TWI666439B - Detection device for detecting leukocyte esterase - Google Patents

Abstract

The invention provides a detection device capable of measuring white blood cell esterase, which comprises: an electrochemical measurement area and a naked eye colorimetric area, wherein the electrochemical measurement area is provided with an electrochemical white blood cell esterase detection test strip, which is based on electrochemical Method A white blood cell esterase content of a fluid sample is measured, which includes an insulating substrate and an electrochemical structure. The electrochemical structure is disposed on the insulating substrate and includes a reaction zone. The reaction zone is coated with a reaction material, wherein the reaction material Includes a buffer solution, a leukocyte esterase reaction substance and an electron conducting substance to detect low and medium to high concentrations of leukocyte esterase content, and in particular to quantitatively detect low concentration of leukocyte esterase content; A colorimetric test strip is provided, which includes a sample detection area, a negative control area, and a positive control area, and is used to visually detect whether the fluid sample contains medium and high concentration leukocyte esterase.

Description

Detection device for detecting leukocyte esterase

The invention relates to a detection device, in particular to a detection device for detecting leukocyte esterase.

Urinary tract infection, commonly known as urethritis, is the inflammation of the urethra after bacteria enter the urethra. Bacteria can enter the bladder through the urethral opening, and can even reach the kidneys, which can cause pyelonephritis. Symptoms of urethritis include a burning sensation during urination, hematuria, cloudy urine, or a foul odor. The diagnostic method can be diagnosed by urinalysis and checking for the presence of nitrite, white blood cells or white blood cell esterase to determine whether the urethra has been infected by bacteria.

Urinary tract infections are the most common bacterial infections, affecting an average of 150 million people each year. Early detection of urinary tract infections is very important in clinical tests. Delayed tests and treatment of urinary tract infections can easily lead to delays in the disease and invasion of the kidneys by bacteria, or more serious complications. There are many different methods for detecting urinary tract infections, for example: urine test strips for blood esterase and nitrite. However, based on the previous integrated analysis results, this test strip is not very accurate, and it is easy because of the color. Misdiagnosis, and the original color of urine caused false negative or false positive results in diagnosis, and quantitative analysis cannot be performed, and the amount of information about the condition is limited; and the white blood cell counting method of urine using a microscope, It is not recommended as a single diagnostic method for urinary tract infections, especially when the symptoms of pyuria are mild; urine culture is currently the most standard urinary tract infection detection method in hospitals, but this method is time-consuming and expensive. Because a urinary tract infection is a A very common bacterial infection, the present invention tends to develop a detection method that is convenient for clinical testing and less expensive, and targets the more sensitive, accurate and quantifiable detection method of leukocyte esterase.

Taiwan Patent No. TWM552595 discloses a detection device for detecting the concentration of nitrite. The device can be used to determine nitrite in a sample by electrochemical method or by colorimetric method. However, the principle of determining nitrite to determine bacterial infection is that bacteria will reduce the protein metabolite nitrate in the urine to nitrite, but the urine is left for too long to cause bacterial growth, so although nitrite is a bacterium There are indicators in the urine. If it is impossible to determine whether the bacteria in the urine are present before or after urination, the detection of nitrate will still result in false negative or false positive conditions.

Therefore, providing a highly accurate detection device that can confirm urethritis can reduce the aforementioned problems, and detecting leukocyte esterase in urine can also be used as a way to determine whether urethritis is caused by bacterial infection. US Patent US5464739A , US46557855A, US6528652B1 and Korean patent No. KR0147861B1 are all based on the detection of leukocyte esterase reactants in a test strip or composition, and there is no quantitative effect; and Taiwan Patent No. TWM457179 discloses an optical detection method White blood cell esterase measurement device, but there is no actual proposal on how to measure white blood cell esterase. Chinese Patent No. CN105890927A and US Patent No. US5776780A are measured by color change and spectrometry. Chinese Patent No. CN101975852A only proposes a test piece to match the spectrum According to the method of measurement, U.S. Patent No. US 7,504,235 proposes photochemical measurement of white blood cell esterase. From this, it can be known that the detection of white blood cell esterase in the prior art is mostly based on optical detection analysis to quantify the white blood cell esterase in the sample, but due to the sensitivity of optical detection Not high, so it cannot meet the requirements for low concentration samples Analysis of the product required, generally commercially available colorimetric test strips can visually identify the minimum contrast to the white blood cell count leukocyte esterase concentration of about 25 ~ 70Leu / μ L, and the reaction time is more than 2 minutes failure, however, in general, number of leukocytes 5Leu / μ L that it is possible urinary tract infection, thereby providing a high sensitivity, high accuracy and quantitative analysis problem can be leukocyte esterase detection apparatus of low concentration samples to be solved in the art.

In order to solve the above problems, the present invention provides a device for electrochemically detecting leukocyte esterase. The leukocyte esterase detection device has high accuracy and quantitative analysis, and includes a low concentration sample of leukocyte esterase, which includes: an electrochemical The measurement area and a naked eye colorimetric area, wherein the naked eye colorimetric area is provided with a colorimetric detection test paper, which allows the user to detect whether the fluid sample contains leukocyte esterase by the naked eye colorimetry, wherein the colorimetric detection test paper includes: a sample detection Area, a negative control area, used to indicate that the fluid sample does not contain leukocyte esterase, and a positive control area, used to indicate that the fluid sample contains leucocyte esterase, where the electrochemical measurement area is provided with an electrochemical A white blood cell esterase detection test strip, the electrochemical white blood cell esterase detection test strip includes an insulating substrate and an electrochemical structure, the insulating substrate having a first surface and a back surface opposite to the second surface, the electrochemical structure Disposed on the first surface, including a reaction zone, coated with a reaction material, wherein the reaction material includes a buffer solution A leukocyte esterase reaction substance and an electron conducting substance; one of the fluid samples enters the reaction zone of the electrochemical structure, performs an electrochemical reaction with the reaction material, and detects an electrical signal caused by the amount of the electrochemical reaction product generated The change is converted into the white blood cell esterase concentration of the fluid sample; the white blood cell esterase detection device detects a white blood cell esterase concentration range of 2-64 μL / mL (equivalent to 0.0102 to 0.3264 U / mL); wherein the white blood cell esterase detection device detects The concentration range of quantified leukocyte esterase is 2-8 μL / mL (equivalent to 0.0102 ~ 0.0408 U / mL).

The insulating substrate is selected from polycarbonate, polyester, polyether, and polyfluorene. Amine, polyurethane, polyimide, polypropylene, polyethylene, polyvinyl chloride, glass, glass fiber, cotton fiber, silica and alumina; the buffer solution is selected from the group consisting of phosphate Group consisting of citrate and acetate buffer solution; the leukocyte esterase reaction substance is an amino ester and a diazonium salt; the electron conducting substance is a conductive metal; the conductive metal is selected from the group consisting of gold , Silver, and copper.

In order to solve the foregoing problem, the present invention provides another urethritis infection detection device, which comprises the leukocyte esterase detection device and a nitrite detection test strip, and the nitrous acid detection test strip may be an electrochemical test strip or a colorimetric Test strip.

In summary, the white blood cell esterase detection device of the present invention has the following advantages: 1. It can quantitatively detect low concentration white blood cell esterase, and the detection concentration can be lower than the lowest white blood cell esterase that can be recognized by the naked eye with commercially available colorimetric test paper. Concentration; 2. Quantitative analysis can be performed, including low concentration range. Generally, commercially available colorimetric test papers can only estimate the approximate number of white blood cells based on rough color patches; 3. Reduce the colorimetric test papers' misjudgment due to the long reaction time, because The colorimetric test paper is invalid after being reacted for more than 2 minutes; 4. The test results are stable, which can avoid misjudgment due to the color of the sample itself; 5. The accuracy is high, the test results can be repeatedly confirmed, and it can be double compared with the test results of nitrite Yes, it is more accurate to infer the situation of urinary tract infection, and to avoid misjudgment of results caused by false negatives or false positives.

1‧‧‧White blood cell esterase detection device

10‧‧‧ Electrochemical measurement area

100‧‧‧ Electrochemical leukocyte esterase test strip

1000‧‧‧ insulated substrate

1000a‧‧‧first surface

1000b‧‧‧Second surface

1002‧‧‧ Electrochemical Structure

1002a‧‧‧Reaction zone

12‧‧‧ naked eye colorimetric area

120‧‧‧ colorimetric test strip

120a‧‧‧sample detection area

120b‧‧‧ negative control area

120c‧‧‧ positive control area

S1 ~ S4‧‧‧step flow

FIG. 1 is a schematic diagram of a leukocyte esterase detection device according to an embodiment of the present invention.

FIG. 2 is an electrochemical leukocyte esterase detection test according to an embodiment of the present invention. Schematic of the tablet.

FIG. 3 is a flowchart of steps according to an embodiment of the present invention.

In order to fully understand the purpose, characteristics and effects of the present invention, the present invention will be described in detail through the following specific embodiments and the accompanying drawings.

In order to obtain the overall consistency of the technical content of the patent of the present invention and to clearly understand the present invention, the definitions of the terms used herein are provided: "Quantitative analysis" here refers to the determination of a test object based on the physical or chemical properties of a test object, such as light absorption, fluorescence, conductivity, etc., using specific instruments or devices, such as electrophoresis, chromatography, etc. Analytical method for the precise content of a component.

"Fluid sample" here refers to a fluid test object that will chemically react with the reaction material in the reaction layer, and its source may be urine, blood, drinking water, vegetable juice, milk or other fluids.

Please refer to FIG. 1, which is a first specific embodiment of the white blood cell esterase detection device 1 of the present invention. In this specific embodiment, the white blood cell esterase detection device 1 includes: an electrochemical measurement area 10 and a naked eye The colorimetric region 12, wherein the electrochemical measurement region 10 is provided with an electrochemical leukocyte esterase detection test strip 100 for electrochemically measuring the content of white blood cell esterase in a fluid sample; and the naked eye colorimetric region 12 is based on the naked eye Observe whether the fluid sample contains leukocyte esterase. The naked eye colorimetric region 12 is provided with a colorimetric detection test paper 120, and the colorimetric detection test paper 120 has three regions, each of which is a sample detection region 120a, for detecting the fluid. The sample, a negative control area 120b, is used to indicate that the fluid sample does not contain white blood cell esterase, and a positive control area 120c is used to indicate that the fluid sample contains white blood cell esterase, which is colored, and the positive control area 120c, The negative control The area 120b and the sample detection area 120a are spaced apart from each other by an appropriate distance or protected by waterproofing to prevent the fluid samples of the sample detection area 120a from flowing to the positive control area 120c and the negative control area 120b and causing interference with each other.

When analyzing the fluid sample, take an appropriate volume, and simultaneously or successively drop to the sample detection area 120a, and the electrochemical leukocyte esterase test strip 100, the reaction time of each colorimetric test strip 120 is about 1-2 The reaction time of the electrochemical leukocyte esterase test strip 100 is about 30 minutes, which can detect an electronic current or resistance signal, and then calculate the result. Leukocyte esterase concentration in a fluid sample. Because the reaction time of the colorimetric detection test paper 120 is short, it can be known whether the fluid sample is a leukocyte esterase positive reaction, and the detection result shows a specific concentration range, which can be compared with the electrochemical leukocyte ester later. Enzyme test strip 100 is quantified and the measured white blood cell esterase concentration is compared to further determine whether the actual quantified measured concentration falls within the concentration range shown on the colorimetric test strip 120. When the colorimetric area is judged to be negative It may be a false negative caused by the low concentration of white blood cell esterase, that is, the specimen has a urinary tract infection at this time. At this time, it can be judged by the electrochemical measurement area at the same time to achieve the purpose of early detection. When the color (such as hematuria or blood) is hidden and the colorimetric area cannot be interpreted, the interpretation of the electrochemical measurement area can be used to avoid false positives or inability to judge due to the color of the sample, and improve the reliability of the test results.

Please refer to FIG. 2, which is a specific embodiment of an electrochemical leukocyte esterase detection test strip 100 in a leukocyte esterase detection device 1 of the present invention. The electrochemical leukocyte esterase detection test strip 100 includes an insulating substrate 1000 and an electrochemical structure. 1002, the insulating substrate has a first surface 1000a and a second surface 1000b facing away from the first surface 1000a; and the electrochemical structure 1002 is disposed on the first surface 1000a and includes a reaction region 1002a, The reaction zone 1002a A reaction material is provided, wherein the reaction material includes a buffer solution, a leukocyte esterase reaction substance, a surfactant, and an electron conducting substance. When a fluid sample enters the reaction zone, it can directly perform an electrochemical reaction with the reaction material.

Among them, a preferred implementation of one of the insulating substrates 1000 in the electrochemical leukocyte esterase detection test strip 100 may be a soft material, such as an insulating polymer material, or a rigid material, such as a ceramic material, glass, or glass. Fiber, etc. Examples of the insulating substrate 1000 include, but are not limited to, polycarbonate, polyester, polyether, polyamide, polyurethane, polyurethane, polypropylene, polyethylene, polyvinyl chloride, glass, glass fiber, and cotton fiber. , Silica, alumina, etc.

The type of buffer solution in the reaction material includes, but is not limited to, phosphate, citrate, acetate buffer solutions, etc., and is used to maintain a specific range of pH; the leukocyte esterase catalytic substance is an amino acid ester. (Amino acid ester) and diazonium salt. Examples of the electron conducting material are conductive metals. The types of the conductive metals include, but are not limited to, gold, silver, and copper. Under constant potential conditions, when the leukocyte esterase in the fluid sample catalyzes the hydrolysis of the amino acid ester, the hydrolyzed product phenylpyrrole (phenylpyrrole) will form a purple azo dye with a diazonium salt. In the example, the conductivity of nano silver was used to measure the change in resistance to infer the content of leukocyte esterase. The reaction formula is as follows: C ₂₀ H ₂₀ N ₂ O ₄ S C ₁₀ H ₈ NO + C ₁₀ H ₁₂ NO ₄ S

_{C 10 H 8 NO + C 10} H 6 N 2 O 4 S - → C 20 H 14 N 3 O 5 S-

In the compounds provided by the present invention, 3- (N-Tosyl-L-alaninyloxy) -5-phenylpyrrole is 3- (N-Tosyl-L-alaninyloxy) -5-phenylpyrrole. For example, the diazonium salt is 1-diazo-2-naphthol-4-sulfonic acid, and other structures are used in this place. The same result is as follows:

Wherein the substituent X of the amino acid ester is one of p-toluenesulfonyl (that is, 3- (N-Tosyl-L-alaninyloxy) -5-phenylpyrrole) or benzyloxycarbonyl; wherein the substituent Y of the amino acid ester is 3- hydroxy-5-phenylpyrrole (that is, 3- (N-Tosyl-L-alaninyloxy) -5-phenylpyrrole) or p-nitrophenol; where the substituent Z of the diazonium salt is NO2, SO3, or H (that is, 1 -Diazo-2-naphthol-4-sulfonic acid).

Compounds using X, Y, and Z substituents can achieve the same effect.

FIG 3 is a flowchart step, operation, taking 10 L [mu] fluid sample, such as urine or blood, dropwise onto the reaction zone of the electrochemical structure Sl, leukocyte esterase will catalyze an electrochemical fluid sample leukocytes ester Enzymatic hydrolysis of amino esters in test strips, wherein a preferred embodiment is the use of 3- (N-toluenesulfonyl-L-alanyloxy-5-phenylpyrrole), which hydrolyzes The product phenylpyrrole will form an azo dye with another reaction material diazonium salt. In a specific embodiment, 1-diazo-2-naphthol-4-sulfonic acid is used as the diazonium salt as an example. After the above reaction, an electrical change will occur, and then the electrical change S2 is detected by an electrical measuring probe instrument (not shown), and the white blood cell esterase concentration can be obtained by computer operation S3. Generate a signal, which can then be displayed on the screen or transmitted to another device S4.

The measured results of 2 to 64 μ L / mL (equivalent to 0.0102 ~ 0.3264U / mL) can be detected by the detecting means of the present invention, the blood cell esterases, and 0 to 8 μ L / mL (equivalent to 0 ~ 0.0408U / mL ) is a linear linear range, so that the leukocyte esterase detection means quantifiable range 2-8 μ L / mL (equivalent to 0.0102 ~ 0.0408U / mL).

In another specific embodiment of the present invention, the white blood cell esterase detection device 1 can further be added with a nitrite detection test strip (not shown in the figure). When the electrochemical measurement area 10 of the white blood cell esterase detection device 1 of the present invention is added At the same time, an electrochemical leukocyte esterase test strip 100 and a nitrite test strip are provided. The nitrite test strip can be an electrochemical test strip or a colorimetric test strip, which can simultaneously measure whether urine contains bacteria and allow white blood cells to The esterase detection and the nitrite detection cooperate with each other to avoid false negatives and false positives caused by a single indicator, so as to increase the accuracy of the detection.

According to the analysis of experimental data, the leukocyte esterase detection device provided by the present invention, wherein the limit of detection (LOD) of the nitrite concentration of the electrochemical nitrite detection test strip is about 1.39x10-7M, and the electrochemical The detection limit of the white blood cell esterase concentration of the white blood cell esterase test strip is about 2 μL / mL (equivalent to 0.0102 U / mL); the detection limit of the nitrite concentration of the colorimetric detection paper is about 1.3x10-5M. The white blood cell ester The detection limit of the enzyme concentration is approximately 4 μL / mL (equivalent to 0.0204 U / mL).

To sum up, the advantages of the white blood cell esterase detection device of the present invention are: 1. It can quantitatively detect low concentration of white blood cell esterase, and the detection concentration can be lower than the lowest white blood cell ester that can be recognized by the naked eye with commercially available colorimetric test paper. Enzyme concentration; 2. Quantitative analysis can be performed, including low concentration ranges. Generally, commercially available colorimetric test papers can only estimate the approximate number of white blood cells based on rough color patches; 3. The test results are stable, which can avoid misjudgment due to the color of the sample itself Situation; 4. Reduce the colorimetric test paper due to the reaction time is too long, resulting in misjudgment of the results, because the colorimetric test paper responds 2 points It will be invalid if it is more than 15 minutes; 5. High accuracy rate, can repeatedly confirm the test results, and double comparison with the test results of nitrite, to avoid false judgment of the results caused by false negatives or false positives, reducing the trouble of repeated testing of the subject, Therefore, a better method for judging urethritis in the art is provided.

The present invention has specifically and fully disclosed the relevant technical content through the preferred embodiments. However, those skilled in the art should understand that this embodiment is only used to describe the present invention. When the patent scope of the present invention cannot be limited by this The illustration is to make the description clear, and it is not drawn according to true proportions, so the present invention is not limited to the appearance and proportions of the diagrams; those who are familiar with this technical field can understand it after understanding the spirit and principles of this creation. Alterations and modifications achieve equivalent purposes, and these changes and modifications should be covered within the scope defined by the scope of patent application as described below.

Claims (9)

A detection device for detecting white blood cell esterase, comprising: an electrochemical measurement area, an electrochemical white blood cell esterase detection test strip is provided, which is used to electrochemically measure the white blood cell esterase content of a fluid sample, and the electrochemical white blood cell esterase The test strip includes: an insulating substrate having a first surface and a second surface, the second surface facing away from the first surface; and an electrochemical structure disposed on the first surface of the insulating substrate, including: A reaction zone is coated with a reaction material, wherein the reaction material includes a buffer solution, a leukocyte esterase reaction substance, and an electron conducting substance; a fluid sample enters the reaction region of the electrochemical structure, and The reaction material undergoes an electrochemical reaction, and the change in the electrical signal caused by the amount of the electrochemical reaction product is detected to convert the white blood cell esterase concentration of the fluid sample; a colorimetric area for the naked eye is provided with a colorimetric detection test paper, which is based on the naked eye ratio To detect whether the fluid sample contains leukocyte esterase, the colorimetric detection test strip includes: a sample detection area for detecting the A body sample; a negative control area for indicating that the fluid sample does not contain white blood cell esterase; and a positive control area for indicating that the fluid sample contains white blood cell esterase; the white blood cell esterase detection device The concentration of leukocyte esterase was in the range of 2-64 μL / mL (equivalent to 0.0102 ~ 0.3264 U / mL). The detection device for detecting leukocyte esterase according to item 1 of the scope of the patent application, wherein the insulating substrate is selected from the group consisting of polycarbonate, polyester, polyether, polyamine, polyurethane, polyimide, polypropylene, A group of polyethylene, polyvinyl chloride, glass, glass fiber, cotton fiber, silica and alumina. The detection device for detecting leukocyte esterase according to item 1 of the scope of the patent application, wherein the buffer solution is selected from the group consisting of a phosphate, citrate and acetate buffer solution. The detection device for detecting leukocyte esterase according to item 1 of the scope of the patent application, wherein the leukocyte esterase reaction substance is an amino acid ester and a diazonium salt. The detection device for detecting leukocyte esterase according to item 1 of the scope of the patent application, wherein the electron conductive substance is a conductive metal. The detection device for detecting leukocyte esterase according to item 5 of the scope of patent application, wherein the conductive metal is selected from the group consisting of gold, silver, and copper. The detection device for detecting white blood cell esterase as described in item 1 of the scope of the patent application, wherein the concentration of the white blood cell esterase detection device for detecting the quantitative white blood cell esterase is 2-8 μL / mL (equivalent to 0.0102 ~ 0.0408 U / mL). A urethritis infection detection device includes the detection device for detecting white blood cell esterase and a nitrite detection test strip as described in item 1 of the scope of patent application; wherein the concentration range of white blood cell esterase detected by the white blood cell esterase detection device is 2-64μL / mL (equivalent to 0.0102 ~ 0.3264U / mL). According to the urethritis infection detection device described in item 8 of the scope of patent application, the nitrous acid test strip may be an electrochemical test strip or a colorimetric test strip.