JP7630831B2 - Rapid quantitative detection system for galactose and uses thereof - Google Patents

Description

The present invention relates to a galactose detection system, and in particular to a system for quickly measuring the concentration of galactose in a biological sample and evaluating the degree of liver dysfunction.

The liver is closely related to the elimination of many drugs that can be eliminated via different metabolic pathways or biliary excretion. Changes in the rate of drug excretion or metabolism caused by liver dysfunction may cause drugs to accumulate or inhibit the formation of active metabolites. Galactose in blood is sensitively correlated with liver dysfunction, and research literature shows that the galactose level in blood is significantly associated with the degree of liver dysfunction. Therefore, the residual function of the abnormal liver can be evaluated according to the galactose level in blood.

A conventional detection method involves intravenous injection of 0.5 g/kg galactose after an 8-hour fast and measuring plasma galactose concentrations 60 minutes later (Tang H.S. et al. (1992) Digestion, 52:222-231; Ranek L. et al. (1983) Clin. Physiol. 3:173-178). The measurement method includes: drawing a measurement curve according to the relationship between different concentrations of standard galactose solution and its light absorption value; adding _HCIO4 into the extracted blood, shaking to mix, then centrifuging to collect the suspension; adding KOH to the suspension, shaking to mix, then centrifuging again to collect the suspension; then adding galactose dehydrogenase to the suspension and placing it in a dark room for 60 minutes to avoid the inaccuracy of color reaction in the preparation of the specimen and the measurement of its light absorption value; and finally finding the concentration value by the measurement curve. However, the detection process is complicated and time-consuming, which requires the use of various agents. Therefore, a long procedure is required to know the detection result.

Taiwan Patent No. I292478 disclosed a method for preparing test specimens for the determination of liver function and sampling test strips. The method also requires injecting galactose into the subject's body and waiting for 60 minutes to measure the concentration of galactose in the blood. The measurement method includes drawing a measurement curve according to the relationship between different concentrations of standard galactose solution and its light absorption value, adding trichloroacetic acid to the test paper, shaking for 30 minutes, then taking out the solvent, adding a solvent containing galactose dehydrogenase therein, shaking for 30 minutes, then adding a dye-forming agent to the obtained solvent, and finally measuring its light absorption value. However, the method is based on galactose injection into the human body and requires preparing test specimens. The detection process is complicated and time-consuming. Therefore, for patients who need to detect galactose, a rapid and simple galactose detection method is needed in the art.

Taiwan Patent No. M488635 discloses a biological test strip, and US Patent No. US971995 discloses a system for detecting hematocrit test, the system includes an electrochemical test strip and a meter. Due to the above prior art, this is a very common technique for monitoring the body condition by electrochemical methods. Due to the instability of enzyme proteins, enzymes cannot be stored in an alkaline environment or in a dry state. Thereby, enzymes are generally stored in an acidic solution, such as stored in an acidic amine sulfate solution with a very short storage time. Therefore, providing a test strip that can be stored in a solid state for a long period of time is another problem to be solved in this field.

The object of the present invention is to provide a system for rapid quantitative detection of galactose, comprising a galactose composition, a test strip or filter paper, and a meter.

The galactose composition contains galactose, a buffer, and 0-99% antioxidants, which enter the human body after metabolism to produce a biological sample.

The test strip or paper contains an enzyme that reacts with the biological sample to generate electrochemical information.

The meter includes a power supply unit for providing a signal. A connector is used to receive the signal provided by the power supply unit and transmit the signal to the test strip or filter paper, where the signal reacting with the electrochemical information generates a corresponding response signal, and the connector transmits the corresponding response signal to the meter. A calculation unit is used to calculate the corresponding response signal. An A/D converter is used to receive the corresponding response signal from the calculation unit and convert the corresponding response signal calculated by the calculation unit into a digital response signal. A processor is used to process the digital response signal. A display device for displaying the digital response signal, and a digital terminal for receiving the digital response signal.

To achieve the above objectives, the buffer is selected from the group including acetate buffer, citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, ascorbic acid buffer, and triethanolamine buffer.

To achieve the above objectives, the antioxidant is selected from the group including vitamin C and/or sodium bisulfite, vitamin A, vitamin E, flavonoids, polyphenols, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and NTA-nitrilotriacetic acid (NTA).

To achieve the above objective, galactose including D-(+)-galactose, L-(-)-galactose, stable isotope galactose, cyclic galactose, or galactose derivatives.

To accomplish the above objectives, the galactose composition is administered orally, by injection, spray, inhalation, buccal, rectal, suppository, or via other medically acceptable methods.

To achieve the above-mentioned objective, the method of oral administration is to have the user ingest the galactose composition in advance, and then measure the content of galactose in the human body by measuring the content of galactose in a biological sample.

To achieve the above-mentioned objective, the method of injection is to inject the galactose composition into the user's body in advance, and then measure the content of galactose in the body's biological sample by measuring the content of galactose in the biological sample.

Another object of the present invention is to provide a test strip, the test strip comprising an insulating substrate, an electrode unit configured on the insulating substrate, a first insulating spacer covering a portion of the electrode unit and including a reaction zone channel provided at a first edge of the insulating spacer, with another portion of the electrode unit being exposed to the reaction zone channel, and

and a second insulating spacer including a second edge, the second insulating spacer covering the reaction zone channel of the first insulating spacer, the first edge of the first insulating spacer, the second edge of the second insulating spacer and the same side edge of the insulating base are all in a convex arc shape, the edge of the insulating base is concave inwardly with respect to a front half portion of the reaction zone channel, the reaction zone channel includes at least a reaction layer, the reaction layer is covered by an electrode unit in the reaction zone channel including at least galactose and a conductive medium, and reacts with the biological sample through an electrochemical reaction, the test strip utilizes the convex tip of the second edge of the second insulating spacer and the concave structure of the insulating base to reduce the cohesive force of the biological sample and rapidly advance the biological sample under the action of capillary action with respect to the front half portion of the reaction zone channel, and the enzyme can oxidize, reduce , decompose or metabolize galactose.

To achieve the above objectives, the test range of galactose in the test strip is 50-2000 μg/ml.

To achieve the above objectives, the insulating substrate is selected from the group consisting of polyvinyl chloride (PVC), glass fiber (FR-4), polyester sulfone, bakelite plate, polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyethylene (PE), polystyrene (PS), glass plate, ceramic, or any combination thereof.

To achieve the above objective, the electrode unit is selected from the group consisting of palladium, platinum, gold colloid, titanium, carbon, silver, copper, gold, and silver.

To achieve the above objective, the reaction layer is selected from the group consisting of an enzyme, a coenzyme, a buffer, a stabilizer, and a surfactant.

To achieve the above objectives, the conductive medium is selected from the group consisting of ferrocene, ferrocenium, methylene blue, tris(acetonitrile)ruthenium trichloride, dihydroxybenzoquinone, phenazine methosulfate, tetrathiafulvalene , tetra-cyano-quino-dimethane, methyl viologen, toluidine blue , 5,6-diamino-1,10-phenanthroline, and 2,2'-bipyridine.

To achieve the above objective, the conductive medium further comprises a metal ion compound, the metal ion compound being selected from the group consisting of MgCl ₂ 'BeCl ₂ 'CaCl ₂ 'SrCl ₂ ', BaCl ₂ , and any one combination thereof.

To achieve the above objectives, the buffer is selected from the group consisting of Tris, Tris-HCl, PBS, MES, CHES, borate, universal buffer mixture (CPB), MOPS, TES, HEPES, TAPSO, tricine, bicine, and TAPS.

To achieve the above objectives, the stabilizer is selected from the group consisting of xylitol, mannitol, polyxylose, araboxylan, mannan, trehalose, PEG, PVA, PEO, methocel, agarose, sol-gel, collagen, chitosan, BSA, casein, neoprotein, amino acid, and any one combination thereof.

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

To achieve the above objectives, the enzymes may be dried, solidified, and stored in a neutral, acidic, or alkaline environment.

Another object of the present invention is to provide a method for implementing a rapid quantitative detection system for galactose in a user environment, the method comprising:
(1) A user takes a galactose composition in advance;
(2) the biological sample is obtained using a biological sampling pen; and
(3) a biological sample is absorbed by a test strip from a biological sampling pen; and
(4) inserting a test strip into a meter; and
(5) The user or specialized medical staff reads the galactose concentration value to determine the user's disease or residual liver function.

To achieve the above objectives, the method may be operated by the subject or by specialized staff.

For the purposes of the above, the disease is neonatal galactosemia.

FIG. 1 is a front view of a rapid detection system for galactose. FIG. 1 is a block schematic diagram of the meter system for the rapid detection system of galactose. 1 shows the accuracy test results for the rapid detection system of galactose. 1 shows the accuracy test results for the rapid detection system for galactose. 1 is a schematic diagram of a test strip structure. This is a typical blood volume analysis using filter paper. 1 is the accuracy test result of galactose under different volumes of test strips. The test results are shown for the test strips on the storage date. 1 shows the test results of a hematocrit evaluation test. 1 shows the test results of a reproducibility evaluation test. This is a kind of correlation between the intravenous injection galactose GSP results and the oral administration galactose OGSP results. Another type of correlation between the intravenous galactose GSP results and the oral galactose OGSP results. 13 is a test strip detection result completed by a semi-automated robotic arm.

The present invention is illustrated in the following embodiments, but is not limited thereto. In other words, all materials used in the present invention are commercially available.

The rapid detection system of galactose shown in FIG. 1a of the present invention adopts an enzyme electrochemical sensing technology. The system mainly adopts a disposable dry enzyme electrode instrument technology, and utilizes galactose or its metabolites metabolized by the liver in the human body by reacting with an enzyme to generate a microcurrent through an electrochemical reaction, and then detects the reading of galactose by measuring the microcurrent. The residual liver function is evaluated according to the reading. The rapid quantitative detection system of galactose of the present invention is not limited to the evaluation of liver function, and can also diagnose diseases related to galactose, such as neonatal galactosemia. In addition, the galactose of the present invention further includes galactose and its derivatives. The biological sample can be blood, saliva, urine, lavage fluid, or any other body fluid.

Embodiment 1: Method of using the rapid quantitative detection system for galactose 1-1 Use of the test strip for galactose detection The galactose test strip shown in FIG. 1b is sealed in an aluminum foil pouch and stored at a temperature of 4° C. to 10° C. (39.2° F. to 51.2° F.). Before use, the test strip needs to be warmed for 20 minutes. After opening, the galactose test strip needs to be used within 30 minutes. After this time, the test strip is discarded and cannot be used again.

1-2 Sample Collection and Preparation The user needs to first drink the oral galactose composition, where the content of galactose is 1%-80%, preferably 4%-40% by weight of the total galactose composition, where buffer solution may not be added or may be added at 0.001%-5% of the total weight, and antioxidant may not be added or may be added at 0.001%-5% of the total weight. A suitable formulation can be prepared by selecting a buffer and an antioxidant and adding the following contents of ingredients: 0.01M-1M antioxidant selected from the group including vitamin C, sodium bisulfite, vitamin A, vitamin E, flavonoids, polyphenols, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and NTA-nitrilotriacetic acid (NTA), and/or 0.01M-1M buffer solution selected from the group including acetate buffer, citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, ascorbate buffer, and triethanolamine buffer, with a pH value adjusted to a range of 4.0-9.0. A stable formulation can be obtained by adding 0.01% citrate buffer and 0.5% sodium bisulfite, resulting in a pH value of 4.5. After drinking the above galactose composition for 60 minutes, the finger is cleaned and wiped with soap and warm water, and then the fingertip is wiped with an alcohol swab before biological sampling. After the fingertip is completely dry, a biological sample is obtained by lightly pricking the fingertip using the biological sampling device, but excessive pressure in the biological sampling area should be avoided.

1-3 Usage Procedure (1) Calibration of the Password Card In order to measure accurate galactose values, the galactose meter should be recalibrated every time a new box of galactose test strips is used. During calibration, only the password card attached to the box can be used, ensure that the password on the password card is the same as the password on the test strip box used for galactose detection, and then insert the contact electrode of the password card into the password card slot of the galactose meter. After inserting a galactose test strip into the test strip slot of the meter, the meter will automatically activate and display "

" is displayed. The user must verify that the password is the same as the password card before removing the password card. The calibration is now complete and the galactose test can be performed.

(2) Galactose Detection The user first washes his/her finger, wipes it thoroughly and then places the biological sampling needle into the biological sampling device at a fixed location. After inserting the galactose test strip into the test strip slot of the meter, the meter will automatically start and display "

" example is displayed. The user verifies that the password on the screen is the same as the password on the test strip box and presses the blood drop symbol "

" flashes on the screen and you may sample a biological sample.

Before sampling the biological sample, wipe the fingertip with an alcohol swab. After the fingertip is completely dry, the biological sample is obtained by lightly pricking the fingertip using a biological sampling device. By allowing the biological sample to lightly touch the biological sample absorption opening of the test strip, the test strip automatically absorbs the biological sample to the reaction zone. The biological sample on the fingertip can be removed when you see that the transparent test window of the test strip reaction zone looks completely red and you hear a "beep" sound. At the end of the test (after about 1 minute), the galactose value is displayed on the screen. Furthermore, the potential of this readout can be transmitted to others, including a physician, via Bluetooth or similar connection via a cell phone or computer.

After testing is complete, the test strip is removed and properly disposed of. The meter will automatically deactivate after three minutes if no continuous testing is performed.

Embodiment 2: Principle and Test of Detection System The present invention mainly provides a system for measuring the galactose content in a biological sample. A user pre-ingests the aforementioned galactose composition. After the galactose composition is metabolized by the liver in the human body, galactose or its metabolites will be present in the blood. A user takes a blood sample from a fingertip and drops the sample onto the test strip claimed by the present invention. Due to the enzyme in the test strip, the enzyme can react with galactose or its metabolites and then generate an electric current through an electrochemical reaction. When the test strip is inserted into the meter of the present invention, the meter detects the amount of galactose in the human body by detecting the current signal in the test strip. This allows the user to monitor the user's health condition. The process of detection is very simple, so that the detection time of galactose can be shortened compared with the prior art with high accuracy and precision.

2 is a block schematic diagram of a galactose detection system according to one embodiment of the present invention. The system includes a test strip 100 and a meter 200. The meter 200 includes a connector 210 connected to the outside, a calculation unit 211 for calculating the concentration, an A/D converter 212, a processor 213, and a display device 214. When the power supply unit 215 applies a signal (the signal is preferably a square wave signal with a frequency of 1 kHz to 22 kHz, and the voltage is 50 mV to 5 V, preferably 300 mV to 800 mV) to the test strip through the connector 210, the galactose or its metabolites in the biological sample and the enzymes in the test strip react through an electrochemical reaction to generate electrochemical information. The signal reacting with the electrochemical information generates a corresponding response signal, and the corresponding response signal is transmitted to the calculation unit 211 of the meter 200 through the connector 210. The calculation unit 211 then calculates a corresponding response signal and outputs the corresponding response signal to the A/D converter 212 to convert the corresponding response signal into a digital response signal that is further processed by the processor 213, and the measurement result is displayed via the display device 214. Furthermore, the digital response signal can be transmitted to the digital terminal 300, such as transmitting the signal of the galactose concentration to a mobile phone or computer via Bluetooth or wirelessly.

2-1 Accuracy Test First, prepare galactose samples with five different concentrations (200 μg/ml, 500 μg/ml, 900 μg/mL, 1200 μg/ml, and 1500 μg/ml, respectively), take 24 groups respectively, add venous blood to them, then use the meter of the present invention to test the concentration values, calculate their average (μg/ml), standard deviation (S.D.), and coefficient of variation (%C.V.), and make a regression analysis chart, where the detection environment is room temperature (25±5°C) and the relative humidity is 20-60%, as shown in Figure 3. The readings of the meter of the present invention have a high correlation coefficient of up to 0.98 with the actual galactose concentration, which indicates high accuracy for the meter of the present invention.

2-2 Precision test First, five different concentrations of galactose samples (200μg/ml, 500μg/ml, 900μg/mL, 1200μg/ml, and 1500μg/ml, respectively) are prepared at room temperature (25±5°C) and 20-60% relative humidity, three groups are taken respectively, and venous blood is added to them, and then the concentration values are tested using the meter of the present invention, and the test is repeated for 8 days, and the average of the coefficient of variation (%C.V.) is calculated (as shown in Figure 4). The data in Figure 4 shows that the average coefficient of variation (%C.V.) of the five samples for 8 days is in the range of 6.5-7.5, which indicates the high precision of the test device.

In light of the above results, the procedure of the galactose detection system of the present invention is simple and rapid. This is because the formula of the galactose composition of the present invention can be quickly metabolized by the liver in the human body, allowing blood or body fluids to contain galactose or its metabolites. Then, a sample is taken at the fingertip. After the sample reacts with the enzyme in the test strip through an electrochemical reaction, the meter detects galactose for only one minute without additional preparation of the test specimen. The procedure reduces the amount of steps for detecting galactose, which further shortens the detection time. Therefore, the present invention provides a rapid, simple, and highly accurate detection galactose method for patients who need to detect galactose.

5 is a schematic diagram of a test strip according to one embodiment of the present 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 test strip contains an enzyme that reacts with galactose or its metabolites and has an electrochemical reaction.

In this embodiment, the insulating substrate 110 has a flat surface with electrical insulation and heat resistance of 40-120°C. The material of the insulating substrate 110 is selected from polyvinyl chloride (PVC), glass fiber (FR-4), polyester sulfone, bakelite plate, polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyethylene (PE), polystyrene (PS), glass plate, ceramic, or any combination of the above-mentioned materials.

As shown in FIG. 5, the electrode unit 120 is constructed on the insulating substrate 110. The electrode unit 120 includes a first end 122 and a second end 124 opposite to each other. In this embodiment, the electrode unit 120 may be constructed of a plurality of electrodes insulated from each other. The material of the electrode unit 120 may be any conductive substance, such as palladium adhesive, platinum adhesive, gold adhesive, titanium adhesive, carbon adhesive, silver adhesive, copper adhesive, gold-silver mixed adhesive, carbon-silver mixed adhesive, or any combination of the conductive materials mentioned above. 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 adhesive silver layer and a conductive carbon powder layer thereon, and the impedance of the conductive carbon powder layer is generally much larger than the impedance of the conductive silver adhesive layer or other metal layer.

The material of the first insulating spacer 130 may include, but is not limited to, polyvinyl chloride (PVC) insulating adhesive tape, ethylene terephthalate insulating adhesive tape, heat-drying insulating varnish, or ultraviolet light-curing insulating varnish. The first insulating spacer 130 covers a portion of the electrode unit 120 (i.e., a portion of the first end 122) and includes a reaction zone channel 134 located on a first edge 132 of the first insulating spacer 130. The first end 122 is exposed to the reaction zone channel 134. A sample (e.g., blood) is suitable for filling the reaction zone channel 134 to carry out a subsequent electrochemical reaction. The two long sides of the reaction zone channel 134 are ladder-shaped, and the width of the reaction zone channel 134 adjacent to the first edge 132 is larger than the width away from the first edge 132.

The reaction zone channel 134 has at least one reaction layer 150 that covers at least one electrode unit 120 in the reaction zone channel 134 and contains at least one galactose and a conductive medium with a sample (such as blood) to generate a chemical reaction. The reaction layer 150 may further include a galactose enzyme measurement region and a conductive medium measurement region.

The composition of the reaction layer 150 can be, but is not limited to, an enzyme, a coenzyme, a conductive medium, a buffer, a stabilizer, and a surfactant, where the conductive medium is used to receive the electrons generated after the active substance reacts with the sample, and conducts the electrons to the meter 200 via the electrode unit 120, and includes, but is not limited to, ferrocene, ferrocenium, methylene blue, tris(acetonitrile)ruthenium trichloride, 2,5-dihydroxybenzoquinone, phenazine methosulfate, tetrathiafulvalene, tetra-cyano-quino-dimethane, methyl viologen, toluidine blue , 5,6-diamino-L, 10-phenanthroline, [M(bpy)3]2+ (M=Ru or Os; BPY=2,2′-bipyridine). In addition, the conductive medium may be a metal ion compound, where the metal ion compound can be dissolved in an aqueous solution in the manner of metal ions under the absorption action between electrons and charges, such as _MgCl2 , _BeCl2 , _CaCl2 , _SrCl2 , BaCl2 _. , or combinations thereof; buffer solutions include, but are not limited to, neutral and alkaline buffer solutions of Tris, Tris-HCl, PBS, MES'CHES, borate, universal buffer mixture (CPB), MOPS, TES, HEPES, TAPSO, lysine, bicine, and TAPS; stabilizers include, but are not limited to, xylitol, mannitol, polyxylose, araboxylan, mannan, trehalose, PEG, PVA, PEO, methocel, agarose, sol-gel, collagen, chitosan, BSA, casein, neoproteins, amino acids, or any one of a combination thereof; surfactants include, but are not limited to, cationic surfactants, anionic surfactants, neutral ionic surfactants, and non-ionic surfactants.

In this embodiment, the second insulating spacer 140 covers the first insulating spacer 130, a part of the electrode unit 120, and a part of the insulating base 110. Because the second insulating spacer 140 completely covers the reaction zone channel 134 of the first insulating spacer 130, the upper, lower, left, and right surfaces of the reaction zone channel 134 are surrounded by the second insulating spacer 140, the insulating base 110, and three wall surfaces of the first insulating spacer 130 beside the reaction zone channel 134 to form a pipe surrounded by a pentahedron. When the sample enters the reaction zone channel 134 through the biological sampling opening, the adhesive force of the biological sample in the reaction zone channel 134 becomes greater than the cohesive force of the biological sample, so that the biological sample can move forward continuously.

In this embodiment, 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 base 110 are all generally convex arc shapes. Furthermore, as shown in FIG. 5, the edge of the insulating base 110 is recessed inward relative to the front half of the reaction zone channel 134. The test strip 100 of this embodiment utilizes the convex tip of the second edge 142 of the second insulating spacer 140 and the recessed structure of the insulating base relative to the front half of the reaction zone channel 134 to reduce the cohesive force of the biological sample and allow the biological sample to move quickly under the action of capillary action. Furthermore, in this embodiment, the second insulating spacer 140 further includes a vent hole 144 located at a position away from the second edge 142, i.e., at the end of the reaction zone channel 134 of the first insulating spacer 130. If the biological sample is blocked by air bubbles and cannot move forward smoothly in the reaction zone channel 134, the air vent 144 is used to release the air in the reaction zone channel 134.

Due to the instability of enzyme proteins, enzymes cannot be stored in an alkaline environment or in a dry state. Therefore, enzymes are generally stored in acidic solutions, such as in acidic amine sulfate solutions with very short storage times. Enzymes cannot be stored in a solid state because they lose activity when dried. However, the test strip in the present invention with the above formulation and structure allows the enzyme to not only be stored in an acidic environment, but also to be solidified and stored in a neutral or alkaline environment. Furthermore, the enzyme with the formulation can retain activity in a dry state and can be stored for a long period of time. Thus, the present invention has broken the previous limitations to ensure that the enzyme can be solidified, dried, and effective to dry the enzyme on the test strip and still be active.

3-1 Detection of detectable volume of test strip Figure 6 is a general filter paper biological volume analysis. The results show that a filter paper fingertip biological sample volume of at least 30 μl can be used to ensure that the error is less than 15%. However, the test strip of the present invention can achieve small biological sample detection. The experimental method is to prepare three different concentrations of galactose samples (200 μg/ml, 900 μg/ml, and 1500 μg/ml, respectively), each of which is 1, 2, 5, 7, and 10 μl in volume (see Figure 7) to detect data values, and then calculate the mean (μg/ml), standard deviation (S.D.), and coefficient of variation (% C.V.), where the acceptable mean C.V value of galactose samples with a concentration of 250 μg/ml or less requires less than 20%, while the acceptable mean C.V. value of galactose samples ranging from 251 to 1500 μg/ml requires less than 20%. 7 shows that the average CV values of the galactose samples having a concentration of 200 μg/ml in each volume are in the range of 3.03-8.15%, which is less than 15%, while the average CV values of the galactose samples having concentrations of 900 μg/ml and 1500 μg/ml in each volume are both in the range of 3.14-6.54%, which is less than 20%. Thus, the test strip of the present invention can detect galactose in volumes of 1 μl or more.

3-2 Testing the Long-term Stability of Test Strips In order to evaluate the working condition of the test strips under harsh environment, the storage days are estimated in an environment of 4°C. Five different concentrations of galactose samples (200 μg/ml, 500 μg/ml, 1200 μg/ml, 900 μg/ml, and 1500 μg/ml, respectively) are prepared, and they are divided into three groups at 30°C, 40°C, and 45°C, respectively, and then the readings of galactose are measured one by one, where the acceptable average CV value of galactose below a concentration of 250 μg/ml is less than 20%, while the acceptable average CV value of galactose within the range of 251-1500 μg/ml needs to be less than 15%, and the correlation coefficient (R) should be greater than 0.9. According to the results in Figure 8, the test strips of the present invention can be stored for 545.32 days (maximum) at 4°C, 30 days at 30°C, 11 days at 40°C, and 7 days at 45°C. The preferred storage environment for the test strips of the present invention is 4°C to 10°C. Here, it is found that the test strips are stable for 180 days at 4°C and 60 days at room temperature. It is estimated that the test strips of the present invention can remain stable for up to 545 days stored at 4°C through accelerated testing.

3-3 Hematocrit Evaluation Test To evaluate whether the test strip can detect different hematocrit (HCT) of samples within the normal range, five different concentrations of galactose biological samples (200 μg/ml, 450 μg/ml, 800 μg/ml, 1150 μg/ml, and 1500 μg/ml, respectively) were prepared, and 20%, 30%, 40%, 50%, and 60% HCT samples were prepared. Then, the readings of galactose were measured one by one. Among them, the acceptable average CV value of galactose below the concentration of 250 μg/ml needs to be less than 20%, while the acceptable average CV value of galactose in the range of 251-1500 μg/ml needs to be less than 15%, and the correlation coefficient (R) should be greater than 0.9. As shown in FIG. 9, the average CV value of galactose in the range of 450-1500 μg/ml needs to be less than 15%, and the correlation coefficient (R) should be greater than 0.9. The average CV value of galactose having a concentration of 200 μg/ml is less than 15%, and the average CV value of galactose having a concentration of 200 μg/ml is less than 20%. Therefore, the test strip of the present invention can detect biological samples with an HCT range of at least 20% to 60%.

3-4 Reproducibility Test To evaluate whether the test results of the rapid quantitative detection system for galactose are reproducible, the following reproducibility test is carried out: Five different concentrations of galactose samples (200 μg/ml, 450 μg/ml, 900 μg/ml, 1200 μg/ml, and 1500 μg/ml, respectively) are prepared for addition to biological samples, where each concentration is tested by three meters, and each meter is tested six times. The acceptable average CV value of galactose below a concentration of 250 μg/ml requires less than 20%, and the acceptable average CV value of galactose in the range of 251 to 1500 μg/ml requires less than 15%. From the results in FIG. 10, the average CV value of galactose samples in the range of 500 to 1500 μg/ml is less than 20%. The V values are within the range of 7.12-9.83%, which is less than 15%, and the average CV of the galactose samples with a concentration of 200 μg/ml is less than 14.58%, which is less than 20%. Therefore, the test results of the rapid quantitative detection system for galactose of the present invention are reproducible.

In light of the aforementioned results, the test strip of the present invention can detect a minimum of 1 μL volume of biological sample. With the aforementioned enzymes and formulations, the test strip can be stored at room temperature for 60 days and at 4° C. for 180 days. It overcomes the obstacle of storage problems. Moreover, the minimum volume of biological sample is 1 μL, thus avoiding the discomfort caused by large wounds per test while maintaining high accuracy of the test results. The present invention provides users with a preferred tool for detecting galactose.

4. Using the detection system to determine liver function 4-1 Comparison between oral galactose OGSP results and intravenous galactose OGSP results As shown in Figures 11 and 12, a total of 127 subjects (56 subjects with normal liver function and 71 subjects with liver dysfunction) are tested to determine the correlation between intravenous galactose OGSP results and oral galactose OGSP results. As suggested in Digestion 1992, 52:222-231, subjects participating in the intravenous galactose GSP test are divided into three groups, where subjects with GSP less than 280 μg/ml are defined as normal liver function group, subjects with GSP in the range of 280-480 μg/ml are defined as moderately impaired liver function group, and subjects with GSP more than 480 μg/ml are defined as severely impaired liver function group. From the results of Figures 10 and 11, the orally administered galactose OGSP value is higher than the intravenously injected galactose OGSP value, and the orally administered galactose OGSP value increases with the degree of liver function impairment, where OGSP and GSP are positively correlated. The orally administered galactose OGSP value of subjects with normal liver function is within the range of 318 ± 27 μg/ml (mean ± standard error) with a minimum value of 18 μg/ml and a maximum value of 887 μg/ml. The orally administered galactose OGSP value of subjects with mild or moderate liver function impairment is within the range of 590 ± 40 μg/ml with a minimum value of 294 μg/ml and a maximum value of 1282 μg/ml. The orally administered galactose OGSP value of subjects with severe liver function impairment is within the range of 777 ± 48 μg/ml with a minimum value of 293 μg/ml and a maximum value of 1499 μg/ml. Table 5 shows the intravenous and oral galactose OGSP results in three groups of subjects, in which the oral galactose OGSP values increase with the degree of liver function impairment. In particular, the oral galactose OGSP values are higher than the intravenous galactose OGSP values. From Figures 11, 12 and Table 5, it can be determined that the oral galactose OGSP values of subjects with normal liver function are mainly in the range of 264-372 μg/ml (mean ± 2*standard error), and the oral galactose OGSP values of subjects with mild or moderate liver function impairment are mainly in the range of 510-670 μg/ml. The oral galactose OGSP values of subjects with severe liver function impairment are mainly in the range of 681-873 μg/ml (mean ± 2*standard error). Even if subject results vary due to individual differences, the oral galactose OGSP value of subjects with normal liver function generally does not exceed 670 μg/ml, and the OGSP value of subjects with impaired liver function generally is greater than 370 μg/ml. Therefore, subjects with OGSP values greater than 370 μg/ml should undergo further liver function tests. In addition to intravenous injection, similar results were obtained by other injections or other administration methods.

Embodiment 5: Neonatal Galactosemia Screening Galactosemia is a genetic disease caused by the fact that patients do not have enough galactose degrading enzyme, and as a result, galactose accumulates in the body. This leads to symptoms such as drowsiness, vomiting, diarrhea, failure of normal growth, and macules. Through screening of the newborn, it can be confirmed that there is no harmful effect on the breast milk of the infant. The galactose meter of the present invention can be used to screen for neonatal galactosemia. The test neonatal galactosemia screening does not rely on the digestion of protein or lactose, but adopts the first biological sample of the infant, so there is no need to collect galactose composition before screening, and the biological sample is sampled from the toe. If the galactose value of the biological sample is detected to be greater than 100 μg/ml, this represents the risk of neonatal galactosemia in the newborn, and further testing is required.

Embodiment 6: Semi-automatic arm motion analysis Figure 13 shows a comparison between the conventional filter paper enzyme analysis and enzyme analysis of the rapid quantitative detection system of galactose carried out by using semi-automatic robot arm in galactose single point method.The analysis is divided into intravenous injection galactose GSP and oral administration galactose OGSP, where the correlation coefficient of the conventional filter paper enzyme analysis and enzyme analysis of the rapid quantitative detection system of galactose of intravenous injection galactose GSP is 0.963, and the correlation coefficient of oral administration galactose OGSP is 0.927.In conclusion, both intravenous injection galactose GSP and oral administration galactose OGSP have a high correlation coefficient of more than 0.9.Therefore, the rapid detection system of galactose of the present invention can be produced through large-scale production.

In summary, the rapid quantitative detection system of galactose provided by the present invention has already been tested for accuracy and precision, and can be used to detect liver function, test for galactose-related diseases, such as newborn screening for galactosemia, and judge the physical condition of medical staff or patients to determine whether further testing is required.

Claims (20)

an enzyme that is galactose dehydrogenase;
a stabilizer for maintaining the stability of the enzyme, the stabilizer comprising trehalose, wherein the galactose dehydrogenase is solidified, dried and remains active;
An insulating substrate;
an electrode unit formed on the insulating base;
a first insulating spacer covering a portion of the electrode unit and including a reaction zone channel disposed at a first edge of the first insulating spacer, another portion of the electrode unit being exposed to the reaction zone channel;
a second insulating spacer having a second edge, the second insulating spacer covering the reaction zone channel of the first insulating spacer, the first edge of the first insulating spacer, the second edge of the second insulating spacer and the same side edge of the insulating base are all convex arc shapes, the side edge of the insulating base is recessed inwardly with respect to a front half portion of the reaction zone channel, the reaction zone channel includes at least a reaction layer, the reaction layer is covered by the electrode unit in the reaction zone channel including at least the enzyme and a conductive medium for reacting with a biological sample by an electrochemical reaction;
For the front half of the reaction zone channel, the convex arc shape of the second edge of the second insulating spacer and the concave structure of the insulating base can be used to reduce the cohesive force of the biological sample and advance the biological sample under the action of capillary action;
the enzyme oxidizes, reduces, degrades or metabolizes galactose,
The enzyme reacts with the biological sample to generate electrochemical information.
Test strips. The insulating substrate is selected from the group consisting of polyvinyl chloride (PVC), glass fiber (FR-4), polyester sulfone, bakelite plate, polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyethylene (PE), polystyrene (PS), glass plate, ceramic, and any combination thereof;
10. The test strip of claim 1 . The electrode unit is selected from the group consisting of palladium, platinum, gold colloid, titanium, carbon, silver, copper, gold, and silver;
10. The test strip of claim 1 . The reaction layer further comprises a coenzyme, a buffer, and a surfactant.
10. The test strip of claim 1 . the conductive medium is selected from the group consisting of ferrocene, ferrocenium, methylene blue, tris(acetonitrile)ruthenium trichloride, dihydroxybenzoquinone, phenazine methosulfate, tetrathiafulvalene, tetracyanoquinodimethane, methyl viologen, toluidine blue, 5,6-diamino-1,10 phenanthroline, and 2,2′-bipyridine;
10. The test strip of claim 1 . the conductive medium further comprises a metal ion compound, the metal ion compound being selected from the group consisting of _MgCl2 , _BeCl2 , _CaCl2 , _SrCl2 , _BaCl2 , and any one combination thereof;
10. The test strip of claim 1 . the buffer is selected from the group consisting of Tris, Tris-HCl, PBS, MES, CHES, borate, CPB, MOPS, TES, HEPES, TAPSO, tricine, bicine, and TAPS;
5. The test strip of claim 4 . The stabilizer is selected from the group consisting of xylitol, mannitol, polyxylose, araboxylan, mannan, trehalose, PEG, PVA, PEO, agarose, sol-gel, collagen, chitosan, BSA, casein, neoproteins, amino acids, and any one combination thereof;
5. The test strip of claim 4 . The surfactant is selected from the group consisting of a cationic surfactant, an anionic surfactant, a neutral ionic surfactant, and a nonionic surfactant.
5. The test strip of claim 4 . The test range of galactose in the test strip is 50 to 2000 μg/ml;
10. The test strip of claim 1 . The enzyme is stored in a neutral, acidic or alkaline environment.
10. The test strip of claim 1 . A rapid quantitative detection system for galactose, comprising:
A test strip according to claim 1;
a galactose composition in the form of a solution, which enters the body and becomes part of the biological sample after metabolism by the liver, the galactose composition comprising galactose, a buffer, and 0.001% to 99% of an antioxidant ;
meter,
The meter,
a power supply unit for providing a signal;
a connector for receiving the signal provided by the power supply unit and transmitting the signal to the test strip, the signal reacting with the electrochemical information to generate a corresponding response signal, the connector transmitting the corresponding response signal to the meter;
a calculation unit for calculating the corresponding response signal;
an A/D converter for receiving the corresponding response signal from the calculation unit and converting the corresponding response signal calculated by the calculation unit into a digital reaction signal;
a processor for processing the digital response signal;
a display device for displaying the digital response signal processed by the processor ;
a digital terminal for receiving the digital response signal processed by the processor ;
A rapid quantitative detection system for galactose. the buffer is selected from the group consisting of an ascorbic acid buffer, a citrate buffer, a phosphate buffer, an acetate buffer, a carbonate buffer, and a triethanolamine buffer;
The system of claim 12 . The antioxidant is selected from the group consisting of vitamin C, sodium bisulfite, vitamin A, vitamin E, flavonoids, polyphenols, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and NTA-nitrilotriacetic acid (NTA);
The system of claim 12 . The galactose includes D-(+)-galactose, L-(-)-galactose, stable isotope galactose, or cyclic galactose.
The system of claim 12 . The galactose composition is administered orally, by injection, spray, inhalation, buccal, rectal, suppository, or other medically acceptable method.
The system of claim 12 . The galactose composition is administered via oral administration before measuring the content of galactose in the biological sample.
17. The system of claim 16 . The galactose composition is administered via injection prior to measuring the content of the galactose in the biological sample.
17. The system of claim 16 . 13. A method for quantitatively detecting galactose in a subject using the system of claim 12 , comprising:
( 1 ) allowing the biological sample obtained from the subject administered a galactose composition using a biological sampling device to be absorbed by the test strip;
( 2 ) inserting the test strip into the meter; and
( 3 ) reading, by a user or professional medical staff, the value of the digital response signal processed by the processor, which indicates the galactose concentration of the subject;
method. the method being operated by the user or by specialized medical staff;
20. The method of claim 19.