CN114846332B

CN114846332B - Biocide compositions compatible with enzyme biosensors and methods of use thereof

Info

Publication number: CN114846332B
Application number: CN201980103233.5A
Authority: CN
Inventors: D.R.康伦; P.帕米迪
Original assignee: Instrumentation Laboratory Co
Current assignee: Werfen North America
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2025-08-26
Anticipated expiration: 2039-10-25
Also published as: CN114846332A; EP4049036A1; WO2021080598A1

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

Biocidal compositions compatible with enzymatic biosensors and methods of use thereof

Technical Field

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

Background

The whole blood intensive care analyzer (WBCCA) plays an important role in the management of critical patients by providing a rapid treatment turnaround time at the point of care. Biocides are key components of WBCCA reagents and inhibit the effect of microbial metabolism on blood analysis results. For example, the normal oxygen content in human blood is very low and any consumption of oxygen by bacteria in the calibration solution can lead to significant errors in the reported oxygen levels. WBCCA have also been developed to include electrochemical biosensor-based metabolite measurements, such as creatinine/creatinine levels in a sample (e.g., a patient's blood), which are important indicators of kidney function. Current creatinine sensors may include enzyme biosensors comprising three enzymes-creatininase, creatinase and sarcosine oxidase, which catalyze the production of glycine, formaldehyde and hydrogen peroxide from creatinine and water, so the final reaction product of hydrogen peroxide (H ₂O₂) 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 a number of parameters, including solution conditions, e.g., pH, temperature, levels of metabolites such as oxygen, and the like. In addition, 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, creatinine biosensors are an important enzyme biosensor example that is inactivated by current biocides. Thus, there is a need for biocide compositions that are compatible with enzymatic biosensors.

Summary of The Invention

The present disclosure provides effective biocidal agents that do not inhibit or prevent the function of a whole blood intensive care analyzer (WBCCA). In particular, the present disclosure provides some effective biocides that do not inhibit or prevent WBCCA from having a sensor function or an enzymatic biosensor function. Furthermore, the present disclosure provides biocidal agents having a Molecular Weight (MW) greater than 320 that do not inhibit or prevent the function of an enzymatic biosensor. In addition, the present disclosure provides some effective biocides that do not inhibit or prevent the function of the enzymatic biosensor. Exemplary potent 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, sulfa, sulfathiazole sodium, sulfadimidine, vantocil IB, and the like. Advantageously, the biocidal agents disclosed herein are capable of effectively neutralizing or destroying unwanted organisms (e.g., bacteria, fungi, etc.) without inhibiting or preventing the function of the enzymatic biosensor. The present disclosure also provides methods of identifying biocides compatible with any 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 a whole blood intensive care analyzer (WBCCA) with an enzymatic biosensor to effectively read biological samples in the presence of biocidal agents that do not adversely affect the function of the enzymatic biosensor.

In one aspect, the present disclosure provides a method for maintaining sensor functionality comprising the steps of adding one or more Sensor Compatible Biocides (SCB) to a solution, and measuring the concentration of one or more analytes with a sensor. In some embodiments, the sensor may be a biosensor, a gas sensor, an ion selective electrode, or a photometric sensor. In some embodiments, the biosensor is an enzymatic biosensor. In some embodiments, the enzyme biosensor may be a creatinine sensor, a creatine sensor, or a combination thereof.

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

In some embodiments, the SCB is a polymyxin selected from the group consisting of polymyxin B, polymyxin E, and combinations thereof.

In some embodiments, the SCB is polymyxin B.

In some embodiments, the SCB is a fluoroquinolone.

In some embodiments, the SCB comprises a sulfone group.

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.

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.

In some embodiments, the SCB comprises at least one β -lactam antibiotic and a polymyxin.

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

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

In one aspect, the present disclosure provides a method of identifying an Enzyme Biosensor Compatible Biocide (EBCB) of 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 antimicrobial efficacy of the biocide 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 assessed based on an enzyme slope, and selecting EBCB according to the enzyme slope.

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

In some embodiments EBCB contains a sulfone group.

In some embodiments EBCB is a β -lactam antibiotic.

In some embodiments, WBCCA sensors are gas sensors, ion-selective electrodes, photometric sensors, and the like.

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

In one aspect, the present disclosure provides compositions comprising one or more Enzyme Biosensor Compatible Biocides (EBCB) selected from the group consisting of levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfa, sulfathiazole sodium, sulfadimethoxine, and Vantocil IB.

In one aspect, the present disclosure provides a composition comprising a first Enzyme Biosensor Compatible Biocide (EBCB), and a second EBCB.

In some embodiments, the first EBCB is selected from the group consisting of levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfanilamide, sulfathiazole sodium, sulfadimidine, and Vantocil IB.

In some embodiments, the second EBCB is selected from the group consisting of levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfanilamide, sulfathiazole sodium, sulfadimidine, and Vantocil IB.

In some embodiments, the first EBCB or the second EBCB is a penicillin selected from amoxicillin, carbenicillin, and benzyl penicillin.

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

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.

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

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

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

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

In some embodiments, the first EBCB or the second EBCB is about 20 to 100mg/L of polymyxin E.

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

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

Definition of the definition

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

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 by the recycling of Adenosine Triphosphate (ATP) that converts Adenosine Diphosphate (ADP) back to ATP by providing phosphate groups. Creatine has the following chemical structure:

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

Ranges may be expressed herein as from "about" one particular value, and/or "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 also be 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 is also to be understood that a number of values are disclosed herein, and that each value is also disclosed herein as "about" that particular value, in addition to the value itself. It should also be understood that throughout this application, data is provided in a variety of different formats, and that the data represents ranges for any combination of endpoints and starting points, and data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than or equal to, and equal to 10 and 15 are considered disclosed and between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed. The ranges provided herein are to be understood as shorthand for all values that fall within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers 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 integers described above, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, specifically considered are "nested sub-ranges" extending from either end of the range. For example, nested subranges of the exemplary ranges 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 another direction.

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

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

Brief Description of Drawings

The following detailed description, given by way of example, is not intended to limit the disclosure to the specific embodiments described, but is best understood when read in conjunction with the accompanying drawings, in which:

FIGS. 1A-1C show the crystal structure reconstruction of the enzyme present in the creatininase sensor. FIG. 1A is a band diagram showing the quaternary structure of a creatininase hexamer known in the prior art. FIG. 1B is a band diagram showing the tertiary structure of the N-terminal domain of creatinase known in the prior art. FIG. 1C is a band diagram showing the tertiary structure of sarcosine oxidase known in the prior art.

FIGS. 2A-2B show graphs of creatinine and creatine slope over time in the presence of MIT or polymyxin B, respectively. Fig. 2A shows a graph of creatinine and creatine slope over time, indicating that the addition of MIT results in a rapid decay of creatinine/creatine slope without recovery over time. Fig. 2B shows a graph of creatinine and creatine slope over time, indicating that the addition of polymyxin B does not cause rapid decay of the creatinine/creatine slope. A steady slope is first established for more than one week before 300mg/L MIT (methylisothiazolinone) or 20mg/L polymyxin B is added to Process Control Solution (PCS) B (e.g., PCS-B).

Fig. 3 shows an exemplary plot of creatinine slope over time in a PCS with polymyxin (left) and gentamicin (right), indicating that the sensor maintains adequate creatinine slope over a full lifetime of three weeks.

Fig. 4A and 4B are graphs showing the effect of colistin in a primary calibration/wash solution in a labeled blood sample (fig. 4A) or clinical specimen (fig. 4B).

Detailed Description

The present disclosure is based, at least in part, on the unexpected discovery that biocidal agents falling within a particular 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 biocidal agents having a Molecular Weight (MW) greater than 320 do not inhibit or prevent the function of enzymes within an enzymatic biosensor. The present disclosure provides a number of specific effective biocidal agents that do not inhibit or prevent the function of enzymes within an enzymatic biosensor, including but not limited to levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfa, sulfathiazole sodium, sulfadimidine, vantocil IB, and the like. Advantageously, the biocidal agents disclosed herein are capable of effectively neutralizing or destroying unwanted organisms (e.g., bacteria, fungi, etc.) without inhibiting or preventing the function of the enzymatic biosensor. The present disclosure also provides methods of identifying effective biocides specific to and compatible with any 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 a whole blood intensive care analyzer (WBCCA) with an enzyme-based biosensor to effectively read biological samples in the presence of biocidal agents that do not adversely affect the function of the enzyme biosensor.

SUMMARY

Historically WBCCA was used only to measure blood gas, electrolytes and CO-blood oxygen saturation, which makes the selection of compatible biocides for these assays relatively simple. However, as the detection methods of WBCCA are extended to include metabolites such as glucose, lactic acid, creatinine, creatine, and the like, identifying biocides compatible with these detection methods becomes difficult because they typically include enzyme-based biosensors and enzymes incorporated into these biosensors are inactivated by most biocides. In the prior art, it has been a significant challenge to identify biocidal agents that are sufficiently powerful to kill all possible bacteria, yeasts and fungi without simultaneously inactivating enzymes incorporated into enzyme biosensors. For example, the prior art solution adopted by medical manufacturers of intensive care analyzers is to change from using chlorinated isothiazolinones (e.g. ProClin300,300) to non-chlorinated isothiazolinones (e.g. Methylisothiazolinone (MIT)). Disadvantageously, non-chlorinated isothiazolinones have higher Minimum Inhibitory Concentrations (MIC), requiring higher concentrations to meet minimum bactericidal requirements and resulting in increased reagent costs.

Whole blood based blood gas analyzers are more susceptible to microbial growth than most other types of clinical analyzers because their calibrator maintains a stable oxygen level reported as partial pressure pO ₂. This presents a serious problem because the reported patient pO ₂ values typically sound very high, e.g., 90mm Hg is a normal pO ₂ result, similar to a normal chloride result of 95mmol/L and much higher than normal ionic calcium, about 1.1-1.2mmol/L, however, these values are fraudulent in that molar and millimoles are important and the normal oxygen content in human blood is only 0.1mmol/L due to poor solubility of oxygen in water. Given that the calibrator is expected to stabilize within 1% (if possible), then an oxygen loss of only 0.001 millimoles or 1 micromolar may cause the accuracy of the oxygen channel on the analyzer to be below ideal. Thus, bacterial contamination within the analyzer may produce significant measurement errors. For example, if a given bacterial colony consumes 10 micromolar of both oxygen and glucose (e.g., in a calibration solution), the analyzer will report a significant error of about 10% of pO ₂, while the error in glucose measurement will be less significant in scale, e.g., from about 5.00mmol/L to 4.99mmol/L, which is relatively insignificant.

The problem of finding effective biocides is exacerbated by the use of different classes of enzymes required for other metabolites such as creatinine and urea or Blood Urea Nitrogen (BUN). Glucose and lactate are most commonly measured with glucose oxidase and lactate oxidase, respectively, while creatinine is measured with two hydrolase-type enzymes (e.g., creatininase and creatinase) and sarcosine oxidase. Unfortunately, some of these enzymes, such as creatininase, are inactivated by biocides of the prior art.

Creatininase, also known as creatinine amidohydrolase or creatinine hydrolase, is a Zn ²⁺ ion-dependent hexamer that catalyzes the hydrolysis of creatinine to creatinine. Creatine enzymes, also known as creatine amino hydrolases, catalyze 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.

Current creatinine sensors in creatine/creatinine systems (e.g., GEM PAK cartridges) include enzyme biosensors that contain 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):

The product hydrogen peroxide (H ₂O₂) was then electrochemically oxidized at a constant polarization potential on a platinum electrode, with the current signal being proportional to the analyte concentration.

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

Creatine and creatinine sensors have a diffusion controlling membrane (also called the adventitia) on top of the enzyme layer. The diffusion-controlled membrane limits the flux of creatinine and creatine substrates into the enzyme layer to ensure that the signal generated by hydrogen peroxide is proportional to the substrate concentration of the sample.

The calibration system of the creatine sensor or the biosensor may involve 2-point calibration based on the following equation:

Δi2= [ cr_cs2 ]. Slope (eq.1)

Δi2 is the current signal measured on the creatine sensor in the first calibration solution (CS 2). [ CR_CS2] is the creatine concentration in the first calibration solution (CS 2). 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.

In accordance with the techniques herein, a calibration system for a creatinine sensor or biosensor may implement a 3-point calibration method. Since the creatinine sensor provides a reading of 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.

3-Point creatinine sensor calibration equation:

Δi2' = [ crea_cs2 ]. Times.slope 1+ [ cr_cs2 ]. Times.slope 2 (eq.2)

Δi3' = [ crea_cs3 ]. Times.slope 1+ [ cr_cs3 ]. Times.slope 2 (eq.3)

Δi2 'and Δi3' are current signals measured on the creatinine sensor in the first calibration solution (CS 2) and the second calibration solution (CS 3), 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.

[ CREA_CS2], [ CREA_CS3], [ CR_CS2] and [ CR_CS3] represent the initial known concentrations of creatinine and creatine in the 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 deduced from eq.2 and 3:

Slope 1= ([ cr_cs3 ]. DELTA.i2 '- [ cr_cs2 ]. DELTA.i3')/([ Creatjcs2 ] [ cr_cs3] - [ Creatjcs3 ] - [ cr_cs2 ]) pA/mg/dL (eq.4)

Slope 2= ([ crea_cs2 ]. DELTA.i3 '- [ crea_cs3 ]. DELTA.i2')/(Creat_ cs2] - [ Creat_ cs3] - [ Creat_ cs2 ]. DELTA.cjcs2 ]) pA/mg/dL (eq.5)

Further complicating the problem, it has been found that the few biocides that have been identified so far that are compatible with glucose and lactate oxidase are incompatible with creatininase, creatinase and sarcosine oxidase, which are commonly used together. In particular, MIT as a commonly used biocide results in rapid inactivation of creatinase and creatinase. Difficulties associated with identifying biocides compatible with proteins such as enzymes or antibodies are discussed in U.S. patent No. 5,506,216, which teaches that proteins can be denatured by such substances. U.S. patent No. 5,506,216 identifies several biocides, including o-phenylphenol, densil P [ dithio-2, 2' -bis (benzamide) ], [1, 2-benzisothiazolin-3-thione ] (Proxel), methylene dithiocyanate, cyanate, hydroxyquinoline, carbendazim [ -methoxycarbonylamino-benzimidazole ] and dazomet [3, 5-dimethyltetrahydro-1, 3, 5-thiadiazine-2-thione ], and finds that these agents can function as biocides that do not interact with proteins, provided they are complexed with cyclodextrin. Consistent with these, few, if any, of these biocides are compatible with creatininase, creatinase and sarcosine oxidase based sensors.

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

In embodiments, the biocide composition compatible with the enzyme biosensor is a combination of a sulfonamide drug and another sulfonamide drug (see, e.g., U.S. patent No. 9,029,118), or any other compatible biocide disclosed herein. In embodiments, the sulfonamide may be present at a concentration of about 0.05g/L to about 20g/L, about 0.3g/L to about 10g/L, about 0.3g/L to about 5g/L, and the like. It is contemplated within the scope of the present disclosure that biocide compositions compatible with enzymatic biosensors may be combinations of sulfonamides with any of the other compounds disclosed herein.

In embodiments, the biocide composition compatible with the enzyme biosensor 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, e.g., U.S. patent No. 3,689,646).

In embodiments, the biocide composition compatible with the enzyme biosensor is a combination of sulfamethoxazole and omeprazole, in a weight ratio of sulfamethoxazole to omeprazole of 5:1 (see, e.g., U.S. patent No.5,135,924).

In embodiments, the biocide composition compatible with the enzyme biosensor is about 125 to about 1,000 μg/ml penicillin (e.g., amoxicillin, carbenicillin, benzyl penicillin, piperacillin, ceftazidime, etc.) and about 5 to 500 μg/ml clavulanic acid (see, e.g., U.S. patent No. 4,526,783). In some embodiments, the penicillin is carbenicillin.

In embodiments, a biocide composition compatible with an enzyme biosensor can include carbenicillin at a concentration of between about 100 and about 1,000 μg/mL and nitrofurantoin at a concentration of 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 can include carbenicillin at a concentration of about 200 μg/mL and nitrofurantoin at a concentration of about 100 μg/mL.

In embodiments, the biocide composition compatible with the enzyme biosensor can include from about 100mg/L to about 1,000mg/L spectinomycin or streptomycin (see, e.g., U.S. patent No. 8,466,345). In embodiments, the biocide composition compatible with the enzyme biosensor may include about 300mg/L of spectinomycin or streptomycin.

In embodiments, the biocide composition compatible with the enzyme biosensor can include from about 100 to about 1,000 μg/ml of ceftazidime (U.S. patent No. 8,501,457).

In embodiments, the biocide composition compatible with the enzyme biosensor can include from about 50 μg/ml to about 1,000 μg/ml of streptomycin (U.S. patent No. 5202427). In embodiments, the biocide composition compatible with the enzyme biosensor can include about 100 μg/ml of streptomycin

In embodiments, the biocide composition compatible with the enzyme biosensor is about 1 and about 100ppm for polymyxin B (U.S. patent No. 5283005).

In an embodiment, the biocide composition compatible with the enzyme biosensor includes about 0.35mg/L of polymyxin B (U.S. patent No. 6368847).

In embodiments, the biocide composition compatible with the enzyme biosensor can include polymyxin E (CAS No. 1066-17-7) at a concentration of between about 5mg/L and about 200mg/L (U.S. patent No. 7960164). In embodiments, the biocide composition compatible with the enzyme biosensor can include polymyxin E (CAS No. 1066-17-7) at a concentration of about 10mg/L, about 15mg/L, about 20mg/L, about 25mg/L, about 30mg/L, about 35mg/L, about 40mg/L, about 45mg/L, or about 50 mg/L.

In embodiments, the biocide composition compatible with the enzyme biosensor is 0.1-0.5% w/v Vantocil IB in aqueous solution (U.S. Pat. No. 6841527).

Kit or analyzer cartridge

The present disclosure also provides kits containing the agents of the present disclosure for use in the methods of the present disclosure. Kits of the present disclosure may include one or more containers containing one or more biocides in one or more solutions (e.g., process Control Solutions (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 the like, including solutions for calibrating and/or measuring creatine and/or creatinine by an enzyme biosensor. In some embodiments, the kit further comprises instructions for use according to the methods of the present disclosure. In some embodiments, these instructions include a description of how to apply the biocide/solution to WBCCA according to any of the methods of the present disclosure. In some embodiments, the instructions include a description of how to install and calibrate the measurement system in the presence of a biocidal agent as disclosed herein.

The instructions typically include information about biocide reagent/solution concentration, reagent/solution ratio, shelf life, etc. The instructions provided in the kits of the present disclosure are typically written instructions for labeling or packaging instructions (e.g., paper contained in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

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

The kits of the present disclosure employ suitable packaging. Suitable packages include, but are not limited to, vials, ampoules, bottles, large glass bottles, jars, flexible packaging (e.g., sealed mylar or plastic bags), foil laminate bags, and the like. Packages for use in combination with specific devices such as GEM PREMIER whole blood analyzers series (Instrumentation Laboratory, bedford, MA) are also contemplated. In certain embodiments, at least one active agent in the agent or solution includes, but is not limited to, levofloxacin, carbenicillin disodium, spectinomycin, piperacillin, ceftazidime, streptomycin, polymyxin B, polymyxin E, sulfa, sulfathiazole sodium, sulfadimidine, vantocil IB, and the like.

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

Reference will now be made in detail to exemplary embodiments of the present disclosure. While the present disclosure will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the present 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 present 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 the application are incorporated herein by reference. Those skilled in the art will recognize that the present disclosure can be practiced with modification to the structures, materials, compositions, and methods disclosed, and that such modifications are considered to be within the scope of the present disclosure.

Example 1 high molecular weight biocides do not inhibit or inactivate enzyme biosensors

The present disclosure finds that polymyxin B and polymyxin E (also known as colistin sulfate) are compatible with ChemSTAT creatinine sensors that measure creatinine using two hydrolase-type enzymes (e.g., creatinase and creatinase) and sarcosine oxidase. When included in the Process Control Solution (PCS) at a concentration sufficient to kill microorganisms (e.g., bacteria, particularly gram-negative bacilli, including various pseudomonas strains), the slope of the sensor remains high enough over 21 days to continue to 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 may result in low recovery of substrate. It is surprising and unexpected that biocides were found to not reduce the slope of such enzyme sensors to a point where they did not meet performance requirements or more often reduce the slope to zero.

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

MIT (methylisothiazolinone)

Cl-MIT (methyl isothiazolinone chloride)

BIT (benzyl isothiazolinone)

MBIT (methyl benzisothiazolinone)

5. Norfloxacin

6. Trimethoprim

Omamide IPBC (iodopropynyl butylcarbamate)

8.Germall Plus

9.Dantogard Plus

FIG. 2A shows a typical plot of creatinine and creatine slope over time when MIT (methylisothiazolinone) is added to PCS at 300mg/L after a reasonably stable slope (e.g., creatinine slope. Gtoreq.400, creatinine slope. Gtoreq.200) is established for more than 1 week. The graph shows that adding an MIT results in all three slopes falling to zero in less than one day. MIT is the most common biocide used for microbial contamination in the blood gas industry and is known to be compatible with oxidase-type enzymes such as glucose oxidase and lactate oxidase. However, FIGS. 2A-2B clearly demonstrate that the hydrolase or sarcosine oxidase is more susceptible to inhibition, resulting in a loss of performance of the creatinine sensor.

In sharp contrast to MIT (up), the present disclosure found that polymyxin B, e.g., 20mg/L (down), had no effect on creatinine or creatine slope (see, e.g., FIG. 2B). After this discovery, several additional rigorous tests were performed to confirm this unexpected enzyme compatibility and the efficacy of both polymyxins in killing pseudomonas.

Fig. 3 shows an exemplary graph of creatinine slope performance over time in PCS using polymyxin (left) and gentamicin (right), showing that the sensor maintains adequate creatinine slope over a full lifetime of three weeks. Importantly, the creatinine slope performance with colistin in the PCS bag (see fig. 3, left) was equivalent to that of the cartridge with the PCS bag protected with gentamicin alone (see fig. 3, right). In this figure, no bacterial contamination was observed on the control or test biocide PCS bags, nor was there any evidence of enzyme inhibition, and therefore performance was similar.

Fig. 4A and 4B are graphs showing the effect of colistin incorporated into a main calibration/wash solution of a blood sample (fig. 4A) or clinical specimen (fig. 4B), which proves to have excellent correlation with the above-mentioned reference method. Example 2 Long term Effect of effective biocides on creatinine and creatine slope

Antimicrobial efficacy was determined by an independent assay and the data are presented in table 1 below. The B bag was protected with 40mg/L colistin sulfate and 40mg/L amikacin, an aminoglycoside currently used at 200mg/L in several GEM cartridge bags.

Table 1. ATCC9027 Pseudomonas aeruginosa, estimated inoculum size was 100,000cfu/mL.

Product(s)	Day 1 CFU/ml	Day 3 CFU/ml	Day 7 CFU/ml	Day 14 CFU/ml
					Flushing solution	20	<1	<1	<1
Blank space	<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

It can be seen that colistin causes a rapid kill of pseudomonas. Amikacin is typically used at 200mg/L and is not known to be effective against gram-negative bacilli at this lower concentration (40 mg/L).

Independent service life evaluations were performed using PCS bags labeled to 36mg/L and the results can be summarized as follows:

Service life of 3 weeks

All 3 cartridges for 12 analytes (pH, pCO ₂、Na⁺、K⁺、Ca⁺⁺、Cl^-, glucose, lactic acid, creatinine, BUN, tCO ₂ and Hct) were verified by calibration and no intelligent quality management errors were detected

All slopes and drifts, including creatinine and creatine, are normal

Glucose in the high-sugar low-oxygen aqueous solution showed no recovery drop (indicating no pO ₂ loss) Glu≥384 mg/dL vs lower limit of about 350mg/dL on all 3 cartridges.

Based on the aqueous control levels 1-5, the linearity of all enzyme sensors (Crea, BUN, glu, lac) is good,

PO ₂ for 3 cartridges was normal;

BloodPanel performance at weeks 1,2 and 3 meets performance requirements according to the total allowed error limits published in the instrument manual.

In general, PCS bags incorporating polymyxin E (40 ppm) were harmless to any sensor.

As described herein, an unusual class of antibiotics was found that was able to kill pseudomonas resistant to gentamicin without inhibiting the activity of hydrolytic enzymes used in the outer membrane of the creatinine sensor on ChemSTAT sensor cards.

Example 3 methods of screening Compounds of enzyme-compatible biocides

Enzyme compatible biocides can be identified in a variety of ways in accordance with the techniques herein.

In one embodiment, the biocide candidate may be injected into, for example, one PCS-B bag after about one week of cartridge life, and any change in sensor slope, particularly any increase in slope decrease rate, may then be observed over the next one or two weeks. During this time, aqueous solutions with high substrate concentrations may be tested to ensure that the enzyme is still able to convert all substrates to products within a defined time (e.g., about one minute).

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

Example 4 identification of biocides compatible with creatinine sensors

The various candidate molecules are screened as described above to determine whether they are compatible with the creatinine sensor, i.e., have a significant impact on the creatinine sensor slope. Table 2 shows the compatibility of the compounds vs, the Molecular Weight (MW) in g/mol and the molecular structure. "failing" means that the compound inhibited the creatinine sensor slope, while "pass" means that there is no significant effect on the creatinine sensor slope.

TABLE 2 summary of slope losses for biocide/antibiotic MW and structural vs creatinine sensors

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 potent 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, sulfa, sulfathiazole sodium, sulfadimidine, vantocil IB, and the like.

Incorporated by reference

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

Equivalents (Eq.)

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 construed as limiting the present disclosure. Various modifications or changes thereto will suggest themselves to those skilled in the art and are encompassed within the spirit and scope of the 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. Furthermore, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A creatine/creatinine system comprising:

A cartridge (cartridge) comprising an enzyme biosensor, the enzyme biosensor comprising:

an enzyme layer comprising creatininase, creatinase and sarcosine oxidase;

A diffusion control membrane on top of the enzyme layer, and

An electrode, and

A solution comprising one or more biocides having a molecular weight greater than 320g/mol, said one or more biocides comprising levofloxacin, polymyxin B and/or polymyxin E.

2. The system of claim 1, wherein the enzyme biosensor is configured to measure a 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-controlling membrane is configured to limit 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 polymyxin E or polymyxin B in the solution is 20 to 100mg/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, polymyxin E, sulfa, sulfathiazole sodium, sulfadimidine, 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, 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 (cartridge).