阅读说明：本技术 用于改进的肌酸酐测量准确度的组合物和方法及其用途 (Compositions and methods for improved accuracy of creatinine measurements and uses thereof ) 是由 徐晓贤 P.帕米迪 D.莱蒙迪 R.马尔霍特拉 M.柯利 于 2019-06-03 设计创作，主要内容包括：本公开内容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。更特别地,本公开内容涉及改进用于测量肌酸酐和肌酸的电化学传感器的测量准确度的组合物和方法。(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 measurement accuracy of electrochemical sensors for measuring creatinine and creatine.)

1. A method of improving the accuracy of a creatinine/creatine measurement system having a calibrated creatine sensor and a calibrated creatinine sensor, the method comprising:

measuring a measured creatine sensor current signal (M Δ I2) with a calibrated creatine sensor for a first biomatrix correction solution (BMCS2) having a known concentration of creatine (CR _ BMCS2) and a known concentration of creatinine (CREA _ BMCS 2);

determining a measured concentration of creatine in BMCS2 (MCR _ BMCS 2);

comparing MCR _ BMCS2 to CR _ BMCS2 to determine creatine sensor biomatrix correction factor (Δ CR);

measuring a measured creatinine sensor current signal (M Δ I2') of BMCS2 with a calibrated creatinine sensor;

determining the measured concentration of creatinine in BMCS2 (MCREA — BMCS 2); and

MCREA _ BMCS2 was compared to CREA _ BMCS2 to determine the creatinine sensor biomatrix correction factor (Δ CREA).

2. The method of claim 1, wherein CR _ BMCS2 and CREA _ BMCS2 have stable values.

3. The method of claim 2, wherein the stabilized value is based on a stabilized ratio of creatine to creatinine in BMCS 2.

4. The method of claim 2, wherein the stabilized value is based on the storage temperature of BMCS 2.

5. The method of claim 1, wherein BMCS2 comprises one or more biomatrix factors selected from the group consisting of: ca⁺⁺，HCO₃ ^-，pH，pCO₂，pO₂Other electrolytes, and metabolites.

6. The method of claim 1, wherein Δ CR is applied to creatine measurements obtained from a biological matrix sample.

7. The method of claim 1, wherein Δ CREA is applied to creatinine measurements obtained from a biological matrix sample.

8. The method of claim 1, further comprising:

measuring a second measured creatine sensor current signal (M2 Δ I2) with a calibrated creatine sensor for a second biomatrix correction solution (BMCS3) having a known concentration of creatine (CR _ BMCS3) and a known concentration of creatinine (CREA _ BMCS 3);

determining the measured concentration of creatine in BMCS3 (M2CR _ BMCS 3);

comparing M2CR _ BMCS3 to CR _ BMCS3 to determine a second creatine sensor biomatrix correction factor (Δ CR');

measuring a measured creatinine sensor current signal (M2 Δ I2') of BMCS3 with a calibrated creatinine sensor;

determining the measured concentration of creatinine in BMCS3 (M2CREA — BMCS 3); and

m2CREA _ BMCS3 was compared to CREA _ BMCS3 to determine creatinine sensor biomatrix correction factor (Δ CREA').

9. The method of claim 8, wherein CR _ BMCS3 and CREA _ BMCS3 have stable values.

10. The method of claim 9, wherein the stabilized value is based on a stabilized ratio of creatine to creatinine in BMCS 3.

11. The method of claim 9, wherein the stable value is based on the storage temperature of BMCS 3.

12. The method of claim 8, wherein BMCS3 comprises one or more biomatrix factors selected from the group consisting of: ca⁺⁺，HCO₃ ^-，pH，pCO₂，pO₂Other electrolytes, and metabolites.

13. The method of claim 1, wherein Δ CR' is applied to a slope of a creatine measurement obtained from a biological matrix sample.

14. The method of claim 8 wherein Δ CR, Δ CR ', Δ CREA and Δ CREA' are applied to slope 1 and slope 2 of creatinine measurements obtained from a biomatrix sample.

15. The method of claim 3 or 10, wherein the stable ratio of creatine to creatinine is about 1.5 to about 2.

16. The method of claim 3 or 10, wherein the stable ratio of creatine to creatinine is 1.5 to 2.

17. The method of claim 3 or 10, wherein BMCS2 or BMCS3 comprises about 2-5mg/dL creatine and about 1-3mg/dL creatinine, respectively.

18. The method of claim 2 or 9, wherein the stable value is stable for at least 8 months.

19. The method of claim 5 or 12, wherein Ca⁺⁺Is between about 0.4 and about 1.6mg/dL and HCO₃ ^-Is between about 10 and about 30 mmol/L.

20. A creatinine/creatine measurement system, comprising:

a calibrated creatine sensor;

a calibrated creatinine sensor;

one or more network interfaces for communicating over a computer network;

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

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

measuring a measured creatine sensor current signal (M Δ I2) with a calibrated creatine sensor for a first biomatrix correction solution (BMCS2) having a known concentration of creatine (CR _ BMCS2) and a known concentration of creatinine (CREA _ BMCS 2);

determining a measured concentration of creatine in BMCS2 (MCR _ BMCS 2);

comparing MCR _ BMCS2 to CR _ BMCS2 to determine creatine sensor biomatrix correction factor (Δ CR);

measuring a measured creatinine sensor current signal (M Δ I2') of BMCS2 with a calibrated creatinine sensor;

determining the measured concentration of creatinine in BMCS2 (MCREA — BMCS 2); and

MCREA _ BMCS2 was compared to CREA _ BMCS2 to determine the creatinine sensor biomatrix correction factor (Δ CREA).

21. The creatinine/creatine measurement system of claim 20, wherein the processes, when executed, are further operable to:

measuring a second measured creatine sensor current signal (M2 Δ I2) with a calibrated creatine sensor for a second biomatrix correction solution (BMCS3) having a known concentration of creatine (CR _ BMCS3) and a known concentration of creatinine (CREA _ BMCS 3);

determining the measured concentration of creatine in BMCS3 (M2CR _ BMCS 3);

comparing M2CR _ BMCS3 to CR _ BMCS3 to determine a second creatine sensor biomatrix correction factor (Δ CR');

measuring a measured creatinine sensor current signal (M2 Δ I2') of BMCS3 with a calibrated creatinine sensor;

determining the measured concentration of creatinine in BMCS3 (M2CREA — BMCS 3); and

m2CREA _ BMCS3 was compared to CREA _ BMCS3 to determine creatinine sensor biomatrix correction factor (Δ CREA').

22. The creatinine/creatine measurement system of claim 22 wherein Δ CR, Δ CR ', Δ CREA, and Δ CREA' are applied to slope 1 and slope 2 of creatinine measurements obtained from a biological matrix sample.

Technical Field

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 measurement accuracy of electrochemical sensors for measuring creatinine and creatine.

Background

The ability to accurately measure creatinine and creatine levels in a patient's blood is important. In particular, serum creatinine is an important indicator of kidney health, as it is excreted unchanged by the kidney and can be easily measured. For example, elevated blood serum creatinine levels are a late-stage marker of chronic kidney disease and are typically only observed when significant kidney damage has occurred. Chronic kidney disease refers to the gradual loss of kidney function. The kidney functions to filter waste and excess fluid from the blood, and these filtered waste and excess fluid are then excreted 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. may accumulate in the body. In the early stages of chronic kidney disease, there may be few signs or symptoms, and progression of the disease may not become apparent until kidney function has been significantly impaired.

Creatinine/creatine in a sample (e.g., blood of a patient) can be measured by an electrochemical sensor. For example, current creatinine sensors may include enzyme-type biosensors that contain three enzymes, creatininase, creatinase, and sarcosine oxidase, which catalyze the production of glycine, formaldehyde, and hydrogen peroxide from creatinine and water. The three enzymes can be immobilized on the surface of a platinum electrode, and hydrogen peroxide (H)₂O₂) Can then be electrochemically oxidized at a constant polarization potential on a platinum electrode and used for measuring in the blood of a patientCreatinine and/or creatine levels. However, in order to determine creatinine and/or creatine concentration in a biological sample, creatinine and creatine sensors need to be calibrated to determine their sensitivity. This can be done by measuring the current response of creatinine and creatine sensors in a calibration solution with predetermined concentrations of creatinine and creatine. After determining the sensitivity of creatinine and creatine sensors, the concentration of creatinine and creatine in any biological sample can be estimated by measuring the current signal of that sample and comparing the measured sensitivity of creatinine and creatine sensors determined by a calibration process. Unfortunately, it is not easy to accurately measure creatinine levels in a biological sample for a variety of reasons. For example, sensor-to-sensor manufacturing variations that occur during biosensor production/manufacture may cause variations in creatinine measurement accuracy that occur on a sensor-to-sensor or sensor device-to-sensor device basis. In addition, changes in the biological sample matrix can also create major challenges for accurately measuring creatinine levels. Accordingly, there is an urgent and unresolved need to determine and develop new methods to improve the accuracy of biosensor calibration for creatinine and/or creatine measurements.

Disclosure of Invention

The present disclosure provides compositions and methods for improving the accuracy of creatinine and creatine in a biosensor.

In one aspect, the present disclosure provides a method of improving the accuracy of a creatinine/creatine measurement system having a calibrated creatine sensor and a calibrated creatinine sensor, the method comprising the steps of: measuring a measured creatine sensor current signal (M Δ I2) with a calibrated creatine sensor for a first biomatrix correction solution (BMCS2) having a known concentration of creatine (CR _ BMCS2) and a known concentration of creatinine (CREA _ BMCS 2); determining a measured concentration of creatine in BMCS2 (MCR _ BMCS 2); comparing MCR _ BMCS2 to CR _ BMCS2 to determine creatine sensor biomatrix correction factor (Δ CR); measuring a measured creatinine sensor current signal (M Δ I2') of BMCS2 with a calibrated creatinine sensor; determining the measured concentration of creatinine in BMCS2 (MCREA — BMCS 2); and comparing MCREA _ BMCS2 to CREA _ BMCS2 to determine the creatinine sensor biomatrix correction factor (Δ CREA).

In one embodiment, CR _ BMCS2 and CREA _ BMCS2 have stable values.

In one embodiment, the stabilized value is based on a stabilized ratio of creatine to creatinine in BMCS 2.

In one embodiment, the stable value is based on the storage temperature of BMCS 2.

In one embodiment, BMCS2 includes one or more biomatrix factors selected from the group consisting of: ca⁺⁺，HCO₃ ^-，pH，pCO₂，pO₂Electrolytes, and metabolites.

In one embodiment, Δ CR is applied to creatine measurements obtained from a biological matrix sample.

In one embodiment, Δ CREA is applied to creatinine measurements obtained from a bio-matrix sample.

In one embodiment, the method further comprises the steps of: measuring a second measured creatine sensor current signal (M2 Δ I2) with a calibrated creatine sensor for a second biomatrix correction solution (BMCS3) having a known concentration of creatine (CR _ BMCS3) and a known concentration of creatinine (CREA _ BMCS 3); determining the measured concentration of creatine in BMCS3 (M2CR _ BMCS 3); comparing M2CR _ BMCS3 to CR _ BMCS3 to determine a second creatine sensor biomatrix correction factor (Δ CR'); measuring a measured creatinine sensor current signal (M2 Δ I2') of BMCS3 with a calibrated creatinine sensor; determining the measured concentration of creatinine in BMCS3 (M2CREA — BMCS 3); and comparing M2CREA _ BMCS3 to CREA _ BMCS3 to determine creatinine sensor biomatrix correction factor (Δ CREA').

In one embodiment, CR _ BMCS3 and CREA _ BMCS3 have stable values.

In one embodiment, the stabilized value is based on a stabilized ratio of creatine to creatinine in BMCS 3.

In one embodiment, the stable value is based on the storage temperature of BMCS 3.

In one embodiment, BMCS3 includes one or more biomatrix factors selected from the group consisting of: ca⁺⁺，HCO₃ ^-，pH，pCO₂，pO₂Electrolytes, and metabolites.

In one embodiment, Δ CR' is applied to the slope of creatine measurements obtained from a biological matrix sample.

In one embodiment, Δ CR ', Δ CREA and Δ CREA' are applied to slope 1 and slope 2 of creatinine measurements obtained from a biomatrix sample.

In one embodiment, the stable ratio of creatine to creatinine is about 1.5 to about 2.

In one embodiment, the stable ratio of creatine to creatinine is 1.5 to 2.

In one embodiment, BMCS2 or BMCS3 includes about 2-5mg/dL creatine and about 1-3mg/dL creatinine, respectively.

In one embodiment, the stable value is stable for at least 8 months.

In one embodiment, Ca⁺⁺Is between about 0.4 and about 1.6mg/dL and HCO₃ ^-Is between about 10 and about 30 mg/dL.

In one aspect, the present disclosure provides a creatinine/creatine measurement system comprising: a calibrated creatine sensor; a calibrated creatinine sensor; one or more network interfaces for communicating over a computer network; a processor coupled to the network interface and the creatine sensor and creatinine sensor and adapted to perform one or more procedures; and a memory configured to store procedures executable by the processor, the procedures operable when executed to: measuring a measured creatine sensor current signal (M Δ I2) with a calibrated creatine sensor for a first biomatrix correction solution (BMCS2) having a known concentration of creatine (CR _ BMCS2) and a known concentration of creatinine (CREA _ BMCS 2); determining a measured concentration of creatine in BMCS2 (MCR _ BMCS 2); comparing MCR _ BMCS2 to CR _ BMCS2 to determine creatine sensor biomatrix correction factor (Δ CR); measuring a measured creatinine sensor current signal (M Δ I2') of BMCS2 with a calibrated creatinine sensor; determining the measured concentration of creatinine in BMCS2 (MCREA — BMCS 2); and comparing MCREA _ BMCS2 to CREA _ BMCS2 to determine the creatinine sensor biomatrix correction factor (Δ CREA).

"control" or "reference" means 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 that from a normal, untreated sample or control sample. Control samples include, for example, creatine solutions, creatinine solutions, and the like. Methods of selecting and testing control samples are within the ability of those skilled in the art. Determining statistical significance is within the ability of those skilled in the art, e.g., the standard deviation value that constitutes the mean of positive results.

As used herein, "creatine (also known as 2- [ carbamimidoyl (methyl) amino ] acetic acid, N-carbamimidoyl-N-methylglycine, or methylguanidinyl acetic acid)" refers to an organic compound that converts Adenosine Diphosphate (ADP) back to ATP, which is the recycling of ATP to generate energy by cells, by supplying phosphate groups. Creatine has the following chemical structure:

as used herein, "creatinine" refers to a by-product of the enzymatic decomposition of creatine, and typically exists in two major tautomeric forms, which are shown below.

Ranges can 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 being "about" the 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 that the data 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 also considered disclosed, as well as 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 should be understood to include any value, combination of values, or subrange from the group comprising 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, e.g., 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 any end point of the range are specifically contemplated. For example, nested sub-ranges of the exemplary range 1 to 50 may 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 thereof, 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.

Even though embodiments are described in different aspects of the disclosure, it is contemplated that any one embodiment described herein may be combined with any other embodiment or embodiments, where applicable or not specifically disclaimed.

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

Drawings

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

figures 1A-1B show two graphs depicting the deviation of creatinine measured by a whole blood creatinine biosensor relative to plasma creatinine on a chemical analyzer. Figure 1A shows creatinine measurements without correction. Figure 1B shows creatinine measurements for the same sample as figure 1A, but with two calibration solutions to correct for creatine and creatinine sensitivity.

Detailed Description

The present disclosure is based, at least in part, on the following findings: one or more biomatrix correction solutions may be used to generate a creatine sensor biomatrix correction factor and a creatinine sensor biomatrix correction factor. The present disclosure provides creatine sensor biomatrix correction factors and creatinine sensor biomatrix correction factors applicable to measured creatine and/or creatinine values in a biomatrix sample,to correct for manufacturing variations from sensor to sensor and inhibitory factors present in the bio-matrix sample (e.g., HCO)₃ ^-，Ca⁺⁺pH, etc.) of the sample.

Overview

Current creatinine sensors in the creatine/creatinine system (e.g., GEM PAK cassette) include enzyme-based biosensors that contain three enzymes. These enzymes are immobilized on the surface of a platinum electrode. The creatinine detection system is based on a cascade of three enzymes (Rx):

product hydrogen peroxide (H)₂O₂) And 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 measurements 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 called the outer membrane) on top of the enzyme layer. The diffusion control membrane restricts the flow of creatinine and creatine substrates (flux) into the enzyme layer to ensure that the signal produced from hydrogen peroxide is proportional to the substrate concentration of the sample.

To determine the respective concentrations of creatinine and creatine in the biological sample(s), the creatinine and creatine sensors need to be calibrated to determine their respective sensitivities. This may be accomplished by comparing the current signals of the creatinine and creatine sensors in a calibration solution that contains predetermined (e.g., known) concentrations of creatinine and creatine. After determining the sensitivity of creatinine and creatinine sensors, the concentration of creatine and creatinine in any biological sample can be estimated by adjusting the measured readings with the results determined from the calibration process.

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

Δ I2 ═ CR _ CS2 gradient (equation 1)

Δ I2 is the current signal measured by 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 concentration of creatine (CR _ CS2), a known concentration of creatinine (CREA _ CS2), and a stable ratio of creatine to creatinine, which may allow a creatine sensor sensitivity (slope) to be established for the creatine sensor.

A calibration system for a creatinine sensor or biosensor may implement a 3-point calibration method. As the creatinine sensor provides readings of both creatinine and creatine in a calibration solution or biological sample containing analytes of both creatinine and creatine, the sensitivity of the creatinine sensor to creatinine (slope 1) or creatine (slope 2) can be determined from equations 2-5 below according to the disclosure defined below. The present disclosure provides that two calibration solutions with different ratios of creatine/creatinine can be used for the 3-point calibration method.

3-point creatinine sensor calibration equation:

Δ I2' ═ CREA _ CS2] slope 1+ [ CR _ CS2] slope 2 (equation 2)

Δ I3' ═ CREA _ CS3] slope 1+ [ CR _ CS3] slope 2 (equation 3)

Δ I2 'and Δ I3' are the current signals measured by 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 an unstable ratio of creatine to creatinine.

[ CREA _ CS2], [ CREA _ CS3], [ CR _ CS2] and [ CR _ CS3] represent the initial known concentrations of creatinine and creatine in calibration solutions CS2 and CS3, respectively. Sensitivity of creatinine sensor to creatinine and creatine, slope 1 (sensor sensitivity to creatinine) and slope 2 (sensor sensitivity to creatine), can be derived from equations 2 and 3:

slope 1 ([ CR _ CS3] × Δ I2 '- [ CR _ CS2] × Δ I3')/([ Creat _ CS2] × [ CR _ CS3] - [ Creat _ CS3] × [ CR _ CS2]) pA/mg/dL (equation 4)

Slope 2 ([ CREA _ CS2] × Δ I3 '- [ CREA _ CS3] × Δ I2')/([ Creat _ CS2] × [ CR _ CS3] - [ Creat _ CS3] × [ CR _ CS2]) pA/mg/dL (equation 5)

CS2 may be formulated at an optimal stable ratio of creatine/creatinine of between about 1.5 and 2, while CS3 may be formulated at a different, unstable ratio of creatine/creatinine (about 10 to 70) which may vary over time from the original ratio at the time of manufacture due to changes in concentration during storage.

The creatinine measurement system described above, when properly calibrated, can quantitatively measure the concentration of creatinine in a biological sample. However, there are several practical problems associated with sensor variations, which arise from typical manufacturing methods, calibration and variations specific to other analyzer operations, and biological sample matrix variations that present significant challenges for accurately measuring creatinine.

For example, the activity of many enzymes may be inhibited or affected by specific chemicals (e.g., ions or gases). For example for converting creatinine to H₂O₂The above three enzyme systems are susceptible (e.g., by inhibition or interference) to many common analytes that may be present in clinical chemistry assays, e.g., HCO₃ ^-And Ca⁺⁺. In addition, for converting creatinine to H₂O₂The three enzyme system of (a) is also sensitive to sample pH. Due to differences in the concentration of such analytes in biological samples between different patient samples or populations, errors may be introduced when measuring creatinine and/or creatine concentrations in biological samples. Prior art solutions to these problems include applying sensor measurement algorithms to address such effects. As added toComplexity, sensor measurement systems are easy to manufacture, which can create variations in sensor-to-sensor variability during the manufacturing process. For example, common variations in sensor outer membrane composition or enzyme membrane thickness can produce significant errors resulting from enzyme inhibition effects due to relative diffusion of analyte(s) relative to inhibitory chemical(s). Unfortunately, enzyme inhibitory effects that can be found within biological samples and produce measurement errors, when present in combination with typical sensor-to-sensor manufacturing variations, are additive; thus, a combination of variable enzyme inhibition effects within the biological sample matrix and sensor-to-sensor manufacturing variability can act in concert (e.g., synergistically) to produce greater measurement error differences.

Calibration matrix

It is common in clinical chemistry analyzers to provide multiple analyte determinations for a single sample measurement. In order to provide results for multiple analytes in a single measurement, up to three bio-matrix calibration solutions with varying levels of analytes may be used for calibration and estimation of sensor sensitivity. For example, to calibrate pH and pCO₂、pO₂Electrolyte or metabolite levels, two calibration reagent solutions with significantly different levels of each analyte may be used. However, to calibrate a creatinine sensor, three calibration reagent solutions with significantly varying levels of analyte may be used. The calibration reagent solution used to calibrate a creatinine sensor according to the present disclosure contains one or more analytes other than creatinine and may be used to calibrate other types of sensors. Some of the one or more analytes present in these calibration reagent solutions may have an inhibitory effect on the ability of the creatinine sensor to measure creatinine. For example, pH, HCO in these reagents₃ ^-And Ca⁺⁺The levels can affect creatinine or creatine sensor calibration and may also have the potential to generate sensor-specific errors in calibration. The error may be due to slight sensor-to-sensor variations and inhibitory effects from the specific analytes described above.

Biological sample matrix

Concentration of creatinine to glucose in a biological sample (e.g., about 65 to about 95mg/dL) or concentration of an analyte having an inhibitory effect on the sensor (e.g., Ca⁺⁺: about 4 to about 6mg/dL or HCO₃-: about 19 to about 26mmol/L) can be typically very low (e.g., about 0.04 to about 1.10 mg/dL). The low concentration of creatinine in the biological sample, the loss of creatinine due to the presence of the diffusion controlling outer membrane, and the conversion process of the three enzymes of the sensor all work in combination, producing very low electrical signals. Conversely, the presence of relatively high concentrations of an analyte that may have an inhibitory effect on creatinine measurements may have a significant impact on such low electrical signals.

One prior art solution to this problem is to use a calibration solution. For example, in this method, the first step is to generate a correction factor for each individual analyte with variable levels of creatinine, creatine, and inhibitor or interfering compounds present in the calibration solution. Disadvantageously, this method requires many assumptions before a simple correction factor can be obtained. In addition, the effect of multiple inhibitors or interfering compounds on the simultaneous interaction of enzymatic activities is not considered. As a result, the effectiveness of this prior art approach is questionable due to the assumptions required. Unfortunately, there is currently no good prior art solution to the practical problem of measuring creatinine levels in biological samples that can exhibit significant sample-to-sample biological matrix variations.

The present disclosure provides compositions and methods for correcting errors that may occur throughout diagnostic cycles of sensor calibration to biological sample matrix measurements. Importantly, the technology herein provides a biomatrix calibration solution designed to match (or pair) with common biological samples containing inhibitory analytes known to affect the activity of the three creatinine/creatine enzyme system. These solutions may have predetermined creatinine and creatine concentrations combined with clinical reference ranges for analytes that have inhibitory or interfering effects on enzymes used in creatinine sensors (e.g., creatininase, creatinase, sarcosine oxidase, etc.), which concentrations are stable due to stable creatine/creatinine ratios or optimized storage conditions. After installation and calibration of the creatinine and/or creatine sensors, the calibration solutions disclosed herein may be measured on the creatinine and/or creatine sensors and the responses to calibration reagents and calibration solution measurements for both creatinine and creatine are obtained and analyzed to identify sensor-specific correction factors. These correction factors can be determined for all subsequent biological samples over the entire life of the sensor (lifetime) without having to repeat this process at each sensor calibration.

The technology herein provides a creatinine measurement system that may include three main steps described in detail below: sensor calibration, establishment of correction factors, and sample measurement.

Sensor calibration

The creatinine measurement system contains both creatinine and creatine sensors. The sensor is periodically subjected to a calibration solution having predetermined creatinine and creatine concentrations throughout the sensor's useful life. After determination, these sensitivities can be used for subsequent sample measurements. In a multi-analyte clinical chemistry analyzer (e.g., GEM PAK cassette), the calibration reagent solution may also contain HCO₃ ^-、Ca⁺⁺And pH, which are known to affect creatinine or creatinase sensors. Since all of these solutions are used as multiple analytes (e.g., HCO)₃-、Ca⁺⁺And pH), they cannot all be formulated to exactly match the biological sample matrix. Thus, the sensor sensitivity obtained from the calibration process is different from the sensitivity measured in the biological sample matrix. In addition, such deviations are inconsistent from sensor to sensor, as slight variations may occur during the manufacturing process of the multi-layer sensor structure.

Creatinine/creatine correction solutions

The technology herein provides one or more specific creatinine/creatine solutions to mimic a biological sample matrix with analytes known to inhibit or interfere with creatinine or creatinase sensors.

The first correction solution may have a predetermined creatinine and creatine concentration in a matrix having inhibitory/interfering factors, e.g., HCO₃ ^-(e.g., at about 20 mM), Ca⁺⁺(e.g., at about 0.8 mM), pH (e.g., at about 7.4), and pCO₂(e.g., at about 30 mmHg), or in HCO₃ ^-(e.g., about 10 to about 30mM), Ca⁺⁺(e.g., about 0.4 to about 1.6mM), pH (e.g., about 7.0 to 8.0), and pCO₂(e.g., about 15 to about 45mmHg), these are concentrations that approximate the normal clinical sample matrix.

A second correction solution similar to the biological matrix but with different levels of creatinine and creatine may also be provided.

These calibration solutions can be stored in sealed reagent bags or ampoules to maintain constant pCO₂And pH level. These solutions can be buffered, supplemented with biocides, and maintained at optimal storage temperatures to ensure stability of the reagents. These calibration solutions can be measured as samples during sensor installation and at the completion of the initial calibration process.

Establishing separate sensor correction factors for creatinine and creatine

During sensor installation and after initial calibration, creatinine/creatine correction solutions can be measured on each creatinine or creatine measurement system to establish sensor sensitivity. The measured creatinine and creatine concentrations from the sensor are compared to predetermined creatinine and creatine levels of the correction solution to establish correction factors for both the creatinine and creatine sensors, respectively.

Alternatively, after sensor installation, two correction solutions may be measured in succession, which may provide a more accurate correction factor due to the measurement of two different levels of creatinine and creatine.

These correction factors provide sensitivity deviations in the biological matrix relative to the sensitivity obtained in the calibration solutions for the particular creatinine and creatine sensors.

According to the techniques herein, the accuracy of a creatinine/creatine measurement system having a calibrated creatine sensor and a calibrated creatinine sensor may be improved by: measuring a measured creatine sensor current signal (M Δ I2) with a calibrated creatine sensor for a first biomatrix correction solution (BMCS2) having a known concentration of creatine (CR _ BMCS2) and a known concentration of creatinine (CREA _ BMCS 2); determining a measured concentration of creatine in BMCS2 (MCR _ BMCS 2); comparing MCR _ BMCS2 to CR _ BMCS2 to determine creatine sensor biomatrix correction factor (Δ CR); measuring a measured creatinine sensor current signal (M Δ I2') of BMCS2 with a calibrated creatinine sensor; determining the measured concentration of creatinine in BMCS2 (MCREA — BMCS 2); and comparing MCREA _ BMCS2 to CREA _ BMCS2 to determine the creatinine sensor biomatrix correction factor (Δ CREA).

To provide a bio-matrix correction value/factor, CR _ BMCS2 and CREA _ BMCS2 may have stable values. For example, such stabilized values may be based on a stabilized ratio of creatine to creatinine in BMCS2, or based on the storage temperature of BMCS 2.

In accordance with the techniques herein, the bio-matrix correction solutions disclosed herein can include, but are not limited to, solutions selected from Ca⁺⁺、HCO₃ ^-、pH、pCO₂、pO₂One or more biomatrix factors of other electrolytes and metabolites.

After obtaining, Δ CR may be applied to creatine measurements obtained from the biomatrix sample, while Δ CREA may be applied to creatinine measurements obtained from the biomatrix sample.

In accordance with the techniques herein, the accuracy of a creatinine/creatine measurement system having a calibrated creatine sensor and a calibrated creatinine sensor may be further improved by including the following additional steps: measuring a second measured creatine sensor current signal (M2 Δ I2) with a calibrated creatine sensor for a second biomatrix correction solution (BMCS3) having a known concentration of creatine (CR _ BMCS3) and a known concentration of creatinine (CREA _ BMCS 3); determining the measured concentration of creatine in BMCS3 (M2CR _ BMCS 3); comparing M2CR _ BMCS3 to CR _ BMCS3 to determine a second creatine sensor biomatrix correction factor (Δ CR'); measuring a measured creatinine sensor current signal (M2 Δ I2') of BMCS3 with a calibrated creatinine sensor; determining the measured concentration of creatinine in BMCS3 (M2CREA — BMCS 3); and comparing M2CREA _ BMCS3 to CREA _ BMCS3 to determine creatinine sensor biomatrix correction factor (Δ CREA').

As discussed above for the first biomatrix correction solution, CR _ BMCS3 and CREA _ BMCS3 may have stable values based on the stable ratio of creatine to creatinine in BMCS3 or the storage temperature of BMCS3 (e.g., 4 ℃).

After obtaining, Δ CR ' may be applied to the slope of the creatine measurements obtained from the biomatrix sample, while Δ CR, Δ CR ', Δ CREA, and Δ CREA ' may be applied to slope 1 and slope 2 of the creatinine measurements obtained from the biomatrix sample.

In embodiments of the present disclosure, the stable ratio of creatine to creatinine is about 1.5 to about 2, or 1.5 to 2.

In embodiments of the present disclosure, BMCS2 or BMCS3 may include about 2-5mg/dL creatine and about 1-3mg/dL creatinine, respectively.

The stable values of the above-described bio-matrix calibration solutions can be stable for at least 8 months according to the techniques herein.

In an embodiment of the present disclosure, Ca⁺⁺Is between about 0.4 and about 1.6mmol/L and HCO₃ ^-Is between about 10 and about 30 mmol/L.

Measurement of creatinine/creatine in biological samples

For each patient sample, the current signals were measured on creatinine and creatine sensors, and then creatinine and creatine concentrations in the patient's sample were estimated using the sensitivity from the latest calibration. A correction factor may then be applied to the measured concentrations to generate final creatinine and creatine measurements.

Alternatively, the correction may be applied to the raw sensitivity obtained from the calibration process. In this method, the correction factors obtained from the calibration solution are directly applied to the sensitivity obtained from the most recent sensor calibration. The sample creatinine and creatine concentrations are then estimated from the sample current signal and the corrected sensitivity.

Reagent kit

The present disclosure also provides kits containing reagents of the present disclosure for use in methods of the present disclosure. Kits of the disclosure may include one or more containers containing reagents of the disclosure (e.g., creatine, creatinine, etc.), and/or may include reagents in one or more calibration solutions containing an analyte, e.g., HCO₃ ^-And Ca⁺⁺. Additionally, the kits of the present disclosure may further include a calibration solution adjusted to a plurality of different pH levels. In some embodiments, the kit further comprises instructions for a method according to this disclosure. In some embodiments, the instructions may include the following instructions: how to apply the reagent/solution to the sensor (e.g., creatine sensor, creatinine sensor, etc.), and how to calculate a variable of interest (e.g., a biological matrix factor such as Ca) according to any method of the present disclosure⁺⁺Correction factor, HCO₃ ^-Correction factor, pH correction factor, pCO₂Correction factor, pO₂Correction factor, electrolyte correction factor(s), metabolite correction factor(s), etc.). In some embodiments, the instructions include instructions for how to install and calibrate a measurement system as disclosed herein.

The instructions typically include information about reagent/solution concentration, reagent/solution ratio (e.g., creatine/creatinine ratio), longevity, 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., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions 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. Instructions may be provided for practicing any of the methods described herein, for example, to install and calibrate a measurement system.

The kit of this disclosure is in a suitable package. Suitable packaging includes, but is not limited to, vials (vitamins), ampoules, bottles (bottles), canisters, flexible packaging (e.g., sealed Mylar (r) or plastic bags), and the like. Packaging is also envisaged for use in combination with specific devices such as the GEM Premier whole blood analyser class (Instrumentation Laboratory, Bedford, MA). In certain embodiments, at least one active agent in the agent or solution is creatine and/or creatinine.

The kit may optionally provide additional components (components), such as buffers and explanatory information. Typically, a kit comprises a container and a label or package insert(s) 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, e.g., Maniatis et al, 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); sambrook et al, 1989, Molecular Cloning, second edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); sambrook and Russell, 2001, Molecular Cloning, third edition (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, volumes 154 And 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, sixth edition, Blackwell Scientific Publications, Oxford, 1988; hogan et al, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); westerfield, m., The zebrafish book. A guide for the laboratory use of Zebrafiadish (Danio relay), (4 th edition, 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 present disclosure also relates to computer systems involved in implementing the methods of the present disclosure relating to both computing and sequencing (sequencing).

The computer system (or digital device) may be used to receive, transmit, display and/or store results, analysis results, and/or generate results and analysis reports. A computer system can 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 CPUs, disk drives, input devices (e.g., a keyboard and/or mouse), and a display device (e.g., a monitor). Data communications, such as the transmission of instructions or reports, may be transmitted over a communications medium to a local server or to a remote location. Communication media may include any means for transmitting and/or receiving 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 review by a receiver (review). The receiver may be, but is not limited to, a stand-alone or electronic system (e.g., one or more computers, and/or one or more servers). The computer system may be integrated with or separate and apart from the creatinine/creatine biosensor system/device.

In some embodiments, a 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 (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 delivered to the computing device via any known delivery means, including for example, communications channels such as telephone lines, the Internet, wireless connections, etc., or via a removable medium such as a computer readable disk, flash drive, etc.

The 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 software. When implemented in hardware (e.g., a creatinine/creatine biosensor system/device), 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 structure may be used in embodiments of the present disclosure. A client-server architecture is a network architecture in which each computer or process on the network is a client or server. A server computer is typically a powerful computer dedicated to managing disk drives (file servers), printers (print servers), or network traffic (web servers). Client computers include PCs (personal computers) or workstations on which users run 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, e.g., any storage device in any computer(s) or the like, such as may be used to implement a database or the like, as shown. Volatile storage media includes dynamic memory, such as the main memory of such a computer platform. 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. Common forms of computer-readable media therefore 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 paper tape, 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 (cartridge), a carrier wave transporting data or instructions, a cable or link transporting such a carrier wave, 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 computer executable code of the present subject matter 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.), and other displays. The computer circuitry is typically housed in a box that includes a plurality of integrated circuit chips, such as microprocessors, memory, interface circuits, and the like. The box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive (e.g., a writable CD-ROM), and other common peripheral devices. An input device (e.g., a keyboard, mouse, or touch-sensitive screen) optionally provides input from a user. The computer may include appropriate software for receiving user instructions, either in the form of a user entering a set of parameter fields (e.g., in a GUI) or in the form of pre-programmed instructions (e.g., pre-programmed for various specific operations).

Reference will now be made in detail to the exemplary embodiments of the present disclosure. While the disclosure will be described in conjunction with the exemplary embodiments, it will be understood that it is 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.

Examples

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

Example 1 deviation tables of whole blood creatinine samples measured by GEM PAK cassette versus plasma creatinine measured by chemical analyzer with and without biomatrix calibration technique.

The bias of the whole blood clinical samples corrected with the two solution matrices (difference in creatinine measured between GEM PAK and reference chemical analyzer) was minimized to meet clinical requirements.

In this study, each test cell contained a creatinine measurement system plus two external calibration solutions. The creatinine measurement system comprised a creatinine sensor, a creatine sensor, three calibration solutions (CS1, CS2, and CS3), and two external biomatrix correction solutions (BMCS2 and BMCS 3). Two methods of establishing creatine and creatinine concentrations in whole blood samples were investigated: option (a), no creatine and creatinine biomatrix correction should be applied for samples measured by GEM PAK; and option (b), creatine and creatinine in the clinical sample of whole blood are corrected based on correction factors established by measuring BMCS2 and BMCS3 at the beginning of the lifetime of each PAK, and then by applying the correction factors to creatine and creatinine slopes throughout the lifetime. Figure 1A shows that the application of option (a) to creatinine and the deviation from creatinine reported for all whole blood clinical samples were clearly negative and somewhat scattered from acceptable clinical criteria (data points in the form of blue diamonds are referred to relative to the dashed line). Figure 1B demonstrates that by applying option (B) to the creatinine sample algorithm, both negative bias in whole blood clinical samples and the dispersion of creatinine reported are significantly reduced across the sample range to meet the desired clinical criteria (again referring to the data points in the form of blue diamonds relative to the dashed line).

Is incorporated by reference

All documents cited or referenced herein, and all documents cited or referenced in the documents cited herein, are incorporated by reference, along with instructions, descriptions, product specifications, and product manuals for any manufacturer of any product in any of the documents mentioned herein or incorporated by reference herein, and may be used in the practice of the present disclosure.

Equivalent forms

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. In view of this, various modifications or changes will occur to those skilled in the art and are intended to be included within the spirit and scope of this application and be considered within the scope of the appended claims. Other advantageous features and functions relating to the systems, methods and processes of the present disclosure will be apparent from the appended claims. Further, those of skill 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 covered by the appended claims.