Abstract:
In accordance with the present invention, active carboxylic acid ester groups are coupled on the surfaces of microspheres so as to reduce protocols for microsphere processing, control side reactions, and stably preserve beads containing active carboxylic acid ester groups. Further, microspheres labeled with at least one fluorescent dye cage in the microspheres, and the microspheres are preserved in lower alcohol.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     1. Field of the Invention  
         [0002]     In general, the present invention is applied to flow cytometry technique. The present invention relates to microspheres, which usually has a diameter of 100 μm or less and is filled with multicolor fluorecent. More specifically, the present invention relates to microspheres that activated esters are held and stabilized on the surface without fluorescence seeping and polymer molecules filled with at least one type of fluorescent dye.  
         [0003]     2. Background Art  
         [0004]     Polymer particles, filled with fluorescent dye, are often used as a marker or indicator in various biomedical assays. “Microspheres” indicates minute particles basically having total diameters within micrometer-size. Microspheres can be analyzed with manual techniques or other methods known in the art. Preferably, automation technologies such as flow cytometry disclosed in U.S. Pat. No. 4,665,024 described below, the patent for which was granted to Mansour et al., can be used for analysis of such particles.  
         [0005]     Hitherto, biomolecules such as proteins (e.g. antigens and antibodies), nucleic acids, peptides, and sugar chains have been coupled to microparticles filled with at least one color of fluorescent dye. They have been used quantitative analysis of biomolecules or genetic polymorphism analysis. A plurality of analytical items can be simultaneously analyzed by a number of microspheres filled with fluorescent dye of different density and coupled with different types of biomolecules.  
         [0006]     The surfaces of microspheres are covered with carboxyl groups. A carboxyl group can form an amide bond with an amino group in a biomolecule via dehydration-condensation reaction. This reaction is used to couple with biomolecules on microspheres. However, since the reactivity of carboxyl groups is low, it is necessary to activate the carboxyl groups in some way. There are various types of reagents capable of activating carboxyl groups. Meanwhile, the aforementioned microspheres are filled with at least one fluorescent dye in which fluorescent dye is prone to seep into organic solvent. Thus, the reaction should be carried out in an aqueous solution. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) is the only reagent capable of activating carboxyl groups in an aqueous solution. In addition, it is necessary for biomolecules to contain primary amino groups so that they react with activated carboxylic acid esters. In particular, biomolecues such as nucleic acids, sugar chains that have no primary amino groups are necessary to introduce primary amino group into those biomolecule prior to the conjugation.  
         [0007]     EDC is a reagent capable of activating carboxyl groups in an acid solvent. The coupling reaction solvent is limited to one used in acidic conditions. However, some types of biomolecules can stably exist only in basic conditions. Thus, the types of biomolecules cannot couple on microspheres. In addition, when nucleic acid couples with microspheres using EDC, EDC may react with amino group of nucleobases under acidic condition.  
         [0008]     The following examples pertain to conventional methods for immobilizing biomolecules on fluorescent stained microparticles: a) method for coupling with a biomolecule after activating carboxyl groups carried on the surface of a microsphere to result in succinimide esters; and b) method for coupling with a biomolecule when simultaneously activating carboxyl groups carried on the surface of a microparticle to result in carbodiimide esters.  
         [0009]     In the method for activating carboxyl groups to result in succinimide esters described in above a), NHS (N-hydroxysuccinimide) and EDC are added to a suspension of microparticles so as to activate carboxyl groups to result in hydroxysuccinimide esters. The method involves the instantaneous immobilization of a biomolecule on a microparticle, the surface of which has been activated.  
         [0010]     In the method for activating carboxyl groups to result in carbodiimide esters described in above b), EDC and biomolecules are simultaneously added to a suspension of microparticles. The method involves the activation of carboxyl groups on the surfaces of microspheres to result in carbodiimide esters such that biomolecules instantaneously react with the active esters, leading to immobilization of the biomolecules on the microspheres.  
         [0011]     Active carboxylic acid ester groups such as hydroxysuccinimide ester groups react with hydroxyl groups in an aqueous solution so as to again become carboxyl groups. This reaction is extensively observed, particularly under basic conditions. However, hydroxysuccinimide ester groups are relatively stable in organic solvent without water.  
         [0012]     Microspheres are filled with fluorescent dyes in which fluorescent dyes are prone to seep into hydrophobic organic solvents such as aromatic hydrocarbon, pyridine, and dioxane.  
         [0013]     JP Patent Publication (Kohyo) Nos. 2001-520323 A and 2002-501184 A described below are known technical literature with regard to methods for producing or using fluorescent stained microparticles.  
       SUMMARY OF THE INVENTION  
       [0014]     When coupling with biomolecules such as proteins (e.g. antigens and antibodies), nucleic acids, sugar chains, and peptides on the surfaces of microparticles (microspheres), EDC and NHS, both of which are unstable in the atmosphere and are handled with difficulty, must be used.  
         [0015]     It is necessary to carry out the reaction whereby biomolecules are coupled on the surfaces of microspheres using EDC under acidic conditions. Thus, for example, peptides that are soluble only in a basic solvent cannot be coupled, resulting in a limitation in terms of the selection of biomolecules that can be coupled.  
         [0016]     In a reaction whereby nucleic acids are coupled on the surfaces of microspheres using EDC, side reactions those may be the reaction of amino group of nucleobases and EDC activated carboxyl group cannot be suppressed, because of stringent reaction conditions. In such case, as a result of a side reaction, nucleic acids that are used for probe are coupled at nonspecific sites or multiple sites so as to largely influence the probe function especially in the single nucleotide polymorphism identification.  
         [0017]     In a reaction whereby biomolecules are coupled on the surfaces of microspheres using EDC and NHS (or sulfo-NHS (N-Hydroxysulfosuccinimide)), since actived carboxylic acid ester groups are unstable, the coupling reaction must be carried out immediately after activation of carboxyl groups. In addition, when 2,3,5,6-tetrafluorophenol and 4-sulfo-2,3,5,6-tetrafluorophenol are used instead of NHS═, resulted carboxyl esters are relatively stable. However, the conjugation reaction between those esters and biomolecule which contain primary amino group is very slow.  
         [0018]     Activated carboxylic acid ester groups can be stable in an organic solvent; however, fluorescent dyes on microspheres become eluted, which ruin the function of microsphere. This is due to the hydrophobicity of fluorescent dyes on microspheres.  
         [0019]     It is an objective of the present invention to provide a technique whereby activated carboxylic acid ester groups are stabilized on the surfaces of microspheres without fluorescent dye seeping so as to simplify protocols for the conjugation between biomolecules and microspheres, extend the type of applicable peptide.  
         [0020]     Inventors of the present invention have found that the above problems can be solved by activating carboxyl groups on the surfaces of microspheres in advance to result in active esters and preserving them in lower alcohol. This has led to the completion of the present invention.  
         [0021]     The present invention relates to stabilization of activated carboxyl group on microspheres which is labeled with at least one fluorescent dye caged into the microspheres, and the microspheres is preserved in lower alcohol.  
         [0022]     In particular, the microspheres are covered with a functional group such as a carboxyl group.  
         [0023]     Further, the carboxyl group is activated to result in hydroxysuccinimide ester.  
         [0024]     The lower alcohol includes 1-butanol, methanol, ethanol, n-propanol, and isopropanol. In the present invention, isopropanol and 1-butanol are more preferable.  
         [0025]     Preferably, in the present invention, the functional group that activates a carboxyl group include a succinimidyl ester group, a sulfo-succinimidyl ester group, a 2,3,5,6-tetrafluorophenol ester group, and a 4-sulfo-2,3,5,6-tetrafluorophenol ester group.  
         [0026]     A microspheres, labeled with at least one type of fluorescent dye, is preserved in lower alcohol such that the activated carboxyl group bonded to microspheres can be stabilized over a long period of time. The microspheres is covered with carboxyl groups that are activated by a succinimidyl ester group, a sulfo-succinimidyl ester group, a 2,3,5,6-tetrafluorophenol ester group, a 4-sulfo-2,3,5,6-tetrafluorophenol ester group, and the like.  
         [0027]     In the reaction of the activated carboxyl group a reaction buffer conducting a coupling reaction are substituted for lower alcohol. Then, the microparticles are coupled with biomolecules on the surfaces of the microspheres. Thus, biomolecules such as antibodies, antigens, nucleic acids, sugar chains, and peptides can be coupled under the respective optimum conditions. In addition, side reactions caused by nucleic acids can be significantly suppressed. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0028]      FIG. 1  shows a flowchart of conventional protocols for activation of the surfaces of microspheres upon protein coupling.  
         [0029]      FIG. 2  shows a flowchart of conventional protocols for protein coupling following activation of the surfaces of microspheres.  
         [0030]      FIG. 3  shows a flowchart of conventional protocols for nucleic acids coupling following activation of the surfaces of microspheres.  
         [0031]      FIG. 4  shows a flowchart of a method for producing microspheres covered with active carboxylic acid esters.  
         [0032]      FIG. 5  shows a flowchart of protocols for microsphere processing for protein coupling in accordance with the present invention.  
         [0033]      FIG. 6  shows a flowchart of protocols for microsphere processing for nucleic acid coupling in accordance with the present invention.  
         [0034]      FIG. 7  shows results obtained by coupling biotinylated homooligonucleotide 20-mers of A, C, G, and T on microparticles using protocols of a conventional method.  
         [0035]      FIG. 8  shows a graph indicating the results of Example 1.  
         [0036]      FIG. 9  shows a graph indicating the results of Example 2.  
         [0037]      FIG. 10  shows a graph indicating the results of Example 3.  
         [0038]      FIG. 11  shows a graph indicating the results of Example 4. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0039]      FIG. 1  shows conventional protocols for activation of the surfaces of microparticles for protein coupling. To couple with proteins on microspheres, carboxyl groups on the surfaces of microparticles are activated to result in active carboxylic acid ester groups using EDC and NHS (or sulfo-NHS). Here, a 0.1M sodium phosphate solution (pH 6.0), a 0.05 M MES (2-(N-morpholino)ethane sulfonic acid) solution (pH 6.0), or the like is used as a buffer for a reaction to form active carboxylic acid ester groups. After the reaction for forming active esters finished, instantaneously, a protein coupling reaction shown in  FIG. 2  must be carried out.  FIG. 2  shows conventional general protocols for a reaction to couple with proteins and active carboxylic acid ester on the surfaces of microspheres. Amino groups of proteins react with active carboxylic acid ester groups, resulting in formation of amide bonds. Thus, proteins were coupled on the surfaces of microspheres. A PBS (phosphate buffered saline: pH 7.4) and 0.05 M MES (pH 6.0) solution or the like is used as a buffer for carrying out a protein coupling reaction. After the coupling reaction finished, blocking and washing are carried out using a PBS-TBN (PBS; 1% BSA (bovine serum albumin); 0.02% Tween (registered trademark: ICI Americas) 20; and 0.05% sodium azide) solution. At the end, processed microspheres are preserved in a PBS-TBN solution.  
         [0040]      FIG. 3  shows conventional protocols for microparticle processing for nucleic acid immobilization. In the case of nucleic acid, it is necessary to modify nucleic acid with a primary amino group via a linker. By allowing EDC to react with carboxylic acid on microsphere, carboxyl groups on microspheres become carbodiimide esters, such that nucleic acid can be coupled on the surfaces of microspheres by reaction of the carbodiimide with the primary amino group introduced to nucleic acids. For this reaction, a 0.1 M MES solution (pH 4.5) is used as a reaction solution. After termination of the coupling reaction, activated surface of microsheres is blocked using a 0.02% Tween 20 solution (registered trademark), and washing is carried out using a 0.1% SDS (sodium dodecyl sulfate) solution. Processed microspheres are preserved in a TE (Tris-EDTA) solution (pH 8.0). Note that a side reaction, whereby coupling occurs at various amino group of nucleobase, cannot be controlled.  
         [0041]      FIG. 4  shows a method for producing microspheres on the surfaces of which active carboxylic acid ester groups are coupled. The reaction for forming active carboxylic acid ester groups is almost the same as that of a conventional method. However, this method significantly differ from that of a conventional method in that the resulted microspheres having active carboxylic acid ester groups are preserved in isopropanol, so as to maintain carboxylic acid ester groups stably.  
         [0042]      FIG. 5  shows protocols for protein processing using microspheres produced in accordance with the present invention. In these protocols, PBS (pH 7.4) or the like is used as a coupling buffer. Thus, by carrying out solvent substitution, proteins can instantaneously be coupled on the surfaces of microspheres. Therefore, the protocols can be significantly reduced compared with those shown in  FIGS. 1 and 2 .  
         [0043]      FIG. 6  shows protocols for nucleic acid processing using microspheres produced in accordance with the present invention. In the protocols, 0.1 M phosphate buffer (pH 8.0) or the like is used as coupling buffer upon coupling of nucleic acid. Since the reaction is carried out under mild conditions, the side reaction that takes place in  FIG. 3  can be suppressed.  
       EXAMPLES  
     Comparative Example 1  
       [0044]     Using conventional protocols, biotinylated homooligonucleotide 20-mers of A, C, G, and T, the ends of which had not been modified with primary amino group, were coupled on microparticles. The processed microparticles are conducted to detect the biotinylated homooligonucleotide using Luminex 100™. The result showed that fluorescent signal of biotinylated homooligonucleotide 20-mers of A, C, and T coupled on the microparticles without modification of primary amino group that were the same as in the case of oligonucleotide with modification of primary amino group ( FIG. 7 ).  
       Example 1  
       [0045]     A coupling reaction was performed on biotinylated oligonucleotide 20-mers, in which primary amino group had not been introduced, using microspheres activated in accordance with the present invention, as shown in  FIG. 6 . The results were compared with those obtained by a conventional method. Then, the side reaction was found to have been significantly suppressed compared with that in the conventional method ( FIG. 8 ).  
       Example 2  
       [0046]     Nucleic acids were immobilized using microspheres produced in accordance with the present invention, as shown in  FIG. 6 . Conjugation of primary amino group introduced in nucleic acids with activated microspheres were performed in the various condition such as before, immediately after, and 3 days, 1 week, and 1 month after substitution using an isopropanol solvent. The experiment was carried out to confirm the presence or absence of fading of fluorescent dyes in isopropanol and the presence or absence of activity retention of active carboxylic acid ester groups ( FIG. 9 ). The identification of bead # 01  and # 97  were performed ordinarily as in those without isopropanol treatment. Therefore, fluorescent dyes do not seep out during the preservation in isopropanol. Even after one month, about half of the activated ester had been maintained.  
       Example 3  
       [0047]     Nucleic acids were immobilized as shown in  FIG. 6 , using microspheres produced in accordance with the present invention. Conjugation of primary amino group introduced in nucleic acids with activated microspheres was performed in the various condition such as before, immediately after, and 3 days after substitution using an ethanol solvent. The experiment was carried out to confirm the presence or absence of fading of fluorescent dyes in ethanol and the presence or absence of activity retention of active carboxylic acid ester groups ( FIG. 10 ). The identification of bead # 6  and # 85  were performed ordinarily as in those without ethanol treatment. However, about half of the activated ester had been degraded even in 3 days.  
       Example 4  
       [0048]     Nucleic acids were immobilized as shown in  FIG. 6 , using microspheres produced in accordance with the present invention. Conjugation of primary amino group introduced in nucleic acids with activated microspheres was performed in the various condition such as before, immediately after, and 5 days after substitution using a 1-butanol solvent. The experiment was carried out to confirm the presence or absence of fading of fluorescent dyes in 1-butanol and the presence or absence of activity retention of active carboxylic acid ester groups ( FIG. 11 ). The identification of bead # 4  and # 85  were performed ordinarily as in those without butanol treatment. After 5 days from preparation, the activity of the ester had not been changed virtually.  
         [0049]     In the present invention, active carboxylic acid ester groups are immobilized on the surfaces of microspheres. Thus, in accordance with the present invention, protocols for microsphere processing can be improved such that side reactions can be controlled and microspheres (beads) having active carboxylic acid ester groups can be stably preserved. As a result, the use of microspheres is promoted in the field of biochemistry.