TW202016537A - A system of rapid quantitative detection galactose and use thereof - Google Patents

Abstract

A rapid quantitative detection system of galactose includes: a galactose composition, including galactose, a buffer, and an antioxidant to generate a body fluid sample after being metabolized in the body; a test paper or a filter paper containing an enzyme to react with the body fluid sample to produce an electrochemical signal; and a measuring instrument including: a power supply unit to provide a signal; a connector to receive the signal provided by the power supply unit and transmit the signal to the test paper or the filter paper. After the signal and the electrochemical signal generate a corresponding reaction signal, the corresponding reaction signal is transmitted to the measuring instrument; a calculation unit to calculate the corresponding reaction signal; an analog-digital converter to receive the calculated corresponding reaction signal and convert the calculated corresponding reaction signal into a digitized reaction signal; a processor to process the digitized reaction signal; a display to display the digitized reaction signal; and a digital terminal machine to receive the digitized reaction signal.

Description

Galactose rapid quantitative detection system and its application

The invention provides a system for quickly detecting the content of galactose in the body, in particular to "measure the concentration of galactose" and evaluate the degree of liver function damage.

The liver is related to the clearance of many drugs. It can clear the original drug or its metabolites through different metabolic pathways or through bile excretion. The drug excretion or changes in metabolic rate caused by liver insufficiency can cause drug accumulation or Impedes the formation of active metabolites in medicines. Because blood galactose has a sensitive correlation with liver dysfunction, and the evidence from the research literature shows that the value of blood galactose has a significant relationship with the degree of liver dysfunction, so it can be evaluated by the value of blood galactose Its residual liver function.

The conventional detection method is that after fasting for 8 hours, 0.5 g/kg of galactose is intravenously injected, and after 60 minutes, the plasma galactose concentration is measured (Tang HSet al (1992) Digestion, 52: 222-231; Ranek L . et al (1983) Clin. Physiol. 3: 173-178). The measurement method is to make a calibration line based on the relationship between the concentration of different galactose standard solutions and their absorbance values; at the same time, the blood drawn is added with HClO _{4 and} mixed by shaking, and the supernatant is taken by centrifugation, and the supernatant is mixed with KOH and mixed After centrifugation, the supernatant was taken and galactose dehydrogenase was added to the supernatant. In order to avoid inaccurate color reaction, it was placed in a dark room for 60 minutes to prepare a specimen, and its absorbance was measured. Calibrate the line to find the concentration value. However, the detection process is complicated and time-consuming, and multiple types of drugs are used, so the time for the subjects to learn the test results is very long.

Taiwan Patent No. I292478 discloses a method of making specimens for liver function determination and sampling test strips. This method also requires injection of galactose into the subject, wait 60 minutes, and measure the concentration of galactose in the blood. The measurement method is to make a calibration curve based on the relationship between the concentration of different galactose standard solutions and their absorbance values; add trichloroacetic acid to the test strips of the sample and shake for 30 minutes; after removing the solvent, add a solvent containing galactose dehydrogenase and shake for 30 Minutes, add the coloring agent, and finally measure the absorbance. However, this method involves the injection of galactose into the human body and the preparation of a sample. The detection process is complicated and time-consuming. Therefore, a fast and simple galactose detection method is needed in the art for patients who need to detect galactose.

Taiwan Patent M488635 discloses a biological test piece that can detect blood glucose; US Patent US971995 discloses a detection system that performs electrochemical blood capacity testing by an electrochemical method. The system includes an electrochemical test piece and a measuring instrument. It can be seen that the electrochemical method Monitoring the state of the body is a common technology. However, there are many difficulties in detecting galactose electrochemically. The reason is that the protein of the enzyme is unstable and cannot be stored in an environment other than an acidic solution, and the storage time is very short. Therefore, it provides a It is another problem to be solved in the field that the test paper is stored in a solid state and stored for a long time while maintaining the detection accuracy.

In order to solve the aforementioned problems, the present invention provides a rapid galactose quantitative detection system, which includes: a galactose composition, including galactose, a buffer and an antioxidant, after being metabolized into the body, an integrated liquid sample is generated; a test paper or a The filter paper contains an enzyme that will react with the body fluid sample to generate an electrochemical signal; and a measuring instrument including: a power supply unit that provides a signal; a connector that receives the signal provided by the power supply unit and The signal is sent to the test On paper or the filter paper, after the signal and the electrochemical signal generate a corresponding reaction signal, the corresponding reaction signal is transmitted to the measuring instrument; a calculation unit calculates the corresponding reaction signal; an analog-to-digital converter, Receiving the corresponding response signal calculated by the calculation unit, converting the calculated corresponding response signal into a digitized response signal; a processor, processing the digitized response signal; a display, displaying the digitized response signal ; The digital response signal can be sent to a digital terminal.

To achieve the foregoing purpose, the buffer is selected from ascorbic acid buffer, citrate buffer, phosphate buffer, phosphate buffer, acetate buffer, carbonate buffer (Carbonate buffer) and triethanolamine buffer (triethanolamine buffer).

To achieve the foregoing purpose, the antioxidant is selected from vitamin C or/and sodium bisulfite, vitamin A, vitamin E, flavonoids, polyphenols, ethylenediaminetetraacetic acid ((Ethylenediaminetetraacetic acid ( EDTA)), diethylenetriaminepentaacetic acid (DTPA), N,N-bis[carboxymethyl]glycine (NTA).

To achieve the foregoing purpose, the galactose comprises D-(+)-galactose, L-(-)-galactose, the stable isotope galactose, cyclic galactose or galactose derivatives.

To achieve the aforementioned objective, the galactose composition is administered orally, by injection, by spray inhalation and buccal, by rectal, suppository, or other clinically suitable means.

To achieve the foregoing purpose, the oral method is to measure the galactose content in the body by allowing the user to use the galactose composition first, and then detecting the galactose content in the body fluid.

In order to achieve the aforementioned purpose, the injection is performed by letting the user first apply the galactose The composition is injected into the body, and then the galactose content in the body fluid is measured by measuring the galactose content in the body fluid.

The present invention provides another object of the invention. A test paper for a rapid quantitative detection system for galactose includes: an insulating substrate; an electrode unit disposed on the insulating substrate; a first insulating spacer covering part of the electrode unit and including A reaction zone flow channel located at a first edge of the first insulating spacer, wherein part of the electrode unit is exposed in the reaction zone flow channel; and a second insulating spacer, including a second insulating spacer, the The second insulating spacer covers the reaction channel of the first insulating spacer, and exhibits a convex arc shape with the first edge of the first insulating spacer and the same side edge of the insulating substrate. A concave structure is present relative to the front section of the reaction channel of the reaction zone, wherein the reaction channel of the reaction zone contains a reaction layer covering the electrode unit located in the reaction channel of the reaction zone and includes an enzyme and a conductive medium for Electrochemical reaction with body fluid samples; wherein the test paper utilizes the convex arc of the second edge of the second insulating spacer and the concave structure of the insulating substrate at the front section of the communication channel of the reaction zone to destroy the cohesion of body fluid , With capillary phenomenon to achieve rapid intake of body fluid samples; where the enzyme can oxidize, reduce, decompose or metabolize galactose.

To achieve the foregoing object, wherein the half-strips of the Lactose test range 50-2000 μ g / ml.

To achieve the foregoing purpose, the insulating substrate is selected from polyvinyl chloride (PVC), glass fiber (FR-4), polyester (polyester suphone), bakelite, polyethylene terephthalate (PET) , Polycarbonate (PC), polypropylene (PP), polyethylene (PE), polystyrene (PS), glass plates and ceramics.

To achieve the foregoing purpose, the electrode unit is selected from the group consisting of palladium glue, platinum glue, gold glue, titanium glue, carbon glue, silver glue, copper glue, gold-silver mixed glue and carbon-silver mixed glue.

To achieve the foregoing purpose, the reaction layer is selected from the group consisting of enzymes, coenzymes, conductive media, buffers and protective agents.

To achieve the aforementioned objective, the conductive medium is selected from the group consisting of ferrocene, ferrocenium, methylene blue, tris(acetonitrile) ruthenium trichloride), 2,5-dihydroxybenzoquinone (2,5-dihydroxybenzoquinone), phenazinemethosulfate (phenazinemethosulfate), tetrathiafulvalene (tetrathiafulvalene), tetracyanoquinodimethane (tetra-cyano-methane quino-dimethane, methyl viologen, toluidine blue, 5,6-diamino-1,10-o-phenanthroline (5,6-diamino-1,10- phenanthroline), [M(bpy)3]2+(M=Ru or Os; BPY=2,2′-bipyridine)).

To achieve the foregoing purpose, the conductive medium may be a metal ion compound selected from the group consisting of MgCl ₂ , BeCl ₂ , CaCl ₂ , SrCl ₂ , BaCl ₂ or a combination thereof.

To achieve the aforementioned purpose, the buffer is selected from the group consisting of Tris, Tris-HCl, PBS, MES, CHES, Borate, Universal buffer mixtures (CPB), MOPS, TES, HEPES, TAPSO, Tricine, Bicine, TAPS Group.

To achieve the foregoing purpose, the stabilizer is selected from the group consisting of xylitol, mannitol, xylan, arabinoxylan, mannan, trehalose, PEG, PVA, PEO, methyl cellulose (Methocel) , Agarose (agarose), sol-gel (sol-gel), collagen (collagen), chitosan (chitosan), BSA, casein, neo protein, amino acid or any combination of them.

To achieve the aforementioned objective, the surfactant is selected from the group consisting of cationic surfactants, anionic surfactants, neutral ionic surfactants, and nonionic surfactants.

To achieve the aforementioned purpose, the enzyme can be immobilized and dried, and stored in a neutral environment of acid and alkali.

The present invention provides another object of the invention, an application of a rapid quantitative detection system for galactose, wherein the rapid quantitative detection system for galactose can be used to determine the concentration of galactose in the body to measure neonatal galactosemia.

To achieve the aforementioned purpose, the rapid galactose quantitative detection system provides monitoring of galactose value and hepatic insufficiency patients or professionals.

To achieve the aforementioned objective, the rapid quantitative detection system for galactose is based on the concentration of galactose to determine the remaining function of the liver.

100‧‧‧Test paper

110‧‧‧Insulated substrate

120‧‧‧electrode unit

122‧‧‧The first end

124‧‧‧The second end

130‧‧‧The first insulating spacer

132‧‧‧First edge

134‧‧‧ Interchange in the reaction area

140‧‧‧Second insulation spacer

142‧‧‧Second Edge

144‧‧‧ vent

150‧‧‧Reaction layer

200‧‧‧ measuring instrument

210‧‧‧Connector

211‧‧‧Calculation unit

212‧‧‧Analog to Digital Converter

213‧‧‧ processor

214‧‧‧Monitor

215‧‧‧Power supply unit

300‧‧‧Digital terminal

Figure 1 is the appearance of the galactose rapid detection system; Figure 2 is the schematic diagram of the galactose rapid detection system; Figure 3 is the accuracy test curve of the galactose rapid detection system; Figure 4 is the precision test of the galactose rapid detection system; Figure 5 is the Schematic diagram of test paper structure; Figure 6 is the volume inspection of filter paper inspection; Figure 7 is the volume inspection of galactose rapid detection system; Figure 8 series of test test paper storage days test; Figure 9 series blood tolerance test; Figure 10 series repeatability test; Figure 11, Figure 12 series of intravenous galactose GSP results and oral galactose OGSP correlation results; Figure 13 series Test paper test results completed by semi-automatic robotic arm.

The present invention is exemplified and illustrated by the following examples, but the present invention is not limited by the following examples. Unless otherwise specified, the materials used in the present invention are commercially available materials.

The galactose rapid quantitative detection system of the present invention uses Electrochemical Sensing Technology (Electrochemical Sensing Technology), and its appearance is shown in Figure 1. The system mainly uses disposable dry enzyme electrode instrument technology, using the metabolism of human liver Galactose or its metabolites react electrochemically with enzymes to generate micro-currents, and then by measuring the micro-currents, the value of galactose is measured, and the remaining condition of the liver function is evaluated by this value, but the galactose in this case is fast The quantitative detection system is not limited to the evaluation of liver function, and can also measure diseases related to galactose, such as galactosemia of neonates, and the galactose described in the present invention further includes galactose and its derivatives.

Example 1 Use method of galactose rapid quantitative detection system

1-1 Use of galactose test paper

The galactose test strips are packaged in aluminum foil bags and stored at 4°C~10°C (39.2°F~51.2°F) and refrigerated. It needs to be warmed for 20 minutes before use, half after opening The lactose test strip needs to be used within 30 minutes. The overdue test strip can no longer be used and must be discarded.

1-2 Sample collection and preparation

The user must first inject or use the galactose composition, wherein the content of the galactose composition is that the galactose content is 1% to 80% of the total amount, and the preferred content state is 4% or 40% of the total amount, without adding or The added buffer dose is 0 or 0.001% to 5%, and the content of no added or added antioxidant is 0 or 0.001 to 5% of the total amount. The above content refers to the weight percentage. Buffer and antioxidant are selected, and the following content can be added to make an appropriate formula; 0.01M~1M antioxidant is selected from vitamin C, sodium bisulfite, vitamin A, vitamin E, flavonoids, polyphenols , Ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentacetic acid (DTPA), N,N-bis[carboxymethyl]glycine (NTA), or select 0.01 M~1M ascorbic acid buffer, citrate buffer, phosphate buffer, acetate buffer, acetate buffer, carbonate buffer and triethanolamine buffer (triethanolamine buffer), adjust the pH value from 4.0 to 9.0. Add 0.01% citrate buffer and 0.5% sodium bisulfite pH 4.5 to obtain a stable formula. After using the above galactose composition for 60 minutes, first wash your fingers with soap and warm water and dry them. Before collecting body fluids, wipe your fingertips with an alcohol cotton pad. After the fingertips are completely dry, use a body fluid collection device to obtain body fluid samples. And should avoid excessively squeezing the body fluid collection part.

1-3 Procedure

(1) Password card correction

In order to measure the correct galactose value, use a new box of galactose test every time When the paper is used, the measuring instrument should be recalibrated. When calibrating, only use the password card attached to the box. And confirm that the password of the password card and the password on the test box used are the same, and then insert the contact electrode of the password card into the slot of the password card of the measuring instrument. After inserting the test paper into the test paper slot of the measuring instrument, the measuring instrument will automatically turn on and the example of "" will be displayed on the screen. The user must confirm that the password is the same as the password card, and then take out the password card, so the calibration is completed, and the galactose test can be performed.

(2) Detection of galactose

”範例，使用者確認螢幕密碼與試紙盒之密碼相同，待螢幕閃爍滴血符號“

”即可採血。 After inserting the test paper into the test paper insertion hole of the measuring instrument, the measuring instrument will automatically turn on and display "

"Example, the user confirms that the screen password is the same as the test box password, and waits for the screen to flash the blood drop symbol"

"You can collect blood.

Before collecting body fluids, wipe your fingertips with an alcohol cotton pad. After completely drying, use a body fluid collection device to obtain body fluid samples. Touch the body fluid lightly to the body fluid suction port of the test paper. The test paper automatically draws the body fluid into the reaction area. When the transparent test window in the reaction area of the test paper fully shows the color of the body fluid and the beep sound is heard, the fingertip body fluid sample can be removed. When the measurement is completed (about 1 minute), the galactose value will be displayed on the screen. After the test is completed, remove the test paper and discard it properly; if the test is not continuous, the meter will automatically shut down after three minutes.

Example 2 Principle and test of detection system

The present invention mainly provides a method for measuring the content of galactose in body fluids by a rapid quantitative detection system for galactose, which allows subjects to use the aforementioned galactose composition, which is metabolized by the human liver to produce galactose in the blood Or its metabolites, the subject collects blood with fingertips to obtain a sample, and the sample is dropped into the test strip of the present invention. Since the test strip contains enzymes, it can electrochemically react with galactose or its metabolites in the blood to generate electric current , Insert the test paper into the measuring instrument of the present invention, the measuring instrument measures the galactose content in the body of the subject by detecting the current signal of the test paper, thereby monitoring Control the health status of the subjects, because the detection process of this method is simple, which can greatly reduce the time it takes to detect galactose in the past, and it does not lose its accuracy.

2 is a schematic block diagram of a galactose detection system according to an embodiment of the present invention. The system includes a test strip 100 and a measuring instrument 200 of the present invention. The measuring instrument 200 includes a connector 210 for external connection, a calculation unit 211 for converting concentration, an analog-to-digital converter 212, a processor 213, and a display 214. When the power unit 215 applies a signal (preferably a square wave signal with a frequency of 1KHz~22KHz; a voltage of 50mV~5V, preferably a voltage of 300mV~800mV) through the connector 210 to the test paper 100, the sample The electrochemical reaction between galactose or its metabolites and the enzyme in the test paper generates an electrochemical message, and the signal reacts with the electrochemical signal to generate a corresponding reaction signal, and transmits the corresponding reaction signal to the measuring instrument through the connector 210 200's calculation unit 211. Then, the calculation unit 211 calculates the corresponding response signal and outputs the corresponding response signal to an analog to digital converter (ADC) 212, and the analog to digital converter 212 converts the corresponding response signal It is a digitized response signal. The digitized response signal is further processed by the processor 213 and/or the measurement result is displayed via the display 214. In addition, the digitized response signal can be transmitted to a digital terminal 300, such as a galactose concentration signal transmitted to a mobile phone or a computer through Bluetooth, wireless signals, etc.

2-1 Accuracy test

First prepare 5 different concentrations of galactose samples (respectively 200μg/mL, 500μg/mL, 900μg/mL, 1200μg/mL and 1500μg/mL), take 24 groups each, and use venous blood as a specific example of body fluid sample Add them separately, then test the concentration value with the measuring instrument requested by the present invention, and calculate the average (μg/mL), standard deviation (SD) and change The coefficient of variation (%C.V.) is made into a regression analysis graph (see Figure 3). The detection environment is room temperature (25±5℃), and the relative humidity is 20% to 60%. As shown in FIG. 3, the measured value of the measuring instrument of the present invention has a high correlation coefficient of up to 0.98 with the actual galactose concentration, which represents the high accuracy of the measuring instrument of the present invention.

2-2 Precision test

First prepare 5 galactose samples with different concentrations (200μg/mL, 500μg/mL, 900μg/mL, 1200μg/mL and 200μg/mL, respectively) when the detection environment is room temperature (25±5℃) and relative humidity is 20% to 60%. 1500μg/mL), take 3 groups of each, and add venous blood as a specific example of body fluid sample respectively, and then test the concentration value with the measuring instrument requested by the present invention, and then repeat for 8 days, and calculate its and variation The average of the coefficient of variation (%CV) (as shown in Figure 4). From the data in Figure 4, the average of the coefficients of variation of the five concentrations for 8 days is between 6.5 and 7.5, indicating that the measuring instrument is highly precise degree.

In summary, the use process of the galactose detection system of the present invention is simple and fast, because the galactose composition of the present invention allows galactose to be rapidly metabolized by the human liver, so that blood or body fluids contain galactose or its metabolism The blood is collected with your fingertips, and the collected sample reacts electrochemically with the enzyme in the test paper and is detected by the measuring instrument of the present invention. It only takes 1 minute to read, no need to make samples again. The step of measuring galactose by the user is greatly reduced, thereby shortening the measurement time. Therefore, the present invention provides a fast, simple, and highly accurate galactose measurement method in the art for patients who need to detect galactose.

Example 3 Test paper

5 is a schematic diagram of a test paper according to an embodiment of the invention. The test strip 100 includes an insulating substrate 110, an electrode unit 120, a first insulating spacer 130 and a second insulating spacer 140, The enzyme contained in the test paper can electrochemically react with galactose or its metabolites in the sample.

The insulating substrate 110 is a base material having a flat surface, having electrical insulation, and a heat resistance capability of 40°C to 120°C. The material may include polyvinyl chloride (PVC), glass fiber (FR-4), and polyester ( polyester suphone), bakelite, polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyethylene (PE), polystyrene (PS), glass plate, ceramic or Any combination of the above materials.

The electrode unit 120 is disposed on the insulating substrate 110 and includes a first end 122 and a second end 124 opposite to each other. The electrode unit 120 may be composed of a plurality of electrodes insulated from each other, and the material may be any conductive substance, such as palladium glue, platinum glue, gold glue, titanium glue, carbon glue, silver glue, copper glue, gold-silver mixed glue, Carbon silver paste, or any combination of the above conductive materials. In one embodiment, the electrode unit 120 is composed of a conductive carbon powder layer or a metal layer. In yet another embodiment, the electrode unit 120 is composed of a conductive silver paste layer and a conductive carbon powder layer located thereon, wherein the impedance of the conductive carbon powder layer is generally much larger than that of the conductive silver paste layer or other metal paste layers.

The material of the first insulating spacer 130 may include but is not limited to polyvinyl chloride insulating tape, ethylene terephthalate insulating tape, heat drying insulating paint or ultraviolet curing insulating paint, which covers part of the electrode unit 120 ( That is, a portion of the first end 122) and includes a reaction zone flow channel 134 at a first edge 132 of the first insulating spacer 130. The first end 122 is exposed in the reaction zone flow channel 134, and a body fluid sample (such as blood) can be filled into the reaction zone flow channel 134 by capillary action to perform subsequent electrochemical reactions. The two long sides of the reaction channel 134 are stepped, and the width near the first edge 132 is greater than the width away from the first edge 132.

The reaction zone flow channel 134 disposed in the first insulating spacer 130 has at least one reaction layer 150, which at least covers the electrode unit 120 in the reaction zone flow channel 134 and includes at least The components of galactase and conductive medium are used to produce chemical reactions with body fluid samples (such as blood). The reaction layer 150 further contains a measurement area of galactase and a conductive medium.

The composition of the reaction layer 150 may be, but not limited to, enzymes, coenzymes, conductive media, buffers, stabilizers, or surfactants. The conductive medium is used to receive the electrons generated after the reaction of the activated substance and the body fluid sample, and conduct the electrons to the measuring instrument 200 through the electrode unit 120, which includes, but is not limited to, ferrocene, ferrocene salt ( ferrocenium), methylene blue, tris(acetonitrile) ruthenium trichloride, 2,5-dihydroxybenzoquinone, phenazinemethosulfate ), tetrathiafulvalene, tetra-cyano-quino-dimethane, methyl viologen, toluidine blue, 5,6-bis Amino-1,10-phenanthroline (5,6-diamino-1,10-phenanthroline), [M(bpy)3]2+(M=Ru or Os; BPY=2,2'-di Pyridine (2,2′-bipyridine)). The conductive medium may be a metal ion compound. The metal ion compound includes metal salts formed by metal ions through the attraction relationship between electrons and charges and can be dissociated in an aqueous solution, but may be but not limited to MgCl ₂ , BeCl ₂ , CaCl ₂ , SrCl ₂ , BaCl ₂ or a combination thereof; the buffer includes but is not limited to Tris, Tris-HCl, PBS, MES, CHES, Borate, Universal buffer mixtures (CPB), MOPS, TES, HEPES, TAPSO, Tricine, Bicine, TAPS Neutral and alkaline buffers; the stabilizer includes but is not limited to xylitol, mannitol, xylan, arabinoxylan, mannan, trehalose, PEG, PVA, PEO, methyl cellulose (Methocel), agarose, sol-gel, collagen, chitosan, BSA, casein, neo protein, amino acid or Any combination thereof; the surfactant, including but not limited to cationic surfactant, anionic surfactant, neutral ionic surfactant or non-ionic surfactant.

In addition, the second insulating spacer 140 of the test paper of the present invention covers the first insulating spacer 130, part of the electrode unit 120, and part of the insulating substrate 110. Since the second insulating spacer 140 completely covers the reaction zone flow channel 134 of the first insulating spacer 130, the upper, lower, left, and right sides of the reaction zone flow channel 134 are respectively separated by the second insulating spacer 140 and the insulating substrate 110 1. The first insulating spacer 130 is surrounded by the three-wall surface beside the flow channel 134 in the reaction zone to form a five-sided closed tube shape. When the body fluid sample enters the reaction zone flow channel 134 through the body fluid sample port of the test paper 100, the adhesion force of the body fluid sample in the reaction zone flow channel 134 will be greater than the cohesion force of the body fluid sample, so that the body fluid sample continues to advance.

Furthermore, the first edge 132 of the first insulating spacer 130, the second edge 142 of the second insulating spacer 140, and the same side edge of the insulating substrate 110 exhibit a convex arc shape. This edge has a concave structure with respect to the front section of the reaction zone flow channel 134. The test strip 100 utilizes the convex structure of the second edge 142 and the concave structure of the insulating substrate 110 with respect to the front section of the reaction zone flow channel 134 to destroy the cohesion of body fluids and to match the capillary Phenomenon to achieve the function of quickly entering body fluid samples. In addition, the second insulating spacer 140 further includes a vent hole 144, which is located farther away from the second edge 142, that is, at the end of the reaction channel 134 of the first insulating spacer 130. The vent hole 144 is used to exhaust the gas in the flow channel 134 of the reaction zone, so as to avoid that the body fluid sample is blocked by the bubbles and cannot smoothly advance in the flow channel 134 of the reaction zone.

Among them, because the protein of galactase in the test paper is unstable, it cannot be stored in an alkaline environment and in a dry state. According to this, galactase is stored in an acidic state and in a solution, but it is stored in an acidic ammonium sulfate solution The time is very short, and it loses its activity once dried, so it cannot be in a solid state. However, through the above formula and structure of the test paper of the present invention, the enzymes in the test paper can not only be stored in an acidic environment, but also in neutral and alkaline It can be cured and preserved in an environmental manner, and at the same time, the enzyme can be kept active in a dry state. It can be stored for a long time. The test paper of Ming Dynasty breaks through the previous limitations with the above formula, so that the enzyme can be fixed and dried, effectively drying the enzyme on the test piece and still maintaining activity.

3-1 Test paper inspection volume inspection

Fig. 6 is a specific example of the body fluid volume analysis of a general filter paper and using blood as a body fluid sample. From the results, it can be seen that at least 30 μL of fingertip blood volume is required to use the filter paper to ensure that the error is less than 15%. The test paper in this case can be tested in a smaller volume. The experimental method is to prepare three concentrations of galactose (200μg/mL, 900μg/mL and 1500μg/mL respectively), each of which will be 1,2,5 , 7 and 10μL capacity to detect the data value (see Figure 7), and each repeated 3 times, and calculate the average (μg/mL), standard deviation (SD) and coefficient of variation (coefficient of variation, %CV) . The acceptable average C.V of galactose whose concentration is below 250μg/mL is less than 20%; and the acceptable average C.V. is less than 15% when the concentration is between 251 and 1500μg/mL. From the results in Figure 7, the average CV of each volume of galactose with a concentration of 200 μg/mL is 3.03 to 8.15%, which is less than 15%; and the average CV of each volume of galactose at 900 μg/mL and 1500 μg/mL is 3.14 to 6.54%. Both are less than 20%, so this test strip can detect galactose with a volume ≧1μL.

3-2 Long-term stability test of test paper

In order to evaluate the test paper under severe environment, it is estimated that it can be stored for 4 days under 4℃ environment. Prepare 5 concentrations of galactose samples (200μg/mL, 500μg/mL, 1200μg/mL, 900μg/mL, and 1500μg/mL), and divide them into 3 groups respectively, which are stored at 30℃, 40℃ and 45℃ Environment, and measure the galactose reading one by one. The acceptable average CV of galactose whose concentration is below 250μg/mL is less than 20%; and the acceptable average CV is less than 15% when the concentration is between 251 and 1500μg/mL. ; The correlation coefficient (R) needs to be greater than 0.9. It can be seen from the results in Fig. 8 that it can be stored for the longest time at 4°C (545.32 days). 30°C is 30 days, 40°C is 11 days, and 45°C is 7 days. The best storage environment for the inventive test paper can be stored at 4℃~10℃. It can be seen that this test strip can now be stored at 180°C for 4 days at room temperature and 60 days at room temperature; the acceleration test is estimated to be 545 days at 4°C.

3-3 Hematocrit Evaluation Test

In order to test whether the test paper can detect blood samples with different red blood cell volume ratio (Hematocrit, HCT) within the normal range. First prepare five different concentrations of galactose blood sample (respectively, 200 μ g / mL, 450 μ g / mL, 800 μ g / mL, 1150μg / mL and 1500μg / mL), and each ready to 20%, 30%, 40%, 50% and 60% of the HCT samples, and measure the galactose reading one by one. The galactose concentration below 250μg/mL, the acceptable average CV is less than 20%; and the concentration is 251 to 1500μg /mL, the acceptable average CV is less than 15%; and the correlation coefficient (R) needs to be greater than 0.9. The results are shown in Figure 9. The average CV of 450~1500μg/mL is less than 15%; the average CV of 200μg/mL is less than 20%, so this test strip can test at least 20%~60% of blood samples in the hemocapacitive range .

3-4 Repeatability test (Repeatability test)

In order to evaluate whether the results of the galactose rapid quantitative detection system are reproducible, a repeatability test is carried out. The test will prepare 5 different concentrations of galactose (200μg/mL, 500μg/mL, 900μg/mL, 1200μg/mL and 1500μg/mL) added to the body fluid sample, each concentration will be tested by 3 measuring instruments, and each measuring instrument will repeat the measurement 6 times. The average C.V. of galactose with a concentration below 250 μg/mL needs to be below 20%, and the average C.V. of galactose with 251-1500 μg/mL needs to be below 15%. Figure 10 shows that the average CV of 500~1500μg/mL is between 7.12~9.83%, less than 15%; the average CV of 200μg/mL is 14.58%, less than 20%, so the results of the rapid quantitative detection system of the present invention have Repeatability.

In summary, the test paper of the present invention can detect a body fluid sample with a minimum volume of 1 μL, and due to the aforementioned enzymes and its formulation, the test paper of the present invention can be stored at room temperature for 60 days and at 4°C for 180 days, which overcomes Known technical barriers to the difficult storage of galactose test paper, and because the testable volume of the test paper of the present invention is at least 1 μL, it can save the subject from the discomfort caused by large wounds during each test, while maintaining the high accuracy of the test results, providing Subjects with galactose are the preferred tool.

Example 4 Judgment of liver function with a rapid quantitative detection system for galactose

4-1 Comparison between the results of oral galactose OGSP and the results of intravenous galactose GSP

Figures 11 and 12 show the correlation between the GSP results of intravenous galactose and OGSP of oral galactose in 127 subjects (56 with normal liver function and 71 with impaired liver function), according to Digest 1992; 52:222- 231 recommends that subjects be divided into 3 groups based on the GSP value of intravenous galactose (GSP). GSP<280μg/ml is defined as normal liver function, and GSP is between 280~480μg/ml The subject was defined as moderately impaired liver function, GSP>480μg/ml. The subject was defined as severely impaired liver function. According to the results in Figures 10 and 11, the OGSP value of oral galactose was more than the GSP value of intravenous galactose High, and the OGSP value of oral galactose increased with the severity of liver function impairment, and OGSP was positively correlated with GSP. The OGSP value of oral galactose in subjects with normal liver function was 318±27μg/ml ( Mean ± standard error SE), with a minimum value of 18 μg/ml and a maximum value of 887 μg/ml; oral galactose OGSP for subjects with mild or moderately impaired liver function is 590 ± 40 μg/ml (mean ± Standard error SE), the lowest value is 294μg/ml, the highest value is 1282μg/ml; the galactose OGSP value of subjects with severely impaired liver function is about 777±48μg/ml (mean±standard error SE), the lowest The value is 293 μg/ml, the highest value is 1499 μg/ml. The results in Table 5 show the results of GSP with intravenous galactose and OGSP with oral galactose in the three groups of subjects. It shows that the OGSP value of oral galactose increases with the severity of liver function impairment. In particular, the OGSP value of oral galactose is higher than the GSP value of intravenous galactose, according to the results in Figures 11, 12 and Table 5. It can be judged that the main range of oral galactose OGSP values (average ± 2 times the standard error SE) of subjects with normal liver function is about 264 to 372 μg/ml, and subjects with mild or moderately impaired liver function The main range of galactose OGSP (mean ± 2 times standard error SE) is about 510 ~ 670 μg/ml, and the main range of oral galactose OGSP (average ± 2 times standard error SE) of subjects with severely impaired liver function is about Between 681μg/ml and 873μg/ml. Even if the results of the subjects vary from individual to individual, the galactose OGSP value of subjects in the normal liver function group usually does not exceed 670 μg/ml, and the OGSP value of subjects with impaired liver function will be greater than 370 μg/ml. Subjects with OGSP values greater than 370 μg/ml should undergo further liver function tests. In addition to the above intravenous injection methods, similar results can be obtained by other injection methods or administration methods.

Example 5 Galactosemia screening in neonates

Galactosemia is a genetic disease, due to the fact that patients do not have enough galactose-degrading enzymes, so galactose accumulates in the body, causing symptoms such as drowsiness, vomiting, diarrhea, abnormal growth and jaundice. Through the newborn screening, it can be determined that the baby will not have it before consuming breast milk Adverse effects, the measuring instrument of the present invention can also be used for neonatal galactosemia screening, because the newborn screening test does not rely on the digestion of protein or lactose, but the first blood sample of the baby, so do This screening does not need to take the galactose composition of the present invention first, and blood is collected from the tip of the toe. When the blood sample results detect that the galactose value is greater than 100μg/ml, it means that the newborn is at risk of galactosemia, which needs to be done Further examination.

Example 6 Operation analysis of semi-automatic arm

Figure 13 shows the comparison of the traditional enzyme filter paper method with the galactose rapid quantitative detection system using the semi-automatic galactose single-point method using a semi-automatic robotic arm analysis, and the two methods of intravenous injection of galactose composition and oral galactose composition, respectively, in which the vein The correlation coefficient of the traditional enzyme filter paper method for the injection galactose composition and the rapid quantitative detection system of galactose in this case was 0.963, while the correlation coefficient of the oral galactose composition was 0.927. Generally speaking, both oral and intravenous injections are highly correlated, and their correlation coefficients are all above 0.9, so the rapid quantitative detection system for galactose of the present invention is available for mass production.

In summary, the galactose rapid quantitative detection system provided by the present invention is tested for accuracy and precision, and can be used to judge liver function and galactose-related diseases, such as galactosemia screen for newborns Inspection, for professionals or patients to judge their physical condition, and according to this to determine whether further examination is required.

100‧‧‧Test paper

200‧‧‧ measuring instrument

210‧‧‧Connector

211‧‧‧Calculation unit

212‧‧‧Analog to Digital Converter

213‧‧‧ processor

214‧‧‧Monitor

215‧‧‧Power supply unit

300‧‧‧Digital terminal

Claims (21)

A rapid quantitative detection system for galactose, including: a galactose composition, including galactose, a buffer and 0~99% antioxidant, into the body and metabolized by the liver to produce an integrated liquid sample; a test paper or a filter paper, including An enzyme, the enzyme reacts with the body fluid sample to generate an electrochemical message; and a measuring instrument, including: a power supply unit to provide a signal; a connector to receive the signal provided by the power supply unit and transmit the signal On the test paper or the filter paper, after the signal and the electrochemical signal produce a corresponding reaction signal, the corresponding reaction signal is sent to the measuring instrument; a calculation unit calculates the corresponding reaction signal; an analog digital The converter receives the corresponding response signal calculated by the calculation unit and converts the calculated corresponding response signal into a digitized response signal; a processor processes the digitized response signal; and a display displays the Digital response signal; wherein the digital response signal can be sent to a digital terminal. For example, the galactose rapid quantitative detection system of the first patent application, wherein the buffer is selected from ascorbic acid buffer (citrate buffer), citrate buffer (phosphate buffer), phosphate buffer (phosphate buffer), Acetate buffer, carbonate buffer (carbonate buffer) and triethanolamine buffer (triethanolamine buffer). For example, the rapid quantitative detection system for galactose in item 1 of the patent application, wherein the antioxidant is selected from vitamin C or/and sodium bisulfite, vitamin A, vitamin E, flavonoids, polyphenols, Ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentacetic acid (DTPA), N,N-bis[carboxymethyl]glycine (NTA). The galactose rapid quantitative detection system as described in item 1 of the patent application scope, wherein the galactose contains at least D-(+)-galactose, L-(-)-galactose, stable isotope galactose, cyclic galactose Or galactose derivatives. The rapid quantitative detection system for galactose as described in item 1 of the scope of patent application, wherein the galactose composition is administered orally or by injection, spray inhalation and oral administration, rectum, suppository, or other clinically suitable means. The rapid quantitative detection system for galactose as described in item 5 of the patent scope, wherein the oral method is to measure the galactose in the body by allowing the user to use the galactose composition first, and then detecting the galactose content in the body fluid content. The rapid quantitative detection system for galactose as described in item 5 of the patent application scope, wherein the injection method is to measure the amount of galactose in the body by letting the user first inject the galactose composition into the body, and then detecting the galactose content in the body fluid Galactose content. A test paper for a rapid quantitative detection system for galactose as described in item 1 of the patent application scope includes: an insulating substrate; an electrode unit, which is arranged on the insulating substrate; A first insulating spacer covering part of the electrode unit and including a reaction zone flow channel at a first edge of the first insulating spacer, wherein part of the electrode unit is exposed in the reaction zone flow channel; and a The second insulating spacer comprises a second edge, the second insulating spacer covers the reaction channel of the first insulating spacer, the second edge of the second insulating spacer is separated from the first insulating spacer The first edge of the sheet and the same side edge of the insulating substrate present a convex arc shape, and the insulating substrate presents a concave structure relative to the front section of the AC channel of the reaction zone, wherein the AC channel of the reaction zone contains a reaction layer, the The reaction layer covers the electrode unit located in the flow channel of the reaction zone and includes an enzyme and a conductive medium for electrochemical reaction with the body fluid sample; wherein the test paper utilizes the outer edge of the second edge of the second insulating spacer The convex arc shape and the concave structure of the insulating substrate at the front section of the communication channel of the reaction zone destroy the cohesion of the body fluid, and match with the capillary phenomenon to quickly enter the body fluid sample; wherein the enzyme can oxidize, reduce, decompose or metabolize galactose. The test paper as described in item 8 of the patent application scope, wherein the insulating substrate is selected from the group consisting of polyvinyl chloride (PVC), glass fiber (FR-4), polyester (polyester suphone), bakelite, and parylene The group consisting of diethyl dicarboxylate (PET), polycarbonate (PC), polypropylene (PP), polyethylene (PE), polystyrene (PS), glass plates and ceramics. The test paper as described in item 8 of the patent application scope, wherein the electrode unit is selected from palladium glue, platinum glue, gold glue, titanium glue, carbon glue, silver glue, copper glue, gold-silver mixed glue and carbon-silver mixed glue The group formed. The test paper as described in item 8 of the patent application, wherein the reaction layer is selected from the group consisting of enzymes, coenzymes, conductive media, buffers, stabilizers and surfactants. The test paper as described in item 8 of the patent application scope, wherein the conductive medium is selected from the group consisting of ferrocene, ferrocenium, methylene blue, triacetonitrile trichloride Ruthenium (tris (acetonitrile) ruthenium trichloride), 2,5-dihydroxybenzoquinone (2,5-dihydroxybenzoquinone), phenazinemethosulfate (phenazinemethosulfate), tetrathiafulvalene (tetrathiafulvalene), tetracyanoquinoline two Methane (tetra-cyano-quino-dimethane), methyl viologen (methyl viologen), toluidine blue (toluidine blue), 5,6-diamino-1,10-o-phenanthroline (5,6- diamino-1,10-phenanthroline), [M(bpy)3]2+(M=Ru or Os; BPY=2,2'-dipyridine (2,2′-bipyridine)). The test paper as described in item 8 of the patent application scope, wherein the conductive medium may be a metal ion compound selected from the group consisting of MgCl ₂ , BeCl ₂ , CaCl ₂ , SrCl ₂ , BaCl ₂ and combinations thereof Group. The test paper as described in item 11 of the patent application scope, wherein the buffer is selected from Tris, Tris-HCl, PBS, MES, CHES, Borate, Universal buffer mixtures (CPB), MOPS, TES, HEPES, TAPSO, A group consisting of Tricine, Bicine and TAPS. The test paper as described in item 11 of the patent application scope, wherein the stabilizer is selected from the group consisting of xylitol, mannitol, xylan, arabinoxylan, mannan, trehalose, PEG, PVA, PEO, Methocel, agarose, sol-gel, collagen, chitosan, BSA, casein, neo protein, A group of amino acids or any combination thereof. The test paper as described in item 11 of the patent application scope, wherein the surfactant is selected from the group consisting of cationic surfactants, anionic surfactants, neutral ionic surfactants and nonionic surfactants. The application of the strip patentable scope of item 8, wherein the half-strips of the Lactose test range 50-2000 μ g / ml. The test paper as described in item 8 of the patent application scope, in which the enzyme can be immobilized and dried, and stored in a neutral environment of acid and alkali. An application such as the rapid quantitative detection system for galactose as claimed in item 1 of the patent scope, wherein the rapid quantitative detection system for galactose can be used to measure the concentration of galactose in the blood to detect neonatal galactosemia. The application as described in item 19 of the patent application scope, wherein the galactose rapid quantitative detection system provides monitoring of galactose value and hepatic insufficiency patients or professionals. The application as described in item 19 of the patent application scope, wherein the rapid galactose quantitative detection system judges the remaining function of the liver based on the concentration of galactose.

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