KR100360069B1 - Analytical apparatus and the determination method of biochemical sample using thereof - Google Patents

Abstract

The present invention relates to an analytical device and a quantitative method comprising a semiconductor laser and a microflow cell, and more particularly, to a simple and highly sensitive analytical device and a quantitative method using a visible light semiconductor laser, a microflow cell and a photodiode. It is about.

In order to solve the problem caused by the change of temperature, current or light reflection in the semiconductor laser used as the light source, the analysis device (spectror) of the present invention is made by double beam detection using a beam splitter to ensure stability and biochemistry. In order to overcome the problem that the amount of sample is very limited and the price of the reagent is high in the analysis of the sample, a thin and long microflow cell was manufactured to enable high sensitivity analysis even with a small amount of the sample.

Description

ANALYTICAL APPARATUS AND THE DETERMINATION METHOD OF BIOCHEMICAL SAMPLE USING THEREOF

The present invention relates to an analytical device and a quantitative method comprising a semiconductor laser and a microflow cell, and more particularly, to a simple and highly sensitive analytical device and a quantitative method using a visible light semiconductor laser, a microflow cell and a photodiode. It is about.

Sugars, along with proteins, nucleic acids, and fats, are one of the four major substances that make up biomolecules, and serve primarily to store or generate energy in vivo. Diabetes is a condition in which sugar concentrations increase abnormally in vivo due to genetic factors or infections. Diabetes is rarely cured after the onset of diabetes and causes various complications. Self-monitering of blood glucose (SMBG) was required for this. Blood glucose self-management is aimed at controlling blood sugar by measuring the concentration of blood glucose in vivo, and for this purpose, accurate measurement must be preceded.

Current methods for quantitative analysis of sugars can be broadly divided into chemistry, electrochemistry, and enzymatic methods. In spectral analysis using light, enzymatic methods are most widely used. Enzyme method is a method of developing a sugar by using an enzyme having a reaction specificity in a chemical reaction. The sugar produces hydrogen peroxide by glucose oxidase, and is purified under a peroxidase catalyst. They react with the chromogenic oxygen acceptor and develop color. Chromogen oxygen receptors include homovanillic acid, o-dianisidine, o-tolidine, and scopoletin. It has one absorption wavelength. Therefore, the sugar can be quantified by measuring the absorbance at this absorption wavelength.

However, in quantifying sugar using such spectroscopic method, the sensitivity of the device is very important for accurate blood glucose measurement. Therefore, many studies are being conducted to increase the sensitivity of the device, and one way to increase the sensitivity of the device is to lengthen the light path through the sample. For the most part, these spectroscopic methods are in good agreement with Lambert-Beer's law, which can increase sensitivity by lengthening the optical path. However, this method is difficult to apply to the actual spectrometer as the light path length increases the amount of sample and the amount of transmitted light decreases. In particular, when quantifying a biological sample such as blood glucose developed by the enzyme method, the amount of samples that can be obtained is very limited, and because expensive enzymes are used as reagents, high sensitivity analysis can be performed even with a small sample. The method is absolutely required.

In particular, semiconductor lasers are characterized by small size, long lifespan, low power consumption, and high output power. Recently, due to mass production, the price is low and the performance is greatly improved. It is becoming. Therefore, when the semiconductor laser is used as a light source of the spectroscope, it is possible to produce a simple type spectrometer that does not require a monochromator by emitting monochromatic light, and 200W xenon used as a light source of a conventional UV / VIS spectrometer Since the lamp can get more power than the intensity of the light passing through the monochromator, high sensitivity analysis is possible with only a photodiode, not a photomultiplier. Currently, such spectroscopy using semiconductor lasers includes laser fluorescence, laser atomic spectroscopy, and laser spectroscopy, and high sensitivity and high sensitivity can be measured by using a semiconductor laser having a large output and a large monochromatic color as a light source.

However, the semiconductor laser still has a problem that the output of the laser is changed by the change of temperature, current or light reflection. In order to solve the shortcomings of the semiconductor laser and to detect more sensitive, various studies are being conducted.

For example, Journal of the Korean Physical Society, vol. 30, no. 2, 1997, pp 431 ~ 433, Kim, Sung-ho and others disclose a method for quantifying proteins using a semiconductor laser colorimeter, an optical fiber, a dipping probe, and the like. Et al. Disclose the analysis of aqueous ammonia using the apparatus (Proc. SPIE., Vol. 3540, pp 91-96, 1998), but the problem of changing the output of the laser due to a change in temperature, current or light reflection. I still have it.

In addition, Kim discloses a method for obtaining a dissociation constant of thymol blue using a semiconductor laser, an optical fiber, and the like (Bulletin of the Korea chemical society, vol. 19, no. 8, 1998). However, it does not solve the above problem.

The present invention is to solve the problems of the prior art as described above, an object of the present invention is to provide a sugar analysis device and a sugar quantification method of a simple structure that is stable to changes in temperature, current, etc., and capable of high sensitivity analysis even with a small amount of samples. To provide.

1 is a schematic view showing a microflow cell according to one embodiment of the present invention,

2 is a schematic view showing a sugar analysis apparatus according to one embodiment of the present invention,

3 is a circuit diagram of a photodiode amplifier circuit usable in one embodiment of the present invention;

4 is a graph showing an oscillation wavelength of a semiconductor laser and an absorption spectrum of a sample according to an embodiment of the present invention;

5 is a graph showing a change in a signal value and a reference value according to the intensity of a light source according to an embodiment of the present invention;

Figure 6 is a graph comparing the stability of the sugar analyzer and the conventional UV / VIS spectrometer according to an embodiment of the present invention,

7 is a graph showing a comparison between the detection interval of the sugar analysis device and the existing UV / VIS spectrometer according to an embodiment of the present invention,

8 is a graph showing a calibration curve of sugars according to an embodiment of the present invention;

Figure 9 is a graph showing the change in absorbance with time of sugar color reaction according to an embodiment of the present invention.

The analysis device of the present invention is characterized by including a semiconductor laser, microflow cell, photodiode and / or beam splitter. The semiconductor laser is preferably a visible light semiconductor laser.

In the analysis device of the present invention, the microflow cell is characterized in that the tubing for inflow and outflow of the sample is inclined at 20 to 70 ° with respect to the glass capillary at both ends of the glass capillary with the inner diameter of 0.1 to 2.0 mm. do.

Referring to a preferred embodiment of the analysis device according to the invention as follows. In the following, an analytical device and a quantitative method for analyzing sugar are disclosed as an example, but the scope of the present invention is not limited to the sugar analytical device and quantitative method.

Micro Flow Cell

One embodiment of the microflow cell used in the analysis apparatus of the present invention is shown in FIG. In the form of this microflow cell, the sample container filled with the sample solution consists of a glass capillary tube (made by Chase, USA) having an inner diameter of 0.1 to 2.0 mm, preferably 1 mm and a length of 33 mm. The tubing in which the sample solution flows in and out (Teflon tubing, manufactured by Upture Sine Ent. Co., Ltd.) is made of aluminum and connected to both ends of the capillary by connecting the tubing to the capillary at a 45 ° The measurement was less influenced by the flow rate, so that the air can escape well. The aluminum assembly serves as a support and may be composed of other materials. On both sides of the assembly, glass windows were attached so that the sample solution injected through the pump (fluid pump, Ismatec, Switzerland) could flow into the mode capillary without counting, and light could be transmitted to analyze the sample solution. . The path length of the microflow cell having the above structure is 34 mm and the internal volume is about 30 μl.

Compared to the cuvette (10 × 10 × 30) used in the existing UV / VIS spectrometer, the microflow cell can theoretically measure 3 to 4 times or more with only about 1/100 samples. .

Analysis device

One embodiment of an analysis device (diode laser / micro-flow cell colorimetric system: DL / MFCS) using a semiconductor laser and a microflow cell of the present invention is shown in FIG. 2. A 633 nm, 5 mW visible light semiconductor laser (LDM145 / 633/5, manufactured by Emarttronic, USA) was used as a light source, and a heat sink holder was made of aluminum to fix heat dissipation to fix the semiconductor laser. The semiconductor laser is driven by receiving power from a D / A converter (PCL-711S PC-multilab card, Advantech, Taiwan) connected to a computer.

The beam emitted from the semiconductor laser is focused through the lens and divided into a signal beam and a reference beam by a beam splitter inclined at 20 to 70 °, preferably 45 °, with respect to the traveling direction of the laser beam. The signal beam passes through the microflow cell, whereby absorption occurs by the color sample injected into the microflow cell, and the remaining beam is detected as an electrical signal by a photodiode detector (SP-45ML). . In addition, since the reference beam is relatively strong compared to the signal beam passing through the microflow cell, the reference beam is attenuated to be equal to the intensity of the laser beam by using a polarizer, and is detected as an electrical signal by using a photodiode. Since the electric signals of the detected signal beam and the reference beam are very weak signals, they are amplified at the same time by attaching an amplification circuit. The signals are converted into digital signals using an A / D converter and transmitted to a computer. To be treated. The amplification circuit of the photodiode used in the present invention is shown in FIG.

According to the analyzing apparatus of the present invention having the above-described configuration, the semiconductor laser having a large output and emitting monochromatic light, preferably a visible light semiconductor laser having a maximum wavelength of 600 to 680 nm, is used as a light source. By using a flow cell, high sensitivity analysis is possible even with a small amount of sample. In addition, a beam splitter is placed between the semiconductor laser and the microflow cell, and the beam splitter is used to make the laser beam a double beam to simultaneously measure the reference value and the signal value. Since the reference value and the signal value are changed at the same time even if the intensity of the light source is changed, by measuring these values simultaneously, there is no influence on the absorbance expressed as the ratio of the reference value and the signal value, thereby further improving the sensitivity of the spectrometer. have. In particular, in the determination of sugars, the reagent developed by o-tolidine using the enzymatic method has an absorption wavelength band (λ _max = 631 nm) between about 520 and 720 nm. Therefore, the reagent developed by using a semiconductor laser emitting a wavelength of 630 nm The absorption wavelength band of the sugar coincides with the oscillation wavelength of the light source, thereby enabling more sensitive sugar analysis.

Sugar quantification method according to the present invention is carried out by a conventional enzyme method using the above-described sugar analysis device of the present invention. That is, a sugar assay sample developed with a color developing enzyme is placed in the microflow cell, and the laser beam is reflected from the semiconductor laser, and the sugar is quantitatively analyzed from the absorbance data of the computer obtained by the above process.

The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

Example 1

reagent

As enzymes to develop sugars, glucose oxidase (G6891, Sigma, USA) and peroxidase (P8415, Sigma, USA) were used, and o-tolidine as a chromogen oxygen receptor. (T35475, Aldrich, USA). Glucose used to prepare the standard solution was purchased from Sigma, and sodium acetate (Duksan, Korea) and acetic acid (Duksan, Korea) were used to prepare buffer solutions. Acid blue 25 (210684, Aldrich, USA) having a maximum absorption wavelength at 630 nm was used as a standard dye solution used for verification of the device.

Measure the performance of the device

end. Measurement of oscillation wavelength of semiconductor laser and absorption spectrum of sample

The light source used in this embodiment was a semiconductor laser having a maximum wavelength at 630 to 640 nm, and a line profile of the light source was obtained by using a diffraction grating and a charged coupled device (CCD). The visible light absorption spectrum of the sample and the acid blue 25 was measured and shown in FIG. 4. As can be seen from Fig. 4, since the output band width of the oscillation wavelength (C) of the semiconductor laser of the present invention is included in the absorption spectrum of the colored glucose (A) or acid blue 25 (B), the semiconductor laser is subjected to the enzymatic method. It can be seen that it is a suitable light source for quantifying glucose or acid blue 25 developed using.

In addition, when the current value supplied to the semiconductor laser is changed, the change in the signal value and the reference value when the output of the semiconductor laser is changed is measured, and this is shown in FIG. 5. As can be seen in FIG. 5, when the output of the semiconductor laser is changed, a relational expression of signal value = 1.058 × reference value-1.343 can be obtained between the signal value and the reference value. The slope value divided by was constant, and the absorbance did not change.

I. Measure stability of the device

The analyzer (hereinafter referred to as "DL / MFCS") and the conventional UV / VIS spectrometer UV-2101PC (Shimatsu, Japan) were measured by measuring the change in absorbance for one hour in a blank state. Stability was measured, and the change in absorbance with time is shown in FIG. 6 and the S / N ratio was calculated. The solid line of FIG. 6 is the result measured by DL / MFCS, and the dotted line is the result measured using the UV / VIS spectrometer. Both devices use double beams, so the change in absorbance with time should be constant, but in practice, it is impossible to remove the complete noise of each spectrometer, which results in a change in absorbance. It is an important factor to decide.

As can be seen in Figure 6, the absorbance was changed from -9 × 10 ^-4 to 1.1 × 10 ^-3 AU for 1 hour in the case of UV / VIS spectrometer, -5.7961 × 10 ^{-4 in} the case of DL / MFCS A change in absorbance of ˜2.7796 × 10 ⁻⁴ AU was shown. Using this result, the standard deviation of noise of each device was calculated. As a result, the standard deviation of absorbance noise measured for 1 hour in the case of the existing UV / VIS spectrometer was 3.9231 × 10 ^-3 , and 1.4520 in the case of DL / MFCS. It appeared as x10 <^-4> . As a result, it can be seen that DL / MFCS is 63% more stable than conventional UV / VIS spectrometer.

All. Determination of detection section

To determine the detection interval, acid blue 25 was dissolved in distilled water to prepare 100 ml of a 400 mg / L (9.6 × 10 ^-4 M) solution, which was then diluted with distilled water to 200 mg / L, 100 mg / L, 50 mg / L. , 20mg / L, 10mg / L , 5mg / L, 2mg / L, 1mg / L, 0.5mg / L, 0.2mg / L, 0.1mg / L, 0.5mg / L, 0.02mg / L (4.8 × 10 - Samples were prepared over a concentration range of 4 M order of ⁸ M), and the absorbance of the solution prepared using the DL / MFCS and the existing UV / VIS spectrometer was measured, and the absorbance according to the concentration was shown. (dynamic range) was determined and shown in FIG.

As can be seen in Figure 7, in the case of a conventional UV / VIS spectrometer concentration range from 0.2 mg / L (4.8 × 10 ^-7 M) to 200 mg / L (4.8 × 10 ^-4 M), the absorbance is 2.1 × Quantitative analysis ranges from 10 ^-3 to 2.3734 AU are shown, and concentration ranges from 0.05 mg / L (1.2 × 10 ^-7 M) to 50 mg / L (1.2 × 10 ^-4 M) for DL / MFCS At, the absorbance ranged from 2.41 × 10 ^-3 to 1.7964 AU, and the correlation coefficient was 0.9992. Both instruments showed a quantitative analysis section over three orders of magnitude, with DL / MFCS being four times thinner than the existing UV / VIS spectrometer.

Example 2

Analysis of sugars using enzyme method

500 mg of glucose was dissolved in distilled water to prepare 100 ml of a 500 mg / dL reference solution, and the solution was diluted with distilled water to prepare a glucose standard solution as shown in Table 1 below.

Standard solution (mg / dL) 50 100 150 200 250 300 350 400 Reference solution (ml) One 2 3 4 5 6 7 8 Distilled water (ml) 9 8 7 6 5 4 3 2

To prepare 100 ml of a buffer solution of pH 4.0 by mixing 18 ml of 0.1 M CH ₃ COONa and 82 ml of 0.1 M CH ₃ COOH at room temperature (25 ° C.), and to make 1200 U / ml of glucose oxidase (GOD) to 10 U / ml, 210 μl of glucose oxidase was diluted with 25 ml of distilled water. Also, to make 310 U / mg of peroxidase (POD) to 40 U / ml, dilute 2 mg of peroxidase with 15.5 ml of distilled water, 0.053 g of o-tolidine in ethanol and 0.01 M o-tolidine solution 25 ml were prepared.

300 μl of 10 U / ml glucose oxidase prepared above, 30 μl of 40 U / ml peroxidase, and 25 μl of 0.01 M o-tolidine were added to eight test tubes, and a 4 ml color developing reagent was prepared by filling the buffer solution. 10 μl of the glucose standard solution prepared in Table 1 was added to eight test tubes containing the prepared color reagents, and reacted for 6 minutes in a 37 ° C. thermostat. The absorbance was measured using DL / MFCS and UV / VIS spectroscopy. . In addition, by using the measured absorbance showing the absorbance according to the concentration was created a calibration curve, which is shown in FIG.

As can be seen in Figure 8, the absorbance increased proportionally with the concentration of the standard solution, the slope was 1.572 × 10 ^-3 , the tangent was 6.306 × 10 ^-4 , the correlation coefficient (correlation coefficient) ) Was 0.992. This result showed similar results in the correlation coefficient showing the linearity when compared with the results obtained using the existing UV / VIS spectroscopy.

In addition, in order to determine the stability according to temperature when developing glucose using enzyme method, 200mg / dL glucose was prepared, and after adding a coloring reagent, the temperature was changed from normal temperature (25 ℃) to 50 ℃ at 5 ℃ intervals. The reaction was observed, and the results are shown in FIG. 9. As can be seen in Figure 9, the reaction rate, time and absorbance vary greatly depending on the temperature, and at 25-30 ° C, the absorbance continues to increase even after 10 minutes after the start of the reaction, indicating that the reaction continues. In the 40 ℃, 45 ℃, 50 ℃ can be seen that the maximum absorbance within 2 to 3 minutes after the start of the reaction, the absorbance drops sharply, the phenomenon that bubbles are generated in the sample by the temperature change during the experiment. At 35 ℃ and 37 ℃ showed the maximum absorbance around 6 minutes after the start of the reaction, the absorbance tended to decrease gradually with time, and reached the maximum absorbance faster at 37 ℃ than 35 ℃. The maximum absorbance was 0.795 at 6 min after the start of the reaction at 37 ℃, and the mean error until 1 min after the maximum absorbance was ± 2.0149 × 10 ^-3, and the mean error up to 2 minutes after was ± 7.7564 × 10 ^-3 The average error of up to 3 minutes was ± 0.0171, and the average error of up to 4 minutes was ± 0.0276. This can be seen that more stable measurement is possible when measured at a temperature and time that can increase the stability of the enzyme reaction in sugar analysis.

As described above, the analysis apparatus according to the present invention is stable to changes in temperature, current, or light reflection by using a beam splitter, and has a problem that the amount of the sample is very limited and the cost of the analysis reagent is high in the analysis of biochemical samples. To overcome this, the use of thin and long microflow cells enables high sensitivity analysis with only a small amount of sample, resulting in about four times better sensitivity with only a hundredth of the sample required for a conventional spectrometer. In addition, the absorbance was measured in the concentration range of 50 ~ 400mg / dL by using the enzyme method to analyze the sample, the results obtained by using the existing UV / VIS spectrometer obtained the same results, the enzyme reaction is stable When measured at temperature and time, sugars can be quantified with better sensitivity.

In addition, the present invention can be applied to other target samples which can be quantified by colorimetric reactions having a wavelength of 600 to 700 nm. That is, colorimetric reaction at 660 nm by phospholipid, sodium chloride and phenol which were colorimetric at 660 nm by reaction of cholesterol, hydrogen peroxide, urea, molybdate and aminonaphthol, which showed colorimetric reaction at 625 nm by sulfosalicylic acid. In addition, the analytical device and the quantitative method of the present invention can be variously used for colorimetric reaction of total lipids, alkaline phosphatase, inorganic phosphorus, ammonia, and protein by the Lowry method.

Claims (5)

A light source includes a semiconductor laser, a microflow cell for receiving a sample, a photodiode and a beam splitter, wherein the beam splitter is located between the semiconductor laser and the microflow cell, and the laser beam from the semiconductor laser is referenced to the signal beam and the reference beam. The photodiode is divided into beams, and the photodiodes are positioned in the advancing direction of the signal beam passing through the microflow cell and the advancing direction of the reference beam, respectively. The analyzing apparatus according to claim 1, wherein the semiconductor laser is a visible light semiconductor laser. According to claim 1, The micro-flow cell is characterized in that the tubing for inflow and outflow of the sample is inclined at 20 ~ 70 ° with respect to the glass capillary, respectively, at both ends of the glass capillary tube with an inner diameter of 0.1 ~ 2.0mm Analysis device. The analysis apparatus of claim 1, wherein the photodiode comprises an amplifying circuit. A method of quantifying a biochemical sample using the device of any one of claims 1 to 4.