CN111077205A - Galactose rapid detection system and application thereof - Google Patents

Abstract

A rapid quantitative galactose detection system, comprising: a galactose composition comprising galactose, a buffer and an antioxidant, which is metabolized in vivo to produce a body fluid sample; a test paper or filter paper containing an enzyme that reacts with the body fluid sample to generate an electrochemical signal; and a gauge, comprising: a power supply unit for providing a signal; the connector receives the signal provided by the power supply unit and transmits the signal to the test paper or the filter paper, and after the signal and the electrochemical signal generate corresponding reaction signals, the corresponding reaction signals are transmitted to the measuring instrument; a calculating unit for calculating the corresponding response signal; the analog-digital converter receives the corresponding reaction signal calculated by the calculating unit and converts the calculated corresponding reaction signal into a digital reaction signal; a processor for processing the digitized response signal; a display for displaying the digitized reaction signal; and a digital terminal for receiving the digitized response signal.

Description

Galactose rapid detection system and application thereof

[ technical field ] A method for producing a semiconductor device

The invention provides a system for rapidly detecting the content of galactose in human blood, in particular to a system for measuring the concentration of galactose and evaluating the degree of liver function damage.

[ background of the invention ]

The liver is associated with clearance (clearance) of many drugs, which can clear the original drug or its metabolites via different metabolic pathways or via biliary excretion, and hepatic insufficiency causing changes in drug excretion or metabolic rate can lead to drug accumulation or prevent drug formation of active metabolites. Since blood galactose has a sensitive correlation with liver function abnormality, and research literature evidence shows that the value of blood galactose has a significant relationship with the degree of liver function abnormality, the residual function of liver function can be evaluated by the value of blood galactose.

A commonly used assay is to inject 0.5g/kg of galactose intravenously after 8 hours fasting and to measure the concentration of galactose in the plasma after 60 minutes (Tang H.S.et al (1992) Digestion,52: 222-; Ranek L.et al (1983) clin. physiol.3: 173-) -178). The measurement mode is that a calibration curve is made according to the relation between the concentrations of different galactose standard solutions and the light absorption values of the galactose standard solutions; simultaneous blood draw with HClO addition₄After shaking and mixing, centrifuging to obtain a supernatant, adding KOH into the supernatant, shaking and mixing, centrifuging again to obtain the supernatant, adding galactose dehydrogenase (galactose dehydrogenase) into the supernatant, placing the mixture in a dark room for 60 minutes to prepare a sample for avoiding inaccurate color reaction, measuring the light absorption value of the sample, and finding out a concentration value according to a calibration curve. However, the detection process is complicated and time-consuming, and requires the use of a variety of reagents, so that the time for the subject to know the detection result is very long.

Taiwan patent No. I292478 discloses a method for preparing a liver function test sample and a sampling test paper. The method also requires injecting galactose into the subject, waiting for 60 minutes, and measuring the concentration of galactose in the blood. The measurement mode is that a calibration curve is made according to the relation between the concentrations of different galactose standard solutions and the light absorption values of the galactose standard solutions; adding trichloroacetic acid into the sample test paper and oscillating for 30 minutes, taking out the solvent, adding a solvent containing galactose dehydrogenase and oscillating for 30 minutes, adding a color developing agent, and finally measuring the light absorption value of the sample test paper. However, the method is to inject galactose into human body, and the detection process is complicated and time-consuming, so that there is a need in the art to provide a rapid and simple galactose detection method for patients requiring galactose detection.

Taiwan patent No. M488635 discloses a biological test strip capable of detecting blood glucose; US971995 discloses a detection system for electrochemically detecting hemocompatibility, which comprises an electrochemical test strip and a measuring instrument, and thus it is known that the electrochemical method for monitoring the body state is a common technique in the prior art, but the electrochemical method for detecting galactose has many difficulties because the protein of enzyme is unstable, cannot be preserved in an environment other than an acidic solution, and has a very short preservation time, so that it is another problem to be solved in the art to provide a test strip which is preserved in a solid state for a long time while maintaining the detection accuracy.

[ summary of the invention ]

In order to solve the foregoing problems, the present invention provides a rapid quantitative galactose detection system, which includes: a galactose composition comprising galactose, a buffering agent and an antioxidant, which is metabolized in vivo to produce a body fluid sample; a test paper or a filter paper containing an enzyme that reacts with the body fluid sample to generate an electrochemical signal; and a measuring instrument, comprising: a power unit for providing a signal; a connector for receiving the signal provided by the power supply unit and transmitting the signal to the test paper or the filter paper, wherein the signal and the electrochemical signal generate a corresponding reaction signal and then transmit the corresponding reaction signal to the measuring instrument; a calculating unit for calculating the corresponding response signal; an analog-to-digital converter for receiving the corresponding response signal calculated by the calculating unit and converting the calculated corresponding response signal into a digital response signal; a processor for processing the digitized response signal; a display for displaying the digitized reaction signal; wherein the digitized response signal can be transmitted to a digital terminal.

To achieve the above object, the buffer is selected from the group consisting of ascorbate buffer (ascorbyl acid buffer), citrate buffer (citrate buffer), phosphate buffer (phosphate buffer), acetate buffer (acetate buffer), carbonate buffer (carbonate buffer) and triethanolamine buffer (triethanolamine buffer).

To achieve the above object, the antioxidant is selected from the group consisting of vitamin C and/or sodium bisulfite (sodium bisulfite), vitamin a, vitamin E, flavonoids, polyphenols, Ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), N-bis [ carboxymethyl ] glycine (NTA).

To achieve the foregoing object, wherein the galactose comprises D- (+) -galactose, L- (-) -galactose, a stable isotope of galactose, cyclic galactose or a galactose derivative.

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

To achieve the above object, wherein the oral administration mode measures the galactose content in the body by allowing the user to drink the galactose composition first, and then measuring the galactose content in the blood.

To achieve the above object, the intravenous injection method is a method for measuring the galactose content in vivo by allowing a user to first inject the galactose composition into the body, and then measuring the galactose content in blood.

The invention provides another object, a test paper of a galactose rapid quantitative detection system, comprising: an insulating substrate; an electrode unit disposed on the insulating substrate; a first insulating spacer covering a portion of the electrode unit and including a reaction zone channel at a first edge of the first insulating spacer, wherein a portion of the electrode unit is exposed to the reaction zone channel; and a second insulating spacer including a second insulating spacer, the second insulating spacer covering the reaction area ac channel of the first insulating spacer, the second insulating spacer and the first edge of the first insulating spacer and the same side edge of the insulating substrate presenting a convex arc shape, the insulating substrate presenting a concave structure relative to the front section of the reaction area ac channel, wherein the reaction area ac channel contains a reaction layer, the reaction layer covering the electrode unit in the reaction area flow channel and including an enzyme and a conductive medium for generating an electrochemical reaction with the body fluid sample; wherein the test paper utilizes the convex arc shape of the second edge of the second insulating spacer and the concave structure of the insulating substrate corresponding to the front section of the channel in the reaction region to destroy the cohesion of the body fluid, and matches with the capillary phenomenon to achieve rapid introduction of the body fluid sample; wherein the enzyme can oxidize, reduce, catabolize or metabolize galactose.

To achieve the above object, the test paper has a galactose test range of 50 to 2000. mu.g/ml.

To achieve the above object, the insulating substrate is selected from the group consisting of polyvinyl chloride (PVC), glass fiber (FR-4), polyester (polyester), bakelite board, polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyethylene (PE), Polystyrene (PS), glass plate, and ceramic.

To achieve the above object, 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 above object, the reaction layer is selected from the group consisting of enzymes, coenzymes, conductive media, buffers and protectors.

To achieve the above object, the conductive medium is selected from the group consisting of Ferrocene (Ferrocene), ferrocenium salt (ferrocenium), methylene blue (methylene blue), triethylenetriamide (ruthenium (tris) (acetonitrile), ruthenaum trichoride), 2,5-dihydroxybenzoquinone (2, 5-dihydrobenzoquinone), phenazine methosulfate (phenazine methosulfate), tetrathiafulvene (tetrathiafulvalene), tetracyanoquinodimethane (tetra-cyanoquinodimethane), methyl viologen (methyl viologen), toluidine blue (toleidine), 5,6-diamino-1,10-phenanthroline (5, 6-diamido-1, 10-phenanthroline), (M BPY)3]2+ (M2 ═ y, 2 '-bipyridine (Ru 2, 2' -bipyridine).

To achieve the above object, the conductive medium can be a metal ionic compound selected from MgCl₂、BeCl₂、CaCl₂、SrCl₂、BaCl₂Or a combination thereof.

To achieve the above object, 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, and TAPS.

To achieve the above object, the stabilizer is selected from the group consisting of xylitol, mannitol, polyxylose, arabinoxylan, mannan, trehalose, PEG, PVA, PEO, methylcellulose (Methocel), agarose (agarose), sol-gel (sol-gel), collagen (collagen), chitosan (chitosan), BSA, casein (casein), regenerated protein (neoprotein), amino acids, or any combination thereof.

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

To achieve the above object, the enzyme can be immobilized and dried, and stored in an acid-base neutral environment.

The invention provides another purpose of the invention, and an application of the galactose rapid quantitative detection system, wherein the galactose rapid quantitative detection system can be used for judging the galactose concentration in vivo to measure the neonatal galactosemia.

To achieve the foregoing objects, the present invention provides a rapid quantitative galactose detection system for monitoring galactose values and measurements by a patient or a professional suffering from hepatic insufficiency.

To achieve the above object, the galactose rapid quantitative determination system determines the remaining liver function according to the concentration of galactose.

[ description of the drawings ]

FIG. 1 is a schematic diagram of a rapid galactose detection system;

FIG. 2 is a schematic diagram of a rapid galactose detection system;

FIG. 3 is a graph of a galactose rapid detection system accuracy test;

FIG. 4 is a schematic view of a test strip configuration;

FIG. 5 graph of a filter paper test volume test;

FIG. 6 is a graph showing the test paper storage days; FIG. 6 shows a graph of a test at 30 ℃ for different days, (b) at 40 ℃ for different days, and (c) at 45 ℃ for different days;

FIG. 7 GSP results for intravenous galactose and OGSP correlation results for oral galactose;

FIG. 8 correlation of GSP results for intravenous galactose with OGSP results for oral galactose;

FIG. 9 is a graph illustrating test paper test results performed by the semi-automated robot;

FIG. 10 shows a schematic diagram of a galactose measuring device with password card calibration;

FIG. 11 shows the detection of galactose by a galactose measurement instrument, which displays the pattern on the screen when the instrument is automatically turned on.

The test paper comprises a test paper 100, an insulating substrate 110, an electrode unit 120, a first end 122, a second end 124, a first insulating spacer 130, a first edge 132, a reaction area AC channel 134, a second insulating spacer 140, a second edge 142, a vent hole 144, a reaction layer 150, a 200 measuring instrument, a 210 connector, a 211 calculating unit, an analog-digital converter 212, a 213 processor, a 214 display, a 215 power supply unit and a 300 digital terminal.

[ detailed description ] embodiments

The present invention is illustrated by the following examples, but the present invention is not limited by the following examples. The materials used in the present invention are, unless otherwise specified, commercially available materials which are readily available.

The present invention relates to a rapid quantitative galactose detection system, which utilizes an enzyme electrochemical sensing Technology (electrochemical sensing Technology), and an appearance schematic diagram thereof is shown in fig. 1. the system mainly adopts a disposable dry enzyme electrode Technology, utilizes galactose metabolized by human liver or its metabolite and enzyme to generate micro current, measures the value of galactose by measuring the micro current, and evaluates the remaining condition of liver function by using the value, but the rapid quantitative galactose detection system is not limited to evaluating liver function, and can also measure related diseases of galactose, such as galactoemia (galactosemia) related to newborn, and the galactose described in the present invention further includes galactose and its derivatives.

Embodiment 1 application method of rapid quantitative galactose detection system

Use of 1-1 galactose test paper

The galactose detection test paper is respectively packaged in aluminum foil bags and stored at the temperature of 4-10 ℃ (39.2-51.2 DEG F) for refrigeration. The temperature needs to be raised for 20 minutes before use, the opened galactose test paper needs to be used within 30 minutes, and the test paper beyond the time can not be used any more and needs to be discarded.

1-2 sample Collection and preparation

The user must inject or use the galactose composition, wherein the content of the galactose composition is 1% to 80% of the total amount, preferably 4% or 40% of the total amount, 0 or 0.001% -5% of the amount of no or no buffer, and 0 or 0.001-5% of the amount of no or no antioxidant, wherein the contents are weight percentages. Selecting a buffering agent and an antioxidant, and adding the following components to prepare a proper formula; 0.01M to 1M antioxidant is selected from the group consisting of vitamin C, sodium bisulfite (sodium bisulfate), vitamin A, vitamin E, flavonoids, polyphenols, Ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), N-bis [ carboxymethyl ] glycine (NTA), or 0.01M to 1M ascorbate buffer, citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, and triethanolamine buffer, the pH value is adjusted to 4.0 to 9.0, and the galactose buffer is added to 0.01% sodium bisulfite (sodium bisulfite), 0.5% sodium bisulfite (sodium bisulfate), and the composition is stabilized at 60 minutes using a pH value of 60.5, the method comprises the steps of firstly cleaning and wiping the fingers with soap and warm water, wiping the fingertips with an alcohol cotton sheet before collecting body fluid, and obtaining a body fluid sample by using a body fluid collecting device after the fingertips are completely dried, wherein excessive squeezing of a body fluid collecting part is avoided.

1-3 application program

(1) Password card correction

To determine the correct galactose value, the galactose meter should be recalibrated each time a new set of galactose strips is used. Only the cryptographic card attached to the box is permitted to be used for correction. And confirming that the password of the password card is the same as the password on the galactose detection test paper box, and then inserting the contact electrode of the password card into the password card slot of the galactose measuring instrument. After inserting the galactose test strip into the test strip slot of the tester, the tester is automatically turned on, and the pattern shown in fig. 10 is displayed on the screen. The user must confirm that the password is the same as the password card, then take out the password card, and then complete the correction, so as to perform the galactose test.

(2) Detection of galactose

After the test paper is inserted into the test paper insertion hole of the measuring instrument, the measuring instrument is automatically started, the pattern shown in figure 11 is displayed on the screen, the user confirms that the screen password is the same as that of the test paper box, and the blood drip symbol flickers on the screen when the screen flickers

Blood can be collected.

Before body fluid is collected, a fingertip is wiped by an alcohol cotton sheet, and after the fingertip is completely dried, a body fluid sample is obtained by a body fluid collecting device. The body fluid is lightly touched to the body fluid sucking port of the test paper, the test paper automatically sucks the body fluid to the reaction area, and the fingertip body fluid sample can be removed after the transparent test window of the test paper reaction area is completely in the body fluid color and beep sound is heard. When the measurement is completed (about 1 minute), the galactose value is displayed on the screen. After the test is finished, taking out the test paper and properly discarding the test paper; if the test is not continuous, the measuring instrument will be automatically shut down after three minutes.

Example 2 detection System principles and testing

The invention mainly provides a rapid quantitative detection system for galactose content in body fluid, which is characterized in that a subject uses the galactose composition, the galactose composition is metabolized by human liver to generate galactose or metabolites thereof in blood, the subject collects blood with fingertips to obtain a sample, the sample is dropped into the test paper of the invention, the test paper contains enzyme which can generate electrochemical reaction with the galactose or the metabolites thereof in the blood to generate current, the test paper is inserted into the measuring instrument of the invention, and the measuring instrument measures the galactose content in the subject through current signals of the test paper, thereby monitoring the health condition of the subject.

FIG. 2 is a schematic block diagram of a galactose detection system according to an embodiment of the present invention, which includes a test strip 100 and a measuring apparatus 200 of the present invention. The measuring instrument 200 includes a connector 210 for external connection, a calculating unit 211 for converting the 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 1 KHz-22 KHz; a voltage of 50 mV-5V, preferably 300 mV-800 mV) to the test strip 100 through the connector 210, galactose or its metabolite in the sample reacts electrochemically with the enzyme in the test strip to generate an electrochemical signal, which reacts with the electrochemical signal to generate a corresponding reaction signal, and the corresponding reaction signal is transmitted to the calculating unit 211 of the measuring instrument 200 through the connector 210. Then, the calculating unit 211 calculates the corresponding response signal and outputs the corresponding response signal to an analog to digital converter (ADC) 212, the ADC 212 converts the corresponding response signal into a digitized response signal, and the digitized response signal is further processed by the processor 213 and/or the display 214 displays the measurement result. In addition, the digitized response signal can be transmitted to a digital terminal 300, such as a mobile phone or a computer via bluetooth, wireless signal, etc.

2-1 accuracy test

First, 5 galactose samples (200. mu.g/mL, 500. mu.g/mL, 900. mu.g/mL, 1200. mu.g/mL and 1500. mu.g/mL) with different concentrations were prepared, 24 groups of galactose samples were taken, venous blood was added as a specific example of a body fluid sample, and the concentration values were measured by the measuring instrument of the present invention, and the mean (. mu.g/mL), standard deviation (S.D) and coefficient of variation (coefficient of variation,% C.V.) were calculated to prepare a regression analysis chart (see FIG. 3). Wherein the detection environment is room temperature (25 + -5 deg.C), and the relative humidity is 20% to 60%. As shown in FIG. 3, the high correlation coefficient between the read value of the tester of the present invention and the actual galactose concentration is as high as 0.98, which indicates that the tester of the present invention has high accuracy.

2-2 precision measurement

Firstly, 5 galactose samples (200 μ g/mL, 500 μ g/mL, 900 μ g/mL, 1200 μ g/mL and 1500 μ g/mL) with different concentrations were prepared under the detection environment of room temperature (25 + -5 deg.C) and the relative humidity of 20% to 60%, 3 groups were respectively taken, venous blood was added as a specific example of the body fluid sample, the concentration value was measured by the measuring apparatus of the present invention, and the procedure was repeated for 8 days, and the average of the concentration value and the average of the coefficient of variation (coefficient evolution,% C.V.) were calculated (as shown in Table 1), and it can be seen from the data in Table 1 that the average of the coefficient of variation of 5 concentrations for 8 days was between 6.5 and 7.5, which indicates that the measuring apparatus has high precision.

TABLE 1 precision test result of galactose rapid detection system

In summary, the galactose detection system of the present invention is simple and fast in use, because the galactose composition formula of the present invention enables galactose to be metabolized rapidly by human liver, so that blood or body fluid contains galactose or its metabolites, then blood is collected by fingertips, and when the collected sample and enzymes in the test paper are subjected to electrochemical reaction and detected by the measuring apparatus of the present invention, only 1 minute of reading time is needed, and no sample is needed to be prepared, so that the steps of measuring galactose of a user are greatly reduced, and the measuring time is further shortened.

EXAMPLE 3 test paper

FIG. 4 is a schematic diagram of a test strip according to an embodiment of the present disclosure. The test strip 100 of the present embodiment includes an insulating substrate 110, an electrode unit 120, a first insulating spacer 130 and a second insulating spacer 140, wherein enzymes contained in the test strip can electrochemically react with galactose or a metabolite thereof in a sample.

The insulating substrate 110 is a substrate having a flat surface, electrical insulation, and heat resistance of 40 to 120 ℃, and may be made of polyvinyl chloride (PVC), glass fiber (FR-4), polyester (polyester), bakelite board, polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyethylene (PE), Polystyrene (PS), glass plate, ceramic, or any combination thereof.

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 thereof may be any conductive material, such as palladium paste, platinum paste, gold paste, titanium paste, carbon paste, silver paste, copper paste, gold-silver paste, carbon-silver paste, or any combination of the above conductive materials. In one embodiment, the electrode unit 120 is formed by a conductive carbon powder layer or a metal layer. In another embodiment, the electrode unit 120 is composed of a conductive silver paste layer and a conductive carbon powder layer thereon, wherein the resistance of the conductive carbon powder layer is generally much greater 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, a polyvinyl chloride insulating tape, a vinyl terephthalate insulating tape, a thermal drying type insulating varnish, or an ultraviolet curing type insulating varnish, which covers a portion of the electrode unit 120 (i.e., a portion of the first end 122) and includes a reaction zone flow channel 134 located at a first edge 132 of the first insulating spacer 130. Wherein the first end 122 is exposed to the reaction area flow channel 134, and the body fluid sample (such as blood) can be filled into the reaction area flow channel 134 by capillary action for performing the subsequent electrochemical reaction. The reaction zone flow channels 134 are stepped on both long sides and have a greater width near the first edge 132 than away from the first edge 132.

The reaction zone channel 134 disposed in the first insulating spacer 130 has at least one reaction layer 150 at least covering the electrode units 120 in the reaction zone channel 134 and containing components at least containing galactosidase and a conductive medium for generating a chemical reaction with a body fluid sample (e.g., blood). Wherein the reaction layer 150 further comprises a galactonase and a conductive medium measuring region.

The composition of the reaction layer 150 may be, but is not limited to, an enzyme, a coenzyme, a conductive medium, a buffer, a stabilizer, or a surfactant. The conductive medium is used to receive electrons generated by the reaction of the active substance and the body fluid sample, and conduct the electrons to the measuring instrument 200 through the electrode unit 120, and includes, but is not limited to, Ferrocene (Ferrocene), ferrocenium salt (ferrocenium), methylene blue (methylene blue), ruthenium trichloride (tris (acetonitrile) nitrile), 2,5-dihydroxybenzoquinone (2,5-dihydroxybenzoquinone), phenazine methyl sulfate (phenazine methosulfate), tetrathiafulvalene (tetrathiafulvalene), tetracyanoquinodimethane (tetracyano-quino-dimehane), methyl viologen (methyviologen), toluidine blue (toluidine blue), 5,6-diamino-1,10-phenanthroline (5, 10-diazepine-1, 3-phenanthrene) (m)]2+ (M ═ Ru or Os; BPY ═ 2,2 '-bipyridine (2, 2' -bipyridine)). The conductive medium can be a metal ionic compound, which contains metal salts formed by the attraction of metal ions by electrons and charges and can be dissociated in aqueous solution, and can be, but is not limited to, MgCl₂、BeCl₂、CaCl₂、SrCl₂、BaCl₂Or a combination thereof; such buffers include, but are not limited to, neutral and alkaline buffers of Tris, Tris-HCl, PBS, MES, CHES, Borate, Universal buffer mixtures (CPB), MOPS, TES, HEPES, TAPSO, Tricine, Bicine, TAPS; the stabilizer includes, but is not limited to, xylitol, mannitol, polyxylose, arabinoxylan, mannan, trehalose, PEG, PVA, PEO, methylcellulose (Methocel), agarose (agarose), sol-gel (sol-gel), collagen (collagen), chitosan (chitosan), BSA, casein (casein), neo protein, amino acids, or any combination thereof; the surfactant includes, but is not limited to, a cationic surfactant, an anionic surfactant, a neutral ionic surfactant, or a nonionic surfactant.

In addition, the second insulating spacer 140 of the test strip of the present invention covers the first insulating spacer 130, a portion of the electrode unit 120, and a portion 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 reaction zone flow channel 134 is surrounded by the second insulating spacer 140, the insulating substrate 110, and the first insulating spacer 130 at three walls beside the reaction zone flow channel 134 to form a tube shape with five closed surfaces. When the body fluid sample enters the reaction area flow channel 134 through the body fluid sampling port of the test paper 100, the adhesion force of the body fluid sample in the reaction area flow channel 134 is greater than the cohesion force of the body fluid sample, so that the body fluid sample continuously advances.

More specifically, 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 form a convex arc, the edge of the insulating substrate 110 is concave relative to the front section of the reaction area flow channel 134, and the test paper 100 utilizes the convex structure of the second edge 142 and the concave structure of the insulating substrate 110 relative to the front section of the reaction area flow channel 134 to break the cohesion of the body fluid and achieve the function of rapidly entering the body fluid sample by capillary phenomenon. Also, the second insulating spacer 140 further includes a vent hole 144 located farther from the second edge 142, i.e., at the end of the reaction zone flow channel 134 of the first insulating spacer 130. The vent 144 is used to exhaust the gas in the reaction area channel 134, so as to prevent the body fluid sample from being blocked by the bubbles and not being able to smoothly advance in the reaction area channel 134.

The galactose enzymes in the test paper are unstable and can not be stored in an alkaline environment and a dry state, so that the galactose enzymes are stored in an acidic state and in a solution, but the storage time in the acidic ammonium sulfate solution is very short, and the galactose enzymes lose activity once being dried, so that the galactose enzymes can not be in a solid state.

3-1 test paper test volume detection

FIG. 5 shows a typical filter paper blood volume analysis, and a specific example of using blood as a body fluid sample, from the results, it can be seen that at least 30 μ L of fingertip blood volume is required to ensure an error of less than 15%. The test paper of the present application can be used for detection in a smaller volume, and the experimental method comprises preparing 3 concentrations of galactose (200 μ g/mL, 900 μ g/mL and 1500 μ g/mL respectively), detecting the data values of each concentration in a volume of 1, 2,5, 7 and 10 μ L (see table 2), repeating the detection for 3 times, and calculating the average (μ g/mL), standard deviation (s.d) and coefficient of variation (coeffient evolution,% C.V.). Wherein galactose at a concentration of 250 μ g/mL or less has an acceptable average C.V of less than 20%; and concentrations of 251 to 1500 μ g/mL, an acceptable average C.V. of less than 15%. As is clear from the results in Table 2, the average C.V of galactose at a concentration of 200. mu.g/mL was 3.03 to 8.15%, less than 15%; on the other hand, the average C.V volume of 900. mu.g/mL and 1500. mu.g/mL of galactose is 3.14 to 6.54%, and both are less than 20%, so that the test strip can detect > 1. mu.L of galactose in volume.

TABLE 2 examination of the volume test results of the galactose rapid test system

3-2 test paper long-term stability test

In order to evaluate the test paper, the number of days of storage at 4 ℃ was estimated under severe conditions.

Galactose samples (200. mu.g/mL, 500. mu.g/mL, 1200. mu.g/mL, 900. mu.g/mL and 1500. mu.g/mL, respectively) were prepared at 5 concentrations, divided into 3 groups, and stored at 30 ℃, 40 ℃ and 45 ℃ in ambient environments, respectively, and their galactose readings were measured one by one. Wherein galactose at a concentration of 250 μ g/mL or less has an acceptable average C.V of less than 20%; and an acceptable average c.v. of less than 15% at a concentration of 251 to 1500 μ g/mL; and the correlation coefficient (R) needs to be greater than 0.9. From the results shown in FIG. 6, it can be seen that the test paper of the present invention can be stored at 4 ℃ for the longest time (545.32 days), 30 ℃ for 30 days, 40 ℃ for 11 days, and 45 ℃ for 7 days, and the test paper of the present invention can be stored in a preferable storage environment at 4 ℃ to 10 ℃. Therefore, the test paper can be stored for 180 days at 4 ℃ and 60 days at room temperature; the accelerated test predicted 545 days of stability at 4 ℃.

3-3 Hematocrit Evaluation Test (Hematocrit Evaluation Test)

Samples of different Hematocrit (HCT) were tested in order to determine whether the test paper could detect different Hematocrit values within the normal range. First, 5 kinds of galactose blood samples (200. mu.g/mL, 450. mu.g/mL, 800. mu.g/mL, 1150. mu.g/mL and 1500. mu.g/mL) with different concentrations were prepared, and HCT samples in the ranges of 20%, 30%, 40%, 50% and 60% were prepared, and their galactose readings were measured one by one. Wherein galactose at a concentration of 250 μ g/mL or less has an acceptable average C.V of less than 20%; and an acceptable average c.v. of less than 15% at a concentration of 251 to 1500 μ g/mL; and the correlation coefficient (R) needs to be greater than 0.9. As shown in tables 3 and 4, the average C.V. values of 450 to 1500. mu.g/mL were less than 15%; the average C.V of 200 ug/mL is less than 20%, so the test strip can test blood samples in the range of at least 20% to 60% hemocompatibility.

TABLE 3 result of hematocrit test

TABLE 4 result of hematocrit test (continue)

3-4 Repeatability test (Repeatability test)

To evaluate whether the results of the galactose detection system were reproducible, a reproducibility test was performed. The test prepared 5 different concentrations of galactose (200. mu.g/mL, 500. mu.g/mL, 900. mu.g/mL, 1200. mu.g/mL and 1500. mu.g/mL, respectively) to be added to the blood samples, each concentration was tested by 3 meters, and each meter was repeated 6 times. The average C.V. of galactose with the concentration of 250 mu g/mL or less needs to be 20% or less, and the average C.V. of 251-1500 mu g/mL needs to be 15% or less. Table 5 shows that the average CV of 500-1500 ug/mL is between 7.12-9.83%, which is less than 15%; the average CV of 200 mug/mL is 14.58 percent and is less than 20 percent, so the result of the galactose rapid quantitative detection system has repeatability.

TABLE 5 repeatability test results

In summary, the test paper of the present invention can detect a bodily fluid sample with a minimum volume of 1 μ L, and due to the above-mentioned enzymes and their formulations, the test paper of the present invention can be stored at room temperature for 60 days and at 4 ℃ for 180 days, overcoming the technical obstacles of the conventional galactose test paper that is not easy to store, and because the test paper of the present invention can detect a volume of 1 μ L, the test paper of the present invention can prevent the subject from discomfort of large wound caused by each detection, and at the same time, maintain the high accuracy of the detection result, and provide a better use tool for the galactose subject.

Example 4 liver function determination Using galactose Rapid quantitative assay System

4-1 comparison of results for oral galactose OGSP with results for intravenous galactose GSP

Fig. 7, 8 are the OGSP correlations of GSP results for iv galactose injection in 127 subjects (normal and impaired liver function 56 and 71) with oral galactose according to Digest 1992; 52: 222-. According to the results of fig. 7 and 8, the OGSP value of oral galactose is higher than the GSP value of intravenous galactose, and the OGSP value of oral galactose increases with the severity of liver function impairment, and the OGSP and GSP are in positive correlation. The oral galactose OGSP value of the subjects in the normal liver function group is 318 +/-27 mu g/ml (mean value +/-standard error SE), the lowest value is 18 mu g/ml, and the highest value is 887 mu g/ml; the oral galactose OGSP value of a subject with mild or moderate impaired liver function is 590 +/-40 mu g/ml (mean value +/-SE standard), the lowest value is 294 mu g/ml, and the highest value is 1282 mu g/ml; the oral galactose OGSP values of about 777 + -48 μ g/ml (mean + -SE standard error) for subjects with severely impaired liver function were found to be 293 μ g/ml at the lowest and 1499 μ g/ml at the highest. Table 6 results show the GSP results of 3 groups of subjects administered with i.v. galactose and OGSP results of oral galactose, which show that the OGSP values of oral galactose increase with the severity of impaired liver function, in particular, the OGSP values of oral galactose are higher than the GSP values of i.v. galactose. From the results shown in FIG. 7, FIG. 8 and Table 6, it can be determined that the subject with normal liver function orally administered galactose OGSP has a main range (mean. + -. 2 times SE standard) of about 264-372 μ g/ml, the subject with mild or moderate liver function orally administered galactose OGSP has a main range (mean. + -. 2 times SE standard) of about 510-670 μ g/ml, and the subject with severe liver function impairment orally administered galactose OGSP has a main range (mean. + -. 2 times SE standard) of about 681 μ g/ml to 873 μ g/ml. Even if the results of the subjects are different from one another due to individual differences, the oral administration of galactose OGSP value of the subjects in the normal liver function group does not exceed 670 mu g/ml, and the OGSP value of the subjects with impaired liver function is greater than 370 mu g/ml, so that the OGSP value of the subjects greater than 370 mu g/ml should be subjected to further liver function tests. Similar results can be obtained with other injection or administration modes than the intravenous injection mode.

TABLE 6 GSP results for subjects intravenously and OGSP results for oral galactose (mean. + -. standard error SE)

P <0.005(ANOVA & LSD analysis)

Example 5 neonatal Galactosemia (Galactospora) screening

Galactosemia is a hereditary disease due to the fact that patients do not have sufficient galactose degrading enzymes, and thus galactose is accumulated in the body, resulting in symptoms of sleepiness, vomiting, diarrhea, abnormal growth, jaundice, and the like. The infant can be determined before eating breast milk via newborn screening without adverse effects. The galactose measuring instrument of the invention can also be used for the galactoemia screening of newborn infants. Since the newborn screening test does not depend on digestion of protein or lactose, but uses the first blood sample of the infant, the newborn need not take the galactose composition of the present invention first, and takes the tiptoe blood, when the value of galactose detected by the blood sample is more than 100 mug/ml, it represents the newborn is at risk of galactosemia, and further examination is needed.

Example 6 semi-automated arm manipulation analysis

Fig. 9 is a graph showing a comparison between a conventional enzyme filter method and a rapid quantitative galactose detection system using a semi-automated robot arm for analyzing galactose by a single-point galactose analysis method, wherein the correlation coefficient between the conventional enzyme filter method and the rapid quantitative galactose detection system is 0.963, and the correlation coefficient between the conventional enzyme filter method and the rapid quantitative galactose detection system is 0.927. Generally speaking, both oral administration and intravenous injection have high correlation, and the correlation coefficient reaches more than 0.9, so the rapid quantitative detection system for galactose of the invention can be used for mass production.

In summary, the present invention provides a rapid and quantitative galactose detection system, which is tested for accuracy and precision and can be used to determine liver function and galactose related diseases, such as, for example, screening of galactoemia of newborn infants, so as to allow medical staff or patients to determine their body status and accordingly determine whether further examination is required.

Claims (21)

1. A rapid quantitative galactose detection system, comprising:

a half lactose composition comprising galactose, a buffering agent and 0-99% of an antioxidant, wherein the half lactose composition enters a body and is metabolized by the liver to generate a body fluid sample;

a test paper or a filter paper containing an enzyme that reacts with the body fluid sample to generate an electrochemical message; and

a meter, comprising:

a power unit for providing a signal;

a connector for receiving the signal provided by the power supply unit and transmitting the signal to the test paper or the filter paper, wherein the signal and the electrochemical signal act to generate a corresponding reaction signal, and then the corresponding reaction signal is transmitted to the measuring instrument;

a calculating unit for calculating the corresponding response signal;

an analog-to-digital converter for receiving the corresponding response signal calculated by the calculating unit and converting the calculated corresponding response signal into a digital response signal;

a processor for processing the digitized response signal; and a display for displaying the digitized reaction signal;

wherein the digitized response signal can be transmitted to a digital terminal.

2. The rapid galactose quantitative determination system according to claim 1, wherein the buffer is selected from the group consisting of ascorbate buffer (ascorbyl buffer), citrate buffer (citrate buffer), phosphate buffer (phosphate buffer), acetate buffer (acetate buffer), carbonate buffer (carbonate buffer), and triethanolamine buffer (triethanolamine buffer).

3. The system of claim 1, wherein the antioxidant is selected from the group consisting of ascorbic acid and/or sodium bisulfite (sodium bisulfate), vitamin A, vitamin E, flavonoids, polyphenols, Ethylenediaminetetraacetic acid (EDTA)), diethylenetriaminepentaacetic acid (DTPA), N-bis [ carboxymethyl ] glycine (NTA).

4. The rapid quantitative galactose detecting system according to claim 1, wherein the galactose comprises at least D- (+) -galactose, L- (-) -galactose, stable isotope galactose, cyclic galactose or galactose derivative.

5. The rapid quantitative galactose detecting system according to claim 1, wherein the galactose composition is administered orally or by injection, by inhalation spray and by buccal, rectal, suppository or other clinically suitable means.

6. The rapid quantitative galactose detecting system according to claim 5, wherein the oral administration is to measure the galactose content in the body by allowing the user to first use the galactose composition and then to detect the galactose content in the body fluid.

7. The rapid quantitative galactose detecting system according to claim 5, wherein the injecting means measures the galactose content in the body by allowing the user to inject the galactose composition into the body first and then detecting the galactose content in the body fluid.

8. A test strip for rapid quantitative detection of galactose according to claim 1, comprising:

an insulating substrate;

an electrode unit disposed on the insulating substrate;

a first insulating spacer covering a portion of the electrode unit and including a reaction zone channel at a first edge of the first insulating spacer, wherein a portion of the electrode unit is exposed to the reaction zone channel; and

a second insulating spacer including a second edge, the second insulating spacer covering the reaction area ac channel of the first insulating spacer, the second edge of the second insulating spacer, the first edge of the first insulating spacer and the same side edge of the insulating substrate presenting a convex arc shape, the insulating substrate presenting a concave structure relative to the front section of the reaction area ac channel, wherein the reaction area ac channel contains a reaction layer, the reaction layer covering the electrode unit in the reaction area flow channel and including a ferment and a conductive medium for generating an electrochemical reaction with a body fluid sample;

wherein the test paper utilizes the convex arc shape of the second edge of the second insulating spacer and the concave structure of the insulating substrate corresponding to the front section of the channel in the reaction region to destroy the cohesion of the body fluid, and matches with the capillary phenomenon to achieve rapid introduction of the body fluid sample;

wherein the enzyme can oxidize, reduce, catabolize or metabolize galactose.

9. The test strip of claim 8, wherein said insulating substrate is selected from the group consisting of polyvinyl chloride (PVC), fiberglass (FR-4), polyester (polyester), bakelite, polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyethylene (PE), Polystyrene (PS), glass plate, and ceramic.

10. The test strip of claim 8, wherein 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.

11. The reagent strip of claim 8, wherein the reaction layer is selected from the group consisting of enzymes, coenzymes, conductive media, buffers, stabilizers, and surfactants.

12. A strip of paper according to claim 8 wherein the conductive medium is selected from the group consisting of Ferrocene (Ferrocene), ferrocenium salts (ferrocornium), methylene blue (methylene blue), ruthenium (tris (acetonitrile) ruthenaum trichloride), 2,5-dihydroxybenzoquinone (2, 5-dihydrobenzoquinone), phenazine methosulfate (phenazine methosulfate), tetrathiafulvene (tetrathiafulvalene), tetracyanoquinodimethane (tetracyano-quinodimethane), methyl viologen (methyphenol), toluidine blue (toluidine eblue), 5,6-diamino-1,10-phenanthroline (5, 6-diamido-1, 10-phenanthroline), [ M (BPY)3]2+ (M, 2 ' -bipyridine), 2 ' -bipyridine (Ru, 2 ' -dipyridyl).

13. The test strip of claim 8, wherein the conductive medium is a metal ion compound selected from the group consisting of MgCl₂、BeCl₂、CaCl₂、SrCl₂、BaCl₂And combinations thereof.

14. The strip of claim 11, wherein said buffer is selected from the group consisting of Tris, Tris-HCl, PBS, MES, CHES, Borate, Universal buffer mix (CPB), MOPS, TES, HEPES, TAPSO, Tricine, Bicine and TAPS.

15. The dipstick of claim 11, wherein the stabilizing agent is selected from the group consisting of xylitol, mannitol, polyxylose, arabinoxylan, mannan, trehalose, PEG, PVA, PEO, methylcellulose (Methocel), agarose (agarose), sol-gel (sol-gel), collagen (collagen), chitosan (chitosan), BSA, casein (casein), neoprotein (neo protein), amino acids, or any combination thereof.

16. The reagent strip of claim 11, wherein the surfactant is selected from the group consisting of cationic surfactants, anionic surfactants, neutral ionic surfactants, and nonionic surfactants.

17. A strip according to claim 8 wherein the galactose test range of the strip is 50 to 2000 μ g/ml.

18. The test strip of claim 8, wherein the enzyme is immobilized and dried and stored in an acid-base neutral environment.

19. Use of the rapid quantitative galactose detection system according to claim 1, wherein the rapid quantitative galactose detection system is used for measuring the concentration of galactose in blood to detect neonatal galactosemia.

20. The use of claim 19, wherein the rapid quantitative galactose detection system provides monitoring of galactose values and measurements by a patient or professional with hepatic insufficiency.

21. The use of claim 19, wherein the galactose rapid quantitative determination system determines the liver residual function by the concentration of galactose.

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