Improved calibration accuracy method for creatinine/creatine sensor

文档序号：863064 发布日期：2021-03-16 浏览：11次中文

阅读说明：本技术 用于肌酸酐/肌酸传感器的改进的校准精度方法 (Improved calibration accuracy method for creatinine/creatine sensor ) 是由徐晓贤普拉萨德·帕米迪大卫·雷蒙迪米克洛斯·埃尔德希于 2019-06-03 设计创作，主要内容包括：本公开涉及用于测量患者血液中的肌酸酐和肌酸的电化学传感器。更具体地,本公开涉及用于改善用于测量肌酸酐和肌酸的电化学传感器的校准精度的组合物和方法。(The present disclosure relates to an electrochemical sensor for measuring creatinine and creatine in the blood of a patient. More particularly, the present disclosure relates to compositions and methods for improving the calibration accuracy of electrochemical sensors for measuring creatinine and creatine.)

1. A method of calibrating a creatinine/creatine measurement system having a creatine sensor and a creatinine sensor, comprising:

measuring a creatine sensor current signal (Δ I2) of a first calibration solution (CS2) with the creatine sensor, the first calibration solution (CS2) having a known creatine concentration (CR _ CS2), a known creatinine concentration (CREA _ CS2), and a stabilization ratio of creatine to creatinine to establish a creatine sensor sensitivity (Slope) of the creatine sensor;

measuring a measured creatine concentration (MCR _ CS3) of a second calibration solution (CS3) with the creatine sensor, the second calibration solution (CS3) having an initial known creatine concentration (CR _ CS3), an initial known creatinine concentration (CREA _ CS3), and a creatine to creatinine instability ratio;

comparing MCR _ CS3 of CS3 with CR _ CS3 to establish a creatine concentration correction for CS 3;

measuring a creatinine sensor current signal of CS 2(Δ I2 ') and a creatinine sensor current signal of CS 3(Δ I3'), a creatinine concentration (CREA _ CS2), a measured creatinine concentration (MCREA _ CS3), and creatine concentrations (CR _ CS2) and (MCR _ CS3) using the creatinine sensors;

determining a first creatinine sensor sensitivity (Slope 1);

determining a second creatinine sensor sensitivity (Slope 2); and

estimating the concentration of creatinine in the sample based on the current signal of the sample and the Slope1 and Slope 2.

2. The method of claim 1, wherein Slope ═ Δ I2/CR _ CS 2.

3. The method of claim 1, wherein Slope1 ═ MCR _ CS3 Δ I2 '-CR _ CS2 Δ I3')/(CREA _ CS2 MCR _ CS 3-MCREA _ CS3 CR _ CS 2).

4. The method according to claim 1, wherein slide 2 ═ CREA _ CS2 Δ I3 '-MCREA _ CS3 Δ I2')/(CREA _ CS2 MCR _ CS 3-MCREA _ CS3 CR _ CS 2).

5. The method of claim 1, wherein the stabilization ratio of creatine to creatinine is about 1.5 to about 2.

6. The method of claim 1, wherein CS2 comprises about 2 to 5mg/dL creatine and about 1 to 3mg/dL creatinine.

7. The method of claim 6, having a creatine to creatinine ratio in CS2 of 1.5 to 2.

8. The method of claim 7, wherein the creatine to creatinine ratio in CS2 is stable for a minimum of 8 months.

9. The method of claim 1, wherein CS3 comprises between about 2mg/dL and about 8mg/dL creatine and between about 0mg/dL and about 1mg/dL creatinine.

10. The method of claim 9, wherein the ratio of creatine to creatinine in CS3 is from about 4 to about 70.

11. The method of claim 1, wherein the creatine concentration correction value is used to correct CREA CS3 based on equimolar creatine to creatinine conversion.

12. A method of calibrating a creatinine/creatine measurement system having a creatine sensor and a creatinine sensor, comprising:

measuring a creatinine sensor current signal (Δ I2 ') of CS2 and a creatinine sensor current signal (Δ I3') of a second calibration solution (CS3) with the creatinine sensor, the second calibration solution (CS3) having an initial known creatine concentration (CR _ CS3), an initial known creatinine concentration (CREA _ CS3), and a creatine to creatinine instability ratio;

determining a first creatinine sensor sensitivity (Slope 1);

determining a second creatinine sensor sensitivity (Slope 2); and

measuring a creatine concentration of a first correction solution (COR1) with the creatine sensor, the first correction solution (COR1) having a known creatine concentration (CR _ COR1), a known creatinine concentration (CREA _ COR1), and a stable ratio of creatine to creatinine;

measuring creatinine concentration of COR1 with the creatinine sensor (CREA — COR 1);

comparing the measured creatine concentration to CR _ COR1 and the measured creatinine concentration to CREA _ COR1 to establish a creatine concentration correction value and a creatinine concentration correction value;

estimating the concentration of creatinine in the sample based on the values of: slope, Slope1, Slope2, creatine correction factors, creatinine correction factors, and creatine and creatinine current signals.

13. The method of claim 12, wherein Slope ═ Δ I2/CR _ CS 2.

14. The method according to claim 12, wherein Slope1 ═ CR _ CS3 ═ Δ I2 '-CR _ CS2 ═ Δ I3')/(CREA _ CS2 ═ CR _ CS 3-CREA _ CS3 ═ CR _ CS 2).

15. The method according to claim 12, wherein slide 2 ═ CREA _ CS2 Δ I3 '-CREA _ CS3 Δ I2')/(CREA _ CS2 ═ CR _ CS 3-CREA _ CS3 ═ CR _ CS 2).

16. The method of claim 12, wherein the stabilization ratio of creatine to creatinine is about 1.5 to about 2.

17. The method of claim 12, wherein CS2 includes about 2 to 5mg/dL creatine and about 1 to 3mg/dL creatinine.

18. The method of claim 17, wherein the ratio of creatine to creatinine in CS2 is 1.5 to 2.

19. The method of claim 18, wherein the creatine to creatinine ratio in CS2 is stable for a minimum of 8 months.

20. The method of claim 12, wherein CS3 comprises between about 2mg/dL and about 8mg/dL creatine and between about 0mg/dL and about 1mg/dL creatinine.

21. The method of claim 20, wherein the ratio of creatine to creatinine in the solution is about 4 to about 70.

22. The method of claim 12, wherein COR1 includes creatine at a concentration between about 0mg/dL and about 2mg/dL and creatinine at a concentration between about 1mg/dL and about 3 mg/dL.

23. The method of claim 12, further comprising the steps of:

measuring a creatine concentration of a second correction solution (COR2) with the creatine sensor, the second correction solution (COR2) having a known creatine concentration (CR _ COR2) and a known creatinine concentration (CREA _ COR2) and a stable ratio of creatine to creatinine; and

creatinine concentration of COR2 was measured using the creatinine sensor (CREA — COR 2).

24. The method of claim 23, wherein Slope1 ═ CR _ CS3 ═ Δ I2 '-CR _ CS2 ═ Δ I3')/(CREA _ CS2 ═ CR _ CS 3-CREA _ CS3 ═ CR _ CS 2).

25. The method according to claim 23, wherein slide 2 ═ CREA _ CS2 Δ I3 '-CREA _ CS3 Δ I2')/(CREA _ CS2 ═ CR _ CS 3-CREA _ CS3 ═ CR _ CS 2).

26. The method of claim 11, wherein the ratio of creatine to creatinine for CS2 is different from the ratio of creatine to creatinine for CS 3.

27. The method of claim 12, wherein the concentration of creatine and creatinine in the first or second correction solutions is maintained at a stability of 95% or better.

28. The method of claim 12, wherein the concentration of creatine and creatinine in the first or second correction solution is maintained by refrigeration.

29. The method of claim 12, wherein a correction factor from the first correction solution or the second correction solution is used to adjust a slope of a creatinine sensor or a creatine sensor.

30. The method of claim 12, wherein the creatine concentration correction value and the creatinine concentration correction value are used to adjust sample results to bias or% correlation.

31. The method of claim 12, further comprising at least one additional calibration solution.

32. A creatinine/creatine measurement system, comprising:

a creatine sensor;

a creatinine sensor;

one or more network interfaces that communicate in a computer network;

a processor coupled to the network interface and the creatine sensor and the creatinine sensor and adapted to perform one or more processes; and

a memory configured to store processing executable by the processor, the processing when executed operable to:

comparing MCR _ CS3 of CS3 with CR _ CS3 to establish a creatine concentration correction for CS 3;

determining a first creatinine sensor sensitivity (Slope 1);

determining a second creatinine sensor sensitivity (Slope 2); and

estimating the concentration of creatinine in the sample based on the current signal of the sample and the Slope1 and Slope 2.

33. A creatinine/creatine measurement system, comprising:

a creatine sensor;

a creatinine sensor;

one or more network interfaces that communicate in a computer network;

a processor coupled to the network interface and the creatine sensor and the creatinine sensor and adapted to perform one or more processes; and

a memory configured to store processing executable by the processor, the processing when executed operable to:

determining a first creatinine sensor sensitivity (Slope 1);

determining a second creatinine sensor sensitivity (Slope 2); and

measuring creatinine concentration of COR1 with the creatinine sensor (CREA — COR 1);

comparing the measured creatinine concentration to CR _ COR1 and the measured creatinine concentration to CREA _ COR1 to establish a creatine concentration correction value and a creatinine concentration correction value; and

Technical Field

The present disclosure relates to electrochemical sensors for measuring creatinine and creatine in a sample. More particularly, the present disclosure relates to compositions and methods for improving the calibration accuracy of electrochemical sensors for measuring (e.g., in the blood of a subject) creatinine and creatine.

Background

The ability to accurately measure creatinine and creatine levels in a patient's blood is an important indicator of kidney (e.g., kidney) health. In particular, serum creatinine is an important indicator of kidney health because it is not excreted by the kidney and is easily measured. For example, elevated levels of serum creatinine are late markers of chronic kidney disease and are typically observed only when severe kidney damage has occurred. Chronic kidney disease refers to a gradual loss of kidney function. The kidney functions to filter waste products and excess fluid from the blood and then expel the filtered waste products and excess fluid in the urine. When chronic kidney disease reaches an advanced stage (e.g., end stage kidney disease), dangerous levels of fluids, metabolites, electrolytes, waste products, etc. accumulate in the body. In the early stages of chronic kidney disease, there may be few signs or symptoms, and the progression of the disease may not become apparent until renal function is significantly impaired.

Creatinine/creatine in a sample (e.g., patient blood) can be measured via an electrochemical sensor. For example, current creatinine sensors may include enzymatic biosensors that include three enzymes: anhydrases, creatininases, and sarcosine oxidases, which catalyze the production of glycine, formaldehyde, and hydrogen peroxide from creatinine and water. These three enzymes may be immobilized on the surface of a platinum electrode, and then hydrogen peroxide (H) may be added₂O₂) Is electrochemically oxidized at a constant polarization potential on a platinum electrode and used to measure creatinine and/or creatine levels in the blood of a patient. However, to determine in a biological sampleThe creatinine and/or creatine concentration of the patient, the creatinine sensor and the creatine sensor need to be calibrated to determine their sensitivity. This can be done by measuring the current response of the creatinine sensor and the creatine sensor with predetermined concentrations of creatinine and creatine in a calibration solution. Once the sensitivity of the creatinine sensor and the creatine sensor is determined, the concentration of creatinine and creatine in any biological sample may be estimated by measuring the current signal of the sample and comparing the measured sensitivity of the creatinine sensor and the creatine sensor determined from the calibration process. Unfortunately, it is not easy to maintain stable creatinine and creatine concentrations in sensor calibration solutions because the hydrolysis of creatinine to creatine is a reversible reaction in aqueous solution. This hydrolysis of creatinine to creatine is accelerated by increasing the storage temperature and/or aging of the calibration solution, and vice versa. Thus, errors in creatinine and creatine concentration values associated with the calibration solution will directly result in errors in the calibration results (i.e., sensor sensitivity), which in turn will propagate errors to all sample results measured using these sensitivities. Therefore, there is a need to identify and develop new methods to improve biosensor calibration accuracy for creatinine and/or creatine measurements.

Disclosure of Invention

The present disclosure provides an electrochemical sensor for measuring creatinine and creatine in a sample. More specifically, the present disclosure provides compositions and methods for improving the calibration accuracy of electrochemical sensors for measuring creatinine and creatine (e.g., blood of a subject).

In one aspect, the present disclosure provides a method of calibrating a creatinine/creatine measurement system having a creatine sensor and a creatinine sensor, the method including the steps of: measuring a creatine sensor current signal (Δ I2) of a first calibration solution (CS2) with a creatine sensor, the first calibration solution (CS2) having a known creatine concentration (CR _ CS2), a known creatinine concentration (CREA _ CS2), and a stabilization ratio of creatine to creatinine, to establish a creatine sensor sensitivity (Slope) of the creatine sensor; measuring a measured creatine concentration (MCR _ CS3) of a second calibration solution (CS3) with a creatine sensor, the second calibration solution (CS3) having an initial known creatine concentration (CR _ CS3), an initial known creatinine concentration (CREA _ CS3), and a creatine to creatinine instability ratio; comparing MCR _ CS3 to CR _ CS3 of CS3 to establish creatine and creatinine concentration correction values for CS3 based on equimolar conversion; measuring a creatinine sensor current signal of CS 2(Δ I2 ') and a creatinine sensor current signal of CS 3(Δ I3'), a creatinine concentration (CREA _ CS2), a measured creatinine concentration (MCREA _ CS3), and creatine concentrations (CR _ CS2) and (MCR _ CS3) using a creatinine sensor; determining a first creatinine sensor sensitivity (Slope 1); determining a second creatinine sensor sensitivity (Slope 2); and estimating the concentration of creatinine in the sample based on the current signal of the sample and Slope1 and Slope 2.

In an embodiment, Slope ═ Δ I2/CR _ CS 2.

In an embodiment, slide 1 ═ MCR _ CS3 Δ I2 '-CR _ CS2 Δ I3')/(CREA _ CS2 MCR _ CS 3-MCREA _ CS3 ═ CR _ CS 2).

In an embodiment, slide 2 ═ CREA _ CS2 Δ I3 '-MCREA _ CS3 Δ I2')/(CREA _ CS2 MCR _ CS 3-MCREA _ CS3 CR _ CS 2).

In embodiments, the stabilization ratio of creatine to creatinine is from about 1.5 to about 2.

In an embodiment, CS2 includes about 2mg/dL to 5mg/dL of creatine and about 1mg/dL to 3mg/dL of creatinine.

In embodiments, the ratio of creatine to creatinine in CS2 is 1.5 to 2.

In the examples, the creatine to creatinine ratio in CS2 was stable for a minimum of 8 months.

In embodiments, CS3 includes between about 2mg/dL and about 8mg/dL creatine and between about 0mg/dL and about 1mg/dL creatinine.

In embodiments, the ratio of creatine to creatinine in CS3 is about 4 to about 70.

In the examples, the creatine concentration correction values are used to correct CREA _ CS3 based on equimolar creatine to creatinine conversion.

In one aspect, the present disclosure provides a method of calibrating a creatinine/creatine measurement system having a creatine sensor and a creatinine sensor, the method including the steps of: measuring a creatine sensor current signal (Δ I2) of a first calibration solution (CS2) with a creatine sensor, the first calibration solution (CS2) having a known creatine concentration (CR _ CS2), a known creatinine concentration (CREA _ CS2), and a stabilization ratio of creatine to creatinine, to establish a creatine sensor sensitivity (Slope) of the creatine sensor; measuring a creatinine sensor current signal (Δ I2 ') of CS2 and a creatinine sensor current signal (Δ I3') of a second calibration solution (CS3) with a creatinine sensor, the second calibration solution (CS3) having an initial known creatine concentration (CR _ CS3), an initial known creatinine concentration (CREA _ CS3), and a creatine to creatinine instability ratio; determining a first creatinine sensor sensitivity (Slope 1); determining a second creatinine sensor sensitivity (Slope 2); measuring a creatine concentration of a first correction solution (COR1) with a creatine sensor, the first correction solution (COR1) having a known creatine concentration (CR _ COR1), a known creatinine concentration (CREA _ COR1), and a stable ratio of creatine to creatinine; measuring creatinine concentration of COR1 using a creatinine sensor (CREA — COR 1); comparing the measured creatinine concentration to CR _ COR1 and the measured creatinine concentration to CREA _ COR1 to establish a creatine concentration correction value and a creatinine concentration correction value; and estimating the creatinine concentration in the sample based on the values of: slope, Slope1, Slope2, and creatine and creatinine current signals.

In an embodiment, Slope ═ Δ I2/CR _ CS 2.

In an embodiment, slide 1 ═ CR _ CS3 ═ Δ I2 '-CR _ CS2 ═ Δ I3')/(CREA _ CS2 ═ CR _ CS 3-CREA _ CS3 ═ CR _ CS 2).

In an embodiment, slide 2 ═ CREA _ CS2 Δ I3 '-CREA _ CS3 Δ I2')/(CREA _ CS2 CR _ CS 3-CREA _ CS3 CR _ CS 2).

In embodiments, the stabilization ratio of creatine to creatinine is from about 1.5 to about 2.

In an embodiment, CS2 includes about 2mg/dL to 5mg/dL of creatine and about 1mg/dL to 3mg/dL of creatinine.

In embodiments, the ratio of creatine to creatinine in CS2 is 1.5 to 2.

In the examples, the creatine to creatinine ratio in CS2 was stable for a minimum of 8 months.

In an embodiment, CS3 includes between about 2mg/dL and about 8mg/dL creatine and between about 0mg/dL and about 1mg/dL creatinine.

In embodiments, the ratio of creatine to creatinine in the solution is from about 4 to about 70.

In an embodiment, COR1 includes creatine at a concentration between about 0mg/dL and about 2mg/dL and creatinine at a concentration between about 1mg/dL and about 3 mg/dL.

In an embodiment, the method further comprises the steps of: measuring a creatine concentration of a second correction solution (COR2) with a creatine sensor, the second correction solution (COR2) having a known creatine concentration (CR _ COR2) and a known creatinine concentration (CREA _ COR2) and a stable ratio of creatine to creatinine; and measuring creatinine concentration of COR2 using a creatinine sensor (CREA — COR 2).

In an embodiment, slide 1 ═ CR _ CS3 ═ Δ I2 '-CR _ CS2 ═ Δ I3')/(CREA _ CS2 ═ CR _ CS 3-CREA _ CS3 ═ CR _ CS 2).

In an embodiment, slide 2 ═ CREA _ CS2 Δ I3 '-CREA _ CS3 Δ I2')/(CREA _ CS2 CR _ CS 3-CREA _ CS3 CR _ CS 2).

In an embodiment, the ratio of creatine to creatinine for CS2 is different from the ratio of creatine to creatinine for CS 3.

In embodiments, the concentration of creatine and creatinine in the first correction solution or the second correction solution is maintained at a stability of 95% or better.

In an embodiment, the concentration of creatine and creatinine in the first correction solution or the second correction solution is maintained by refrigeration.

In an embodiment, the correction factor from the first correction solution or the second correction solution is used to adjust the slope (slope) of the creatinine sensor or the creatine sensor.

In an embodiment, a creatine concentration correction value and a creatinine concentration correction value are used to adjust the sample results to a bias or% correlation.

In embodiments, the method further comprises at least one additional calibration solution.

In one aspect, the present disclosure provides a creatinine/creatine measurement system including: a creatine sensor; a creatinine sensor; one or more network interfaces that communicate in a computer network; a processor coupled to the network interface and the creatine sensor and creatinine sensor and adapted to perform one or more processes; and a memory configured to store processing executable by the processor, the processing when executed operable to:

measuring a creatine sensor current signal (Δ I2) of a first calibration solution (CS2) with a creatine sensor, the first calibration solution (CS2) having a known creatine concentration (CR _ CS2), a known creatinine concentration (CREA _ CS2), and a stabilization ratio of creatine to creatinine, to establish a creatine sensor sensitivity (Slope) of the creatine sensor; measuring a measured creatine concentration (MCR _ CS3) of a second calibration solution (CS3) with a creatine sensor, the second calibration solution (CS3) having an initial known creatine concentration (CR _ CS3), an initial known creatinine concentration (CREA _ CS3), and a creatine to creatinine instability ratio; comparing MCR _ CS3 of CS3 with CR _ CS3 to establish a creatine concentration correction for CS 3; measuring a creatinine sensor current signal of CS 2(Δ I2 ') and a creatinine sensor current signal of CS 3(Δ I3'), a creatinine concentration (CREA _ CS2), a measured creatinine concentration (MCREA _ CS3), and creatine concentrations (CR _ CS2) and (MCR _ CS3) using a creatinine sensor; determining a first creatinine sensor sensitivity (Slope 1); determining a second creatinine sensor sensitivity (Slope 2); and estimating the concentration of creatinine in the sample based on the current signal of the sample and Slope1 and Slope 2.

measuring a creatine sensor current signal (Δ I2) of a first calibration solution (CS2) with a creatine sensor, the first calibration solution (CS2) having a known creatine concentration (CR _ CS2), a known creatinine concentration (CREA _ CS2), and a stabilization ratio of creatine to creatinine, to establish a creatine sensor sensitivity (Slope) of the creatine sensor; measuring a creatinine sensor current signal (Δ I2 ') of CS2 and a creatinine sensor current signal (Δ I3') of a second calibration solution (CS3) with a creatinine sensor, the second calibration solution (CS3) having an initial known creatine concentration (CR _ CS3), an initial known creatinine concentration (CREA _ CS3), and a creatine to creatinine instability ratio; determining a first creatinine sensor sensitivity (Slope 1); determining a second creatinine sensor sensitivity (Slope 2); measuring a creatine concentration of a first correction solution (COR1) with a creatine sensor, the first correction solution (COR1) having a known creatine concentration (CR _ COR1), a known creatinine concentration (CREA _ COR1), and a stable ratio of creatine to creatinine; measuring creatinine concentration of COR1 using a creatinine sensor (CREA — COR 1); comparing the measured creatine concentration to CR _ COR1 and the measured creatinine concentration to CREA _ COR1 to establish a creatine concentration correction value and a creatinine concentration correction value; and estimating the creatinine concentration in the sample based on the values of: slope, Slope1, Slope2, and creatine and creatinine current signals.

By "control" or "reference" is meant a standard of comparison. In one aspect, as used herein, a sample or subject that is "altered as compared to a control" is understood to have a level that is statistically different from a sample from a normal, untreated, or control sample. Control samples include, for example, creatine solution, creatinine solution, and the like. Methods of selecting and testing control samples are within the ability of those skilled in the art. Determination of statistical significance is within the ability of those skilled in the art, e.g., the number of standard deviations from the mean that constitute a positive result.

As used herein, "creatine (also known as 2- [ carbamoyl (methyl) amino ] acetic acid, N-carbamoyl-N-methylglycine, or methylguanidinoacetic acid) refers to an organic compound that produces energy for cells by providing a phosphate group to cycle Adenosine Diphosphate (ADP) back to ATP for Adenosine Triphosphate (ATP). Creatine has the following chemical structure:

as used herein, "creatinine" refers to the enzymatic breakdown by-product of creatine and is typically present in two major tautomeric forms, as shown below.

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It will also be understood that a number of values are disclosed herein, and that each value is also disclosed herein as "about" that particular value, in addition to the value itself. It should also be understood that throughout this application, data is provided in a number of different formats and represents a range of endpoints and starting points and any combination of data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than or equal to, and equal to 10 and 15 are considered disclosed and between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed. Ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. For subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, the nesting subranges of the exemplary ranges 1 to 50 can include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in another direction.

Other features and advantages of the disclosure will be apparent from the following description of the preferred embodiments of the disclosure, and from the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated by reference. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Any one of the embodiments described herein may be combined with any other embodiment or embodiments, even if the embodiments are described in different aspects of the disclosure, where applicable or not explicitly disclaimed.

These and other embodiments are disclosed and/or encompassed by the following detailed description.

Drawings

The following detailed description, given by way of example and not intended to limit the disclosure solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which:

fig. 1A-1B show two graphs depicting the deviation of creatinine measured by a whole blood creatinine biosensor relative to plasma creatinine on a (vs.) chemical analyzer in two study groups using an analysis package (cartidge) with the same CS3 reagent under different storage conditions: the data points in the green circle are the assay packages with CS3 reagent stored at 25 ℃ for 4 months, while the data points in the blue diamonds are the assay packages with CS3 reagent stored at 15 ℃ for 8 months. Fig. 1A shows creatinine measurements without real-time creatine correction, while fig. 1B shows creatinine measurements where creatine and creatinine are corrected based on real-time measurement of creatine by a creatine sensor according to exemplary embodiments of the present disclosure. The dashed line provides an acceptable deviation limit (e.g., total allowable error, TEa) when comparing test results from whole blood analysis of creatinine values relative to plasma reference values.

Fig. 2A-2B show two graphs depicting the deviation of creatinine measured by a whole blood creatinine biosensor relative to plasma creatinine on a chemical analyzer. Figure 2A shows creatinine results without the on-board measurement of creatine and two correction solutions. Figure 2B shows creatinine results from the same sample but with onboard measurements of creatine and two correction solutions according to an exemplary embodiment of the present disclosure.

Fig. 3 is a process flow diagram for on-board calibration of a creatine sensor and a creatinine sensor according to an exemplary embodiment of the present disclosure.

Fig. 4 is a process flow diagram for external calibration of creatine sensors and creatinine sensors according to an exemplary embodiment of the present disclosure.

Fig. 5 shows a simplified procedure for on-board calibration of creatine sensors and creatinine sensors according to an exemplary embodiment of the present disclosure.

Fig. 6 shows a simplified procedure for external calibration of creatine sensors and creatinine sensors according to an exemplary embodiment of the present disclosure.

Detailed Description

The present disclosure is based (at least in part) on the following findings: the 3-point calibration process can be used to accurately calibrate creatinine sensors in creatine and creatinine biosensor systems that can be implemented at room temperature. Advantageously, the present disclosure provides compositions and methods that enable accurate creatine/creatinine sensor calibration using either internal calibration solutions or external calibration solutions, which in turn allow accurate detection of creatine and/or creatine in a biological or laboratory sample.

Overview

Current creatinine sensors (e.g., GEM PAK analysis package) in the creatine/creatinine system include enzymatic biosensors containing three enzymes. These enzymes are immobilized on the surface of a platinum electrode. The creatinine detection system is based on the following three enzyme cascades (Rx):

product hydrogen peroxide (H)₂O₂) Is then electrochemically oxidized at a platinum electrode at a constant polarization potential, and the current signal is proportional to the analyte concentration.

The presence of creatine in clinical samples necessitates an additional sensor for creatine measurement to correct the creatine response of creatinine sensors. The creatine sensor includes only reactions (2) and (3) of the above enzyme cascade.

Both creatine and creatinine sensors have a diffusion control membrane (also referred to as the outer membrane) on top of the enzyme layer. The diffusion control membrane limits the flux of creatinine substrates and creatine substrates (creatinine and creatine substrates) into the enzyme layer to ensure that the signal generated by hydrogen peroxide is proportional to the substrate concentration of the sample.

To determine the respective concentrations of creatinine and creatine in a biological sample, the creatinine sensor and the creatine sensor need to be calibrated to determine their respective sensitivities. This may be accomplished by comparing the readings of the creatinine sensor and the creatine sensor in a calibration solution containing predetermined (e.g., known) concentrations of creatinine and creatine. Once the sensitivity of the creatinine sensor and the creatine sensor is determined, the concentration of creatine and creatinine in any biological sample can be estimated by adjusting the measured readings using the results determined from the calibration process.

Theoretically, the creatinine measurement system as described above can quantitatively measure the concentration of creatinine in a biological sample. However, there are several practical problems associated with reagent stability, which presents challenges for accurately measuring creatinine. For example, as shown in the following figure, the hydrolysis of creatinine to creatine is a reversible process in aqueous solution that may vary in a manner dependent on temperature and solution medium until an optimal stable ratio of creatine/creatinine is reached (e.g., about 1.5 to 2). When the creatine/creatinine ratio is above or below the optimal range, creatine will be converted to creatinine or creatinine will be converted to creatine. Thus, the concentration of creatinine and creatine in the calibration solution will vary over time during storage from the original factory-dispensed concentration, as shown in rx.4.

Disadvantageously, this makes it difficult to accurately calibrate the creatinine sensor.

In addition, conversion between creatinine and creatine under storage conditions will alter the creatine/creatinine ratio until an optimal stabilization ratio is reached. Unfortunately, this introduces calibration errors into the sensor measurements, and these errors become more pronounced over time as the calibration solution ages. This results in limited shelf life and overall assay performance of the reagent. One prior art solution to this problem is to confirm through experimentationShelf life decay curve (e.g., equation [ CR _ CS ]) for known storage temperatures]＝[CR_CS]₀-a lifetime-b lifetime ^2, wherein [ CR _ CS ^2]₀Is the creatine concentration value initially manufactured to be assigned to the calibration solution, constants a and b are determined by experimentation, and lifetime is the shelf life of the calibration solution) to account for the conversion of creatinine to creatine in the standard solution during storage. By using this decay curve, real-time corrections can be correlated to factory-assigned creatinine/creatine concentrations based on the length of storage. A limitation of this approach is that calibration based on the decay curve assumes that the calibration solution is exposed to a constant temperature over time. Unfortunately, this assumption is inaccurate because the calibration solution/reagent may be exposed to temperatures that can sometimes fluctuate dramatically over time (e.g., during transfer between facilities). In this case, errors may be introduced in sensor calibration if the decay curve used is based on a calibration solution that suffers significant temperature fluctuations that affect the ability of the creatinine sensor to accurately report creatinine concentrations in a calibration solution/reagent (e.g., creatine/creatinine solution) that is stored for, for example, 8 months or longer.

Other prior art methods to minimize creatinine/creatine conversion include refrigerating the reagent (e.g., creatine/creatinine powder or solution), limiting the shelf life of the calibration solution to weeks, and/or preparing the reagent/calibration solution on-site at the customer's factory by mixing creatinine and creatine powders into the solution just prior to use. Unfortunately, all of these prior art methods are impractical for point-of-care (point-of-care) use at a clinical testing site.

The technology herein addresses the above-listed prior art problems of creatine/creatinine corrected solution instability (e.g., conversion of creatine/creatinine in a corrected solution during storage) by minimizing the variation in measured biological creatinine levels. As further described below, the following steps may be implemented to account for the effects of calibration errors due to variations in the creatine/creatinine ratio in the calibration solution during transportation and/or storage:

1. the creatine sensor is installed and calibrated.

2. The creatine concentrations of the second calibration solution (e.g., CS3) and additional calibration solutions not used for creatine sensor calibration are measured to assess changes in the concentration of the second calibration solution during transport and storage.

3. Since creatine is assumed to be converted to creatinine or creatinine is assumed to be converted to creatine, changes in creatine concentration (in molar equivalent concentrations) relative to factory-assigned values are used to correct for changes in creatinine concentration during storage.

4. The creatine and creatinine corrections are applied to a second calibration reagent for creatinine prior to completion of the creatinine sensor calibration process, and the same corrections are continued throughout the life of the sensor.

The technology herein provides a system for calibrating a creatine and/or creatinine biosensor.

A calibration system for a creatine sensor or creatine biosensor may involve a 2-point calibration based on the following equation:

Δ I2 ═ CR _ CS2 × Slope (equation 1)

Δ I2 is the current signal measured on the creatine sensor in the first calibration solution (CS 2). [ CR _ CS2] is the concentration of creatine in the first calibration solution (CS 2). As discussed in detail below, CS2 may have a known creatine concentration (CR _ CS2), a known creatinine concentration (CREA _ CS2), and a steady ratio of creatine to creatinine, which enables the creatine sensor sensitivity (Slope) of the creatine sensor to be established.

In accordance with the techniques herein, a calibration system for a creatinine sensor or a creatinine biosensor may implement a 3-point calibration method. Since the creatinine sensor provides readings of creatinine and creatine in a biological sample or a calibration solution containing two analytes, the sensitivity of the creatinine sensor to creatinine (Slope1) or creatine (Slope2) can be determined according to the following equations 2 to 5 of the present disclosure, as defined below. The present disclosure provides that two calibration solutions with different ratios of creatine/creatinine can be used in a 3-point calibration method.

3-point creatinine sensor calibration equation:

Δ I2' ═ CREA _ CS2] · Slope1+ [ CR _ CS2 ]. Slope2 (equation 2)

Δ I3' ═ CREA _ CS3] · Slope1+ [ CR _ CS3 ]. Slope2 (equation 3)

Δ I2 'and Δ I3' are the current signals measured on the creatinine sensors in the first calibration solution (CS2) and the second calibration solution (CS3), respectively. CS3 may have an initial known creatine concentration (CR _ CS3), an initial known creatinine concentration (CREA _ CS3), and a creatine to creatinine instability ratio.

[ CREA _ CS2], [ CREA _ CS3], [ CR _ CS2] and [ CR _ CS3] indicate the initial known concentrations of creatinine and creatine in calibration solutions CS2 and CS3, respectively. The sensitivity of the creatinine sensor to creatinine Slope1 (sensor sensitivity to creatinine) and the sensitivity to creatine Slope2 (sensor sensitivity to creatine) can be derived by equations 2 and 3.

Slope1 ═ ([ CR _ CS3 ]. DELTA.i 2 '- [ CR _ CS2 ]. DELTA.i 3')/([ Creat _ CS2 ]. DELTA.cr _ CS3] - [ Creat _ CS3 ]. DELTA.cr _ CS2]) pA/mg/dL (equation 4)

Slope2 ═ ([ CREA _ CS2 ]. DELTA.i 3 '- [ CREA _ CS3 ]. DELTA.i 2')/([ Creat _ CS2 ]. DELTA.cr _ CS3 ]. DELTA.creat _ CS3 ]. DELTA.cr _ CS2]) pA/mg/dL (equation 5)

CS2 may be formulated with an optimal stability ratio of creatine/creatinine of between about 1.5 and 2, while CS3 may be formulated with a different creatine/creatinine instability ratio (about 10 to 70) that will vary over time from the original ratio at the time of manufacture due to concentration variations during storage. This variation in creatine and creatinine concentrations from the original manufacturing stage introduces calibration errors, and hence sample measurement errors, if not corrected for.

In accordance with the present disclosure, the calibration solutions CS2 and CS3 may be used in "on-board" applications (e.g., within a biosensor device or an analysis package).

As described herein, the present disclosure also provides such correction solutions: it is designed to be stable at factory-dispensed creatinine and creatine concentrations, either through an appropriate creatine/creatinine ratio or through optimal storage conditions. Such correction solutions may be used in "external" applications, such as, for example, during initial calibration of creatinine and/or creatine sensor systems. Once these correction solutions are measured with separate creatinine and/or creatine sensors, deviations from the measured creatinine and creatine concentrations can be determined by comparison to factory-assigned creatinine and creatine concentrations, which are then used as correction factors for all subsequent biological samples. The correction factor can be used to minimize the effect of any residual error in creatinine and myoacid number assignment for a particular assay package, and can be used continuously throughout the life of the sensor.

The novel creatinine/creatine sensor calibration system described herein may include the steps of: 1) correcting the creatine/creatinine concentration of a calibration solution that may not have a stable creatinine/creatine ratio; 2) calibrating the sensor; 3) establishing a correction factor; and 4) performing a sample measurement. These steps will be described in detail below:

1. it was established that the creatinine and creatine concentrations of the calibration solution were not at the optimal creatine/creatinine ratio. In this exemplary creatinine/creatine sensor calibration system, there are three calibration solutions: calibration solution 1(CS1), calibration solution 2(CS2), and calibration solution 3(CS 3). CS1 contained no creatinine or creatine and was used for baseline measurements (e.g., blank control). Both CS2 and CS3 contain creatinine and creatine concentrations in varying proportions. CS2 has a creatine/creatinine ratio of about 1.5 to 2, which is stable and can maintain factory-assigned creatinine and creatine concentrations for at least 8 months throughout shelf life without significant change. CS3 has a different creatine/creatinine ratio compared to CS2, which is unstable and will change over time upon storage (e.g., by slowly converting from creatine to creatinine during storage of the solution). When creatinine and creatine sensors are installed and exposed to a calibration solution that has aged with the shelf life of the solution but is within specified solution storage temperature conditions (15 ℃ -25 ℃), creatine sensor sensitivity can be accurately determined by measuring the current in CS2 and applying the factory-dispensed creatine concentration of CS 2. The measured current on the creatine sensor and the creatine sensitivity are then used to measure the creatine concentration in CS3 and/or the external correction solution. The concentration of creatine in CS3 that has been converted to creatinine can be derived from the difference between factory-assigned creatine concentration values and the measured creatine concentration values in CS 3. The increase in creatinine in CS3 can then be estimated by using the molar equivalent concentration of creatine concentration changes in CS3 as a correction to the factory-dispensed creatinine concentration. Thus, both creatinine and creatine concentrations in CS3 are determined in real time during initial sensor calibration from the beginning of sensor installation without applying an attenuation curve (e.g., by means of an equation).

2. And (6) calibrating the sensor. Each creatinine measurement system includes both a creatinine sensor and a creatine sensor. And periodically calibrating the sensor using a calibration solution throughout the lifetime of the sensor. The sensitivity of the creatinine sensor and the creatine sensor can be calculated as described in the background section previously. These sensitivities can then be used for subsequent sample measurements.

The sensitivity of the creatine sensor was determined by measuring the current in the CS2 solution and the factory-assigned CS2 creatine concentration, as shown in equation 1 above.

Creatinine sensitivity was measured by the following procedure (sensitivity to creatinine was Slope1 and sensitivity to creatine was Slope2 as defined in equations 3 and 4): measuring the current in CS2 and CS 3; comparing these measurements (measurements) to factory assigned creatinine and creatine concentrations in CS 2; and applying the real-time correction to factory-assigned creatinine and creatine concentrations in CS3 at sensor installation and initial calibration as shown in equations 2 through 5 above.

3. In addition, external calibration may be accomplished using one or more calibration solutions. For example, two correction solutions, e.g., a first correction solution (COR1) and a second correction solution (COR2) with stable creatine/creatinine concentrations, may be used to correct any residual errors from the calibration accuracy. These solutions have stable predetermined creatinine and creatine concentrations (e.g., the creatinine and creatine concentrations of COR1 may range from about 0mg/dL to 2mg/dL and 1mg/dL to 3mg/dL, respectively, while the creatinine and creatine concentrations of COR2 may range fromAbout 0mg/dL to 1mg/dL and 2mg/dL to 8mg/dL, respectively). For example, these solutions can be sealed in reagent bags or ampoules and designed to maintain a constant pCO₂pH level and creatinine/creatine level. These solutions may be buffered and/or contain biocides to ensure stability. It is important that the creatinine and creatine concentrations of these two solutions remain stable during their use. This can be achieved in a number of ways known to the skilled person. For example, an external calibration solution may be sealed in an ampoule to maintain repeatability of use in calibrating the sensor from assay package to assay package. In yet another example, the correction solution may be refrigerated and/or also packaged in ampoules.

Once the sensor calibration is complete and the sensitivity is established, the creatinine/creatine correction solution can be measured on each creatinine measurement system at the beginning of its use. Creatinine and creatine concentrations measured by the sensor are compared to factory-assigned creatinine and creatine levels and used to establish correction factors for the creatinine sensor and the creatine sensor, respectively.

Alternatively, two correction solutions may be measured continuously at the beginning of the life span and the correction factor may be determined more accurately based on two different creatinine and creatine concentration levels. These correction factors provide correction for residual errors from the calibration process, including shelf life decay for that particular analysis package.

Since these correction solutions have precise predetermined creatinine and creatine concentrations and remain stable and outside the creatinine measurement system, they can be used as independent checks of the calibration accuracy of the creatinine measurement system. Thus, a tolerance range for accepting or rejecting the creatinine measurement system is established based on the creatinine and creatine report results from this analysis package of calibration solutions.

4. Creatinine/creatine is measured in a biological sample. For each patient sample, the current signal was measured on a creatinine sensor and a creatine sensor. After the dc reading, the sensitivity from the latest calibration was used to estimate creatinine and creatine concentrations. Finally, correction factors may be applied to obtain final creatinine and creatine results.

In an alternative embodiment, when two calibration solutions are used, the calibration may be applied to the sensitivity obtained from the calibration process. In this method, the correction factors for the sensitivities obtained from the two correction solutions from the initial calibration can be applied directly to the sensitivity from the latest sensor calibration. The sample creatinine and creatine concentrations are then estimated based on the sample readings and corrected sensitivity.

Fig. 5 shows an example of a simplified procedure for on-board calibration of a creatine sensor and a creatinine sensor according to an exemplary embodiment of the present disclosure. For example, a non-generic, specially configured device (e.g., a GEM Premier analyzer) may execute the program 100 by executing, for example, stored instructions. The procedure 100 may begin at step 105 and continue to step 110, where, as described in more detail above, in the apparatus, there is a creatine sensor, a creatinine sensor, one or more network interfaces in communication in a computer network, a processor coupled to the network interfaces and the creatine sensor and creatinine sensor and adapted to perform one or more processes, and a memory configured to store the processes executable by the processor and to provide on-board calibration of the creatine sensor and creatinine sensor.

In step 110, the device measures a creatine sensor current signal (Δ I2) of a first calibration solution (CS2) with a creatine sensor, the first calibration solution (CS2) having a known creatine concentration (CR _ CS2), a known creatinine concentration (CREA _ CS2), and a stabilization ratio of creatine to creatinine, to establish a creatine sensor sensitivity (Slope) of the creatine sensor.

In step 115, the device measures the measured creatine concentration (MCR _ CS3) of a second calibration solution (CS3) with a creatine sensor, the second calibration solution (CS3) having an initial known creatine concentration (CR _ CS3), an initial known creatinine concentration (CREA _ CS3), and a creatine to creatinine instability ratio.

The simplified procedure may then proceed to step 120, where the apparatus compares MCR _ CS3 of CS3 with CR _ CS3 to establish creatine and creatinine concentration correction values for CS3 based on equimolar conversion (equivalent molar conversion) in step 120.

In step 125, the device measures the creatinine sensor current signal of CS 2(Δ I2 ') and the creatinine sensor current signal of CS 3(Δ I3'), the creatinine concentration (CREA _ CS2), (MCREA _ CS3), and the creatine concentrations (CR _ CS2) and (MCR _ CS3) with the creatinine sensors. In step 130, the apparatus determines a first creatinine sensor sensitivity (Slope1) and a second creatinine sensor sensitivity (Slope 2).

In step 135, the device then estimates the concentration of creatinine in the sample based on the current signal of the sample and the Slope1 and Slope 2. The process 100 then terminates at step 140.

Fig. 6 shows a simplified procedure for external calibration of creatine sensors and creatinine sensors according to an exemplary embodiment of the present disclosure. For example, a non-generic, specially configured device (e.g., a GEM Premier analyzer) may execute the program 200 by executing, for example, stored instructions. The procedure 200 may begin at step 205 and continue to step 210, where the apparatus has a creatine sensor, a creatinine sensor, one or more network interfaces in communication in a computer network, a processor coupled to the network interfaces and the creatine sensor and creatinine sensor and adapted to perform one or more processes, and a memory configured to store the processes executable by the processor and to provide on-board calibration of the creatine sensor and creatinine sensor, as described in more detail above.

In step 210, the device measures a creatine sensor current signal (Δ I2) of a first calibration solution (CS2) with a creatine sensor, the first calibration solution (CS2) having a known creatine concentration (CR _ CS2), a known creatinine concentration (CREA _ CS2), and a stabilization ratio of creatine to creatinine, to establish a creatine sensor sensitivity (Slope) of the creatine sensor.

In step 215, the device measures a creatinine sensor current signal (Δ I2 ') of CS2 and a creatinine sensor current signal (Δ I3') of a second calibration solution (CS3) with a creatinine sensor, the second calibration solution (CS3) having an initial known creatine concentration (CR _ CS3), an initial known creatinine concentration (CREA _ CS3), and a creatine to creatinine instability ratio.

The simplified procedure may then proceed to step 220 where the device determines a first creatinine sensor sensitivity (SLOPE1) and a second creatinine sensor sensitivity (SLOPE2) in step 220.

In step 225, the device measures the creatine concentration of the first correction solution (COR1) with a creatine sensor, the first correction solution (COR1) has a known creatine concentration (CR _ COR1), a known creatinine concentration (CREA _ COR1), and a stable ratio of creatine to creatinine, and measures the creatinine concentration of COR1 with a creatinine sensor (CREA _ COR 1).

In step 230, the device measures creatinine concentration of COR1 with a creatinine sensor (CREA — COR 1).

In step 235, the device then compares the measured creatine concentration to CR _ COR1 and the measured creatinine concentration to CREA _ COR1 to establish a creatine concentration correction value and a creatinine concentration correction value.

In step 240, the device estimates creatinine concentration in the sample based on the values of SLOPE, SLOPE1, SLOPE2, creatine correction factor, creatinine correction factor, and creatine and creatinine current signals. The routine 200 then terminates at step 245.

The present disclosure addresses the problem of creatinine and creatine sensor sensitivity bias due to shelf life decay of the calibration solution. As described in the working examples below, the techniques herein have reduced the reporting bias for creatinine in clinical samples and extended the shelf life of the assay package to at least 5 months. It is well known that the conversion of creatinine to creatine over shelf life will affect the sensitivity of the sensor if not properly handled. The present disclosure is also applicable to creatinine measurement systems using calibration solutions that are not at an optimal stable creatine/creatinine ratio.

Reagent kit

The present disclosure also provides kits comprising the reagents of the present disclosure for use in the methods of the present disclosure. Kits of the present disclosure may include one or more containers containing the reagents of the present disclosure (e.g., creatine, creatinine, etc.) and/or may contain reagents in one or more solutions (e.g., CS1, CS2, CS3, calibration solutions (e.g., COR1 and COR2, etc.)) used to calibrate the creatine and/or creatinine sensors. In some embodiments, the kit further comprises instructions for use of the methods according to the present disclosure. In some embodiments, the instructions include a description of how to apply the reagent/solution to a sensor (e.g., a creatine sensor, a creatinine sensor, etc.) and how to calculate a variable of interest (e.g., Δ I2, Δ I2 ', Δ I3', Slope1, Slope2, etc.) according to any of the methods of the present disclosure. In some embodiments, the instructions include a description of how to install and calibrate the measurement systems disclosed herein.

The instructions typically include information about reagent/solution concentrations, reagent/solution ratios (e.g., creatine/creatinine ratios), shelf life, and the like. The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the reagent/solution can be used to calibrate any of a variety of creatine and/or creatinine sensors for use in a measurement system as described herein. For example, instructions for practicing any of the methods described herein can be provided to install and calibrate a measurement system.

The kits of the present disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, ampoules, bottles, jars, flexible packaging (e.g., sealed mylar or plastic bags), and the like. Packaging for use in conjunction with specific devices, such as the GEM Premier whole blood analyzer family (instrumentation laboratories, bedford, massachusetts), is also contemplated. In certain embodiments, at least one active agent in the agent or solution is creatine and/or creatinine.

The kit may optionally provide other components, such as buffers and explanatory information. Typically, a kit includes a container and a label or package insert on or associated with the container.

The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and transgenic biology, which are within the skill of the art. See, for example: maniatis et al, 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); sambrook et al, 1989, Molecular Cloning,2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); sambrook and Russell,2001, Molecular Cloning,3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); ausubel et al, 1992), Current Protocols in Molecular Biology (John Wiley & Sons, included periodic updates); glover,1985, DNA Cloning (IRL Press, Oxford); anand, 1992; guthrie and Fink, 1991; harlow and Lane,1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); jakoby and patan, 1979; nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); transformation And transformation (B.D. Hames & S.J. Higgins eds. 1984); culture Of Animal Cells (r.i. freshney, Alan r.loss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the threading, Methods In Enzymology (Academic Press, Inc., N.Y.); gene Transfer Vectors For Mammarian Cells (J.H.Miller and M.P.Calos eds.,1987, Cold Spring Harbor Laboratory); methods In Enzymology, Vols.154and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer And Walker, eds., Academic Press, London, 1987); handbook Of Experimental Immunology, Volumes I-IV (D.M.Weir and C.C.Blackwell, eds., 1986); riott, Essential Immunology,6th Edition, Blackwell Scientific Publications, Oxford, 1988; hog et al, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); westerfield, M.A. The zebrafish book.A. guide for The laboratory use of zebrafish (Danio relay), (4th Ed., Univ.of Oregon Press, Eugene, 2000).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The disclosure also relates to computer systems related to performing the methods of the disclosure in relation to computing and ordering.

The computer system (or digital device) may be used to receive, transmit, display and/or store results, analyze results, and/or generate reports of results and analysis. A computer system may be understood as a logical device that can read instructions from a medium (e.g., software) and/or a network port (e.g., from the internet), which can optionally be connected to a server having a fixed media. The computer system may include one or more of a CPU, a disk drive, an input device such as a keyboard and/or mouse, and a display (e.g., monitor). Data communication such as transmission of instructions or reports may be accomplished through a communication medium to a server at a local or remote location. A communication medium may include any device that transmits and/or receives data. For example, the communication medium may be a network connection, a wireless connection, or an internet connection. Such connections may provide for communication over the world wide web. It is contemplated that data related to the present disclosure may be transmitted over such a network or connection (or any other suitable means for transmitting information, including but not limited to mailing a physical report such as a print) for receipt and/or for viewing by a recipient. The recipient may be, but is not limited to, a person or an electronic system (e.g., one or more computers and/or one or more servers).

In some embodiments, the computer system may include one or more processors. The processor may be associated with one or more controllers, computing units, and/or other units of the computer system or embedded in firmware as desired. If implemented in software, the routines may be stored in any computer readable memory (such as RAM, ROM, flash memory, magnetic disk, laser disk, or other suitable storage medium). Likewise, the software may be transferred to the computing device via any known transfer method, including, for example, over a communication channel such as a telephone line, the internet, a wireless connection, or via a removable medium such as a computer readable disk, flash drive, or the like. Various steps may be implemented as various blocks, operations, tools, modules, and techniques, which in turn may be implemented in hardware, firmware, software, or any combination of hardware, firmware, and/or hardware. When implemented in hardware, some or all of the blocks, operations, techniques, etc., may be implemented in, for example, a custom Integrated Circuit (IC), an Application Specific Integrated Circuit (ASIC), a field programmable logic array (FPGA), a Programmable Logic Array (PLA), etc.

A client-server, relational database architecture may be used in embodiments of the present disclosure. The client-server architecture is a network architecture that: wherein each computer or process on the network is a client or server. Server computers are typically powerful computers dedicated to managing disk drives (file servers), printers (print servers), or network traffic (web servers). Client computers include a PC (personal computer) or workstation on which a user runs applications, and example output devices as disclosed herein. Client computers rely on server computers to obtain resources such as files, devices, and even processing power. In some embodiments of the present disclosure, the server computer processes all database functions. The client computer may have software that handles all front-end data management and may also receive data input from a user.

A machine-readable medium, which may include computer-executable code, may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer, etc., such as may be used to implement the databases shown in the figures. Volatile storage media includes dynamic memory, such as the main memory of such computer platforms. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media can take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during Radio Frequency (RF) and Infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch-cards, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

The subject computer executable code may be executed on any suitable device that may include a processor including a server, a PC, or a mobile device such as a smartphone or tablet. Any controller or computer optionally includes a monitor, which may be a cathode ray tube ("CRT") display, a flat panel display (e.g., active matrix liquid crystal display, etc.), or the like. The computer circuitry is typically housed in a box that includes a number of integrated circuit chips, such as microprocessors, memory, interface circuits, and the like. The box may also optionally include a hard disk drive, a floppy disk drive, a high capacity removable drive (such as a writable CD-ROM), and other common peripheral elements. An input device such as a keyboard, mouse, or touch sensitive screen optionally provides input from a user. The computer may include suitable software for receiving user instructions, which may be in the form of user inputs into a set of parameter fields, for example in a GUI, or pre-programmed instructions, for example, pre-programmed for various different specific operations.

Reference will now be made in detail to exemplary embodiments of the present disclosure. While the disclosure will be described in conjunction with the exemplary embodiments, it will be understood that they are not intended to limit the disclosure to those embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the disclosure as defined by the appended claims. Standard techniques known in the art or those specifically described below are utilized.

Examples of the invention

The present disclosure is further illustrated by the following examples, which should not be construed as limiting. The contents of all references cited in this application, as well as the published patents and patent applications, are incorporated herein by reference. Those skilled in the art will recognize that the present disclosure may be practiced with modification to the disclosed structures, materials, compositions, and methods, and such modifications are considered within the scope of the present disclosure.

Example 1: performance of a whole blood creatinine sample measured by a creatinine sensor relative to plasma creatinine measured by a chemical analyzer.

In this study, each test analysis package contained a creatinine measurement system consisting of a creatinine sensor, a creatine sensor, and three calibration solutions (CS1, CS2, CS3), where both sets of analysis packages had the same reagents, but the CS3 reagent was subjected to different storage conditions: the data points in the green circles are the assay packages with CS3 reagent stored at 25 ℃ for 4 months, while the data points in the blue diamonds are the assay packages with CS3 reagent stored at 15 ℃ for 8 months. Two methods of establishing CS3 creatine and creatinine concentrations for creatinine calibration were investigated: option (a) which does not make real-time creatine correction to CS 3; option (b), as described in this disclosure [ step 1, page 10 ], corrects creatine and creatinine in CS3 based on creatine measured in real time by a creatine sensor. Figure 1A shows that the deviation of reported creatinine between the two CS3 groups is significant by applying option (a) to creatinine calibration (see data points between green circles and blue diamonds). Figure 1B demonstrates that by applying option (B) to creatinine calibration, the deviation in reported creatinine between the two CS3 groups was significantly reduced across the entire sample range to meet the designed clinical specifications (again see data points between green circles and blue diamonds).

Example 2: deviation plot of whole blood creatinine samples measured by GEM PAK assay package versus plasma creatinine measured by chemical analyzer with and without current technology.

In this study, each test analysis package contained a creatinine measurement system plus two external calibration solutions as in the previous example. The creatinine measurement system consisted of a creatinine sensor, a creatine sensor and three calibration solutions (CS1, CS2 and CS 3). All test assay packages had the same CS3 reagent stored for 3 months at ambient conditions. Two methods of establishing CS3 creatine and creatinine concentrations for creatinine calibration and calibration corrections were investigated: option (a) that does not apply real-time creatine correction to CS3 and does not apply secondary correction; and option (b), creatine and creatinine in CS3 are corrected based on creatine measured in real time by a creatine sensor, and then corrected a second time at the beginning of the analysis package lifetime based on the measurements of the two external correction solutions (as described in this disclosure starting from steps 1-4 on pages 10-13). Figure 2A shows, by applying option (a) to creatinine calibration, that the deviation of creatinine reported in all blood samples is clearly negatively biased and somewhat dispersed with respect to acceptable clinical indicators (see green circles data points versus dashed lines). Figure 2B shows that by applying option (B) to creatinine calibration, both the negative bias and dispersion of creatinine reported in blood samples are significantly reduced across the entire sample range to meet the designed clinical specifications (again see green circles data points versus dashed lines).

Incorporation by reference

All documents cited or referenced herein and all documents cited or referenced in the documents cited herein, as well as any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein, or any document incorporated by reference herein, are incorporated by reference herein, and may be employed in the practice of the present disclosure.

Equivalents of

It should be understood that the detailed examples and embodiments described herein are given by way of illustration only, and are not to be taken by way of limitation of the present disclosure. Various modifications or changes based on the detailed examples and embodiments described herein will be suggested to those skilled in the art, and are intended to be included within the spirit and purview of this application and scope of the appended claims. Other advantageous features and functions associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

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