) -propranolol, which is present in its free basic form (137 mg, 0.53 mmol) is dissolved in 40 ml of acetonitrile in a 150 mm> <øZS mm glass fl ash of borosilicate equipped with a screw cap. Then methacrylic acid (113 mg, h.) Fr * -fa-r ^ t is added; 1.31 mmol), trimethylolpropane trimethacrylate (684 mg, 2.02 mmol) and azobisisobutyronitrile (28 mg, 3% by weight of the monomer). The solution is purified with a weak de-fate of argon for 5 minutes and the bottle is sealed during the argon-fate. The polymerization is carried out by placing the glass fl ash of borosilicate in a water bath, heated to 60 ° C, for 24 hours. After the polymerization, the particles are collected by centrifugation. The template is removed by solvent extraction, bath procedure, with methanol containing 10% acetic acid (v / v). This is repeated until the template can no longer be detected in the washing solution by spectrometric measurement methods. Finally, the polymeric particles are washed with acetone and dried in a desiccator dryer.

Example 2: Preparation of non-embossed control nanoparticles Non-embossed control nanoparticles are prepared by the same method as described in Example 1 but with the difference that no propanolol is added to the prepolymerization mixture.

Example 3: Electrospinning of embossed nanobres Polystyrene (molecular weight 230.00 (_) g minor), LIO-diphenylantiacen and Triton X-100 are dissolved in methyl ethyl ketone. The resulting solution contains 12.5% by weight of polystyrene, 1% by weight of 1,10-diphenylanthracene and 0.6% by weight of Triton X-100. Embossed nanoparticles prepared according to Example 1 (6.25% by weight of the solution) are added to the solution before the mixture is treated in an ultrasonic bath. This is done to obtain a homogeneous suspension of nanoparticles. The mixture is poured into a syringe inserted into a qi 0.8-0.9 mm spinneret connected to a high voltage of 20 kV (HV Power Supply, Gamma High Voltage Research, Ormond, FL). A grounded aluminum foil placed at a distance of 15-25 cm from the spinneret is used as the receiving electrode.

Continuous composite beams are collected on the aluminum foil in the form of a fragile mat (Figure 4). The resulting nano attan berrnattan is then dried in a vacuum cannon. ïíšß fi BTH! -Eïï Example LQ: Electrospinning of control nanofibers Using the same technique as described in Example 3, control - nanofibers are produced The only difference is that non-embossed control nanoparticles, prepared according to Example 2, are used instead of embossed nanoparticles. Figure 5 shows SEM images of the obtained control nanofibers.

Example 5: Measurement of proximity scintillation The proximity scintillation calculation is made with a ß-radiation counter Rackbeta 1219 (LKB Wallac, Sollentuna, Sweden). In a number of micro-centrifuge tubes of polypropylene, nanotubes prepared according to Examples 3 and 4 are immersed in a solution of 25 mM citrate buffer (pH 6.0) and acetonitrile (50:50, v / v). 3 H-labeled (S) -propanolol (0.246 pmol, specific activity: 555GBq mmol, NEN Life Science Products, Inc. Boston, MA, USA) is added and the previously used solution is made up until the total volume of 1 mL is obtained. The microcentric tubes are incubated at room temperature overnight with gentle agitation on a shaking table. After incubation, the tubes are transferred to 6-mL insert vials which are placed in 20 mL of standard vials and rinsed for 1 min. Figure 6 shows the detected scintillation signal when different amounts of nanobres are exposed to tritium-labeled (S) -propranolol. Proximity scintillation assay of (S) -propranolol To produce dose-response curves for the measurement of (Sj-propranolol in unknown samples, use the same procedure as that described in Example 5 except that various amounts of unlabeled (S ') -propranolol or similar drugs are added to the incubation solution before incubation.

The amount of nanobres used is fixed at 2 mg. The presence of unlabeled propanolol reduces the scintillation signal, resulting in a sigmoid (sigma-shaped) dose-response-1-surf. Figure 7 shows the dose-response curve for (S) -propranolol and other similar drugs. The nanobres used are prepared in Example 3. As shown, the cross-reaction when tested against other similar drugs is always less than 10%.

Brief Description of the accompanying Figures Figure 1 shows a sehematic sketch of the electrospinning method used for the production of scintillation fibers containing molecularly imprinted particles;

Figure 3 shows the chemical structure of some representative organic senintillators.

Figure 4 shows SEM images of electrospun nanoparticles containing molecularly embedded nanoparticles.

Figure 5 shows SEM images of electrospurx nanoparticles containing control nanoparticles.

Figure 6 shows the scintillation signal detected when different amounts of nanoparticles are exposed to tritime-labeled Sypropranolol. eng. counts per rriinute).

Figure 7 shows the dose-response curve for the replacement of 3H- (S ') -propranolol from the nanometers containing molecularly imprinted nanoparticles. The mass of the nano-brema used was 2 mg.

Comparative analytes: (S-propranolol hydrochloride (filled square), (m-propranolol hydrochloride (open circle) acebutolol hydrolide chloride (open triangle), pindolol (open square), metopronolol (+) - tartrate salt (filled circle), atenolol (filled triangle).

Claims (12)

(fg Patentkrav 1. A method for producing scintillation particles containing molecularly particle particles. Said scintillation fibers are prepared by electrospinning of a polymer solution mixed with molecularly embossed particles. A method according to Claim 1 wherein the polymer solution contains at least one kind of scintillator. A method according to Claims 1 and 2 wherein said scintillator is either an organic or an Inorganic scintillator. Said organic scintillator includes but is not limited to 1,10-diphenylanthracene p-terphenyl, Zβ-diphenyloxazole, 1,4-bis (5-phenyloxazole-Z-ybenbenzene, 1,4-bis (2-methylstyrene) benzene, and 1,1 , 4,4, -tetraphenyl-1,3-butadiene A method according to Claim 1 wherein the size of said molecularly embossed particles is between 5 nanometers and 10 micron grains. A method according to Claim 1 wherein the diameter of said scintillation fis is between 5 nanometers and 50 micrometers. A method according to Claims 1 and 4, wherein said molecularly embossed particles are prepared by one of the following polymerization methods: precipitation polymerization, emulsion polymerization, mini-emulsion polymerization, microemulsion polymerization, dispersion polymerization, suspension polymerization. A method according to Claims 1 and 4, wherein said molecularly embossed particles are obtained by mechanical grinding of larger molecularly embossed monoliths. A method according to Claims 1 and 2 wherein said polymer solution contains one or typer your types of polymers. Said polymers can be dissolved in the electrospinning solution and are selected from synthetic polymers or bio-macromolecules including proteins, DNA and RNA molecules. A method according to Claims 1, 2 and 8 wherein at least one of said polymers contains aromatic side groups. 10. l0. A method according to Claims 1, 2 and 8 wherein one of said polymers contains covalently bonded organic scintillators. A method according to Claims 1, 8 and 9 wherein said polymer having an annular structure with aromatic side groups is polystyrene or polyvinyltoluene. Use of said scintillations prepared according to Claims 1-9 in applications for proximity scintillation assays.