Systems and methods for measuring liver enzyme levels in blood

Abstract

A disposable test kit for determining the concentration of one or more liver enzymes in a blood sample comprises: a box body having a plurality of chambers, at least one of the plurality of chambers containing a reactant mixture that reacts when catalyzed by one of the liver enzymes ALT, AST, ALP or GGT in the blood sample to form a reaction solution; a removable box cover connected to the box body; and a test strip module connected to the box body. The test strip module contains at least one analyte test strip configured to receive a portion of the reaction solution and electrochemically measure at least one analyte, the at least one analyte being a reaction product in the reaction solution.

Description

Background of the Invention 1. Technical Field

[0002] The present invention generally relates to detection techniques for measuring enzyme activity. In particular, the present invention relates to detection techniques for measuring liver enzyme activity. 2. Background technology

[0004] Aminotransferases are enzymes that the liver uses to make glycogen. Glycogen is the storage form of glucose. Any glucose that is not used immediately will be converted to glycogen and stored in cells for future use. Most of the glycogen will be stored in the liver, while the rest will be stored in skeletal muscle, the glial cells of the brain, and other organs.

[0005] Liver enzymes are substances that are normally present in high concentrations in the liver. Some of the more important liver enzymes include alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), and alkaline phosphatase (ALP). Any increase in the level of one of these liver enzymes present in the blood can be a sign of liver problems.

[0006] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are two important enzymes for studying signs of liver problems. If both are used together, the comparison of ALT and AST can help to distinguish liver toxicity, liver disease or liver damage.

[0007] ALT and AST are enzymes mainly present in the liver, but they also exist in red blood cells, heart cells, muscle tissue and other organs (such as pancreas and kidney). Specifically, AST is present in various tissues, including liver, brain, pancreas, heart, kidney, lung and skeletal muscle. If any of these tissues is damaged, AST will be released into the bloodstream. Although the AST level that rises indicates that the tissue is damaged, it is not liver-specific. On the other hand, ALT is mainly present in the liver. Any rise in ALT is a direct indicator of liver damage.

[0008] ALP is an enzyme in your blood that helps break down proteins. The body uses ALP for a variety of processes, and it plays a particularly important role in liver function and bone development. By using an ALP test, the amount of this enzyme circulating in a person's blood can be measured. Slightly irregular ALP levels are usually nothing to worry about. Severely abnormal levels can indicate a serious underlying medical condition, usually one related to the liver, bones, or gallbladder. Since ALP is most abundant in the bones and liver, elevated ALP levels in the blood are often a sign of a liver or bone condition. Blockage or damage to the liver can cause ALP levels to rise. This can also occur if bone cell activity increases.

[0009] Abnormal ALP levels usually indicate one of the following conditions: gallstones, cholecystitis (inflammation of the gallbladder), liver cancer, abnormal and noncancerous growths of the liver, cirrhosis (scarring of the liver), hepatitis (inflammation of the liver usually due to infection), gallbladder cancer, overuse of medications that harm the liver, excessive alcohol consumption, poor nutrition (especially deficiencies of vitamin D, calcium, protein, magnesium, and zinc), and bone cancer.

[0010] GGT is an enzyme found in many organs of the body, with the highest levels found in the liver. Normally, GGT levels are low in the blood. When any of the bile ducts that carry bile from the liver to the intestines is blocked by, for example, a tumor or stone, GGT is often the first liver enzyme to rise in the blood. This makes it the most sensitive liver enzyme test for detecting bile duct problems. However, the GGT test is not very specific and cannot be used to distinguish between various causes of liver damage because it rises with many types of liver disease, such as liver cancer and viral hepatitis, as well as other non-liver diseases, such as acute coronary syndrome. Therefore, the GGT test itself is not recommended for routine use. However, the GGT test itself can be used in conjunction with other tests and determine the cause of high ALP levels. In liver disease, both GGT and ALP are elevated, but only ALP is elevated in diseases that affect bone tissue. Therefore, GGT can be used as a follow-up to high ALP to help determine if the high ALP result is due to liver or bone disease. If the GGT test result is at a normal GGT level, it may indicate a problem with the bones. On the other hand, a high GGT level may indicate a problem with the liver or bile ducts. GGT levels can sometimes be elevated in response to small amounts of alcohol. Higher GGT levels are more common in chronic heavy drinkers than in people who drink less than 2 to 3 drinks a day or in people who drink large amounts occasionally (binge drinkers). The GGT test can be used to evaluate someone for acute or chronic alcohol abuse.

[0011] The current method for testing ALT, AST, ALP, and GGT requires a blood sample. A phlebotomist or nurse obtains a blood sample from a vein in the patient's inner elbow using a needle and a plastic tube attached to the end of the needle to draw a small amount of blood. The blood sample is then sent to a clinical laboratory for analysis.

[0012] Normal laboratory values ​​are measured in international units per liter (IU / L) and vary depending on a person's body mass index and the individual laboratory reference values. Typically, the normal reference range for adults is 8 to 48 IU / L for AST, 7 to 55 IU / L for ALT, 20 to 140 IU / L for ALP, and 0 to 30 IU / L for GGT. The upper limit of this range is used to determine how high the level of liver enzymes is. AST or ALT levels provide valuable assistance primarily in the diagnosis of liver disease. The AST / ALT ratio can provide valuable clues about what is happening and whether the problem is acute or chronic. The AST / ALT ratio is a calculated value that compares the AST and ALT levels in the blood. Depending on which value is elevated and the degree of elevation, the disease involved can be clearly indicated.

[0013] For example, an AST / ALT ratio of less than 1 suggests nonalcoholic fatty liver disease. An AST / ALT ratio of 1 suggests acute viral hepatitis or drug-related liver toxicity. An AST / ALT ratio above 1 suggests cirrhosis. An AST / ALT ratio greater than 2:1 suggests alcoholic liver disease. Using the ratio alone is not enough. The magnitude of the increase is important.

[0014] For example, in alcoholic fatty liver disease, AST will often exceed eight times the upper limit of normal, and ALT will exceed five times the upper limit of normal. If both AST and ALT exceed four times their respective upper limits of normal, this indicates the presence of non-alcoholic fatty liver disease. In the case of acute viral hepatitis, AST and ALT are both 25 times their respective upper limits of normal. In the case of chronic hepatitis C, AST and ALT may be 2 to 10 times the upper limit of normal. If AST and ALT exceed more than 50 times their respective upper limits of normal, this indicates ischemic liver disease.

[0015] There are several detection techniques that can be used to measure AST and ALT, which are detailed in the journal article entitled "Detection Techniques for Aspartate Aminotransferase (AST / GOT) and Alanine Aminotransferase (ALT / GPT)" (Sensors, 2006, 6, pp. 756-782). These include colorimetric analysis, spectrophotometric analysis, chemiluminescence, chromatography, fluorescence and ultraviolet absorption, radiochemical analysis, and electrochemical techniques.

[0016] The limitation of colorimetric analysis is that it requires the preparation of calibration curves and the stopping of enzyme reactions. Spectrophotometric analysis requires additional instruments such as spectrophotometers, 3-T Sigma scanners, etc., and is therefore not suitable for bedside instant applications or home testing systems. In addition, spectrophotometry cannot give reliable results when the serum is icteric or seborrheic or contains hemolysates, which is common in clinical samples. In chemiluminescence, there are some chemiluminescence inhibitors, such as superoxide dismutase or N-nitro-L-arginine methyl ester hydrochloride, the presence of which requires the reduction or elimination of these inhibitors before detection. Chromatography has its own problems. Chromatographic separation of the reaction mixture is more complicated due to the presence of compounds with very similar chromatographic behavior and very different hydrophobicity and charge from each other. Optimization of test conditions is very important for the determination of two enzymes in a single serum sample.

[0017] Fluorescence and UV absorption techniques are also problematic. Many environmental factors, including pH, ionic strength, noncovalent interactions, light intensity, temperature, etc., can affect the results using these techniques. Strict reagent, container, and equipment conditions are also required. Otherwise, nonspecific fluorescence or fluorescence quenching can affect the results. Electrochemical techniques include detection using unmodified gold electrodes, detection using pyruvate oxidase-modified electrodes, detection using glutamate oxidase-modified electrodes, and detection using L-lactate biosensors for GOT and GPT. However, electrochemical measurements often have poor resolution of interferon signals present in serum samples, which can be attributed both to the properties of the electrocatalytic surface and to the relatively low protein loading of the enzyme immobilized on the electrode. Summary of the invention

[0018] A recent attempt to develop point-of-care transaminase tests is described in a May 2013 article by Nira R. Pollock et al., National Institutes of Health, in Clinical Gastroenterology & Hepatology titled, “Point-of-Care Paper-Based Fingertip Transaminase Tests: A Low-Cost ‘Lab-on-a-Chip’ Technology for Developing Countries.” The authors describe a 3D device made by layering patterned paper into a postage-stamp-sized device that can perform two separate tests on a single clinical sample in 15 minutes; one area measures AST and the other measures ALT. The device also includes three control areas to ensure proper device performance. The AST assay chemistry (based on the sulfonation of methyl green) results in a visual transition from blue to pink. The ALT assay (based on a peroxidase chemistry) produces a red dye in the presence of high ALT levels. However, the device is not yet available for clinical use.

[0019] Therefore, there is a need for a point-of-care analyzer device that can measure liver enzymes and provide reliable and accurate results.

[0020] The object of the present invention is to provide a bedside point-of-care device and method for measuring blood liver enzyme levels.

[0021] Another object of the present invention is to provide a bedside instant system and method for measuring blood liver enzyme levels using electrochemical techniques.

[0022] Another object of the present invention is to provide a bedside instant system and method for measuring ALT and / or AST and / or ALP and / or GGT in blood.

[0023] The object of the present invention is to provide reliable and accurate results when determining ALT and / or AST and / or ALP and / or GGT in a blood sample using electrochemical techniques.

[0024] The present invention achieves these and other objects by providing, in one embodiment, a disposable test cartridge for use in a point-of-care system to determine the concentration of one or more liver enzymes in a blood sample.

[0025] In one embodiment of the present invention, the test box comprises a box body, a removable box cover and a test strip module. The box body has a plurality of chambers, wherein each of the plurality of chambers has an opening at the top of the box body. At least one of the plurality of chambers comprises a reactant mixture, which can react chemically when mixed with a blood sample, thereby forming a reaction solution comprising at least one reaction product. The reactant mixture comprises a chemical substance that has specificity for the catalysis of one of the liver enzymes ALT, AST, ALP or GGT in the blood sample. The removable box cover is removably connected to the box body and covers a plurality of chambers. The removable box cover also has a capillary receiving hole and a capillary element that is removably inserted into the box cover. The capillary element extends into a chamber in the plurality of chambers when the removable box cover is connected to the box body. The capillary element is configured to obtain the blood sample by capillary action when the capillary element contacts the blood sample. The test strip module is directly connected to the cartridge body and contains at least one analyte test strip configured to receive a portion of the reaction solution and measure at least one analyte, wherein the at least one analyte is at least one reaction product.

[0026] In another embodiment of the present invention, the reactant mixture comprising chemical substances specific for the determination of ALT comprises L-alanine and alpha-ketoglutarate.

[0027] In another embodiment of the present invention, the reactant mixture comprising chemical substances specific for the determination of AST comprises L-aspartic acid and α-ketoglutarate.

[0028] In another embodiment, the reactant mixture comprising chemicals specific for the measurement of AST comprises L-aspartate, alpha-ketoglutarate, and oxaloacetate decarboxylase.

[0029] In another embodiment, the reactant mixture comprising a chemical substance specific for the determination of ALP comprises sodium phenyl phosphate or a derivative thereof. The following reaction represents the chemical structure of the derivative of the reactant, namely sodium phenyl phosphate and the reaction product phenol and its derivatives.

[0030]

[0031] Wherein R1 and R2 can be -H, -R, -OR, -NO2, -COOR, -SO3H, etc. For the test data disclosed later, R1 and R2 are -H.

[0032] In another embodiment, the reactant mixture containing chemical substances specific for the determination of GGT contains L-glutamic acid γ-aniline and its derivatives and glycylglycine and its derivatives. The following reaction represents the chemical structure of the derivatives of the reactants (i.e., L-glutamic acid γ-aniline and the reaction product aniline and its derivatives).

[0033]

[0034] Wherein X1 and X2 can be -H, -R, -OR, -NO2, -COOR, -SO3H, etc.

[0035] In another embodiment, the test strip module comprises an analyte test strip configured to measure pyruvate or L-glutamate when at least one chamber of the plurality of chambers comprises a chemical specific for catalysis of one of the liver enzymes ALT or AST.

[0036] In another embodiment, a test strip module comprises an analyte test strip configured to measure phenol or a derivative thereof when at least one chamber of the plurality of chambers comprises a chemical catalyzed by a liver enzyme ALP.

[0037] In another embodiment, a test strip module comprises an analyte test strip configured to measure aniline or a derivative thereof when at least one chamber of the plurality of chambers comprises a chemical species catalyzed by the liver enzyme GGT.

[0038] In another embodiment, a test strip module comprises an analyte test strip having a reagent matrix disposed on a working electrode, the reagent matrix comprising one of pyruvate oxidase, glutamate oxidase, or glutamate dehydrogenase.

[0039] In another embodiment, a test strip module comprises an analyte test strip having a reagent matrix disposed on a working electrode, the reagent matrix not comprising an enzyme or a mediator.

[0040] In another embodiment, an analyte test strip has an electrical contact end, a sample end, and a sample receiving port spaced a predetermined distance from the sample end.

[0041] In one embodiment, the sample end includes an exhaust port and a sample channel between the sample receiving port and the exhaust port.

[0042] In one embodiment, a test strip module comprises two or more analyte test strips, wherein at least two analyte test strips of the two or more analyte test strips measure the same analyte.

[0043] In one embodiment, a test strip module comprises two or more analyte test strips, wherein each of the two or more analyte test strips measures a different analyte.

[0044] In one embodiment, a method for determining the concentration of one or more liver enzymes in a blood sample is disclosed. The method includes: providing a blood sample receiving chamber; providing a reagent chamber containing a predetermined chemical substance, which is catalyzed by one of the liver enzymes ALT, AST, ALP or GGT in the blood sample; obtaining a blood sample and placing at least a portion of the blood sample in the blood sample receiving chamber; diluting the blood sample to form a diluted blood sample; placing a predetermined amount of the diluted blood sample in the reagent chamber; allowing the reaction between the predetermined chemical substance and the diluted blood sample to continue for a predetermined period of time to form a reaction solution containing at least one reaction product; transferring a portion of the reaction solution to an analyte test strip capable of measuring the amount of at least one reaction product in the reaction solution; measuring the amount of at least one reaction product present in the reaction solution; correlating the amount of at least one reaction product in the reaction solution with the amount of one of the liver enzymes ALT, AST, ALP or GGT in the blood sample.

[0045] In one embodiment, the measuring step further comprises measuring the amount of one of pyruvate, glutamate, phenol (or a derivative thereof), or aniline (or a derivative thereof) in the reaction solution.

[0046] In one embodiment, the correlating step further comprises correlating the amount of one of pyruvate, glutamate, phenol, or aniline in the reaction solution with the amount of ALT, AST, ALP, or GGT in the blood sample.

[0047] In one embodiment, the measuring step further comprises measuring at least one reaction product using an electrochemical measurement technique.

[0048] In one embodiment, the measuring step further comprises measuring at least one of the reaction products using amperometry to determine ALT, AST, ALP, or GGT.

[0049] In one embodiment, the measuring step comprises measuring at least one reaction product using linear sweep voltammetry to determine the ALP.

[0050] In another embodiment, a disposable test box kit for measuring one or more liver enzyme concentrations in a blood sample in a bedside instant analyzer is disclosed. The kit includes a box body, a removable box cover and a test strip module. The box body has a plurality of chambers, wherein each of the plurality of chambers has an opening at the top of the box body. At least one of the plurality of chambers contains a reactant mixture, which can react chemically when mixed with a blood sample, thereby forming a reaction solution containing at least one reaction product. The reactant mixture contains a chemical substance that is specific to the catalysis of one of the liver enzymes ALT, AST, ALP or GGT in the blood sample. A disposable pipette tip is arranged in another of the plurality of chambers. The removable box cover is removably connected to the box body and covers the plurality of chambers. The removable box cover also has a capillary receiving hole and a capillary element removably inserted into the box cover. The capillary element extends into one of the plurality of chambers when the removable box cover is connected to the box body. The capillary element is configured to obtain a blood sample by capillary action when the capillary element is removed from the disposable test box and contacts the blood sample. The test strip module is directly connected to the cartridge body and comprises at least one analyte test strip configured to receive a portion of the reaction solution and measure at least one analyte, wherein the at least one analyte is at least one reaction product.

[0051] In another embodiment, a kit includes a disposable test cartridge and instructions for use of a capillary element. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is a perspective view of one embodiment of the present invention showing a point-of-care analyzer and a disposable test cartridge for a point-of-care analyzer.

[0053] Figure 2 yes Figure 1 An enlarged cross-sectional view of a disposable test cartridge of FIG. 1 is shown, showing a test strip module, a capillary wiper, and a disposable pipette tip.

[0054] Figure 3 yes Figure 1 An exploded perspective view of a disposable test cartridge.

[0055] Figure 4 yes Figure 1 Exploded side view of a disposable test cartridge.

[0056] Figure 5 yes Figure 1 A top plan view of a box body of a disposable test box.

[0057] Figure 6 yes Figure 5 Magnified view of the defined area C.

[0058] Figure 7 yes Figure 1 A top plan view of a box body of a disposable test box.

[0059] Figure 8 It is along Figure 7 An enlarged cross-sectional view of the sensor module taken along line AA in FIG.

[0060] Fig. 9 It is along Figure 7 An enlarged cross-sectional view of the sensor module taken along line BB in FIG.

[0061] Fig.10 yes Figure 1 A left side front view of the box body of the disposable box.

[0062] Fig.11 yes Fig.10 An enlarged view of the area defined by rectangle D.

[0063] Fig.12 yes Figure 1 An enlarged front view of one embodiment of an analyte sensor strip of a disposable cartridge is shown.

[0064] Fig.13 yes Fig.12 An enlarged top plan view of an analyte sensor strip is shown.

[0065] Fig.14 yes Fig.12 An exploded perspective view of an analyte sensor strip is shown in FIG.

[0066] Fig.15 is an enlarged perspective view of another embodiment of an analyte sensor strip of a disposable test cartridge.

[0067] Fig.16 yes Fig.15 An enlarged top plan view of an analyte sensor strip is shown.

[0068] Fig.17 is a graphical representation of the data in Table 1 showing the results of ALT sensor current versus ALT concentration using a pyruvate strip sensor.

[0069] Fig.18 is a graphical representation of the data in Table 2 showing the results of ALT sensor current versus ALT concentration using a glutamate strip sensor.

[0070] Fig.19 is a graphical representation of the data in Table 3, showing the results of AST sensor current versus AST concentration using a pyruvate sensor.

[0071] Fig. 20 is a graphical representation of the data in Table 4 showing the results of ALP sensor current versus ALP concentration using a current sensor.

[0072] Fig.21 is used Fig. 20 Graph of the linear sweep (ie, sweep) voltammogram response of the current sensor to the ALP current-voltage.

[0073] Fig. 22 is used Fig. 20 A graphical representation of the amperometric response of the current sensor to the ALP sensor current-time.

[0074] Fig.23 is a graphical representation of the data in Table 5, showing the results of GGT sensor current versus GGT concentration using an amperometric sensor. DETAILED DESCRIPTION

[0075] Figure 1 One embodiment of a disposable test cartridge 10 for measuring various liver enzymes in a bedside instant analyzer 300 is shown. The test cartridge 10 includes a cartridge body 20, a cartridge cover 40, a capillary element 60, a capillary wiper 80 (not shown), and a test strip module 200 having one or more analyte test strips 230, 430. The capillary element 60 can be removed from the cartridge cover 40.

[0076] Now go to Figure 2, a cross-sectional view of a disposable test cartridge 10 is shown, showing a capillary element 60, a capillary wiper 80, a cartridge cover 40 and a test strip module 200. The capillary element 60 includes a capillary element body 62 having a top body surface 64, a capillary element finger 66 that defines a capillary element volume 68, a top body opening 70 that is in communication with the capillary element volume 68, and a capillary 72 extending from a finger end 66a. The cross-sectional area of ​​the capillary element volume 68 decreases from the top body opening 70 to the finger end 66a. It should be understood that the capillary element volume 68 is open and extends continuously through the capillary 72 from the top body opening 70. The capillary element volume 68 may have a continuous taper or a stepped taper or a plurality of concentric reduced diameters. The capillary wiper 80 has an upper portion 82 and a tapered lower portion 88. As can be seen, the capillary 72 extends through the tapered lower portion 88 of the capillary wiper 80 and into one of the plurality of chambers 22 of the cartridge body 20. The test strip module 200 is connected to the cartridge body 20. In this embodiment, the test strip module 200 is connected to the front wall 24 of the cartridge body 20. The test strip module 200 can be physically connected by fasteners, adhesive coatings, interlocking structures, etc., or can be molded as part of the cartridge body 20 to form a unitary structure. It is contemplated that the test strip module 200 can also be connected along the side of the cartridge body 20.

[0077] Figure 3 and Figure 4 yes Figure 1 An exploded view of the disposable test cartridge 10 is shown. Figure 3 is a front perspective view of the test box 10, and Figure 4 is a side cross-sectional view of the test box 10. The box body 20 has a plurality of chambers 22 extending below the top surface of the box body, each chamber having an opening 26. The plurality of chambers 22 are aligned in a row one after another between the front wall 24 of the box body and the rear wall 23 of the box body. However, this arrangement is not important. Each of the plurality of chambers 22 has a specific purpose, such as receiving a test sample, containing chemical reagents suitable for a specific test, one or more calibration standards, a solution for diluting a blood sample, etc. For example, a combination of a disposable test box 10 and a bedside instant analyzer can provide the following test results, including but not limited to: ALT, AST, ALP, GGT, HbA1C, eAG, total cholesterol, HDL cholesterol, triglycerides, LDL cholesterol, cholesterol / HDL ratio, non-HDL cholesterol, urine albumin, urine creatinine, and albumin / creatinine ratio. More specifically and with reference to Figure 4, the multiple chambers 22 of the box body 20 are arranged in the following order. Chamber 22a includes a disposable pipette tip 25, and chamber 22b is a blood sample receiving chamber into which a capillary is inserted and in which a blood sample is deposited. It is also a chamber for diluting a blood sample. Chambers 22c to 22e contain various reagents for determining a set of predetermined test results, and chamber 22f generally contains a buffer solution or other saline solution for diluting a blood sample deposited in chamber 22b. Figure 3 and Figure 4 Not shown are foil seals that cover all openings 26 of the plurality of chambers 22 to prevent contamination of the chambers 22 and contamination of reagents contained within certain chambers.

[0078] The capillary wiper 80 has an upper portion 82 and a tapered lower portion 88. The upper portion 82 and the tapered lower portion 88 are tubular, which means that the capillary wiper 80 defines a capillary wiper volume 94. The lower volume portion 96 is adapted to accommodate the capillary element fingers 66 and a portion of the capillary 72.

[0079] The box cover 40 has a cover top surface 42, which has a plurality of sunken cover sides 44 forming a cover recess 46. Extending upward from the cover top surface 42 is a cover extension 48 that also forms a recess 46a of the cover recess 46. The cover extension 48 has a stepped extension 50 with an extension top surface 52, and a capillary receiving hole 54 that is communicated with the part 46a of the cover recess 46. The cover tube extension 56 extends from the capillary receiving hole 54 to a predetermined distance in the recess 46a. The cover tube extension 56 cooperates with the upper part 82 of the capillary wiper 80. The cover recess 46 accommodates the top 28 of the box body 20 and is locked in place by a releasable tab 28a. In this embodiment, the box cover 40 also has a rear side extension 58 with a rear extension end 58a. The rear side extension 58 extends along and adjacent to the box body rear wall 23, and the rear extension end 58a is received in the cover receiving groove 29 on the box body 20. The rear extension 58a has a small point 58b (at Figure 3 ), the bedside instant analyzer 300 uses a sharp point to pierce the foil-covered openings 26 of the plurality of chambers 22 during use of the disposable test cartridge 10. The analyzer 300 uses a pipette tip 25 to dilute the sample and select a predetermined amount of the diluted sample for mixing with the test reagent that forms the test product after the test reagent reacts with the diluted blood sample for a predetermined period of time. The pipette tip 25 is also used to remove a predetermined amount of the test product to utilize one or more analyte strips 230, 430 (such as the strip module 200) Figure 13-14 As will be appreciated, once the capillary 72 and sample are inserted into and assembled with the cartridge cover 40, the disposable test cartridge 10 is then inserted into the analyzer 300, which removes the cartridge cover 40 and uses the rear extension end 58a to pierce the foil seal.

[0080] As previously described, capillary element 60 has an element body 62, a top body surface 64, depending capillary element fingers 66 defining a capillary element volume 68, a top body opening 70 communicating with the capillary element volume 68, and a capillary tube 72 extending from finger end 66a. Capillary tube 72 has an optionally tapered distal tube end 72a.

[0081] The sensor strip module 200 includes a module body 210 and one or more analyte sensor strips 230. The one or more analyte sensor strips 230 are single-use and disposable. The module body 210 includes: a module body platform 212 for receiving one or more analyte sensor strips 230; and a plurality of retaining devices 214 for capturing one or more analyte sensor strips 230 and retaining them in corresponding strip receiving areas 224 on the body platform 212.

[0082] Reference now Figure 5 , showing a top view of the cartridge 20 and the module body 210. In this embodiment, a releasable tab 28a is shown on the opposing side walls 27a, 27b of the cartridge 20, which locks the cover 40 in place after the cover 40 is assembled to the cartridge 20. The releasable tab 28a serves to allow the bedside instant analyzer 300 to remove the cover 40 from the test cartridge 10 once the test cartridge 10 is inserted into the analyzer 300 to test a blood sample previously deposited into the chamber 22 prior to inserting the test cartridge 10, and also to allow the cover 40 to be re-applied after the test is completed so that the test cartridge 10 can be removed and disposed of. In this embodiment, the module body 210 is connected to the cartridge body front wall 24 of the cartridge 20 adjacent to the first strip module wall 216 of the module body 210. Figure 6 yes Figure 5 2 is an enlarged view of the area defined by ellipse C in FIG. In this embodiment, the module body 210 has a first strip module wall 216, a second strip module wall 218, a module front wall 220, a module rear wall 222, a module body platform 212, and a plurality of strip holders 214. In this illustration, the module body platform 212 is divided into three areas. Each of the three areas is configured to receive and retain an analyte test strip. Therefore, in this embodiment, the module body 210 is capable of holding three analyte test strips for indirectly measuring ALT and / or AST and / or ALP and / or GGT in a blood sample.

[0083] Figure 72 is a top view of the box body 20 and the module body 210. In the top view of the box body 20, multiple chambers 22, a first box body wall 27a, a second box body wall 27b, multiple cover retaining tabs 28a, a box body front wall 24, a first strip module wall 216, a second strip module wall 218, a front strip module wall 211a, a rear strip module wall 211b, and module body sight lines A and B are shown. Figure 8 It is along Figure 7 2 is an enlarged cross-sectional view of the module body 210 taken along line AA in FIG. As can be more clearly seen, the module body platform 212 is recessed from the top strip module surface 210a, and the strip retainer 214 has a hooked end 214a that is adapted to receive the longitudinal edge 236 of the analyte sensor strip 230. The module body platform 212 also includes a plurality of platform openings 212a through which a corresponding one of the plurality of strip retainers 214 extends above the module body platform 212. Fig. 9 It is along Figure 7 BB line in FIG. 2 is an enlarged cross-sectional view of the module body 210. It should be noted that when Figure 8 The hooked ends 214a of the plurality of strip retaining devices 214 face in opposite directions in this view when compared to the hooked ends 214a shown. This is intended to allow the two opposing longitudinal edges 236 (eg, Figure 12-13 236) is secured to the module body platform 212 within the strip receiving area 224. It is contemplated that one side of each strip receiving area 224 may have a flange (not shown) that captures a portion of one longitudinal edge 236 of the analyte sensor strip 230, while one or more strip retaining devices 214 capture a portion of the opposing longitudinal edge 236.

[0084] Fig.10 is a perspective front view of the box body 20, and Fig.11 yes Fig.10 An enlarged perspective view of the area C defined in FIG. Fig.11 More clearly shown are the strip retainers 214 extending through the platform opening 212a and the hooked ends 214a above the recessed body platform 212. The plurality of strip retainers 214 are all resilient to allow limited flexing when the analyte sensor strip 230 is inserted into the strip receiving area 224 so that each test strip retainer 214 returns to its initial position, i.e., resting the analyte sensor test strip 230 against the module body platform 212 in the test strip receiving area 224.

[0085] Sensor construction

[0086] Reference now Fig.12 and 13, shows one embodiment of an analyte sensor strip 230. The analyte sensor strip 230 is a multi-layered, one-piece sensor strip having an electrode end 232, an electrode end 232a, an electrical contact end 234, an electrical contact end 234a, and opposing longitudinal edges 236. In this embodiment, the electrode end 232 includes a sample inlet 238 for receiving a portion of a reaction solution, a test strip test chamber 240, and an exhaust port 242. The electrical contact end 234 has two or more electrical coupling pads 244 that are in electrical contact with respective electrodes located within the strip test chamber 240. In this embodiment, the analyte sensor strip has a length of approximately 30.2 mm (1.1 inches) and a width of approximately 5.5 mm (0.22 inches).

[0087] Fig.14 yes Fig.12 and 13 FIG. 2 is an enlarged exploded perspective view of an embodiment of an analyte sensor strip 230 of FIG. As shown, the one-piece sensor strip of this embodiment has four strip layers. The analyte sensor strip 230 includes a base layer 250, an insulating and electrode defining layer 270, a channel forming layer 290, and a cover layer 310. All layers of the analyte sensor strip 230 are made of a dielectric material, preferably a plastic. Examples of preferred dielectric materials are polyvinyl chloride, polycarbonate, polysulfone, nylon, polyurethane, nitrocellulose, cellulose propionate, cellulose acetate, cellulose acetate butyrate, polyester, polyimide, polypropylene, polyethylene, and polystyrene.

[0088] Base layer 250 has a conductive layer 252 on which three conductive paths 254, 256, and 258 are defined. Conductive paths 254, 256, and 258 may be formed by scribing or scoring conductive layer 252. In an alternative, base layer 250 may be a dielectric material on which conductive paths 254, 256, and 258 are screen printed. A sheet of gold mylar may be used and cut into the following shapes: Fig.14 The shape shown thereby forms the base layer 250 of the analyte sensor strip 230 .

[0089] Scratching or scoring the conductive layer 252 can be accomplished by mechanically scribing the conductive layer 252 sufficiently to produce at least two independent conductive paths 254, 256 and optionally a third conductive path 258. The preferred scribing or scoring method of the present invention is accomplished by using a CO2 laser, a YAG laser, or an excimer laser. The score line is thin, but sufficient to form two or more separate electrical paths. The conductive layer 252 can be made of any conductive material, such as gold, tin oxide / gold, palladium, other precious metals or their oxides or carbon film components. The preferred conductive material is gold or tin oxide / gold. Additional score lines 264 (not to scale; for illustration purposes only) can be formed along the outer edge of the base layer 250 to avoid possible static problems that may cause noise signals. However, it should be understood that the score lines 264 are not necessary for the function of the analyte sensor strip 230.

[0090] The strip insulation and electrode defining layer 270 (also referred to as the reagent retaining layer 270) has: a first electrode opening 272 exposing a portion of the first conductive path 258; a second electrode opening 274 exposing a portion of the second conductive path 256; and optionally a third electrode opening 276 exposing a portion of the third conductive path 254. Fig.14 As shown, electrode openings 272, 274 and 276 are aligned with each other and have a spacing of about 0.03 inches (0.64 mm) between adjacent openings, but this spacing is not critical. The circular openings are for illustrative purposes only. It should be understood that the shape and size of the openings are not critical. The sizes of the circular openings do not have to be substantially equal, as long as the ratio of the surface areas remains substantially constant.

[0091] Reagent holding layer 270 is made of plastic material, preferably can be purchased from the medical grade single-sided adhesive of Adhesive Research company of Glen Rock, Pennsylvania or Global Instruments Corporation (GIC) (Taiwan, China).The acceptable thickness for adhesive tape of the present invention is in the scope of about 0.001 inch (0.025mm) to about 0.005 inch (0.13mm).Preferred thickness is about 0.003 inch (0.075 millimeter).It should be understood that it is not necessary to use adhesive tape.Reagent holding layer 270 can be made of plastic sheet, and can be coated with pressure-sensitive adhesive, photopolymer, ultrasonically bonded on base layer 250, screen printed on base layer 250, or 3D printed on base layer 250, to obtain the result identical with using above-mentioned polyester tape.

[0092] The three electrode openings 272, 274 and 276 define electrode wells W, R and B, respectively, and retain chemical reagents forming a working electrode (W), a reference electrode (R) and a blank electrode (B). To determine ALT and AST, the electrode well W carries an enzyme capable of catalyzing a reaction involving an enzyme substrate or a substrate of the enzyme catalytic reaction, and a mediator capable of transferring electrons between the enzyme catalytic reaction and the working electrode to generate a current representing the activity of the enzyme or substrate (representing the liver enzyme to be measured) and at least one polymer binder. The substrate electrode well B has similar chemical properties to the electrode well W, but without an enzyme. One or more chemical components (such as additional polymers, stabilizers and fillers) may be optionally included in the reagent matrix. The reference reagent matrix is ​​loaded into the electrode hole R.

[0093] For the determination of ALP or GGT, the electrode well W is not loaded with enzyme or mediator, and the reason is discussed later. In other words, the electrode well W does not contain the enzyme and mediator for the determination of ALP or GGT.

[0094] Preferably, the reference matrix comprises at least a chemical oxidant, such as a redox mediator in reduced form, a redox mediator in oxidized form, or a mixture of redox mediators in reduced and oxidized forms. For example, when using a preferred conductive coating, potassium ferrocyanide or potassium ferrocyanide, or a mixture of potassium ferrocyanide and potassium ferrocyanide can be loaded to make the reference electrode work. The mixture of potassium ferrocyanide and potassium ferrocyanide can be prepared so that the concentration of potassium ferrocyanide is in the range of up to about 10%, while the concentration of potassium ferrocyanide is in the range of up to about 5%. In an alternative, the reference electrode (electrode well R) can be loaded with an Ag / AgCl layer (e.g., by applying Ag / AgCl ink or by sputter coating (a) Ag layer, then chlorinating Ag, or (b) Ag / AgCl layer) or other reference electrode materials that do not require a redox mediator to function properly. It should be noted that the positional arrangement of the working electrode, reference electrode, and blank electrode in the channel is not critical to obtaining usable results from the sensor.

[0095] The channel forming layer 290 has a channel notch 292 located at the electrode end 232. The length of the channel notch 292 is such that when the channel forming layer 40 is laminated to the reagent retaining layer 270, the electrode regions W, R and B are within the space defined by the channel notch 292. The length, width and thickness of the channel notch 292 define the capillary chamber volume. The channel forming layer 290 is laminated to the insulating and electrode defining layer 270. Similar to the insulating and electrode defining layer 270, the channel forming layer 290 can be made of a plastic sheet and can be coated with a pressure sensitive adhesive, a photopolymer, ultrasonically bonded to the insulating and electrode defining layer 270, screen printed to the insulating and electrode contour layer 270, or 3D printed to the insulating and electrode contour layer 270.

[0096] Channel forming layer 290 is made of plastic material, preferably can be purchased from Adhesive Research company of Glen Rock, Pennsylvania or global instrument company (Taiwan region) medical grade double-sided pressure-sensitive tape. The thickness of adhesive tape is preferably in the range of about 0.001 inch (0.025mm) to about 0.010 inch (0.25mm). Channel recess 292 can be made by laser or punching (preferred method). The length of channel recess 292 not including fluid sample opening 312 is about 0.22 inch (5.68mm) to about 0.250 inch (6.35mm), width is about 0.05 inch (1.28mm) to about 0.07 inch (1.778mm), thickness is about 0.0039 inch (0.1mm) to about 0.009 inch (0.225mm). It should be understood that the thickness and size of channel recess 292 are not critical.

[0097] The cover layer 310 laminated to the channel-forming layer 290 has a fluid sample opening 312 spaced apart from the electrode end 232a of the analyte sensor strip 230. The cover layer 310 forms a vent 242 at the electrode end 232a to ensure that when the reaction product sample is placed in the fluid sample opening 312, the sample in the strip test chamber 240 will completely cover the electrode areas W, R, and B. The preferred material of the cover layer 310 is a polyester film. In order to promote the capillary action of the sample fluid in the test chamber 240, it is desirable that the polyester film has a highly hydrophilic surface on the portion of the polyester film that forms the strip test chamber 240. Transparent films purchased from 3M or GIC can be used.

[0098] Reference now Fig.15 and 16 , another embodiment of an analyte sensor strip 430 is shown. In this embodiment, the analyte sensor strip 430 has a laminate 432, a sample receiving well 434, and an electrical contact end 436. The laminate 432 has a base layer 450 and a cover 460. The cover 460 has a sample opening 462, which forms the sample receiving well 434 when combined with the base layer 450. The base layer 450 has at least three electrical paths 452, 454 and 456, which have a first portion exposed at the electrical contact end 436 to connect to a sensor measuring device or module (not shown), and a second portion exposed through the sample receiving well 434. It will be understood that the layout of the scored electrical paths in this embodiment (analyte test strip 430) is modified from the electrical paths in the previously disclosed embodiment (analyte test strip 230) to accommodate three electrode regions W, R and B in the sample receiving well 464. In Fig.16 An example of such an arrangement is shown in FIG by dashed lines defining one embodiment.

[0099] The second portion of the electrical paths 452, 454, and 456 exposed through the sample receiving well 434 produces at least a working electrode W, a reference / counter electrode R, and an optional blank electrode B. As described above for measuring ALT and AST, the first reagent matrix comprises an enzyme capable of catalyzing a reaction involving an enzyme substrate or a substrate with the enzyme catalyzed reaction, a mediator capable of transferring electrons transferred between the enzyme catalyzed reaction and the working electrode to produce a current representing the activity of the enzyme or substrate, and at least one polymer binder, and is disposed on the working electrode W. The second reagent matrix is ​​disposed on the optional blank electrode B and comprises a chemical composition similar to that of the electrode well W, but without the enzyme and the mediator. As described above, one or more chemical components (e.g., additional polymers, stabilizers, and fillers) may optionally be included in the reagent matrix. The reference / counter electrode R may comprise any reference material previously disclosed.

[0100] For the measurement of ALP or GGT, electrode well W was not loaded with enzyme or mediator, the reasons are discussed later.

[0101] In an embodiment of the present invention, the sample receiving well 434 serves both as a sample inlet and as a sample chamber for receiving reaction products.

[0102] It should be understood that the conduit path in any embodiment disclosed herein can be made of any non-corrosive metal. As is well known to those of ordinary skill in the art, carbon deposits (e.g., carbon paste or carbon ink) can also be used as conduit paths. It should also be understood that when a blank electrode B is optionally included in the analyte sensor strip 230, 430 for measuring ALT or AST, the blank electrode is used to correct any current generated by interferents in the blood sample. For measuring ALP or GGT, it is mainly used to determine any faults in the sample flowing into the test chamber.

[0103] Chemical reagents for analyte test strips for the determination of ALT or AST

[0104] Enzymes

[0105] The analyte test strip 230 of the present invention contains at least a chemical reagent in the reagent matrix of the working electrode W, which consumes pyruvate or glutamate according to the enzyme used in the reagent matrix. The chemical reagent is one of pyruvate oxidase (which can selectively react with pyruvate in the reaction solution present in the test box 10 during use) or glutamate oxidase or glutamate dehydrogenase (which can selectively react with L-glutamate in the reaction solution present in the test box 10 during use). The blank electrode B is not necessary in the present invention, and the sensor strip 230, 430 can only contain the electrode W and the electrode R. The present invention can optionally include a blank electrode B for eliminating background current that may be caused by possible interfering chemicals in the blood sample. As previously described, the use of such a blank electrode B helps to better distinguish the current caused by the pyruvate or L-glutamate complex mediator (as the case may be) and the current caused by other oxidizable substances in the reaction product. Commercially available pyruvate oxidase is available from a variety of sources including, but not limited to, Sigma-Aldrich (now known as MilliporeSigma), AG Scientific, Inc., Sekisui Diagnostics, and Toyoba USA, Inc.

[0106] Commercially available L-glutamate oxidase can be purchased from various sources, including but not limited to Sigma-Aldrich, CosmoBio USA, and Sorachim SA. Commercially available glutamate dehydrogenase can be purchased from various sources, including but not limited to Sigma-Aldrich, MyBioSource.com, Lee Biosolutions, Inc., and BBI Solutions. The concentration of these chemical reagents in the reagent matrix of the sensor strip is in the range of about 0.1% to 10%, preferably in the range of 0.5% to 2%.

[0107] Chemical oxidants

[0108] The working electrode W, the optional blank electrode B and the reference electrode R include a chemical oxidizing agent (such as a redox mediator), unless the reference electrode R is a reference electrode for a substance that does not require a chemical oxidizing agent, for example, an Ag-AgCl reference electrode. When a redox mediator is used, it is preferred to use an oxidized form of the redox mediator. It is also desirable that the reduced form of the mediator can be electrochemically oxidized at the electrode surface under an applied potential. It is also desirable that the mediator is stable in the reagent matrix. When used in a reference electrode, it is also desirable that the mediator can enable the reference electrode to function properly. The redox mediator can be selected from, but is not limited to, various metal compounds and organic redox compounds. Examples of acceptable redox mediators include, but are not limited to, various metal compounds and organic redox compounds. Acceptable redox mediators include potassium (or sodium) ferrocyanide, ferrocene and its derivatives, ruthenium compounds (such as hexaamineruthenium (III) chloride and its derivatives) and osmium complexes, 1,10-phenanthroline-5,6-dione, Medora Blue, tetrathiafulvalene 7,7,8,8-tetracyanoquinodimethane, tetrathiafulvalene, TCNQ, hydroquinone, dichlorophenol diphenol, p-benzoquinone, o-phenylenediamine, 3,4-dihydroxybenzaldehyde, indoxyl, phenazine, phenothiazine, 2,6-dichloroindoxyl, toluidine blue O, etc. The preferred mediator is 1,10-phenanthroline-5,6-dione. The concentration of 1,10-phenanthroline-5,6-dione in the reagent matrix is ​​preferably about 0.1% to about 5% (w / w) of the reagent matrix, and preferably about 0.1% to 0.5%.

[0109] Enzyme cofactors

[0110] The enzyme cofactors included in the reagent matrix of the analyte sensor strips 230, 430 are the organic cofactors thiamine triphosphate (TPP) and flavin adenine dinucleotide (FAD) and the inorganic cofactor Mg 2+ (In case of pyruvate oxidase). If glutamate dehydrogenase is used, a cofactor of NAD is used. The concentration of each cofactor in the reagent matrix is ​​approximately 0.25% (w / w).

[0111] Polymer Binders

[0112] The polymer used as a binder in the reagent matrix should have sufficient water solubility and should also be able to stabilize and bind all other chemicals in the reagent to the conductive surface layer of the electrode area. Suitable polymers include (but are not limited to) low and high molecular weight polyethylene oxide (PEO), polyethylene glycol, polyvinyl pyrrolidone, starch, methylcellulose, hydroxypropyl cellulose, polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC) and polyamino acids. The reagent binder can be a single polymer or a combination of polymers, preferably in a concentration range of about 0.02% (w / w) to about 7.0% (w / w). The preferred binder in the reagent matrix of the present invention is a combination of polyethylene oxide (PEO) and methylcellulose. The molecular weight of PEO ranges from thousands to millions and can be purchased from Scientific Polymer Products in New York, USA. The concentration of PEO in the reagent matrix is ​​preferably about 0.04% (w / w) to about 2% (w / w). The concentration of methylcellulose, available under the trade name Methocel 60HG (Cat. No. 64655, Fluka Chemicals, Milwaukee, WI) in the reagent matrix is ​​preferably in the range of about 0.02% (w / w) to about 5% (w / w).

[0113] Surfactants

[0114] Surfactants can be optionally included in the reagent matrix to facilitate the distribution of the reagent matrix into the electrode area. When the sample fluid enters the sample channel of the analyte test strip, the surfactant also helps to quickly dissolve the dried chemical reagent. The amount and type of surfactant are selected to ensure the aforementioned functions and avoid denaturation of the enzyme. The surfactant can be selected from (but not limited to) various anionic, cationic, nonionic and zwitterionic detergents. Examples of acceptable surfactants are polyoxyethylene ether, Tween 20, sodium cholate hydrate, hexadecylpyridinium chloride monohydrate and CHAP. The preferred surfactant is polyoxyethylene ether. More preferably, it is tert-octylphenoxypolyethoxyethanol, and can be purchased under the trade name Triton X-100. The concentration of the surfactant in the reagent matrix is ​​preferably about 0.01% (w / w) to about 2%.

[0115] Buffer

[0116] Optionally, the buffer can be present in a dry form in the sensor strip of the present invention. The amount of the buffer is sufficient to substantially maintain the pH of the reagent matrix. Examples of suitable buffers include phosphate buffers, citrate buffers, TRIS buffers, etc. The pH of the buffer is preferably in the range of about 5.0 to about 8.5.

[0117] Bulking Agent

[0118] Optional fillers of water-soluble and inactive ingredients are preferably added to the reagent mixture / matrix. When the electrode forming layer is used to contain the reagent matrix, so that the electrode openings in the electrode forming layer will not capture bubbles when the sample fluid fills the capillary channel, it is advantageous to use fillers. Various sugars can be added to the reagent mixture / matrix, such as trehalose, galactose, glucose, sucrose, lactose, mannitol, mannose, fructose, sucrose, lactose, lactitol, sorbitol, xylitol, nicotinamide, maltose, etc., as long as they do not react with other ingredients and are not active on the electrode surface. The filler can be a chemical substance or a combination of chemical substances. The amount of the filler when included in the reagent mixture / matrix is ​​in the range of about 1% to about 15% (w / w).

[0119] Chemical reagents for analyte test strips for the determination of ALP or GGT

[0120] Polymeric binders, surfactants, fillers, but no enzymes or mediators

[0121] The polymer binder, surfactant, and filler are the same as those listed above for the analyte test strips for the determination of ALT and AST. It is important to note that no enzyme or mediator is used in the reagent matrix for the analyte sensor strips for the determination of ALP and GGT. The reason for this is that the reaction products measured for the ALP and GGT determinations can be directly oxidized at the electrode surface, thus no substrate and mediator for the reaction products are required to generate current.

[0122] Buffer

[0123] A buffer is present in the reagent matrix for the analyte sensor strip to measure ALP, and the pH range of the buffer is 7 to 12. Preferably, the buffer provides a pH range of 10 to 11. Examples of such a buffer include diethanolamine buffer, AMP (2-amino-2-methyl-1-propanol) buffer, and the like.

[0124] A buffer is also present in the reagent matrix for the analyte sensor strip to measure GGT, and the pH range of the buffer is 7 to 9. Preferably, the buffer provides a pH of 8.3. Examples of such buffers include phosphate buffers, TRIS buffers, citrate buffers, etc. The preferred buffer is TRIS buffer.

[0125] Chemical reagents for measuring ALT, AST, ALP or GGT in a disposable test chamber

[0126] ALT Reagent

[0127] To measure the amount of liver enzyme ALT in a blood sample, reagents are deposited in at least one of the plurality of chambers 22 of the disposable test cartridge 10, thereby forming a reactant mixture during assembly of the disposable test cartridge. For ALT, the chamber 22 includes the following reagents: L-alanine, alpha-ketoglutarate, a polymer binder, a surfactant, a buffer, a filler (such as a sugar), and a preservative.

[0128] The concentration of L-alanine is in the range of 0.01 M to 2 M. The preferred concentration is in the range of 0.1 M to 1 M, and the most preferred concentration is in the range of 0.2 to 0.5 M. For the test data published subsequently, the concentration used was 0.3 M.

[0129] The concentration range of α-ketoglutarate is 0.01 to 2 M. The preferred concentration range is 0.05 M to 1 M, and the most preferred concentration range is 0.1 M to 0.5 M. For the tests disclosed subsequently, the concentration used was 0.2 M.

[0130] The polymer used as adhesive in the reaction mixture should have sufficient water solubility, and should also be able to stabilize and compound all other chemical substances in the reaction mixture. Suitable polymers include, but are not limited to, polyethylene oxide (PEO), polyethylene glycol, polyvinyl pyrrolidone, starch, methylcellulose, hydroxypropyl cellulose, polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC), fish gelatin, chitosan, gum and polyamino acid. The polymer adhesive can be a single polymer or a combination of polymers. For PEO, the molecular weight of PEO is in the range of thousands to millions, available from ScientificPolymer Products in New York, USA. The concentration of PEO in the reaction mixture is preferably about 0.04% (w / w) to about 2%. According to the trade name Methocel 60HG (Catalog No. 64655, Fluka Chemicals, Milwaukee, Wisconsin, USA), the concentration of methylcellulose available in the reagent layer composition is preferably in the range of about 0.02% (w / w) to about 5%.

[0131] The surfactant is selected from (but not limited to) various anionic, cationic, nonionic and zwitterionic detergents. Examples of acceptable surfactants are polyoxyethylene ether, Tween 20, sodium cholate hydrate, hexadecylpyridinium chloride monohydrate and CHAPS (zwitterionic detergent). Preferred surfactants are polyoxyethylene ether. More preferably, the surfactant is tolylphenoxypolyethoxyethanol, and can be purchased under the trade name Triton X-100. The concentration of TritonX-100 in the reaction mixture is preferably about 0.01% (w / w) to about 2%.

[0132] Add a buffer to the reaction mixture. A pH in the pH range of 5 to 11 is acceptable. The preferred pH range is 7 to 8. For test data published later, a pH of 7.4 was used. Acceptable buffers for the determination of ALT include phosphate buffer, trisodium buffer, citrate buffer, and the like.

[0133] Optional fillers of water-soluble and inactive ingredients are preferably added to the reagent mixture. Once the blood sample is added to the chamber 22 containing the reaction mixture, the use of fillers is conducive to reducing the trend of capturing bubbles in the reaction mixture. Various sugars (such as trehalose, galactose, sucrose, lactose, mannitol, mannose, fructose, lactitol, sorbitol, xylitol, nicotinamide, maltose, starch, etc.) can be added to the reaction mixture as fillers, as long as they do not react with other ingredients. Fillers can be a combination of chemicals or chemicals. The amount of fillers in the reactant mixture is about 1% to about 15% (w / w).

[0134] Since preassembled disposable boxes are usually stored for several months before use, it is encouraged to add at least one preservative. The preservative provides long-term stability for the reactant mixture in the preassembled disposable box. The concentration of the preservative (e.g., Prolin (which is a proteinogenic amino acid)) included in the reactant mixture is in the range of 0.05% to 2%.

[0135] Reagents for AST

[0136] To measure the amount of liver enzyme AST in a blood sample, reagents are deposited in at least one of the plurality of chambers 22 of the disposable test cartridge 10, thereby forming a reactant mixture during assembly of the disposable test cartridge. For AST, the chamber 22 contains the following reagents: L-aspartic acid, α-ketoglutaric acid, a polymer binder, a surfactant, a buffer, a filler (such as a sugar), a buffer, a preservative, and optionally oxaloacetate decarboxylase.

[0137] The concentration of L-aspartic acid is in the range of 0.01 M to 2 M. A preferred concentration is in the range of 0.1 M to 1 M, and a most preferred concentration is in the range of 0.2 to 0.5 M. For the test data subsequently disclosed, a concentration of 0.5 M was used.

[0138] The concentration range of alpha-ketoglutarate is 0.01 to 2 M. The preferred concentration range is 0.05 M to 1 M, and the most preferred concentration range is 0.1 M to 0.5 M. For the tests disclosed subsequently, the concentration used was 0.25 M.

[0139] The concentration of oxaloacetate decarboxylase ranged from 50 IU / mL to 500 IU / mL. For the subsequently disclosed tests, the concentration used ranged from 100 IU / mL to 150 IU / mL.

[0140] The polymer binder, surfactant, optional filler and preservative are the same as those listed above for the ALT assay.

[0141] A buffer is added to the reaction mixture. A pH in the pH range of 5 to 11 is acceptable. The preferred pH range is 7 to 9. For the test data disclosed subsequently, a pH of 8 was used. Acceptable buffers for the determination of ALT include phosphate buffer, TRIS buffer, citrate buffer, and the like.

[0142] Reagents for ALP

[0143] To measure the amount of liver enzyme ALP in a blood sample, reagents are deposited in at least one of the plurality of chambers 22 of the disposable test cartridge 10, thereby forming a reactant mixture during assembly of the disposable test cartridge. For ALP, the chamber 22 contains the following reagents: sodium phenyl phosphate or its derivatives, a polymer binder, a surfactant, a buffer, a filler (such as a sugar), a buffer, and a preservative.

[0144] The concentration of sodium phenyl phosphate ranged from 100 mM to 300 mM. For the test data published subsequently, the concentration used was 200 mM.

[0145] The polymer binder, surfactant, optional filler and preservative are the same as those listed above for the ALT assay.

[0146] A buffer is added to the reaction mixture. A pH in the pH range of 7 to 12 is acceptable. The preferred pH range is 10 to 11. Acceptable buffers for determining ALP include diethanolamine buffer, AMP (2-amino-2-methyl-1-propanol) buffer, and the like.

[0147] Reagents for GGT

[0148] To measure the amount of liver enzyme GGT in a blood sample, reagents are deposited in at least one of the plurality of chambers 22 of the disposable test cartridge 10, thereby forming a reactant mixture during assembly of the disposable test cartridge. For GGT, the chamber 22 contains the following reagents: L-glutamic acid gamma-aniline or a derivative thereof, glycylglycine, a polymer binder, a surfactant, a buffer, a filler (such as a sugar), a buffer, and a preservative.

[0149] The concentration of L-glutamic acid γ-aniline ranged from 10 mM to 30 mM. For the test data published subsequently, the concentration used was 20 mM.

[0150] The concentration of glycylglycine ranged from 200 mM to 400 mM. For the test data published subsequently, the concentration used was 300 mM.

[0151] The polymer binder, surfactant, optional filler and preservative are the same as those listed above for the ALT assay.

[0152] A buffer is added to the reaction mixture. A pH in the pH range of 5 to 11 is acceptable. The preferred pH range is 7 to 9. For the test data disclosed subsequently, a pH of 8.3 was used. Acceptable buffers for the determination of ALT include phosphate buffer, TRIS buffer, citrate buffer, and the like.

[0153] General procedure for determination of ALT, AST, ALP and GGT in blood samples

[0154] The following is a general test procedure for measuring certain liver enzymes, which is used to measure liver enzymes such as ALT, AST, ALP and GGT. The test includes obtaining a predetermined amount of capillary blood, diluting a predetermined amount of capillary blood, transferring the predetermined amount of diluted capillary blood into a chamber 22 containing a reactant mixture for measuring a specific liver enzyme, resuspending the diluted capillary blood in the reactant mixture, mixing the diluted capillary blood with the reactant mixture, and reacting the resulting mixture of the diluted capillary blood and the reactant mixture for a predetermined time to form a reaction solution. At the end of the predetermined reaction time, a predetermined amount of the reaction solution is extracted by a pipette in the bedside instant analyzer 300 and deposited onto the analyte test strip 230, 430 on the test strip module 200 to measure the amount of a specific analyte in the reaction solution. The analyte test strip 230, 430 can determine which analyte is directly related to the determination of the specific liver enzyme.

[0155] More specifically, a lancet is used to prick the patient's finger to obtain a blood sample. A disposable test cartridge 10 is selected for the particular test to be performed. In this case, it is a disposable test cartridge 10 capable of testing for the presence of one of the four liver enzymes ALT, AST, ALP, and GGT. The specific test cartridge 10 has at least one analyte sensor strip 230, 430 connected to the test strip module 200 for measuring the analyte. Each disposable test cartridge 10 is coded for the type of liver enzyme assay and the type of analyte sensor strip 230, 430.

[0156] The capillary element 60 is separated from the disposable test cartridge 10. The finger tips 66a of the capillary element fingers 66 contact the blood drop produced on the finger by puncturing the patient's skin. A portion of the blood drop enters the capillary element 60 by capillary action. The capillary element 60 is inserted and reconnected to the disposable test cartridge 10 in the same direction as when it was removed from the disposable test cartridge 10 before obtaining the blood sample. Once reconnected, the disposable test cartridge 20 is ready to be inserted into the bedside instant analyzer 300. Once inserted, the bedside instant analyzer 300 is activated.

[0157] When in the bedside instant analyzer 300, the capillary blood sample residing in the capillary element 60 is sucked into the chamber 22 in which the capillary element 60 is suspended by the analyzer 300. The cover 40 is removed from the disposable test box 20, and a predetermined amount of a predetermined diluent (i.e., a buffer or saline solution) remaining in different chambers 22 of the disposable test box 10 is removed by a pipette and deposited in the chamber 22 containing the blood sample. A sufficient amount of diluent is added to the chamber 22 containing the blood sample to obtain a dilution ratio in the range of 1:1 to 1:50. The preferred range of the dilution ratio is 1:2 to 1:20. The most preferred range of the dilution ratio for the analyzer 300 is 1:5 to 1:10. For the test data shown, the dilution ratio used is 1:10. It is understood that the dilution ratio of the diluent is relatively large, or it is equal to the amount of capillary blood in the chamber. After dilution, a predetermined amount of the diluted blood sample is transferred into the chamber 22 of the disposable test cartridge 10 containing the reactant mixture, which is used to determine the specific liver enzyme activity in the diluted blood sample. Although each disposable test cartridge 10 can be dedicated to a single liver enzyme, it is conceivable that the disposable test cartridge 10 can include tests for other liver enzymes disclosed herein. It is also contemplated that additional analyte test strips for determining other liver enzymes will also be incorporated into the disposable test cartridge 10, which is configured to test two or more liver enzymes in a blood sample.

[0158] Determination of ALT in blood samples

[0159] In the presence of ALT as a catalyst, L-alanine and α-ketoglutarate react to produce pyruvate and L-glutamate. This is represented by equation 1:

[0160]

[0161] After dilution, a predetermined amount of the diluted blood sample is moved into the chamber 22 of the disposable test cartridge 10, which contains a reactant mixture for determining ALT activity in the blood sample. The amount of the predetermined amount of diluted blood sample is in the range of 5 to 100 microliters. Preferably, the amount of the predetermined amount of diluted blood sample is in the range of 10 to 50 microliters. For the test data obtained, the predetermined amount of the diluted blood sample is in the range of 15 to 30 microliters. The diluted capillary blood is mixed with the reactant mixture, and the resulting mixture of the diluted capillary blood and the reactant mixture is reacted for a predetermined time to form a reaction solution. For the determination of ALT, the reaction time is in the range of 5 minutes to 60 minutes. The preferred reaction time is in the range of 10 minutes to 30 minutes. The more preferred reaction time is in the range of 20 minutes to 30 minutes. For the data obtained below, the reaction time is 25 minutes at a temperature of 40-45 degrees Celsius.

[0162] An analyte test strip 230 for measuring pyruvate in the reaction solution is used for testing. The reaction of the pyruvate test strip uses pyruvate oxidase in the sensor strip reagent matrix. The reaction equation is as follows:

[0163]

[0164] Med ox is the oxidized form of the mediator, while Med red It is the reduced form of the mediator.

[0165] The bias potential on the working electrode and the reference electrode of the pyruvic acid sensor ranges from 100mv to 500mv, preferably 300mV to 400mV. The voltage value within this range is not critical, but must be kept constant. The bias potential on the working electrode and the reference electrode causes the mediator to change from a reduced state to an oxidized state on the electrode surface, thereby generating a current according to the concentration of pyruvic acid in the reaction product.

[0166] As can be seen from Equation 1, the analyte sensor strip for measuring glutamate can also be used in place of the pyruvate sensor strip. The glutamate test strip reaction uses glutamate oxidase or glutamate dehydrogenase in the sensor test strip reagent matrix. The reaction equation is as follows:

[0167]

[0168] Med ox is the oxidized form of the mediator, while Med red It is the reduced form of the mediator.

[0169] The bias potential on the working electrode and the reference electrode of the glutamate sensor ranges from 100mv to 500mv, preferably 300mV to 400mV. Similar to the pyruvic acid sensor, the voltage value in this range is not critical, but must be kept constant. The bias potential on the working electrode and the reference electrode causes the mediator to change from a reduced state to an oxidized state on the electrode surface, thereby generating a current according to the concentration of glutamate in the reaction product.

[0170] ALT concentration values ​​in blood samples were obtained using a blood analyzer known as the Dimension RxL Chemistry Analyzer, sold by Siemens Medical Diagnostics, Tarrytown, N.Y. Test data from analyte test strips were obtained using a CH Instruments potentiostat (Model CHI 812B or Model CHI 660A).

[0171] Test data for ALT determination using pyruvate sensor

[0172] To determine the linear response between blood ALT concentration and the pyruvate test strip-based response, three different ALT concentration levels were tested. The concentration levels were 61 IU / L, 135 IU / L, and 468 IU / L. Samples were prepared for these ALT levels and 10 current measurements were performed for each ALT concentration. For each measurement, a new pyruvate sensor strip was used. Table 1 lists the test data obtained for determining ALT.

[0173] Table 1

[0174]

[0175] Fig.17 The measured current response of the working electrode (i.e., the pyruvate oxidase-based electrode) to different ALT concentrations is shown. The current response is linear with the pyruvate concentration over the entire ALT concentration range tested. The ALT concentration is directly proportional to the pyruvate concentration.

[0176] Test data of ALT measurement using glutamate sensor

[0177] To determine the linear response between blood ALT concentration and the glutamate test strip-based reaction, three different ALT concentration levels were used in the test. The concentration levels were 61 IU / L, 269 IU / L, and 468 IU / L. Samples were prepared for these ALT levels and 3 current measurements were performed for each ALT concentration. For each measurement, a new glutamate sensor strip was used. Table 2 shows the test data obtained for the determination of ALT using the glutamate sensor strip.

[0178] Table 2

[0179]

[0180] Fig.18 The measured current response of the working electrode (ie, the glutamate dehydrogenase-based electrode) to different ALT concentrations is shown. The current response is linear with glutamate concentration over the entire ALT concentration range tested. ALT concentration is directly proportional to glutamate concentration.

[0181] Determination of ALT in blood samples

[0182] In the presence of AST as a catalyst, L-aspartate and α-ketoglutarate react to produce oxaloacetate and L-glutamate. This is represented by equation 2:

[0183]

[0184] In this case, glutamate sensing test strips are used to measure AST, and the reactions discussed above are used to represent that glutamate oxidase or glutamate dehydrogenase is included in the reagent matrix of the glutamate test strip. However, a pyruvate sensor is preferred because its results are more accurate. To use a pyruvate test strip, additional compounds are required, as described below.

[0185] Under the action of oxaloacetate decarboxylase, the oxaloacetate product in equation 2 can also undergo further reactions to produce pyruvate and carbon dioxide. This is represented by equation 3:

[0186] Oxaloacetate + oxaloacetate decarboxylase → pyruvate + CO2 Equation 3

[0187] Thus, if oxaloacetate decarboxylase is present in the reaction mixture in cartridge 22, pyruvate is formed and the pyruvate test strip can be used to measure the pyruvate present in the reaction solution. The concentration of AST in the blood sample is directly proportional to the measured pyruvate concentration.

[0188] After dilution, a predetermined amount of the diluted blood sample is moved into the chamber 22 of the disposable test cartridge 10, which contains a reactant mixture for determining ALT activity in the blood sample. The amount of the predetermined amount of diluted blood sample is in the range of 5 to 100 microliters. Preferably, the amount of the predetermined amount of diluted blood sample is in the range of 10 to 50 microliters. For the test data obtained, the predetermined amount of the diluted blood sample is in the range of 15 to 30 microliters. The diluted capillary blood is mixed with the reactant mixture, and the resulting mixture of the diluted capillary blood and the reactant mixture is reacted for a predetermined time to form a reaction solution. For the determination of ALT, the reaction time is in the range of 5 minutes to 60 minutes. The preferred reaction time is in the range of 10 minutes to 30 minutes. The more preferred reaction time is in the range of 20 minutes to 30 minutes. For the data obtained below, the reaction time is 25 minutes at a temperature of 40-45 degrees Celsius.

[0189] As previously discussed with respect to measuring AST, when oxalate decarboxylase is present in the reactant mixture of the disposable test cartridge 10, an analyte test strip 230 for measuring pyruvate in the reaction solution is used for testing. The reaction of the pyruvate test strip uses pyruvate oxidase in the sensor strip reagent matrix. The reaction equation is as previously discussed with respect to the pyruvate test strip.

[0190] As previously disclosed for the ALT assay, AST concentration values ​​in blood samples were obtained using a blood analyzer known as the DimensionRxL Chemistry Analyzer, sold by Siemens Medical Diagnostics, Inc., Tarrytown, N.Y. Test data from analyte test strips were obtained using a CH Instruments potentiostat (Model CHI 812B or Model CHI 660A).

[0191] Test data of AST determination using pyruvate sensor

[0192] To determine the linear response between blood AST concentration and the reaction based on the pyruvic acid test strip, three different AST concentration levels were used for testing. The concentration levels were 25 IU / L, 196 IU / L, and 698 IU / L. Samples were prepared for these AST levels, and 10 current measurements were performed for each ALT concentration. For each measurement, a new pyruvic acid sensor strip was used. Table 3 lists the test data obtained for determining AST.

[0193] Table 3

[0194]

[0195] Fig.19 The measured current response of the working electrode (i.e., the electrode based on pyruvate oxidase) to different AST concentrations is shown. In the entire AST concentration range tested, the current response is a linear relationship with the pyruvate concentration. The AST concentration is directly proportional to the pyruvate concentration (glutamate when using glutamate oxidase or glutamate dehydrogenase).

[0196] Determination of ALT in blood samples

[0197] In the presence of ALP as a catalyst, sodium phenyl phosphate or its derivatives react to produce phenol or its derivatives and phosphate. This is represented by equation 4:

[0198]

[0199] As explained above for the determination of ALT and AST, after dilution, a predetermined amount of the diluted blood sample is transferred into the cavity 22 of the disposable test cartridge 10, which contains a reactant mixture for determining the ALP activity in the blood sample. The amount of the predetermined amount of diluted blood sample is in the range of 5 to 100 microliters. Preferably, the amount of the predetermined amount of diluted blood sample is in the range of 10 to 50 microliters. For the test data obtained, the predetermined amount of the diluted blood sample is in the range of 15 to 30 microliters. The diluted capillary blood is mixed with the reaction mixture, and the resulting mixture of the diluted capillary blood and the reaction mixture is reacted for a predetermined length of time. For the determination of ALT, the reaction time is in the range of 5 minutes to 60 minutes. The preferred reaction time is in the range of 10 minutes to 30 minutes. The more preferred reaction time is in the range of 20 minutes to 30 minutes. For the data obtained below, the reaction time is 25 minutes at a temperature of 40-45 degrees Celsius.

[0200] Unlike the sensor strips used in the ALT and AST assays, no enzyme or mediator is required in the reagent matrix of the working electrode. This is because the reaction product phenol observed in Equation 4 above is electrochemically oxidized directly at the electrode surface.

[0201] ALP concentration values ​​in blood samples were obtained using a blood analyzer known as the Dimension RxL Chemistry Analyzer, sold by Siemens Medical Diagnostics, Tarrytown, N.Y. Test data from analyte test strips were obtained using a CH Instruments potentiostat (Model 812B or Model 660A).

[0202] Test data of ALP measurement using current sensor

[0203] To determine the linear response between blood ALP concentration and the response of the current measurement test strip, three different ALP concentration levels were tested. The concentration levels were 72 IU / L, 178 IU / L, and 458 IU / L. Samples were prepared for these ALP levels and 10 current measurements were performed for each ALP concentration. For each measurement, a new current sensor strip was used. Table 4 lists the test data obtained for the determination of ALP.

[0204] Table 4

[0205]

[0206] Fig. 20 The current response of the working electrode (i.e., current sensing electrode) to different ALP concentrations is shown. The current response is linear with the phenol concentration over the entire ALP concentration range tested. The ALP concentration is proportional to the phenol concentration.

[0207] Fig.21 is a linear sweep voltammogram of the current sensor, showing the current traces for the three ALP concentration standards described above. The linear sweep voltammogram shows that the potential between the working electrode and the reference electrode is swept from 0mV to 500mV. This is a non-fixed potential sensor technology. For all current tests using linear sweep voltammograms, the analyte sensor strips 230, 430 undergo a current sweep from 0mV to 500mV. When using this technique, the current reading can be obtained when the sweep reaches 350mV because the current separation between the concentration levels of ALP is the largest.

[0208] Fig. 22is an amperometric response plot of current versus time for three concentrations of ALP. This amperometric technique uses a fixed potential (i.e., bias potential) between the working electrode and the reference electrode. The fixed potential is similar to the potential disclosed above for measuring ALT or AST. This graph is used to determine when to record the current measurement after the sample is introduced to the current sensor strip. For all current tests using the amperometric technique to measure ALP, the current measurement was recorded 10 seconds (i.e., the "time period") after the sample covered the working electrode and the reference electrode. When the sample covered both the working electrode and the reference electrode at the same time, there was a large change in the current measurement. This marked the beginning of the 10 second time period. In this graph, the start time of the 10 seconds begins at the moment when the current change is large. As shown in Fig. 22 As shown, this occurs at approximately 2.5-3 seconds. This means that the current recording time according to this graph will be approximately 12.5-13 seconds. From a practical point of view, the timer will start when the instrument senses a large current change, and a current record will be registered ten seconds after the timer starts. From a practical point of view, the timer will start when the instrument senses a large current change, and a current record will be registered 10 seconds after the timer starts.

[0209] Determination of ALT in blood samples

[0210] In the case of GGT as a catalyst, L-glutamic acid-γ-aniline and glycylglycine react to produce L-glutamylglycine and aniline. This is represented by Equation 5:

[0211]

[0212] As explained above for the ALP assay, after dilution, a predetermined amount of the diluted blood sample is moved into the chamber 22 of the disposable test cartridge 10, which contains a reactant mixture for determining GGT activity in the blood sample. The amount of the predetermined amount of diluted blood sample is in the range of 5 to 100 microliters. Preferably, the amount of the predetermined amount of diluted blood sample is in the range of 10 to 50 microliters. For the test data obtained, the predetermined amount of the diluted blood sample is in the range of 15 to 30 microliters. The diluted capillary blood is mixed with the reaction mixture, and the resulting mixture of the diluted capillary blood and the reaction mixture is reacted for a predetermined time. For the determination of ALT, the reaction time is in the range of 5 minutes to 60 minutes. The preferred reaction time is in the range of 10 minutes to 30 minutes. The more preferred reaction time is in the range of 20 minutes to 30 minutes. For the data obtained below, the reaction time is 25 minutes at a temperature of 40-45 degrees Celsius.

[0213] As with the sensor strips for measuring ALP, no enzyme or mediator is required in the reagent matrix of the working electrode. This is because the reactant product aniline observed in Equation 5 above is electrochemically oxidized directly on the electrode surface.

[0214] ALP concentration values ​​in blood samples were obtained using a blood analyzer known as the Dimension RxL Chemistry Analyzer, sold by Siemens Medical Diagnostics, Tarrytown, N.Y. Test data from analyte test strips were obtained using a CH Instruments potentiostat (Model 812B or Model 660A).

[0215] Test data of GGT measurement using current sensor

[0216] To determine the linear response between blood GGT concentration and the amperometric test strip-based response, tests were performed using 3 different GGT concentration levels. The concentration levels were 52 IU / L, 153 IU / L, and 430 IU / L. Samples were prepared for these GGT levels and 10 amperometric measurements were performed for each ALT concentration. For each measurement, a new amperometric test strip was used. Table 5 lists the test data obtained for the determination of ALP.

[0217] Table 5

[0218]

[0219] Fig.23 The measured current response of the working electrode (i.e., the current sensing electrode) to different GGT concentrations is shown. The current response is linear with the aniline concentration over the entire GGT concentration range tested. The GGT concentration is directly proportional to the aniline concentration.

[0220] Although the preferred embodiments of the present invention have been described herein, the above description is illustrative only. Those skilled in the relevant art may make further modifications to the invention disclosed herein, and all such modifications are deemed to be within the scope of the invention defined in the claims.

Claims

1. A disposable test kit for measuring the concentration of one or more liver enzymes in a blood sample in a bedside instant analyzer, the disposable test kit comprising: A box body having a plurality of chambers, each of the plurality of chambers having an opening at the top of the box body, and at least one of the plurality of chambers containing a reactant mixture, the reactant mixture being capable of chemically reacting when mixed with a blood sample to form a reaction solution containing at least one reaction product, the reactant mixture containing a chemical substance having specificity for catalysis of liver enzymes ALP or GGT in the blood sample; a removable cartridge cover connected to the cartridge body and covering the plurality of chambers, the removable cartridge cover having a capillary receiving hole and a capillary element removably inserted into the cartridge cover, the capillary element being configured to obtain a blood sample; and A test strip module is directly connected to the cartridge body and contains at least one analyte test strip configured to receive a portion of the reaction solution and measure at least one analyte, the at least one analyte being at least one reaction product.

2. The disposable test kit according to claim 1, wherein: The reactant mixture comprising chemicals specific for the determination of ALP comprises sodium phenyl phosphate or a derivative thereof.

3. The disposable test kit according to claim 1, wherein: The reactant mixture containing chemicals specific for the determination of GGT includes L-glutamic acid γ-aniline and its derivatives and glycylglycine.

4. The disposable test kit according to claim 1, wherein: The test strip module includes an analyte test strip configured to measure phenol or a derivative thereof when at least one chamber of the plurality of chambers contains a chemical catalyzed by a liver enzyme ALP.

5. The disposable test kit according to claim 1, wherein: The test strip module includes an analyte test strip configured to measure aniline or a derivative thereof when at least one chamber of the plurality of chambers contains a chemical species catalyzed by the liver enzyme GGT.

6. The disposable test kit according to claim 1, wherein: The test strip module includes an analyte test strip having a reagent matrix disposed on a working electrode, the reagent matrix including one of pyruvate oxidase, glutamate oxidase, or glutamate dehydrogenase.

7. The disposable test kit according to claim 1, wherein: The test strip module includes an analyte test strip having a reagent matrix disposed on a working electrode, the reagent matrix not including an enzyme or a mediator.

8. The disposable test kit according to claim 1, wherein: The analyte test strip has an electrical contact end, a sample end, and a sample receiving port spaced a predetermined distance from the sample end.

9. The disposable test kit according to claim 8, wherein: The sample end includes an exhaust port and a sample channel located between the sample receiving port and the exhaust port.

10. The disposable test kit according to claim 1, wherein: The test strip module comprises two or more analyte test strips, wherein at least two analyte test strips of the two or more analyte test strips measure the same analyte.

11. The disposable test kit according to claim 1, wherein: The test strip module contains two or more analyte test strips, wherein each of the two or more analyte test strips measures a different analyte.

12. A method for determining the concentration of one or more liver enzymes in a blood sample, the method comprising: Provide a blood sample receiving room; providing a reactant chamber containing a predetermined chemical substance, which is catalyzed by liver enzymes ALP or GGT in the blood sample; obtaining a blood sample and placing at least a portion of the blood sample into a blood sample receiving chamber; diluting the blood sample to form a diluted blood sample; placing a predetermined amount of diluted blood sample into the reaction chamber; allowing a reaction between a predetermined chemical substance and the diluted blood sample to continue for a predetermined period of time, thereby forming a reaction solution comprising at least one reaction product; transferring a portion of the reaction solution to an analyte test strip capable of measuring the amount of at least one reaction product in the reaction solution; measuring an amount of at least one reaction product present in the reaction solution; The amount of at least one reaction product in the reaction solution is correlated to the amount of the liver enzyme ALP or GGT in the blood sample.

13. The method according to claim 12, wherein: The measuring step further includes measuring the amount of phenol or aniline in the reaction solution.

14. The method according to claim 13, wherein: The correlating step further includes correlating the amount of phenol or aniline in the reaction solution with the amount of ALP or GGT in the blood sample, respectively.

15. The method according to claim 12, wherein: The measuring step further comprises measuring at least one reaction product using an electrochemical measurement technique.

16. The method according to claim 12, wherein: The measuring step further comprises measuring at least one of the reaction products using amperometry to determine ALP or GGT.

17. The method according to claim 12, wherein: The measuring step includes measuring at least one of the reaction products using linear sweep voltammetry to determine the ALP.

18. A disposable kit for use in a bedside analyzer to determine the concentration of one or more liver enzymes in a blood sample, the disposable kit comprising: Disposable test kit, including A box body having a plurality of chambers, each of the plurality of chambers having an opening at the top of the box body, and at least one of the plurality of chambers containing a reactant mixture, the reactant mixture being capable of chemically reacting when mixed with a blood sample to form a reaction solution containing at least one reaction product, the reactant mixture containing a chemical substance having specificity for catalysis of liver enzymes ALP or GGT in the blood sample; a removable cartridge cover connected to the cartridge body and covering the plurality of chambers, the removable cartridge cover having a capillary receiving hole and a removable capillary element removably inserted into the cartridge cover, the capillary element being configured to obtain a blood sample; a disposable pipette tip disposed in one of the plurality of chambers; and A test strip module is directly connected to the cartridge body and contains at least one analyte test strip configured to receive a portion of the reaction solution and measure at least one analyte, the at least one analyte being at least one reaction product.

19. The disposable cartridge kit of claim 18, further comprising instructions for use of the disposable test cartridge and the capillary element.

20. A method for electrochemically determining the concentration of one or more liver enzymes in a blood sample using a bedside instant analyzer, the method comprising: Provide bedside instant analyzers; A disposable test cartridge for use in a point-of-care analyzer is obtained, the disposable test cartridge comprising A box body having a plurality of chambers, each of the plurality of chambers having an opening at the top of the box body, and at least one of the plurality of chambers containing a reactant mixture, the reactant mixture being capable of chemically reacting when mixed with a blood sample to form a reaction solution containing at least one reaction product, the reactant mixture containing a chemical substance having specificity for catalysis of liver enzymes ALP or GGT in the blood sample; a removable cartridge cover connected to the cartridge body and covering the plurality of chambers, the removable cartridge cover having a capillary receiving hole and a removable capillary element removably inserted into the cartridge cover, the capillary element being configured to obtain a blood sample; a disposable pipette tip disposed in one of the plurality of chambers; and a test strip module directly coupled to the cartridge body and comprising at least one analyte test strip configured to receive a portion of the reaction solution and measure at least one analyte, the at least one analyte being at least one reaction product; removing the capillary element from the removable cartridge cover, receiving a blood sample in the capillary element, and placing the capillary element containing the blood sample back into the removable cartridge cover; The disposable test cartridge is placed into a point-of-care analyzer; and Start the test cycle in the point-of-care analyzer.

21. The method according to claim 20, wherein: The starting of a test cycle in the bedside instant analyzer includes: aspirating the blood sample from the capillary element to the bottom of the chamber directly below the capillary element; Remove the cover from the box body; Connecting the pipette tip to the automated pipetting device within the point-of-care analyzer; diluting the blood sample by aspirating a predetermined amount of diluent from another chamber of the plurality of chambers and depositing the predetermined amount of diluent into the chamber containing the blood sample to form a diluted blood sample; removing a predetermined amount of the diluted blood sample and depositing the predetermined amount of the diluted blood sample into another chamber of the plurality of chambers containing a reactant compound of a chemical substance to form a reaction solution by aspirating the predetermined amount of the diluted blood sample, wherein the chemical substance is specific to catalysis of liver enzyme ALP or GGT in the blood sample; After a predetermined reaction time, removing a predetermined amount of the reaction solution and depositing it on at least one analyte test strip to test at least one analyte; measuring the amount of the at least one analyte in the reaction solution; The amount of at least one analyte in the reaction solution is correlated with the amount of liver enzyme ALP or GGT in the blood sample.

22. The method according to claim 20, wherein: The measuring step includes measuring the amount of phenol or aniline in the reaction solution.

23. The method according to claim 22, wherein: The measuring step includes measuring at least one of the reaction products using amperometry to determine ALP or GGT.

24. The method according to claim 22, wherein: The measuring step includes measuring at least one of the reaction products using linear sweep voltammetry to determine the ALP.

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