Abstract:
A capillary electrophoresis method for identification and analyzing collagen quantitatively, which is used to identify and quantify collagen in a sample, comprises the steps of: (a) dissolving a collagen-containing sample to form a sample solution; (b) preparing a capillary with an inner wall thereof having a positively-charged layer; (c) introducing the sample solution into the capillary filled with an analytical buffer solution; and (d) driving the sample solution to pass through the capillary. The present invention also discloses an assay kit implementing the abovementioned method. The method of the present invention does not need the purifying pre-treatment and cracking the collagen-containing sample but directly performs the capillary electrophoresis analysis of collagen. Therefore, the present invention can shorten the time for analyzing collagen quantitatively.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to a method for identification and analyzing collagen quantitatively, particularly to an assay kit for using capillary electrophoresis method to identify and analyze collagen quantitatively. 
       BACKGROUND OF THE INVENTION 
       [0002]    Collagen extensively and massively exists in the extracellular matrix of connective tissues of animals. A research report (Moseley et al., 2004, Br J Dermatol 150:401-413) points out that collagen may function as a biological marker of disease activity or therapy prognosis. Another research report (Ruszczak, Z., 2003, Adv Drug Deliv Rev 55:1595-1611) points out that collagen scaffold may function as the regenerative environment of cells. Thus, how to accurately detect the quantity of collagen and estimate the rates of reconstruction and degradation of collagen becomes an important subject for the clinical application of collagen. 
         [0003]    The conventional methods for quantitatively analyzing collagen include the colorimetric analysis (Stegemann et al., 1967, Clin Chim Acta 18:267-273; Moore, S., 1968, J Biol Chem 243:6281-6283), the high performance liquid chromatography (HPLC), the liquid chromatography tandem mass spectrometry (MS) (Ikeda et al., 1993, J Chromatogr 621:133-138; Kindt et al., 2000, Anal Biochem 283:71-76), and the enzyme linked immunosorbent assay (ELISA) (Bellon, G., 1985, Anal Biochem 150:188-202). However, the abovementioned methods are very complicated and expensive. Therefore, they are not ideal methods for identifying and analyzing collagen quantitatively. 
         [0004]    Compared with the abovementioned methods, the capillary electrophoresis is a simpler method for analyzing collagen quantitatively (Deyl, Z &amp; Adam, M, 1989, J Chromatogr 488:161-197; Novotna et al., 1996, J Chromatogr B Biomed Appl 681:77-82; Deyl et al., 1997, J Chromatogr B Biomed Sci Appl 689:181-194; Chalmers, et al., 1999, J Chromatogr Sci 37:443-447). The capillary electrophoresis covers both advantages of electrophoresis and chromatography and can be automated. Therefore, it has been widely used to analyze and identify molecules. In capillary electrophoresis, a voltage is applied to the sample containing different molecules inside a capillary, and different molecules are separated by different electrophoretic mobility and electro-osmotic flow thereof. The silanol groups on the inner wall of the capillary will be dissociated and slightly negatively charged in the solution inside the capillary, particularly in an acidic solution. The negative charges will attract the cations and make the cations distributed on the capillary. When a voltage is applied to the capillary, the cations attached to the inner wall of the capillary will be attracted to the negative pole. The aggregated cations result in viscosity, which will drag the entire solution inside the capillary toward the negative pole and cause a bulk flow of the solution, i.e. the so-called electroosmotic flow. Because of the electrophoretic mobility difference and the electroosmotic flow, the capillary electrophoresis method has a high resolution and a high separation effect. Further, the capillary electrophoresis needs only a small amount of sample because the capillary functions as the electrophoresis path. 
         [0005]    In the conventional capillary electrophoresis technology for analyzing collagen quantitatively, the sample should be purified, extracted, and then processed with cyanogen bromide (CNBr) (Deyl, Z &amp; Adam, M, 1989, J Chromatogr 488:161-197; Deyl et al., 1997, J Chromatogr B Biomed Sci Appl 689:181-194). The cyanogen bromide enables the cracking reaction of the methionine on the amino acid sequences of protein, and the products of the cracking reaction are analyzed with the capillary electrophoresis method. The collagen cracking reaction is time-consuming and prolongs the process of the collagen analysis. The cyanogen bromide is a toxic material and needs processing and disposing carefully. Further, the pretreatments, such as extraction and cracking, should inevitably reduce the total amount of collagen and thus affect the quantitative result of the capillary electrophoresis analysis. 
       SUMMARY OF THE INVENTION 
       [0006]    The primary objective of the present invention is to simplify the analysis process of collagen using the capillary electrophoresis method and shorten the analysis time. To achieve the abovementioned objective, the present invention proposes a capillary electrophoresis method for identification and analyzing collagen quantitatively in a sample. The method of the present invention comprises steps: dissolving a collagen-containing sample in an acidic solution to form an acidic sample solution; preparing a capillary with the inner wall thereof coated with positively-charged compounds and preferably with an amine group; introducing the acidic sample solution into the capillary containing an analytical sodium dihydrogen phosphate buffer solution; and driving the acidic sample solution to pass through the capillary. 
         [0007]    The present invention also proposes an assay kit for the abovementioned method. The assay kit of the present invention comprises an acidic solution used to dissolve the sample to form a sample solution; a capillary with the inner wall thereof coated with a layer of a positively-charged compound and preferably with an amine group; and an analytical buffer solution used to analyze the sample in the capillary. 
         [0008]    In comparison to the conventional capillary electrophoresis technology for analyzing collagen quantitatively, the present invention does not need to pre-treat the sample with cyanogen bromide but dissolve the sample directly in an acidic solution, whereby the present invention can greatly reduce the operation time for collagen identification and quantification, and whereby the present invention is exempt from the extraction process and the cracking process, wherefore less collagen is lost in the analysis process, and more accurate result is attained by the capillary electrophoresis method of the present invention. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0009]      FIG. 1A  is a diagram showing the linear regression of the peak areas obtained in detecting collagen with concentrations between 0 and 60 ng according to the present invention; 
           [0010]      FIG. 1B  is a diagram showing the linear regression of the peak areas obtained in detecting collagen with concentrations between 0 and 300 ng according to the present invention; and 
           [0011]      FIG. 2  is an electrophoresis diagram obtained in simultaneously detecting collagen and other types of proteins according to the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0012]    The present invention provides a capillary electrophoresis method for identification and analyzing collagen quantitatively, wherein a capillary electrophoresis system is used to separate and analyze the sample. The capillary electrophoresis system has an autosampler and at least one kind of wavelength detectors, such as an ultraviolet detector with choice of the detection wavelength. The autosampler includes at least one capillary or micro-channel. The capillary is filled with an analytical buffer solution and equipped with a driving-voltage device at two ends thereof. The operation of the autosampler includes a capillary flushing step, a pneumatic sample injection step, and a sample movement step within the capillary. 
         [0013]    The buffer solution used in the present invention contains sodium dihydrogen phosphate, which can effectively dissolve the collagen in the sample, whereby the capillary electrophoresis analysis can have stable results. The buffer solution may further contain the following components: other alkali phosphate solutions, alkali chloride solutions (such as a hydrochloric acid/potassium chloride solution), amino acid solutions (such as a glycine/hydrochloric acid), citric acid solutions (such as a citric acid/sodium citrate), formic acid solutions, acetic acid solutions (such as sodium acetate/acetic acid solution), propionic acid solutions, butyric acid solutions, oxalic acid solutions, tartaric acid solutions, Shikimic acid solutions, and o-phthalic acid solutions (such as a potassium hydrogen phthalate/hydrochloric acid solution). However, the present invention does not limit the buffer solution to have to contain the abovementioned components or only contain the abovementioned solutions. 
         [0014]    The capillary used in the present invention may be a capillary having a positively-charged layer coated on the inner wall thereof, particularly an amine capillary having a polymerized amine compound coated on the inner wall thereof. The amine compound may be a primary amine compound or a material containing ammonium ions. The flushing or pneumatic injection of the capillary will be undertaken in the autosampler of the capillary electrophoresis system. 
         [0015]    The collagen sample used in the present invention is sourced from the collagen purified in the laboratory, a commercial available collagen or the collagen from connective tissues of human beings or animals. The connective tissues may come from skin, intestine, cartilage, tendon, sponge bone, compact bone, and so on. The animal tissue is washed and then cut into pieces. The cut tissue is processed for water content analysis to obtain a dried sample of the animal tissue. The dried sample is freeze-ground to obtain a sample powder. Alternatively, the dried sample is dissolved in an appropriate acidic solution to obtain a sample solution. 
         [0016]    To define the range in which the capillary electrophoresis method of the present invention can detect, the samples containing different concentrations of a single type of collagen or multiple types of collagens are respectively injected into the capillaries for analysis. The peak areas are obtained by integration and the linear relationship of concentrations is determined to establish the standard curve. 
         [0017]    The technical contents of the present invention are described below in details. However, the description is only to demonstrate the present invention but not to limit the scope of the present invention. 
         [0018]    In the specification of the present invention, the Beckman P/ACE TE  MDQ instrument system is used to exemplify the capillary electrophoresis system. The instrument system includes an autosampler, a UV (ultraviolet) detector able to select the detection wavelength, a full-wavelength range PDA (Photodiode Array) detector, and an LIF (Laser Induced Fluorescence) detector. The light source of the UV detector and the full-wavelength range PDA detector is a deuterium lamp emitting light having a wavelength from 190 to 360 nm and the former is equipped with four filters for the wavelengths of 200 nm, 214 nm, 254 nm and 280 nm. The light source of the LIF detector is an argon laser having a wavelength of 488 mm and a diode laser having a wavelength of 635 nm. The autosampler has at least one capillary (or micro-channel) filled with an analytical buffer solution and a driving-voltage device arranged at two ends thereof. 
         [0019]    Below are described the embodiments of the present invention and the test results. 
       Embodiment I: the Sensitivity of the Capillary Electrophoresis Method and the Standard Curve Analysis 
       [0020]    To define the range the capillary electrophoresis method of the present invention can detect and to establish the standard curve for collagen analysis, different amounts of type I collagen are injected into an amine capillary (a product of Beckman USA and having an inner diameter of 50 μm, an outer diameter of 360 μm and a length of 65 cm) with an injection pressure of 2 psi and an injection time of 6 seconds. The type I collagen samples have amounts of 276.8, 207.6, 138.4, 83.6, 76.14, 69.21, 55.37, 54.58, 47.46, 41.53, 35.59, 33.22, 27.76, 23.73, 17.79, 13.76, 11.86, 6.29, 4.88, 3.46, 2.76, 1.73, 1.37, 1.09, 0.9, 0.46, 0.27, 0.16, 0.13, 0.09, 0.06, 0.05 ng respectively. The capillary electrophoresis analyses are undertaken in a sodium dihydrogen phosphate buffer solution having a concentration of 50 mM and an acidity of pH2.5, at a temperature of 25° C., under an electrophoresis voltage of −25 KV, and with an UV absorption wavelength of 214 nm. The peak areas are obtained by integration and the linear relationship of concentrations is determined to establish the standard curve. 
         [0021]    The results of the abovementioned tests are shown in  FIG. 1A  and  FIG. 1B . The maximum and minimum amounts of collagen detected by the capillary electrophoresis method of the present invention are 276.8 ng and 0.05 ng respectively. Therefore, the capillary electrophoresis method of the present invention has a larger detection range than the conventional technology. Furthermore, a linear relationship is obtained between the peak area and the collagen content. When the sample containing 0.05 ng of collagen is tested for 20 times successively, the average value of peak areas, the standard deviation, and the relative standard deviation of the peak areas are 14379, 940 and 6.5% respectively. Therefore, the capillary electrophoresis method of the present invention has high reproducibility and reliability. As the relative standard deviation in detecting 0.05 ng of collagen is lower than 15%, 0.05 ng may be used as the detection sensitivity of the capillary electrophoresis method of the present invention. 
       Embodiment II: Collagen Specificity Analysis of the Capillary Electrophoresis Method 
       [0022]    Prepare a sample of 10 μL of 1 mg/mL collagen solution containing phenylmethanol, wherein the collagen includes type I, II, III, IV, and V. Prepare a sample of a mixed solution of 10 μL of 1 mg/mL albumin and protein mixture (Beckman Cat. No. 477436), wherein the latter contains ribonuclease A, cytochrome C and lysozyme. The abovementioned samples are injected into an amine capillary (a product of Beckman USA and having an inner diameter of 50 μm, an outer diameter of 360 μm and a length of 65 cm) with an injection pressure of 2 psi and an injection time of 6 seconds. The capillary electrophoresis analysis of the abovementioned samples is undertaken in a sodium dihydrogen phosphate solution having a concentration of 50 mM and an acidity of pH2.5, at a temperature of 25° C., under an electrophoresis voltage of −25 KV, and with an UV absorption wavelength of 214 nm. The analysis result is shown in  FIG. 2 . The peaks of phenylmethanol, collagen, ribonuclease A, albumin, cytochrome C and lysozyme sequentially appear in the migration time axis, which means that the capillary electrophoresis method of the present invention has a pretty high specificity in identifying collagen and can effectively measure the total amount of collagen. 
       Embodiment III: Quantitative Analysis of Collagen in Connective Tissue 
       [0023]    The connective tissues used in the tests may come from skin, intestines, cartilage, tendon, sponge bone or compact bone of pigs. All the connective tissues are divided into four groups of samples. The four groups of samples are analyzed respectively with (a) a hydroxyproline colorimetric reaction method, (b) a first capillary electrophoresis method for hydroxyproline detection, (c) a second capillary electrophoresis method for hydroxyproline detection, and (d) the capillary electrophoresis method of the present invention for collagen analysis. 
         [0024]    Firstly, the redundant fat and muscle is removed from the connective tissues of pigs. Next, the connective tissues are flushed with pure water and a phosphate solution to remove blood and dirt. The connective tissues are then cut into tiny pieces (about 1 mm 3 ). Next, the cut tissues are weighed to get the wet weights thereof and then lyophilized for about 24 hours or more to get constant dry weights, whereby the water content of tissue samples is learned, and dried tissue samples are obtained. Next, the dried tissue samples are freeze-ground into powders with a cryogenic grinder (a product of SPEX CertiPrep, Inc.). The powders are then dissolved in a 0.5M acetic solution and the mixture is agitated with a homogenizer (a product of Polytron®, Kinematica AG, Switzerland) and a sonicator (a product of Quantrex 280H, Ultrasonices, L&amp;R Manufacturing, Co., USA) to attain homogeneous collagen solutions with appropriate concentration. Then, the collagen solutions are stored at a temperature of 4° C. for analysis. 
       (A) Hydroxyproline Colorimetric Reaction Method 
       [0025]    The connective tissues of pigs are freeze-ground into powders. The powders are decomposed in a 6N hydrochloric acid solution by a ratio of 0.5 mg sample and 0.5 ml hydrochloric acid solution at 110° C. for 24 hours. The product is then mixed with chloramines T and the Ehrlich&#39;s reagent for a colorimetric reaction (according to Huang-Lee, LLH &amp; Nimni, ME, 1993, Biomed Eng Appl Basis Comm 5: 664-675). The products of the colorimetric reaction are analyzed with an enzyme-linked immunosorbent assay device (the ELISA reader and the VERSAmax microplate reader, Molecular Devices, USA). The content of hydroxyproline is calculated from the absorbance at a wavelength of 550 nm in comparison to a standard curve. The total amount of collagen is estimated according to the ratio that hydroxyproline conventionally exists in collagen. The calculation formula is collagen concentration=hydroxyproline concentration X 7.46. 
       (B) A First Capillary Electrophoresis Method for Hydroxyproline Detection 
       [0026]    The connective tissues of pigs are freeze-ground into powders. The powders are decomposed in a 6N hydrochloric acid solution by a ratio of 0.5 mg sample and 0.5 ml hydrochloric acid solution at 110° C. for 24 hours. The decomposed product is vacuum-dried in the presence of sodium chloride to remove hydrochloric acid. The dried product is re-dissolved in a 0.5M sodium hydrogen carbonate buffer solution by a ratio of 0.5 mg dried product: 0.5 ml buffer solution. The re-dissolution liquid is mixed with a fresh 0.02M dansyl chloride/acetone solution by a volume ratio of 1:1, and the mixture solution is placed in a 65° C. dry bath incubator for 40 minutes to enable a derivative reaction. After the derivative reaction, the sample solution is injected into an uncoated capillary (a product of Beckman USA and having an inner diameter of 50 μm and a length of 60.2 cm) with an injection pressure of 0.5 psi and an injection time of 20 seconds. The capillary electrophoresis analysis is undertaken in a borate/phosphate buffer solution, at a temperature of 25° C., under an electrophoresis voltage of 27 kV, and, with an UV absorption wavelength of 214 nm, wherein the ratio of (0.02M borate/phosphate, 0.1M SDS, pH9): methanol=9:1. The result of the capillary electrophoresis analysis is compared with a hydroxyproline calibration curve to obtain the hydroxyproline content in the sample. The total amount of collagen is estimated according to the ratio that hydroxyproline conventionally exists in collagen. 
       (C) A Second Capillary Electrophoresis Method for Hydroxyproline Detection 
       [0027]    The connective tissues of pigs are freeze-ground into powders. The powders are decomposed in a 6N hydrochloric acid solution by a ratio of 0.5 mg sample and 0.5 ml hydrochloric acid solution at 110° C. for 24 hours. After the decomposition reaction, sodium hydroxide is added to neutralize the hydrochloric sample solution. The neutralized sample solution is mixed with a 0.5M sodium hydrogen carbonate buffer solution by a volume ratio of 1:1. The buffered sample solution is mixed with a fresh dansyl chloride/acetone solution by a volume ratio of 1:1, and the mixture solution is placed in a 65° C. dry bath incubator for 40 minutes to enable a derivative reaction. After the derivative reaction, the sample solution is injected into an uncoated capillary (a product of Beckman USA and having an inner diameter of 50 μm and a length of 60.2 cm) with an injection pressure of 0.5 psi and an injection time of 20 seconds. The capillary electrophoresis analysis is undertaken in a borate/phosphate buffer solution, at a temperature of 25° C., under an electrophoresis voltage of 27 kV, and, with an UV absorption wavelength of 214 nm, wherein the ratio of (0.02M borate/phosphate, 0.1M SDS, pH9): methanol=9:1. The result of the capillary electrophoresis analysis is compared with a hydroxyproline calibration curve to obtain the hydroxyproline content in the sample. The total amount of collagen is estimated according to the ratio that hydroxyproline conventionally exists in collagen. 
       (D) Capillary Electrophoresis Method of the Present Invention for Collagen Analysis 
       [0028]    The connective tissues of pigs are freeze-ground into powders. The powder is dissolved in a 0.5M acetic solution, and the mixture of the powder and the acetic solution is homogenized. The homogenized tissue solution is injected into an amine capillary (a product of Beckman USA and having an inner diameter of 50 μm, an outer diameter of 360 μm and a length of 65 cm) with an injection pressure of 2 psi and an injection time of 6 seconds. The capillary electrophoresis analysis is undertaken in a sodium dihydrogen phosphate buffer solution having a concentration of 50 mM and an acidity of pH2.5, at a temperature of 25° C., under an electrophoresis voltage of −25 KV, and with an UV absorption wavelength of 214 nm, whereby collagen can be directly identified. The content of collagen is worked out from the area of the peak. 
         [0029]    The test results of the abovementioned four quantitative analysis methods for collagen in connective tissues are shown in Table 1. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Water 
                   
                   
                   
                   
               
               
                   
                 content 
                 Method 
                 Method 
                 Method 
                 Method 
               
               
                   
                 (%) 
                 (A) (%) 
                 (B) (%) 
                 (C) (%) 
                 (D) (%) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Pig Skin 
                 61.9 ± 0.6 
                 23.7 ± 2.7 
                 20.7 ± 2.8 
                 22.3 ± 1.5 
                 23.8 ± 2.9 
               
               
                 Pig Intestine 
                 84.9 ± 0.8 
                  1.1 ± 0.3 
                  1.1 ± 0.3 
                  1.0 ± 0.2 
                  0.8 ± 0.2 
               
               
                 Pig Cartilage 
                 70.1 ± 0.7 
                 14.6 ± 1.2 
                 12.5 ± 0.7 
                 14.5 ± 0.7 
                 10.1 ± 2.9 
               
               
                 Pig Tendon 
                 61.2 ± 0.6 
                 32.9 ± 2.6 
                 29.0 ± 4.3 
                 28.7 ± 0.4 
                 30.5 ± 2.7 
               
               
                 Spongy 
                 34.5 ± 2.2 
                  4.1 ± 1.5 
                  4.1 ± 1.1 
                  4.1 ± 0.7 
                  4.1 ± 0.8 
               
               
                 Bone 
               
               
                 Compact 
                 21.6 ± 1.4 
                 15.2 ± 0.9 
                 12.9 ± 3.1 
                 11.8 ± 0.6 
                 15.1 ± 4.0 
               
               
                 Bone 
               
               
                   
               
             
          
         
       
     
         [0030]    From the quantitative analysis results of collagen in connective tissues taken from the same source, the collagen contents detected by different analysis methods have negligible variation. Compared with the conventional technology that indirectly obtains the collagen content via analyzing the hydroxyproline content, the capillary electrophoresis method of the present invention can detect the collagen and measure the collagen content directly. 
         [0031]    As the capillary electrophoresis method for analyzing collagen of the present invention is exempt from the pre-treatment of purification, extraction, and processing with cyanogen bromide used in the conventional technology, the present invention can greatly shorten the time for analysis. From the embodiments described above, it is known that the capillary electrophoresis method of the present invention can detect a large range of collagen concentration and quantify the total amount of collagen in tissue. Furthermore, the method of the present invention has high specificity for collagen identification and sensitivity when the collagen is mixed with other types of proteins. Therefore, the present invention has improvements over the conventional technology and possesses novelty and non-obviousness—the conditions for a patent. Thus, the inventor files the application for a patent. It will be appreciated if the patent is approved fast. 
         [0032]    The present invention has been described in detail with the embodiments. However, the embodiments are only to exemplify the present invention but not to limit the scope of the present invention. Any equivalent modification or variation according to the spirit of the present invention is to be also included within the scope of the present invention.