Biocide compositions compatible with enzyme biosensors and methods of use thereof

Abstract

The present disclosure relates to biocide compositions compatible with enzyme biosensors and methods of use thereof. More particularly, the present disclosure relates to biocide compositions compatible with enzymes used to measure creatine and creatinine levels.

Description

Field of the Invention

[0001] The present disclosure relates to biocide compositions compatible with enzyme biosensors and methods of use thereof. More particularly, the present disclosure relates to biocide compositions compatible with enzymes used to measure creatine and creatinine levels. Background of the Invention

[0003] Whole blood critical care analyzers (WBCCA) play an important role in the management of critically ill patients by providing rapid treatment turnaround times at the point of care. Biocides are key components of WBCCA reagents, inhibiting the effects of microbial metabolism on blood analysis results. For example, normal oxygen levels in human blood are very low, and any consumption of oxygen by bacteria in the calibration solution can result in significant errors in reported oxygen levels. WBCCAs have also evolved to include electrochemical biosensor-based metabolite measurements, such as creatinine / creatine levels in a sample (e.g., a patient's blood), which are important indicators of kidney function. Current creatinine sensors may include enzymatic biosensors containing three enzymes—creatininase, creatinase, and sarcosine oxidase—which catalyze the production of glycine, formaldehyde, and hydrogen peroxide from creatinine and water, so that the final reaction product, hydrogen peroxide (H2O2), can be electrochemically oxidized to measure creatinine and / or creatine levels in a sample (e.g., a patient's blood). An important consideration in the design and use of WBCCA enzyme biosensors is that the catalytic activity of the biosensor enzyme depends on multiple parameters, including solution conditions such as pH, temperature, levels of metabolites such as oxygen, and the like. Furthermore, such enzymes are also greatly affected by the presence of any inhibitors. Unfortunately, enzymes used in enzyme biosensors are often inhibited or inactivated by current biocides. For example, the creatinine biosensor is an example of an important enzyme biosensor that is inactivated by current biocides. Therefore, there is a need for biocide compositions that are compatible with enzyme biosensors. SUMMARY OF THE INVENTION

[0005] The present disclosure provides effective biocides that do not inhibit or prevent the function of whole blood critical care analyzers (WBCCA). Specifically, the present disclosure provides some effective biocides that do not inhibit or prevent WBCCA with sensor function or enzyme biosensor function. In addition, the present disclosure provides biocides that do not inhibit or prevent enzyme biosensor function with a molecular weight (MW) greater than 320. In addition, the present disclosure provides some effective biocides that do not inhibit or prevent enzyme biosensor function. Exemplary effective biocides that do not inhibit or prevent the function of enzyme biosensors include but are not limited to levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfonamides, sulfathiazole sodium, sulfadimethoxine, Vantocil IB, etc. Advantageously, the biocides disclosed herein can effectively neutralize or destroy harmful organisms (e.g., bacteria, fungi, etc.) without inhibiting or preventing the function of enzyme biosensors. The present disclosure also provides a method for identifying a biocide compatible with any one of a variety of enzyme biosensors. In addition, the present disclosure also provides methods of using the disclosed biocides.The compositions and methods provided herein are important because they allow whole blood critical care analyzers (WBCCAs) with enzyme biosensors to effectively read biological samples in the presence of biocidal agents that do not negatively affect the function of the enzyme biosensor.

[0006] In one aspect, the present disclosure provides a method for maintaining sensor functionality comprising the steps of: adding one or more sensor-compatible biocides (SCBs) to a solution; and measuring the concentration of one or more analytes with a sensor. In some embodiments, the sensor can be a biosensor, a gas sensor, an ion-selective electrode, or a photometric sensor. In some embodiments, the biosensor is an enzyme biosensor. In some embodiments, the enzyme biosensor can be a creatinine sensor, a creatine sensor, or a combination thereof.

[0007] In some embodiments, the SCB is an antibiotic having a molecular weight greater than about 320 g / mol.

[0008] In some embodiments, the SCB is a polymyxin selected from polymyxin B, colistin, and combinations thereof.

[0009] In some embodiments, the SCB is polymyxin B.

[0010] In some embodiments, the SCB is a fluoroquinolone.

[0011] In some embodiments, the SCB comprises a sulfone group.

[0012] In some embodiments, the solution is a biological sample, a process control solution (PCS), a calibration solution, a quality control solution, a conditioning solution, or a cleaning solution.

[0013] In some embodiments, the SCB is a beta-lactam antibiotic selected from the group consisting of amoxicillin, ampicillin, carbenicillin, cefazolin, cefepime, cefoxitin, ceftazidime, clavulanic acid, imipenem, oxacillin, penicillin, and piperacillin.

[0014] In some embodiments, the SCB includes at least one beta-lactam antibiotic and a polymyxin.

[0015] In some embodiments, the solution further comprises a beta-lactamase inhibitor.

[0016] In some embodiments, the SCB comprises a polymyxin and a fluoroquinolone.

[0017] In one aspect, the present disclosure provides a method for identifying an enzyme biosensor-compatible biocide (EBCB) for an enzyme biosensor, comprising the steps of measuring a stable enzyme biosensor activity of the enzyme biosensor in a solution for a period of time; adding one or more biocides to the solution containing the enzyme biosensor; determining the antimicrobial efficacy of the biocides in the solution; measuring the enzyme biosensor activity in response to the one or more biocides for another period of time, wherein the enzyme biosensor activity is evaluated based on an enzyme slope; and selecting an EBCB based on the enzyme slope.

[0018] In some embodiments, the biocide is one or more antibiotics having a molecular weight greater than about 350 g / mol.

[0019] In some embodiments, the EBCB contains a sulfone group.

[0020] In some embodiments, the EBCB is a β-lactam antibiotic.

[0021] In some embodiments, the WBCCA sensor is a gas sensor, an ion selective electrode, a photometric sensor, or the like.

[0022] In some embodiments, the WBCCA sensor is an enzyme biosensor, optionally a creatinine / creatine sensor.

[0023] In one aspect, the present disclosure provides a composition comprising one or more enzyme biosensor compatible biocides (EBCBs) selected from the group consisting of levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfonamides, sulfathiazole sodium, sulfadimethoxine, and Vantocil IB.

[0024] In one aspect, the present disclosure provides a composition comprising: a first enzyme biosensor-compatible biocide (EBCB); and a second EBCB.

[0025] In some embodiments, the first EBCB is selected from the group consisting of levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, colistin, sulfonamides, sulfathiazole sodium, sulfadimethoxine, and Vantocil IB.

[0026] In some embodiments, the second EBCB is selected from levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfonamide, sulfathiazole sodium, sulfadimethoxine, and Vantocil IB.

[0027] In some embodiments, the first EBCB or the second EBCB is a penicillin selected from amoxicillin, carbenicillin, and benzylpenicillin.

[0028] In some embodiments, the concentration of penicillin is from about 12.5 to about 500 μg / ml.

[0029] In some embodiments, the first EBCB is carbenicillin at a concentration of about 5 to about 800 μg / ml and the second EBCB is nitrofurantoin at a concentration of about 1 to about 200 μg / mL.

[0030] In some embodiments, the first EBCB or the second EBCB is about 15 to about 1500 mg / L spectinomycin.

[0031] In some embodiments, the first EBCB or the second EBCB is about 10 to about 500 mg / L of ceftazidime.

[0032] In some embodiments, the first EBCB or the second EBCB is about 10-500 mM streptomycin.

[0033] In some embodiments, the first EBCB or the second EBCB is about 20 to 100 mg / L polymyxin B.

[0034] In some embodiments, the first EBCB or the second EBCB is about 20 to 100 mg / L colistin.

[0035] In some embodiments, the first EBCB or the second EBCB is 0.1-0.5% w / v Vantocil IB in aqueous solution.

[0036] In some embodiments, the first EBCB is colistin and the second EBCB is levofloxacin.

[0037] definition

[0038] "Control" or "reference" refers to a standard of comparison. In one aspect, as used herein, a sample or subject that is "altered compared to a control" is understood to have a level of activity that is statistically different from that of a sample derived from a normal, untreated, or control sample. Control samples include, for example, creatine solution, creatine solution, and the like. Methods for selecting and testing control samples are within the skill of those skilled in the art. Determination of statistical significance is within the skill of those skilled in the art, for example, the number of standard deviations from the mean that constitute a positive result.

[0039] 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 generates energy for cells by recycling adenosine triphosphate (ATP) by donating a phosphate group to convert adenosine diphosphate (ADP) back into ATP. Creatine has the following chemical structure:

[0040]

[0041] As used herein, "creatinine" refers to the enzymatic breakdown byproduct of creatine and generally exists in two major tautomeric forms, as shown below.

[0042]

[0043] Ranges may be expressed herein as from "about" a particular value, and / or "about" another particular value. When such ranges are expressed, another aspect includes from a particular value and / or to another particular value. Similarly, when values ​​are expressed as approximations, by using the antecedent "about," it is understood that the particular value forms another aspect. It should also be understood that the endpoints of each range are significantly related to, and independent of, the other endpoint. It should also be understood that many values ​​are disclosed herein, and in addition to the value itself, each value is also disclosed herein as "about" that particular value. It should also be understood that throughout the application, data is provided in a variety 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 should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed, as well as between 10 and 15. It should also be 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. Scope provided herein should be understood as the abbreviation of all values ​​in this scope.For example, 1 to 50 scopes are understood to include any numeral, combination of numerals or subranges selected from 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, and all intermediate decimal values ​​between the above-mentioned integers, such as for example 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8 and 1.9.About subrange, specifically contemplate " nested subrange " extending from any endpoint of scope. For example, nested sub-ranges of the exemplary range of 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 the other direction.

[0044] Where applicable or not specifically disclaimed, any of the embodiments described herein is contemplated to be capable of being combined with any other embodiment or embodiments, even if the embodiments are described under different aspects of the disclosure. For example, it is expressly contemplated within the scope of the present disclosure that an effective biocide may be used alone or as a combination of two or more effective biocides.

[0045] These and other embodiments are disclosed and / or encompassed by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The following detailed description, which is given by way of example but is not intended to limit the disclosure to only the specific embodiments described, can be best understood in conjunction with the accompanying drawings, in which:

[0048] Figures 1A-1C A crystal structure reconstruction of the enzyme present in the creatinine sensor is shown. Figure 1A is a ribbon diagram showing the quaternary structure of the creatininase hexamer known in the art. Figure 1B is a ribbon diagram showing the tertiary structure of the N-terminal domain of creatinase known in the prior art. Figure 1C is a ribbon diagram showing the tertiary structure of sarcosine oxidase known in the art.

[0049] Figures 2A-2B Graphs showing the changes in creatinine and creatine slope over time in the presence of MIT or polymyxin B, respectively. Figure 2A A graph showing the creatinine and creatine slopes over time shows that the addition of MIT resulted in a rapid decay in the creatinine / creatine slope that did not recover over time. Figure 2B A graph showing the creatinine and creatine slopes over time shows that the addition of polymyxin B does not cause a rapid decay of the creatinine / creatine slope. Before adding 300 mg / L MIT (methylisothiazolinone) or 20 mg / L polymyxin B to process control solution (PCS) B (e.g., PCS-B), a stable slope was first established for over a week.

[0050] Figure 3 Example graphs showing the change in creatinine slope over time in PCS with polymyxin (left) and gentamicin (right) are shown, demonstrating that the sensor maintains an adequate creatinine slope over its full service life of three weeks.

[0051] Figure 4A and 4B is shown in spiked blood samples ( Figure 4A ) or clinical specimens ( Figure 4B ) in the main calibration / cleaning solution. Detailed Description of the Invention

[0053] The present disclosure is based, at least in part, on the unexpected discovery that biocides falling within a specific molecular weight range do not inhibit or prevent the function of enzymes and enzyme biosensors. In particular, the present disclosure provides the unexpected and surprising discovery that biocides with a molecular weight (MW) greater than 320 do not inhibit or prevent the function of enzymes in enzyme biosensors. The present disclosure provides a number of specific and effective biocides that do not inhibit or prevent the function of enzymes in enzyme biosensors, including but not limited to levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfonamides, sulfathiazole sodium, sulfadimethoxine, Vantocil IB, etc. Advantageously, the biocides disclosed herein can effectively neutralize or destroy harmful organisms (e.g., bacteria, fungi, etc.) without inhibiting or preventing the function of enzyme biosensors. The present disclosure also provides methods for identifying effective biocides that are specific for and compatible with any of a variety of enzyme biosensors. In addition, the present disclosure also provides methods for using the disclosed biocides. The compositions and methods provided herein are important because they allow whole blood critical care analyzers (WBCCAs) having enzyme-based biosensors to effectively read biological samples in the presence of biocidal agents that do not negatively impact the function of the enzyme biosensor.

[0054] Overview

[0055] Historically, WBCCA has only been used to measure blood gases, electrolytes, and CO2-oxygen saturation, which makes the selection of compatible biocides for these assays relatively simple. However, when the detection methods of WBCCA were expanded to include metabolites such as glucose, lactate, creatinine, creatine, etc., it became difficult to identify biocides compatible with these detection methods because they typically include enzyme-based biosensors, and the enzymes incorporated into these biosensors are inactivated by most biocides. In the prior art, it has been a major challenge to determine a biocidal agent that is powerful enough to kill all possible bacteria, yeasts, and fungi without simultaneously inactivating the enzymes incorporated into the enzyme biosensor. For example, the prior art solution adopted by medical manufacturers of critical care analyzers is to switch from using chlorinated isothiazolinones (e.g., ProClin 300) to using non-chlorinated isothiazolinones (e.g., methylisothiazolinone (MIT)). Unfortunately, non-chlorinated isothiazolinones have higher minimum inhibitory concentrations (MICs), requiring higher concentrations to meet the minimum bactericidal requirements and resulting in increased reagent costs.

[0056] Whole-blood-based blood gas analyzers are more susceptible to microbial growth than most other types of clinical analyzers because their calibrators maintain a stable oxygen level, reported as partial pressure pO2. This presents a serious problem because reported patient pO2 values ​​often sound large. For example, 90 mm Hg is a normal pO2 result, similar to a normal chloride result of 95 mmol / L, and much higher than the normal ionized calcium, approximately 1.1-1.2 mmol / L. However, these values ​​are deceptive because it is the moles and millimoles that matter, and because oxygen is poorly soluble in water, the normal oxygen content in human blood is only 0.1 mmol / L. Given that calibrators are expected to be stable to within 1% (if possible), a loss of just 0.001 millimoles or 1 micromole of oxygen could make the oxygen channel on the analyzer less accurate than ideal. Therefore, bacterial contamination within the analyzer can produce significant measurement errors. For example, if a given bacterial colony consumes 10 micromoles of both oxygen and glucose (e.g., in a calibration solution), the analyzer will report a significant error in pO2 of about 10%, while the error in the glucose measurement will be proportionally less significant, e.g., from about 5.00 mmol / L to 4.99 mmol / L, which is relatively insignificant.

[0057] The use of different types of enzymes required for other metabolites such as creatinine and urea or blood urea nitrogen (BUN) has exacerbated the problem of finding effective biocides. Glucose and lactic acid are most often measured with glucose oxidase and lactate oxidase, respectively, while creatinine is measured with two hydrolytic enzymes (e.g., creatinase and creatinase) and sarcosine oxidase. Unfortunately, some of these enzymes, such as creatinase, are inactivated by the biocides of the prior art.

[0058] Creatinase, also known as creatinine amide hydrolase or creatinine hydrolase, is a Zn 2+ An ion-dependent hexamer that catalyzes the hydrolysis of creatinine to creatine. Creatinase, also known as creatine aminohydrolase, catalyzes the hydrolysis of creatine to sarcosine and urea. Sarcosine oxidase requires FAD (flavin adenine dinucleotide) and catalyzes the oxidative demethylation of sarcosine (N-methylglycine) to glycine.

[0059] Current creatinine sensors in creatine / creatinine systems (e.g., GEM PAK cartridges) include enzyme biosensors containing these three enzymes immobilized on the surface of a platinum electrode. The creatinine detection system is based on the following three enzyme cascade reactions (Rx):

[0060]

[0061]

[0062]

[0063] The product hydrogen peroxide (H2O2) is then electrochemically oxidized on a platinum electrode at a constant polarization potential, and the current signal is proportional to the analyte concentration.

[0064] The presence of creatine in clinical samples requires an additional sensor for creatine measurement to correct the creatinine sensor's creatine response. The creatine sensor only includes reactions (2) and (3) of the above enzyme cascade.

[0065] The creatine and creatinine sensor has a diffusion-controlling membrane (also called an outer membrane) on top of the enzyme layer. The diffusion-controlling membrane limits the flux of creatinine and creatine substrates into the enzyme layer, ensuring that the signal generated by hydrogen peroxide is proportional to the substrate concentration in the sample.

[0066] The calibration system for a creatine sensor or biosensor may involve a 2-point calibration based on the following equation:

[0067] ΔI2 = [CR_CS2] * slope (Eq. 1)

[0068] ΔI2 is the current signal measured on the creatine sensor in the first calibration solution (CS2). [CR_CS2] is the creatine concentration in the first calibration solution (CS2). CS2 may have a known concentration of creatine (CR_CS2), a known concentration of creatinine (CREA_CS2), and a stable creatine to creatinine ratio, which makes it possible to establish the creatine sensor sensitivity (slope) of the creatine sensor.

[0069] According to the teachings herein, a calibration system for a creatinine sensor or biosensor can implement a three-point calibration method. Since the creatinine sensor provides readings for both creatinine and creatine in a biological sample or calibration solution containing two analytes, the sensitivity of the creatinine sensor to creatinine (Slope 1) or creatine (Slope 2) can be determined according to the present disclosure from equations 2-5 below, as defined below. The present disclosure provides that two calibration solutions with different creatine / creatinine ratios can be used in a three-point calibration method.

[0070] 3-point Creatinine Sensor Calibration Equation:

[0071] ΔI2′=[CREA_CS2]*Slope1+[CR_CS2]*Slope2 (Eq. 2)

[0072] ΔI3′=[CREA_CS3]*Slope1+[CR_CS3]*Slope2 (Eq. 3)

[0073] ΔI2′ and ΔI3′ are the current signals measured on the creatinine sensor 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 creatine to creatinine ratio.

[0074] [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. The sensitivity of the creatinine sensor to creatinine and creatine, slope 1 (sensor sensitivity to creatinine), and slope 2 (sensor sensitivity to creatine) can be derived from Eq. 2 and 3:

[0075] Slope 1=([CR_CS3]*ΔI2'-[CR_CS2]*ΔI3') / ([Creat_CS2]*[CR_CS3]–[Creat_CS3]*[CR_CS2])pA / mg / dL (Eq.4)

[0076] Slope 2 = ([CREA_CS2]*ΔI3'–[CREA_CS3]*ΔI2') / ([Creat_CS2]*[CR_CS3]–[Creat_CS3]*[CR_CS2])pA / mg / dL (Eq.5)

[0077] Furthermore, to further complicate the issue, the few biocides identified to date that are compatible with glucose and lactate oxidase have been found to be incompatible with the commonly used creatinase, creatinase, and sarcosine oxidase. In particular, MIT, a commonly used biocide, rapidly inactivates creatinase and creatinase. The difficulties associated with identifying biocides that are compatible with proteins, such as enzymes or antibodies, are discussed in U.S. Patent No. 5,506,216, which notes that proteins can be denatured by such substances. U.S. Patent No. 5,506,216 identified several biocides, including o-phenylphenol, Densil P [dithio-2,2'-bis(benzamide)], [1,2-benzisothiazolin-3-thione] (Proxel), methylene dithiocyanate, cyanate esters, hydroxyquinoline, carbendazim [-methoxycarbonylamino-benzimidazole], and dazomet [3,5-dimethyltetrahydro-1,3,5-thiodiazine-2-thione], and found that these agents could function as biocides that did not interact with proteins, but only when they were complexed with cyclodextrin. Consistent with this, few, if any, of these biocides were compatible with sensors based on creatininase, creatinase, and sarcosine oxidase.

[0078] Without wishing to be bound by theory, the creatininase biosensor active site consists of a narrow hydrophobic cleft (Yoshimoto et al., 2004. Journal of Molecular Biology), and it is believed that interaction of the creatine substrate with the creatininase enzyme can be prevented by small non-polar molecules that denature the enzyme, such as occurs in the biocide compositions disclosed herein.

[0079] In an embodiment, the biocide composition compatible with the enzyme biosensor is a combination of a sulfonamide with another sulfonamide (see, e.g., U.S. Patent No. 9,029,118), or any other compatible biocide disclosed herein. In an embodiment, the sulfonamide may be present at a concentration of about 0.05 g / L to about 20 g / L, about 0.3 g / L to about 10 g / L, about 0.3 g / L to about 5 g / L, and the like. It is contemplated within the scope of the present disclosure that the biocide composition compatible with the enzyme biosensor may be a combination of a sulfonamide with any other compound disclosed herein.

[0080] In embodiments, a biocide composition compatible with enzyme biosensors is a combination of about 100 to about 1,000 micrograms per milliliter of sulfathiazole and about 20 to about 2,000 micrograms per milliliter of quinacrine hydrochloride (see, eg, US Pat. No. 3,689,646).

[0081] In an embodiment, the biocide composition compatible with the enzyme biosensor is a combination of sulfadimethoxine and ormeprim in a weight ratio of sulfadimethoxine to ormeprim of 5:1 (see, eg, US Pat. No. 5,135,924).

[0082] In an embodiment, a biocide composition compatible with an enzyme biosensor is about 125 to about 1,000 μg / ml of a penicillin (e.g., amoxicillin, carbenicillin, benzylpenicillin, piperacillin, ceftazidime, etc.) and about 5 to 500 μg / ml of clavulanic acid (see, e.g., U.S. Patent No. 4,526,783). In some embodiments, the penicillin is carbenicillin.

[0083] In embodiments, a biocide composition compatible with an enzyme biosensor may include carbenicillin at a concentration between about 100 and about 1,000 μg / ml and nitrofurantoin at a concentration between about 100 and about 500 μg / mL (see, e.g., U.S. Patent No. 5,741,663). In embodiments, a biocide composition compatible with an enzyme biosensor may include carbenicillin at a concentration of about 200 μg / mL and nitrofurantoin at a concentration of about 100 μg / mL.

[0084] In embodiments, a biocide composition compatible with an enzyme biosensor may include about 100 mg / L to about 1,000 mg / L spectinomycin or streptomycin (see, e.g., U.S. Patent No. 8,466,345). In embodiments, a biocide composition compatible with an enzyme biosensor may include about 300 mg / L spectinomycin or streptomycin.

[0085] In embodiments, a biocide composition compatible with an enzyme biosensor may include about 100 to about 1,000 μg / ml of ceftazidime (US Pat. No. 8,501,457).

[0086] In embodiments, a biocide composition compatible with an enzyme biosensor may include about 50 μg / ml to about 1,000 μg / ml of streptomycin (U.S. Pat. No. 5,202,427). In embodiments, a biocide composition compatible with an enzyme biosensor may include about 100 μg / ml of streptomycin.

[0087] In embodiments, the biocide composition compatible with the enzyme biosensor is between about 1 and about 100 ppm for polymyxin B (US Pat. No. 5,283,005).

[0088] In an embodiment, the biocide composition compatible with the enzyme biosensor includes about 0.35 mg / L of polymyxin B (US Pat. No. 6,368,847).

[0089] In an embodiment, a biocide composition compatible with an enzyme biosensor may include polymyxin E (CAS No. 1066-17-7) (U.S. Patent No. 7,960,164) at a concentration between about 5 mg / L and about 200 mg / L. In an embodiment, a biocide composition compatible with an enzyme biosensor may include polymyxin E (CAS No. 1066-17-7) at a concentration of about 10 mg / L, about 15 mg / L, about 20 mg / L, about 25 mg / L, about 30 mg / L, about 35 mg / L, about 40 mg / L, about 45 mg / L, or about 50 mg / L.

[0090] In an embodiment, the biocide composition compatible with the enzyme biosensor is 0.1-0.5% w / v Vantocil IB (US Pat. No. 6,841,527) in aqueous solution.

[0091] Reagent kit or analyzer cartridge

[0092] The present disclosure also provides a kit containing a medicament of the present disclosure for the method of the present disclosure. The kit of the present disclosure may include one or more containers, which are included in one or more biocides in one or more solutions (e.g., process control solution (PCS), including but not limited to PCS-A, PCS-B, PCS-C, PSC-D, etc.). Exemplary containers may include bags, glass ampoules (e.g., sold as quality control solutions), and etc., which include solutions for calibrating and / or measuring creatine and / or creatinine by enzyme biosensors. In some embodiments, the kit also includes instructions for use according to the method of the present disclosure. In some embodiments, these instructions include a description of how biocide / solution is applied to WBCCA according to any method of the present disclosure. In some embodiments, the instructions include a description of how to install and calibrate a measurement system in the presence of a biocide as disclosed herein.

[0093] The instructions typically include information regarding biocide agent / solution concentration, agent / solution ratios, shelf life, etc. The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a piece of paper included with the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0094] 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 with the measurement systems described herein. Instructions for practicing any of the methods described herein, eg, installing and calibrating the measurement system, can be provided.

[0095] The kit of the present disclosure adopts suitable packaging. Suitable packaging includes but is not limited to vials, ampoules, bottles, large glass bottles, wide-mouth bottles, flexible packaging (such as sealed polyester film or plastic bags), foil laminated bags, etc. It is also considered that the packaging used in combination with specific equipment such as GEM Premier whole blood analyzer series (Instrumentation Laboratory, Bedford, MA) is used. In certain embodiments, at least one active agent in reagent or solution includes but is not limited to levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfonamides, sulfathiazole sodium, sulfadimethoxine, Vantocil 1B, and etc.

[0096] The kit may optionally provide additional components, such as buffers and interpretative information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0097] Reference will now be made in detail to the exemplary embodiments of the present disclosure. Although the present disclosure will be described in conjunction with the exemplary embodiments, it should be understood that the present disclosure is not intended to be limited to those embodiments. On the contrary, it is intended to cover substitutions, modifications, and equivalents that may be included within the spirit and scope of the present disclosure as defined by the appended claims. Example

[0098] The present disclosure is further illustrated by the following examples, which should not be construed as limiting. The contents of all references cited throughout the application and published patents and patent applications are incorporated herein by reference. Those skilled in the art will recognize that the present disclosure can be implemented by variations of the disclosed structures, materials, compositions, and methods, and that these variations are considered to be within the scope of the present disclosure.

[0099] Example 1: Large molecular weight biocides do not inhibit or inactivate enzyme biosensors

[0100] The present disclosure discovered that polymyxin B and polymyxin E (also known as colistin sulfate) are compatible with the ChemSTAT creatinine sensor, which measures creatinine using two hydrolase enzymes (e.g., creatininase and creatinase) and sarcosine oxidase. When included in a process control solution (PCS) at concentrations sufficient to kill microorganisms (e.g., bacteria, particularly Gram-negative bacilli, including various strains of Pseudomonas), the sensor's slope remained high enough over 21 days to consistently allow accurate measurement of creatinine in human blood and aqueous control solutions, even at the upper end of the linear range where reduced enzyme activity could result in low substrate recovery. The discovery that the biocides did not reduce the slope of this enzyme sensor to a point where performance requirements were not met or, more often, reduced the slope to zero was surprising and unexpected.

[0101] The most commonly used biocides inactivate creatine / creatinine biosensors. For example, a partial list of some antimicrobial agents that irreversibly inhibit creatininase, creatinase, and sarcosine oxidase includes the following:

[0102] 1.MIT (methylisothiazolinone)

[0103] 2. Cl-MIT (Methylisothiazolinone Chloride)

[0104] 3. BIT (Benzylisothiazolinone)

[0105] 4.MBIT (methylbenzisothiazolinone)

[0106] 5. Norfloxacin

[0107] 6. Trimethoprim

[0108] 7. Omacide IPBC (iodopropynyl butylcarbamate)

[0109] 8.Germall Plus

[0110] 9.Dantogard Plus

[0111] Figure 2A Typical curves of the slopes of creatinine and creatine over time are shown when MIT (methylisothiazolinone) is added to PCS at 300 mg / L after establishing reasonably stable slopes (e.g., slope ≥400 for creatinine and slope ≥200 for creatine) for more than 1 week. The figure shows that the addition of MIT causes all three slopes to drop to zero in less than a day. MIT is the most common biocide used in the blood gas industry for microbial contamination and is known to be compatible with oxidase enzymes such as glucose oxidase and lactate oxidase. However, Figures 2A-2B It clearly indicates that the hydrolase or sarcosine oxidase is more susceptible to inhibition, resulting in the performance loss of the creatinine sensor.

[0112] In stark contrast to MIT (top), the present disclosure found that polymyxin B, for example at 20 mg / L (bottom), had no effect on creatinine or creatine slope (see e.g. Figure 2B ). This discovery was followed by several additional rigorous tests to confirm this unexpected enzyme compatibility and the efficacy of the two polymyxins in killing Pseudomonas.

[0113] Figure 3 Example graphs showing the creatinine slope performance over time in PCS using colistin (left) and gentamicin (right) demonstrate that the sensor maintains an adequate creatinine slope over its full three-week service life. Importantly, the sensor maintains an adequate creatinine slope over its full three-week service life using colistin (see Figure 3 , left) and a cartridge with a PCS bag protected with gentamicin only (see Figure 3 In this figure, no bacterial contamination was observed on either the control or test biocide PCS bags, nor was there any evidence of enzyme inhibition, thus performance was similar.

[0114] Figure 4A and 4B is shown to spike blood samples ( Figure 4A ) or clinical specimens ( Figure 4B ) in the primary calibration / wash solution, which demonstrates excellent correlation with the reference method described above. Example 2: Long-term effects of effective biocides on creatinine and creatine slopes

[0115] Antimicrobial efficacy was determined by independent assays and the data are presented below in Table 1. Bag B was protected with 40 mg / L colistin sulfate and 40 mg / L amikacin, an aminoglycoside currently used at 200 mg / L in several GEM cartridge bags.

[0116] Table 1: ATCC9027 Pseudomonas aeruginosa, estimated inoculum size of 100,000 cfu / mL.

[0117] product CFU / ml on day 1 CFU / ml on day 3 CFU / ml on day 7 CFU / ml on day 14 flushing solution 20 <1 <1 <1 blank <1 <1 <1 <1 Plate control No growth No growth No growth No growth Pipette control No growth No growth No growth No growth Positive control Growth Growth Growth Growth

[0118] It can be seen that colistin causes rapid killing of Pseudomonas.Amikacin is typically used at 200 mg / L and is not known to be effective against Gram-negative bacilli at this lower concentration (40 mg / L).

[0119] An independent service life evaluation was conducted using PCS bags spiked to 36 mg / L and the results can be summarized as follows:

[0120] 3-week service life

[0121] For 12 analytes (pH, pCO2, Na + , K + , Ca ++ 、Cl - All three cartridges (glucose, lactate, creatinine, BUN, tCO2, and Hct) passed calibration verification with no Smart Quality Management errors detected

[0122] All slopes and excursions, including creatinine and creatine, appeared normal

[0123] • Glucose in the high glucose, low oxygen aqueous solution showed no decrease in recovery (indicating no pO2 loss): Glu ≥ 384 mg / dL on all 3 cartridges vs. a lower limit of approximately 350 mg / dL.

[0124] Based on aqueous control levels 1-5, all enzyme sensors (Crea, BUN, Glu, Lac) showed good linearity.

[0125] · pO2 of 3 cartridges was normal;

[0126] The BloodPanel performance for Weeks 1, 2, and 3 met the performance requirements based on the total allowable error limits published in the instrument manual.

[0127] Overall, PCS bags spiked with colistin (40 ppm) did not cause any damage to any of the sensors.

[0128] As described herein, an unusual class of antibiotics was discovered that is able to kill gentamicin-resistant Pseudomonas bacteria without inhibiting the activity of the hydrolase used in the outer membrane of the creatinine sensor on the ChemSTAT sensor card.

[0129] Example 3: Method for Screening Compounds for Enzyme-Compatible Biocides

[0130] According to the teachings herein, enzyme-compatible biocides can be identified in a variety of ways.

[0131] In one embodiment, a biocide candidate can be injected into, for example, a PCS-B bag after about a week of cartridge life, and any changes in the sensor slope, particularly any increase in the rate of slope decrease, can be observed over the next week or two. During this time, an aqueous solution with a high substrate concentration can be tested to ensure that the enzyme is still able to convert all substrate to product within the specified time (e.g., about one minute).

[0132] In one embodiment, multiple cartridges assembled with, for example, PCS-B bags containing candidate biocides can be tested over an extended period of time (e.g., 20-30 days) corresponding to the effective cartridge life (which may depend on the type of cartridge being tested). In this case, the cartridge begins a hydration process that typically lasts about 50 minutes with the biocide already in the solution bag (e.g., PCS-B). Typically, at least 3 cartridges can be tested, and a control cartridge that does not contain the test substance in the solution bag (e.g., PCS-B) can also be included. Over the course of 3 weeks, in addition to running aqueous controls for which an acceptable range has been established, whole human blood can also be tested to simulate normal customer use. At the end of the test period, the slope patterns of all sensors (enzyme biosensors, ion selective sensors, gas sensors, and physical sensors such as conductivity sensors) are checked to ensure that they remain within predetermined performance limits and that they do not exhibit excessive electronic drift.

[0133] Example 4: Identification of biocides compatible with creatinine sensors

[0134] As described above, various candidate molecules were screened to determine whether they were compatible with the creatinine sensor, i.e., whether they had a significant effect on the creatinine sensor slope. Table 2 lists the compounds by compatibility, molecular weight (MW) in g / mol, and molecular structure. A "fail" rating indicates that the compound inhibited the creatinine sensor slope, while a "pass" rating indicates that the compound had no significant effect on the creatinine sensor slope.

[0135] Table 2. Summary of biocide / antibiotic MW and structure vs. creatinine sensor slope loss

[0136]

[0137]

[0138]

[0139]

[0140] The above data indicate that biocidal agents having a molecular weight (MW) greater than 320 do not inhibit or prevent the function of the enzyme biosensor. Exemplary effective biocidal agents that do not inhibit or prevent the function of the enzyme biosensor include, but are not limited to, levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfonamides, sulfathiazole sodium, sulfadimethoxine, Vantocil IB, and the like.

[0141] Incorporated by Reference

[0142] All documents cited or referenced herein, and all documents cited or referenced in documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any documents incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of the present disclosure.

[0143] equivalent

[0144] It should be understood that the detailed examples and embodiments described herein are given by way of example only for illustrative purposes and are in no way to be considered as limitations of the present disclosure. Various modifications or changes thereof will be suggested to those skilled in the art and are included within the spirit and scope of this application and are considered to be within the scope of the appended claims. Additional 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 determine many equivalents to the specific embodiments of the present disclosure described herein using only routine experimentation. These equivalents are intended to be covered by the following claims.

Claims

1. A creatine / creatinine system comprising: A cartridge comprising an enzyme biosensor comprising: an enzyme layer comprising creatininase, creatinase, and sarcosine oxidase; a diffusion control membrane on top of the enzyme layer; and electrodes; and A solution comprising one or more biocides having a molecular weight greater than 320 g / mol, the one or more biocides comprising levofloxacin, polymyxin B and / or colistin.

2. The system of claim 1, wherein the enzyme biosensor is configured to measure the concentration of one or more analytes in the solution comprising the one or more biocides.

3. The system of claim 1, wherein each of the one or more biocides of the solution has a molecular weight greater than 320 g / mol.

4. The system of claim 1, wherein the diffusion control membrane is configured to limit the flux of substrate into the enzyme layer.

5. The system of claim 1, wherein the solution is in contact with the enzyme layer.

6. The system of claim 1, wherein the one or more biocides is levofloxacin.

7. The system of claim 1, wherein the enzyme biosensor comprises a creatinine sensor.

8. The system of claim 1, wherein the one or more biocides are polymyxin B and / or polymyxin E.

9. The system of claim 8, wherein the concentration of the colistin or polymyxin B in the solution is 20 to 100 mg / l.

10. The system of claim 1, wherein the one or more biocides further comprise one or more of: carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, colistin, sulfonamides, sulfathiazole sodium, sulfadimethoxine, or polyhexamethylene biguanide hydrochloride.

11. The system of claim 1 , wherein the one or more biocides further comprise one or more of amoxicillin, ampicillin, carbenicillin, cefazolin, cefepime, cefoxitin, ceftazidime, clavulanic acid, imipenem, oxacillin, penicillin, or piperacillin.

12. The system of claim 1, wherein the solution is a biological sample, a process control solution, a calibration solution, a quality control solution, a conditioning solution, or a cleaning solution.

13. The system of claim 1, wherein the enzyme biosensor is in a cartridge.

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