Non-enzyme based detection method for electronic monitoring of biological indicator

A sterilization indicator system and method of using the system to determine efficacy of a sterilization process. The system may include a vial having an optional first compartment and a second compartment comprising a growth medium comprising one or more of a disaccharide, an oligosaccharide or a polysaccharide capable of conversion to a monosaccharide by germinating spores of the one or more species of microorganism, the vial being free of the monosaccharide prior to use; a strip including two or more electrodes to oxidize the monosaccharide and to carry a resulting electrical signal, and an apparatus to detect and measure the electrical signal resulting from the oxidation. Spores of a suitable biological indicator may be disposed in the first compartment and/or on the strip.

TECHNICAL FIELD

The present invention relates to biological indicators for testing the efficacy of sterilization processes, more specifically, to non-enzyme-based methods for monitoring such biological indicators, in which such monitoring can be carried out electronically.

BACKGROUND

One of the most important classes of indicators are the biological indicators (BI). Biological indicators provide the highest degree of assurance that sterilization conditions were met within the processor or processed load itself. This type of indicator is meant to represent the worst case for the processing system by providing an extremely high number of highly resistant organisms to that particular process within or on the indicator. Usually bacterial spores are the organism of choice for monitoring sterilization systems.

Biological indicators typically consist of microorganisms inoculated onto a carrier material. The microorganisms are typically bacterial spores that are known to be very resistant to the particular sterilization medium in which they are to be used. The carrier is placed into a sterilization cycle along with the medical device load. Following completion of the cycle the biological indicator is incubated and monitored for growth for up to seven days. Growth of a biological indicator indicates that the sterilization process was not adequate to attain complete sterilization and that the medical device load needs to be reprocessed before use. No growth of a biological indicator confirms that conditions within the sterilizer were adequate to kill at least the number of bacterial spores loaded onto the indicator (e.g., 10 bacterial spores) and therefore provides a level of assurance that the medical device load is sterile. Unfortunately many medical devices are actually used prior to the user knowing the results of the full incubation. Thus, there is a need in the hospital setting for detection of viable biological indicator spores in the shortest possible timeframe.

Historically, the detection of viable biological indicators relied on visual means of detection. The growth and multiplication of viable organisms can be seen/detected as evidenced by turbidity in the growth media. This turbidity can take days to become noticeable. Another visual and more common means of detection is with a colorimetric pH indicator. As viable organisms begin to metabolize and use up the nutrient sources such as sugars that are provided in the growth media, they excrete acidic waste products. As these acidic waste products accumulate in the growth media, the pH of the system is lowered resulting in a color change of the growth media if a pH indicator is present. Detection by this means usually takes 18-48 hours.

More recently, fluorescence has been used to detect the activity of enzymes that are produced by the organisms of interest by adding a fluorogenic enzymatic substrate to the growth media. This newer methodology lessens the incubation time from days to hours. However, the main limitation for reducing the incubation time beyond that seen for the fluorescence methodology is the inherent background fluorescence that naturally occurs with many components of the biological indicator including the plastic vials and growth media. Authentic, detectable signals must be high enough to be distinguishable over this inherent native background fluorescence. Therefore to increase the sensitivity of the system one needs to either reduce the background fluorescence (noise) or move to a different technology that has higher sensitivity (signal).

Thus, in the prior and current art, biological indicators rely on colorimetric or fluorometric means to determine viability. Detection is limited by the need for the generated signals, whether colorimetric or fluorometric, to be above substantial background levels. This has resulted in detection times for viable organisms on the order of hours to days in order for sufficient signal to be accumulated to be detectable above background levels. It would be beneficial for both hospitals and patients for the detection time of viable organisms in biological indicators to be on the order of minutes or less.

SUMMARY

One such method that permits control over the signal to noise ratio is electrical detection. The monitoring of changes in the electrical properties of systems is a sensitive means to monitor for other changes within that system. The resulting electric outputs can then be conditioned by electronic means to provide amplified and filtered signals that are directly proportional to the reagent generating them.

The present invention provides a rapid detection of viable microorganisms of a biological indicator using a non-enzyme-based electronic detection method to detect the accumulation of simple sugars such as glucose resulting from the enzymatic breakdown of complex sugars. The non-enzyme-based system of the present invention includes a combination of at least one naturally occurring glycosidase present in a viable spore and electrodes adapted to oxidize a simple sugar, such as glucose, produced by the glycosidase in the viable spore. The incubation or growth medium provided for the spores post-sterilization is provided free of the simple sugar and with a complex sugar, such as a disaccharide or a polysaccharide. The glycosidase reacts with the complex sugar added to the growth media and breaks it down into the simple sugars, including, for example, at least one glucose. The simple sugar product is then exposed to the electrodes on a strip that are adapted to selectively oxidize glucose which produces an electron transfer as part of its oxidation of the simple sugar. The quantity of the electron transfer is proportional to the amount of the simple sugar produced by viable spores. The transfer of free electrons in the oxidation can be monitored and measured electronically. If the electronic monitoring detects electron transfer, it means that at least some spores are viable and have survived the sterilization process, and so showing the sterilization was not efficacious. The electrodes may be those used in standard, state of the art blood glucose monitoring devices, and in fact, known state of the art blood glucose monitoring devices can be readily adapted for use in determining the presence of any glucose or simple sugar in the combined media. Thus, the present invention allows the determination of the efficacy of the sterilization process by a very specific, very sensitive method that can be simply carried out and measured electronically. In one embodiment, the system and process uses standard, state of the art devices designed for use in monitoring blood glucose levels in diabetic patients.

Thus, in one embodiment, the present invention relates to a sterilization indicator system, including:

spores of one or more species of microorganism;

a vial comprising an optional first compartment and a second compartment containing a growth medium comprising one or more of a disaccharide or a polysaccharide capable of conversion to a monosaccharide by germinating spores of the one or more species of microorganism, the vial being adapted to combine contents of the second compartment with the spores for analysis after the vial has been exposed to the sterilant;

a strip comprising two or more electrodes adapted to oxidize the monosaccharide and to carry an electrical signal resulting from the oxidation of the monosaccharide, wherein the two or more electrodes are adapted to provide contact with the combined contents of the second compartment and the spores during and/or after incubation; and

an apparatus linked or linkable to the two or more electrodes and adapted to detect and measure the electrical signal resulting from electron transfer when the monosaccharide is oxidized by the two or more electrodes,

wherein the system is free of the monosaccharide prior to exposure to a sterilant.

In one embodiment, the first compartment is absent, the spores are disposed on the strip, and the strip initially is separate from the second compartment.

In one embodiment, the first compartment is present, the spores are disposed on the strip, and the strip initially is separate from both the first compartment and the second compartment.

In one embodiment, the first compartment is present, the spores are disposed in the first compartment, and the strip initially is separate from both the first compartment and the second compartment.

In one embodiment, the first compartment is present, the spores are disposed in the first compartment, and the strip initially is in the first compartment.

In one embodiment, the first compartment is present, the spores are disposed on the strip, and the strip initially is in the first compartment.

In one embodiment, the first compartment is present, the spores are disposed in the first compartment, and the strip initially is in the second compartment.

In one embodiment, the monosaccharide is glucose.

In one embodiment, the one or more species of microorganism comprises one or both ofGeobacillus stearothermophilusandBacillus atrophaeus.

In one embodiment, the apparatus is a glucose reader.

In one embodiment, the two or more electrodes are linkable to a glucose reader by a wireless link.

In one embodiment, the disaccharide is maltose that is converted to glucose by a glucosidase produced by or present in the germinating spores.

In one embodiment, the at least two electrodes comprise graphite, graphene, carbon, carbon nanotubes, gold, platinum, palladium, silver, nickel or copper or a combination or alloy of any two or more thereof.

Thus, the present invention provides an elegant and simple solution to the problem of rapidly determining the efficacy of a sterilization process, and provides an apparatus specially adapted for such use.

It should be appreciated that for simplicity and clarity of illustration, elements shown in the Figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to each other for clarity. Further, where considered appropriate, reference numerals have been repeated among the Figures to indicate corresponding elements.

Furthermore, it should be appreciated that the process steps and structures described below may not form a complete process flow for producing an end-useable sterilization indicator. The present invention can be practiced in conjunction with apparatus and processing techniques currently used in the art, and only so much of the commonly practiced process steps are included as are necessary for an understanding of the present invention.

DETAILED DESCRIPTION

The biological indicators described in the following rely on a new mechanism to detect spore viability. The invention described here utilizes an electronic signal that is generated based on the accumulation of a monosaccharide, e.g., glucose, that results from the ability of viable organisms to break down complex sugars. Electronic detection methods based on glucose have been readily available for years for monitoring the glucose levels in the blood of diabetic patients. Until now, however, there has been neither use of nor suggestion to adapt these electronic detection mechanisms as a means to detect viable organisms following sterilization processes.

Most organisms have inherent capabilities to break down complex sugars (such as maltose) into simple sugars (such as glucose) in order that the more useful simple sugar molecule can be utilized as an energy source by the organism. The same basic reactions utilized in monitoring glucose levels in blood can be adapted to detect viable organisms, such as spores, that may survive a sterilization cycle. In one embodiment of the present invention, the same electronic glucose monitors may be adapted for use in monitoring efficacy of sterilization processes. Unlike blood glucose monitoring, however, where glucose is prevalent and is measured directly, in the present invention, initially no glucose is present, and glucose is only obtained from an undetectable complex carbohydrate molecule that must first be acted upon by a viable organism before glucose is released and able to be detected. If no viable organisms survive the sterilization conditions, there is no glucose at all present to be detected. Thus, in the present invention, the simple sugar, usually glucose, is never present unless and until a viable microorganism that has survived the sterilization conditions being monitored breaks down a complex carbohydrate to form the simple sugar. If and when this breakdown occurs, it may be detected and measured in accordance with the present invention.

As viable spores begin to germinate (a fundamental life activity) they produce and release enzymes that enable them to break down the complex sugars into more readily usable simple sugars such as glucose. By formulating a medium that is high in complex sugars, such as disaccharides, oligosaccharides and/or polysaccharides (selected on the basis that they can be broken down by the active enzymes of viable test organisms selected for the sterilization indicator) an increase in monosaccharide, e.g., glucose, would be expected upon exposing viable spores to this medium and this increasing concentration of the monosaccharide in the growth media can be detected electronically by electrodes adapted to oxidize the monosaccharides. Under the conditions of sterilization, spores are killed and any enzymes the spores may have possessed prior to the sterilization will be destroyed. Therefore, an increase in the monosaccharide detected after exposure of these spores to the medium of this invention means that the organisms are viable (proof of life). The spore mediated conversion of complex sugars to glucose represents the first step in detecting viable organisms according to the process of the present invention. The second step is the oxidation of any thus-produced monosaccharide by the two or more electrodes, and the third step is detection of any electrical signal accompanying the oxidation.

The organisms of most interest for monitoring sterilization processes areGeobacillus stearothermophilusandBacillus atrophaeus. Germinating spores of both organisms produce enzymes, including, for example, alpha-glucosidase, which can break down the complex sugar maltose into two glucose molecules. This exemplifies just one means to achieve the first step in the present invention. Other spore produced enzymes may also be used to break down other complex sugars, e.g., disaccharides, oligosaccharides or polysaccharides, to produce monosaccharides such as glucose.

The monosaccharide, e.g., glucose, can then be oxidized by the electrodes to achieve the second step in the present invention. The monosaccharide may be detected by non-enzymatic glucose biosensors using, for example, a fixed potential, chronoamperometric method for the direct electrochemical determination of newly formed monosaccharide molecules. Suitable sweeping potential methods are known to those skilled in the art. For example, C. Fang, C. Yi, Y. Wang, Y. Cao and X. Liu, inBiosens. Bioelectron.24 (2009) 3164 disclose a molecularly imprinted polymer that can be used. Other methods of detection by non-enzymatic glucose biosensors are known in the art as well.

Thus the process is dependent upon the first step in the process, in which any germinating spores produce an enzyme capable of breaking down complex carbohydrates, e.g., maltose or lactose, to more simple sugars, e.g., glucose. Failure to achieve production of the end product of the first step (as will be the case if the spores are killed) prevents detection of the simple sugar in the second step. So, in the absence of any product of the first reaction (e.g., glucose converted from maltose) no signal will result or be observed. In this case, absence of any signal derived from oxidation of a monosaccharide such as glucose would mean the monitored sterilization was successful.

Examples of suitable oligosaccharides are fructo-oligosaccharides, galacto-oligosaccharides, mannan-oligosaccharides, gum arabic, guar gum and guar hydrolysate.

Other suitable disaccharides, oligosaccharides and/or polysaccharides may be known to those of skill in the art, and may also be useful with the present invention.

The present invention utilizes an electronic signal that relies on detecting the emergence of simple sugars that are generated from the breakdown of complex sugars when in the presence of germinating spores of indicator organisms. Electronic detection methods based on sugars have been readily available for years for monitoring the glucose levels in the blood of diabetic patients. Until now, however, these electronic detection mechanisms have not been used or suggested for use as a means to detect viable organisms following sterilization processes. It is enabled here by the realization that conditions can be set so that in the absence of viable, surviving spores, no monosaccharide, e.g., glucose, is present in the system and none will be detected, while at the same time providing a very sensitive measurement of any of the monosaccharide that does become present due to the presence of germinating spores. The present invention thus takes advantage of the fact that most living organisms have inherent capabilities to break down complex sugars (such as maltose) into simple sugars (such as glucose) in order that the more useful glucose molecule can be utilized as an energy source by the organism. Glucose monitoring through enzyme-based electronic detection methods (e.g. glucose oxidase based biosensors) have been readily available for years to monitor the glucose levels in the blood of diabetic patients. These same basic methods for monitoring glucose levels in blood can be altered and adapted to detect viable organisms, such as spores, that may survive a sterilization cycle. As viable spores begin to germinate, they produce enzymes that enable them to break down complex sugars into more readily usable simple sugars such as glucose. By formulating a medium that is high in complex sugars (selected on the basis that they can be broken down by the active enzymes of viable selected organisms), an increase in glucose would be expected upon exposing viable spores to this medium and incubating. Under the conditions of sterilization, spores are killed and any of the spore derived enzymes it may already possess will be destroyed.

Therefore, an increase in glucose detected after exposure of these spores to this medium confirms that the organisms are viable. The germinating-spore-mediated conversion of complex sugars into simple sugars is directly detected in the electrocatalytic process of the present invention. This provides a heretofore unavailable method for the detection of viable indicator spores without the requirements for exogenously added enzyme or any added signal generating chemicals.

Thus, in one embodiment, the present invention relates to a sterilization indicator system, including spores of one or more species of microorganism; a vial comprising an optional first compartment and a second compartment containing a growth medium comprising one or more of a disaccharide, an oligosaccharide or a polysaccharide capable of conversion to a monosaccharide by germinating spores of the one or more species of microorganism, the vial being adapted to combine contents of the second compartment with the spores for analysis after the vial has been exposed to a sterilant; a strip comprising two or more electrodes adapted to oxidize the monosaccharide and to carry an electrical signal resulting from the oxidation of the monosaccharide, wherein the two or more electrodes are adapted to provide contact with the combined contents of the second compartment and the spores during and/or after incubation; and an apparatus linked or linkable to the two or more electrodes and adapted to detect and measure the electrical signal resulting from electron transfer when the monosaccharide is oxidized by the two or more electrodes, in which the system is free of the monosaccharide prior to exposure to the sterilant. Since the system is free of the monosaccharide prior to exposure to the sterilant, any electrical signal which is detected and measured would indicate the presence of viable, germinating spores, and this would then indicate that the sterilization process failed to fully sterilize the load, since the indicator contained surviving spores.

The electrodes suitable for use with the present invention include a variety of electrode types, sensitivities, linear ranges, limits of detection and suitability for deposition by Inkjet, silk screen or similar methods. The composition and physical form of these electrodes define them as electrocatalysts and include, but are not limited to: porous or intercalated layers (e.g. comprising one or more of copper, nickel, silver, platinum and gold), graphite, graphene, carbon, carbon nanotubes, nanoparticles (e.g. comprising for example copper and carbon nanotubes, doped platinum and metal films), unmodified metals such as copper, nickel, silver, platinum and gold, chemically modified electrodes, alloys (e.g., nickel-titanium, nickel-copper and nickel-chromium-iron), electrochemically deposited forms such as (e.g., copper oxides and bimetallic gold/copper) and polymers and composites (e.g., polymeric nickel-oxide and gold/Nafion®). Reported sensitivities range from 2×10−4to over 200 mA per mM (milliamp per millimole) and the linear ranges are as high as 4 to 5 orders of magnitude. Limits of detection can be achieved as low as 2×10−8mM. Though many of these physical-electric properties are far below what is needed of blood glucose determination, they are ideally suited for monitoring glucose generation in the presence of a small number of surviving indicator spores.

Non-enzymatic glucose sensors can be operated using, for example sweeping potential methods such as linear sweep, and cyclic and square wave voltammetry. The process of non-enzymatic electrocatalysis occurs when the analyte (e.g., newly emergent glucose) adsorbs to the electrode surface forming a bond which alternately adsorbs and desorbs catalyzing the oxidation of glucose and inducing the transfer of electrons. It is this flow of energy which is proportional to the quantity of glucose in the system and is thus the means by which the presence of viable spores may be detected.

The following exemplary reactions depict functions of the enzyme naturally occurring in any germinating spores that survive the sterilization process, and of the oxidation by the electrodes in the strip added to the incubation/recovery medium in accordance with an embodiment of the present invention in which maltose is the complex sugar (disaccharide) and glucose is the simple sugar or monosaccharide:

When a voltage differential is applied across a working electrode and a reference electrode, the working electrode becomes polarized and an oxidizing current resulting from the electron transfer is produced. This oxidizing current can be measured and is proportional to the amount of simple sugar present is the system after the sterilization and incubation, when none was present prior to the sterilization and incubation.

This invention utilizes organisms that are resistant to the sterilization process to be monitored in conjunction with a specialized medium that has been formulated with complex sugars, e.g., disaccharides, oligosaccharides and/or polysaccharides that can be reduced by viable spores to monosaccharides such as the simple sugar glucose. The increases in the simple sugar levels in the system if viable spores are present can thus be monitored electronically.

In one embodiment, the at least two electrodes comprise graphite, graphene, carbon, carbon nanotubes, gold, platinum, palladium, silver, nickel or copper or a combination or alloy of any two or more thereof. Other suitable electrode materials, as known in the blood glucose monitoring arts, can be used, as will be understood by the skilled person.

As used herein, the phrases “an electrical signal resulting from the oxidation of the monosaccharide”, and “an electrical signal resulting from the oxidation of the glucose” mean that the electrical signal is that signal which results from the oxidation, as compared to a background signal, such as might be provided by use of a reference electrode. Thus, while there may be some threshold electrical signal between the electrodes, even in the absence of the oxidation, it is the signal resulting from the oxidation that is of interest and is measured in the present invention.

Referring now to the drawings,FIGS. 1 and 2show a sterilization indicator system10useful with a first exemplary embodiment of the present invention. The following descriptions are provided to generally inform how an exemplary sterilization system works, and are not intended to limit the invention. The indicator system10comprises a cap20that is mountable on a container30. The container30includes a closed, bottom end31and an open, upper end, and defines an interior space34. The cap20has an outer wall22, an open, lower end, and a closed, upper end23. The cap also includes an inner wall (or walls)24disposed interior of the cap's outer wall, forming a separate wall, and defining an inner chamber26. The inner chamber26includes an opening25adjacent to the bottom end of the wall(s)24. The chamber26contains a fluid50, and the cap20includes a breakable barrier40disposed about the opening25of the chamber26to encapsulate the fluid50within the chamber26.

In the embodiment illustrated inFIGS. 1 and 2, the indicator system is configured for the cap20to be mounted to the container30in a snap-fit relationship. In other embodiments, not shown, the indicator system may be configured for the cap to be mounted to the container in a threaded relationship in which the cap is engaged with the container by threads and the system is activated by rotating the cap with respect to the container, i.e., by screwing the cap further onto the container. As shown inFIGS. 1 and 2, the container30includes an annular projection32forming a ridge or lip adjacent or near the upper end of the container. The cap20includes an annular projection29forming a ridge or lip adjacent the bottom of the cap. The cap20may be mounted onto the container30by sliding the ridge29of the cap over the ridge32of the container. The ridge32of the container30engages the ridge29on the cap20to prevent the cap20and container30from decoupling. The cap20and container30may be sized such that the ridge32exerts a sufficient amount of pressure against the cap20to prevent the cap20from sliding downward without applying an external downward force to the cap20. In this way, the breakable barrier40may be kept spaced apart from the edges38of puncture members36so the breakable barrier40does not contact and/or is not broken by the puncture members until such time as desired to activate the indicator.

As shown inFIGS. 1 and 2, the container30is adapted to break the breakable barrier40. The containers include one or more projections36(which may also be referred to herein as “puncture members”) having an edge38adapted to break or puncture the breakable barrier40when the cap20with the breakable barrier40is moved downward toward and the barrier40contacts the edge38of projection36. The puncture member36is shown as being integral with and extending up from the bottom wall37of the container. In another embodiment, not shown, puncture members36may extend both from the side wall35and from the bottom wall37.

To evaluate a sterilization process, a calibrated concentration of microorganisms is disposed within the interior34of the container30. The microorganisms may be disposed directly on the walls35of the container or may be provided on a support member (e.g., support member70) that is disposed within the container30or on electrodes that may be variously located, as described below with respect toFIGS. 6-11. The sterilization indicator is then assembled by mounting the recovery medium-filled cap20on the container30. The cap20may be mounted by snap-fitting the cap20onto the container30as described above, or, for example, by a threaded mounting. With reference toFIG. 1, the recovery medium-filled cap20is mounted on the container30in a first, non-activated (or open) position such that the breakable barrier40remains intact and is not punctured by the puncture members36. Desirably, in the first, non-activated position, the breakable barrier40is positioned away from and does not contact the edges38of the puncture members36.

With the indicator10assembled such as shown inFIG. 1, the sterilization indicator then can be subjected to a sterilization process. The cap20is shown as having apertures28through which a sterilant vapor may enter and flow into indicator system. The sterilant enters the cap through the apertures28(into the space between the outer wall22and the inner wall24) and flows into the container30through a space60defined between the exterior surface of the inner wall24on the cap20and the inner surface of the wall35on the container30. The sterilant vapor flows into the container30and acts upon the microorganisms of the biological indicator, in this embodiment.

After the sterilization process is completed, the sterilization indicator may be activated by moving the cap20downward toward the container30to a second (or closed or activated) position, which is illustrated inFIG. 2. The cap20is moved downward by applying a sufficient downward force or pressure on the cap20. As the cap20is moved downward, the breakable barrier40is brought into contact with the edge38of the puncture member36, and eventually moved into a position such that the edge38of the puncture member36punctures or penetrates the breakable barrier40. When the breakable barrier40is punctured, the opening25of the chamber26is exposed, and the liquid recovery medium50drains into the interior region34of the container30and into contact with the microorganisms as shown inFIG. 2.

As shown inFIGS. 1 and 2, in this embodiment, the inner surface of the cap20includes a second annular projection27, and the cap may be moved downward to a position such that the upper portion of the projection27engages the bottom of ridge32on the container30, and the cap20is held in the second, closed/activated position. The second, closed/activated position may serve to hold the cap20in a sealed relationship with the container30, which may prevent additional microorganisms from entering the system. Use of the projections27is optional, in other embodiments of the sterilization system. U.S. Pat. No. 5,770,393 illustrates other suitable configurations, and this patent is incorporated herein by reference for its teachings relating to configurations of cap and container. In another alternative embodiment, the inner surface of the cap20and the outer surface of the container30may be threaded, and the cap20may be moved into and maintained in a closed position by screwing the cap20onto the container30, in which the cap20may be threaded as shown, e.g., in U.S. Pat. No. 8,173,388 B2, which may be consulted for additional details on this embodiment of the vial, and which is hereby incorporated herein by reference for its teachings relating to the vial and cap configuration of this and the foregoing embodiments. All of these alternative configurations are within the scope of the present invention.

As described above, the cap20in the embodiment illustrated inFIGS. 1 and 2is shown as having the aperture28to allow for the ingress of the vapor sterilant into the indicator. It will be appreciated, however, that the cap need not be provided with such a feature. The number, size, shape, and/or location of the aperture(s) may be selected as desired, with consideration of the particular sterilant with which the sterilization indicator is to be used. For example, the location, shape, and size of the apertures in the cap and/or the container may be selected to provide a tortuous path for the entrance and exit of the sterilization vapor between the microorganisms and the surrounding environments. The tortuous path may also serve to inhibit or prevent contamination from external agents, and to make certain that an adequate amount of sterilant is available. By including the tortuous path, it is more likely that the entire load will be exposed to the sterilant thereby killing any extant microorganisms before the test organism in the sterilization indicator is killed.

Apertures may be provided in the container in addition to or as an alternative to providing apertures in the cap. If apertures are not provided in the cap, the inner wall(s) need not be located to provide a space between the inner wall of the cap and the inner surface of the container. Additionally, if apertures are provided in the container, they should be located such that the growth medium does not leak or spill out through such apertures when the indicator is activated and the barrier is broken.

FIG. 3depicts an indicator10in which an aperture80is formed in the sidewall35of the container30at an appropriate position, in addition to the apertures28in the cap20. The aperture shown inFIG. 3is in the sidewall35of the container30be near the top of the container30, in the vicinity of the edge38of the puncture member36, to avoid leakage or spilling after activation. As can be seen fromFIG. 3, after activation, the aperture80at this location will be covered by the cap20in the activated position. It is noted that the indicator10shown inFIG. 3includes the aperture28in the cap20, but this is not necessary. In one embodiment (not shown), the container30includes the aperture80and is used with a cap similar to the cap20, but which does not include an aperture such as the aperture28. Thus, an aperture can be provided either in the cap or in the container, or in both the cap and the container.

After the sterilization process has been completed, the cap20is pressed or twisted downward such that the edge38of the puncture member36penetrates and breaks the breakable barrier40releasing the growth medium in the space26to mix with and incubate with any of the biological indicator microorganisms that may have survived the sterilization process. The recovery medium50may comprise an aqueous medium or aqueous solution that provides for germination, metabolism and subsequent grow out of organisms as required. The aqueous medium or aqueous solution may be buffered.

The sterilization indicator10is then incubated for a sufficient period of time to allow microorganism viability to be determined. During incubation, any viable microorganisms will begin to metabolize and germinate, and this metabolism and germination includes activity by the enzymes to break down the disaccharide. oligosaccharide or polysaccharide to produce a monosaccharide, for example to break down maltose to produce glucose. In accordance with the present invention, the glucose “byproduct” is then available to be oxidized by the electrodes that are provided, which oxidation is detected via the electrical signal produced by the two or more electrodes described herein.

FIG. 4is a schematic depiction of an electro-conductive strip400containing three electrodes402a,402band402csuitable for use in an embodiment of the present invention. The strip400further includes electronics404adapted to provide electrical communication between the electrodes402a,402band402c, and an apparatus linked or linkable to the electrodes that is adapted to detect and measure the electrical signals resulting from electron transfer when any glucose present is oxidized by the electrodes. As disclosed and described, the electrodes402a,402band402care capable of acting upon a monosaccharide, such as glucose, to oxidize the monosaccharide and produce a detectable electron transfer. As described, the at least two electrodes, may include two electrodes that participate in the oxidation, while the third electrode may function as a reference electrode. Other embodiments, not shown, may include a different number of electrodes. For example, the reference electrode may be omitted, or an additional electrode or pair of electrodes may be added. The electronics404may include any appropriate electrical connection between the electrodes and an external apparatus that detects and measures any electrical signals generated. Such connections may include, but are not limited to, hard wiring, physical electrical contacts, e.g., spring-loaded or jacks, Ethernet, Bluetooth, 802.11, wireless local area networks (WLANs), WiFi, WiMax and the like, or any other wired or wireless communication type known in the art.

FIG. 5is a schematic depiction of a test incubator/reader for use in an embodiment of the present invention. The test incubator/reader may include electrical connections suitable to connect to the three electrodes described with respect toFIG. 4via one of the connections described. The test incubator/reader may include heating and atmosphere controls to provide an appropriate temperature and atmosphere for incubation of the combined contents of the first compartment and the second compartment of the sterilization indicator. The test incubator/reader may further include electronic circuitry adapted to detect and measure any electrical signal generated when the enzyme provided on the electrodes converts a monosaccharide, e.g., glucose, to reaction products including free electrons, in accordance with the present invention.FIG. 6provides an example of a suitable arrangement for the test incubator/reader depicted inFIG. 5.

FIG. 6is a highly schematic cross-sectional view of an exemplary sterilization indicator during incubation in an exemplary test incubator/reader600, with the strip containing the electrodes in place. The test incubator/reader depicted inFIG. 6includes a lower container602and a cap or lid604. As shown inFIG. 6, disposed in the test incubator/reader600is a sterilization indicator vial606, in which the recovery/incubation medium608has been combined with spores of a selected test organism, e.g.,Geobacillus stearothermophilus, following a sterilization process which is being subjected to efficacy determination in accordance with an embodiment of the present invention. The test incubator/reader600is equipped with an electro-conductive strip610, similar to that ofFIG. 4, which has been inserted into the combined contents of the first and second compartments in the container602, in the test incubator/reader600in accordance with an embodiment of the present invention. The test incubator/reader600further includes hard-wired electrical connections612between the three electrodes on the strip610and the electrical circuitry used to detect any electrical activity generated by the enzymatic conversion of simple sugars to their reaction products. As described with respect toFIG. 4, the test incubator/reader600may communicate with the strip610via any appropriate method.

FIG. 6is representative of an embodiment of a vial606in which there is only one compartment, e.g., in which the optional first compartment is absent, the spores are disposed on the strip610, and the strip610initially is separate from the second compartment. As shown inFIG. 6, the initially separate strip610has been placed into the growth medium608which contains one or more of a disaccharide, an oligosaccharide and a polysaccharide capable of conversion to a monosaccharide by any germinating spores of the one or more microorganism, should any of the microorganisms have survived the sterilization process. In the embodiment in which the first compartment is absent, the growth medium608is not necessarily subjected to the sterilization process; in this embodiment, it is only necessary that the strip610, with the spores disposed on it, be subjected to the sterilization conditions. Of course, appropriate steps should be take to assure that the growth medium is free of any microorganisms capable of producing simple sugars from more complex sugars, and thus, it also may be sterilized or subjected to the same sterilization conditions as is the strip.

FIG. 7is a schematic depiction of a sterilization indicator system including a vial700in which the spores are in the first compartment and the incubation medium and the strip are in the second compartment, in accordance with an embodiment of the present invention. As shown inFIG. 7, the vial700includes a cap702and a container704. The container704forms a first compartment, and the cap702includes a second compartment containing an incubation or recovery medium706. In the embodiment ofFIG. 7, spores of a suitable microorganism are located within the first compartment, and in this embodiment, the spores are immobilized on a carrier708. The container704includes a puncture member710, which is placed to puncture a breakable barrier712, when the vial700is to be activated for incubation after it has been subjected to a sterilization process. The sterilization indicator system further includes an electro-conductive strip714which has two or more electrodes, in this embodiment, three electrodes, which is initially located in the second compartment, i.e., the cap702. The strip714is substantially similar to the strip described above with respect toFIG. 4, but is not limited thereto. As described with respect toFIG. 4, the strip714includes suitable electronics to provide a link between the electrodes and an apparatus adapted to detect and measure the electrical signal resulting from electron transfer when any simple sugar present, e.g., glucose, is oxidized by the electrodes.FIG. 7is an example of an embodiment in which the first compartment is present, the spores are disposed in the first compartment, and the strip initially is in the second compartment.

FIG. 8is a schematic depiction of a sterilization indicator system having a vial800in which the spores and the strip are in the first compartment, but are separate from each other, and the incubation medium is in the second compartment, in accordance with an embodiment of the present invention. As shown inFIG. 8, the vial800includes a cap802and a container804. The container804forms a first compartment, and the cap802includes a second compartment containing an incubation or recovery medium806. Spores of a suitable microorganism are located within the first compartment, in one embodiment, the spores are immobilized on a carrier808. The container802includes a puncture member810, which is placed to puncture a breakable barrier812, when the vial800is to be activated for incubation after it has been subjected to a sterilization process. The sterilization indicator system further includes a strip814which has two or more electrodes, in this embodiment, three electrodes, which is located in the first compartment. The strip814is substantially similar to the strip described above with respect toFIG. 4, but is not limited thereto. As described with respect toFIG. 4, the strip814includes suitable electronics to provide a link between the electrodes and an apparatus adapted to detect and measure the electrical signal resulting from electron transfer when any simple sugar present, e.g., glucose, is oxidized by the electrodes.FIG. 8is an example of an embodiment in which the first compartment is present, the spores are disposed in the first compartment, and the strip initially is in the first compartment.

FIG. 9is a schematic depiction of a sterilization indicator system having a vial900in which the spores are in the first compartment and the incubation medium is in the second compartment, and the strip is separate from the vial, in accordance with an embodiment of the present invention. As shown inFIG. 9, the vial900includes a cap902and a container904. The container804forms a first compartment, and the cap902includes a second compartment containing an incubation or recovery medium906. Spores of a suitable microorganism are located within the first compartment in one embodiment, the spores are immobilized on a carrier908. The container902includes a puncture member910, which is placed to puncture a breakable barrier912, when the vial900is to be activated for incubation after it has been subjected to a sterilization process. The sterilization indicator system further includes a strip914which has two or more electrodes, in this embodiment, three electrodes, which is located separate from the vial900. The strip914is substantially similar to the strip described above with respect toFIG. 4, but is not limited thereto. As described with respect toFIG. 4, the strip914includes suitable electronics to provide a link between the electrodes and an apparatus adapted to detect and measure the electrical signal resulting from electron transfer when any simple sugar present, e.g., glucose, is oxidized by the electrodes.FIG. 8is an example of an embodiment in which the first compartment is present, the spores are disposed in the first compartment, and the strip initially is separate from both the first compartment and the second compartment.

FIG. 10is a schematic depiction of a sterilization indicator system having a vial1000in which the spores are on the strip and the strip is in the first compartment, and the incubation medium is in the second compartment, in accordance with an embodiment of the present invention. As shown inFIG. 10, the vial1000includes a cap1002and a container1004. The container1004forms a first compartment, and the cap1002includes a second compartment containing an incubation or recovery medium1006. Spores of a suitable microorganism are located within the first compartment on a strip1014, in this embodiment. The container1002includes a puncture member1010, which is placed to puncture a breakable barrier1012, when the vial1000is to be activated for incubation after it has been subjected to a sterilization process. The sterilization indicator system further includes the strip1014which has two or more electrodes, in this embodiment, three electrodes, which is located in the first compartment. The strip1014is substantially similar to the strip described above with respect toFIG. 4, but is not limited thereto, except that in this embodiment, the strip1014also holds the spores. As described with respect toFIG. 4, the strip1014includes suitable electronics to provide a link between the electrodes and an apparatus adapted to detect and measure the electrical signal resulting from electron transfer when any simple sugar present, e.g., glucose, is oxidized by the electrodes.FIG. 10is an example of an embodiment in which the first compartment is present, the spores are disposed on the strip, and the strip initially is in the first compartment.

FIG. 11is a schematic depiction of a sterilization indicator system having a vial1100in which the spores are on the strip and the strip is separate from the vial, nothing is in the first compartment, and the incubation medium is in the second compartment, in accordance with an embodiment of the present invention. As shown inFIG. 11, the vial1100includes a cap1102and a container1104. The container1104forms a first compartment, and the cap1102includes a second compartment containing an incubation or recovery medium1106. The container1102includes a puncture member1110, which is placed to puncture a breakable barrier1112, when the vial1100is to be activated for incubation after it has been subjected to a sterilization process. The sterilization indicator system further includes a strip1114which has two or more electrodes, in this embodiment, three electrodes, which is located separate from the vial1100. Spores of a suitable microorganism are located on the strip1114, in this embodiment. The strip1114is substantially similar to the strip described above with respect toFIG. 4, but is not limited thereto, except that in this embodiment, the strip1114also holds the spores. As described with respect toFIG. 4, the strip1114includes suitable electronics to provide a link between the electrodes and an apparatus adapted to detect and measure the electrical signal resulting from electron transfer when any simple sugar present, e.g., glucose, is oxidized by the electrodes.FIG. 11is an example of an embodiment in which the first compartment is present, the spores are disposed on the strip, and the strip initially is separate from both the first compartment and the second compartment.

FIG. 12is a schematic depletion of a device for receiving the signal produced by the reactive electrode, converting it to a digital signal and transferring it to a microcontroller. As will be understood by the skilled person, the differential signal obtained from the reactive electrode in the test strip, as modified and measured by the transimpedance amplifier, is fed to an analog digital converter (ADC) and then to the microcontroller/microprocessor unit (MCU/MPU), which in turn outputs the results to a display by which the user can determine the outcome of the test, and whether the sterilization was successful.

Suitable non-enzymatic glucose readers may be selected by those skilled in the art for use with the present invention. Such readers that are commercially available include the Accu-Chek® from Roche Diagnostics, the FreeStyleLite® from Abbott Diabetes Care, and the OneTouch Ultra® from Johnson & Johnson. A laboratory development test system is the EC epsilon electrochemical workstation (Bioanalytical Systems, Inc.). See also, the Electrochemistry Encyclopedia, “Electrochemical Blood Glucose Test Strips for People with Diabetes”, Ben Feldman, October, 2009, for additional information on blood glucose readers.

Thus, in one embodiment, the present invention further provides a method for determining the efficacy of a sterilization process. In accordance with this embodiment, the method includes the following steps:

exposing the sterilization indicator system of any preceding claim to a sterilant in a sterilization process;

combining contents of the first compartment and the second compartment, and incubating the combined contents;

contacting the strip with the combined contents before and/or during the incubating and oxidizing any monosaccharide with the two or more electrodes;

linking the apparatus to the two or more electrodes;

with the apparatus linked to the two or more electrodes, detecting and measuring any electrical signal produced by the two or more electrodes from the oxidizing; and

determining efficacy of the sterilization process based on the presence or absence of the electrical signal.

The foregoing method can be carried out according to the details provided in the foregoing written description and in conjunction with the knowledge of persons skilled in the art. While some small amount of testing and experimentation may be needed to optimize the production of a suitable sterilization indicator system and the conduction of a suitable sterilization process employing the disclosed sterilization indicator system, any such testing or experimentation will be only minimal.

In the present invention the defection of viable organisms is performed through the monitoring of an electronic signal resulting from the emergence of a simple sugar in a population of germinating spores of the organisms. Only viable spores that have survived the sterilization conditions can express this activity and so appearance of a simple sugar is a positive indicator of viable spores (a FAIL condition when evaluating sterilizer performance). Conversely, the absence of the simple sugar under these test conditions is an indication that no spores in the biological indicator remain viable (a PASS condition for effective sterilizer performance).

In one embodiment, the present invention uses or adapts for use a glucose meter, such as one used for the non-enzymatic determination of blood glucose concentrations for diabetes patients. There is, however, one crucial difference between the present invention and the known use of such glucose meters, and that relates to the concentration of the monosaccharide, e.g., glucose, that is determined. Blood glucose concentrations generally range from about 2 mM (millimolar) to about 30 mM. The concentrations of the monosaccharide, e.g., glucose, determined in accordance with the present invention are much lower, approaching the limits of the existing instrumentation. As is known, the limit of detection when using, for example, CuO nanowire modified electrodes and a fixed potential chronoamperometric method is about 0.0005 mM, or 4 logs lower than standard blood glucose concentrations. As is known, the limit of detection when using molecularly imprinted polymers grafted onto gold electrodes (“MIP Au”) is about 2×10−6mM, or 6 logs lower than standard blood glucose concentrations, when using sweeping potential methods of electronic monitoring. The foregoing limits of detection are disclosed in Electrochemical Non-Enzymatic Glucose Sensors: A Perspective and an Evaluation, Kathryn E Toghill and Richard Compton;Int. J. Electrochem. Sci.,5 (2010) 1246-1301. While there are examples of greater sensitivities, the foregoing clearly demonstrates the significant departure of the present invention from existing commercial methods.

In accordance with embodiments of the present invention, the limits of detection of concentrations of the monosaccharide, e.g., glucose, that would indicate the presence of viable, germinating spores that survived the sterilization process are vastly improved over the prior art sterilization indicators and with respect to blood glucose determinations. The improved detection limits provide for much faster determinations of the efficacy of the sterilization process being tested. This is because, should the sterilization process have failed, and spores survived, as the spores begin germinating, the initial concentrations of the monosaccharide will be very low. By enabling the detection of very low concentrations of the monosaccharide, with the present invention, the time delay before any monosaccharide is detected may be reduced to minutes or even seconds, as compared to the hours to days required by conventional sterilization indicators. This represents an important and significant improvement with respect to conventional sterilization indicators.

One factor that may possibly limit the lowest level that can be detected by the present invention is the purity of the disaccharide, oligosaccharide or polysaccharide used in the growth medium. If these higher saccharides are impure and contain significant quantities of the associated monosaccharide, it may be necessary to further purify the disaccharide. Alternatively or in addition, blank samples can be analyzed to determine a baseline, using the same growth medium but without any spores, dead or alive. In such case, the electrical signal resulting from electron transfer when the monosaccharide is oxidized by the electrodes would be that signal that exceeds whatever baseline signal is obtained using the blank samples.

The present invention, in one embodiment, relates to the use ofGeobacillus stearothermophilusspores, which are the accepted indicator organism for the evaluation of sterilizer cycles using steam and vaporous hydrogen peroxide. The present invention, in another embodiment, relates to the use ofBacillus atrophaeusspores, which are the accepted indicator organism for sterilizers using ethylene oxide and dry heat as sterilant. The present invention is unique in the use of an added oligosaccharide (e.g. maltose) as the substrate for naturally occurring, spore associated glycosidase (e.g. alpha-glucosidase) to generate increasing levels of simple sugars, e.g., glucose, as the analyte whose possible emergence from viable spores post-sterilization is monitored in evaluating the efficacy of the relevant sterilization process. The absence of emerging levels of the simple sugar is the indication that the spores of the sterilization indicator are no longer viable and thus evidence of a successful sterilization process. Conversely, the presence of emerging levels of the simple sugar, which is the by-product of viable, germinating spores, is the indication of the failure of the sterilizer to produce the conditions necessary for a successful sterilization. This same process could be used with other spore associated enzymes (e.g., beta-galactosidase) to convert other di-, oligo- or polysaccharides (e.g., lactose) to generate the analyte simple sugar, e.g., glucose. Since the presence of active spore-associated enzymes, or their production after the sterilization event under evaluation, occurs very early in the germination process, the indicator organisms still have all the morphological and membrane properties of spores, although at the point at which the simple sugar is detected, the spores are just beginning to germinate. In a preferred embodiment, the supplemented carbohydrate is maltose which has particular benefits. As the first benefit, the disaccharide does not interact with the electrode to produce a signal without interaction with viable, germinating spores expressing active enzyme, e.g., glucosidase. As another benefit, maltose provides two molecules of glucose rather than one, which is the case for many other potential complex sugars like lactose. One could use other naturally occurring enzymes from the spore like beta-galactosidase and a corresponding substrate like lactose (produces one glucose molecule and one galactose molecule) to achieve the same end—increasing the presence of glucose in the test solution, in either case, the product of the first step (e.g., glucose) would interact with the electrode biosensor of the cited example to produce the detectable signal of the present invention. A third benefit is the ability to use a hand held glucose biosensor for the detection and evaluation of results leading to a PASS or FAIL designation with regard to remaining viable indicator spores. Use of such a biosensor allows for read times of from seconds to minutes which is also new to the field of sterilization cycle monitoring.

An important advantage of the present invention is the faster read time for biological indicators. Additionally, because the signal is measured electronically there is the opportunity for signal conditioning and amplification. Furthermore, with a simple algorithm correlating the direct relationship of oxidizing current to quantity of simple sugar no interpretation of PASS and FAIL will be required by the user. Another advantage over similar technology is that it is independent of any added enzymes that add cost, reduce shelf life and subject the performance of the biological sensor to rate limiting diffusion and enzyme kinetics influences.

The application of non-enzyme glucose detection in blood testing (for which there is a great deal of available data) is influenced by inhibitors found in blood and serum and must be used at physiological conditions including neutral pH. Because these inhibitors are not present in spore preparations used in the sterilization indicator system of the present invention, and because much broader ranges of temperature, pH, ionic strength and other conditions can be used with spores, such conditions can be adjusted to optimize the electrocatalytic needs of the electrode system without regard of the constraints of maintaining physiological conditions. For example, much greater sensitivity can be achieved at values of pH that would not be possible in other enzyme based biological indicator systems.

While the principles of the invention have been explained in relation to certain particular embodiments, these embodiments are provided for purposes of illustration. It is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading the specification. Therefore, it is to be understood that the invention disclosed herein is intended to cover such modifications as fall within the scope of the appended claims. The scope of the invention is limited only by the scope of the claims.