WO2000057178A2

WO2000057178A2 - Cell detection using small volume elements

Info

Publication number: WO2000057178A2
Application number: PCT/US2000/006408
Authority: WO
Inventors: Geoffrey A. Dafforn; Rajesh D. Patel; Nurith Kurn
Original assignee: Dade Behring Inc.
Priority date: 1999-03-19
Filing date: 2000-03-13
Publication date: 2000-09-28
Also published as: WO2000057178A3

Abstract

Disclosed is a system for detecting living cells in a fluid comprising or suspected of comprising the living cells. In one aspect, the system comprises: a) a first implementation adapted to receive the fluid and to render at least a portion of any living cells in the fluid suitable for concentration; b) a second implementation adapted to receive at least a portion of the fluid from the first implementation, wherein the second implementation comprises an array of individual elements adapted to concentrate the living cells from the fluid and to reduce diffusion of the fluid between each element, the system being further adapted to disperse the living cells from the first implementation into each of the individual elements; and c) a detector operably linked to each individual element in the array, the detector being capable of registering elements having at least one living cell therein, wherein, detection of cells by the system is indicative of the presence of the living cells in the fluid. Additionally disclosed are methods for using the system to detect living cells in a fluid of interest such as blood or a blood-related product such as plasma.

Description

CELL DETECTION USING SMALL VOLUME ELEMENTS

FIELD OF THE INVENTION The present invention relates to a system and methods for detecting cells.

In one aspect, the invention relates to a system for detecting living cells in a fluid by concentrating the cells from the fluid and dispersing individual cells into small volume elements. Also provided are methods for detecting the living cells in the fluid. The present invention has a variety of applications including the detection of microorganisms in a biological fluid such as blood.

BACKGROUND OF THE INVENTION

There has been substantial interest in detecting cells and especially microorganisms in food, cosmetics, industrial effluents, and pharmaceutical samples. A variety of detection methods have been developed that employ optical, electrochemical, biological, biochemical and/or physical detection strategies. See generally Hobson, N.S. et al. (1996) in Microbial Detection, Biosensors & Bioelectronics, 11 , 455, Rechnitz, G. A (1990J J. of Biotechnology 15, 201 ; and references cited therein.

Specific attention has been focused on detecting microorganisms in clinical samples. For example, standard microbiological tests have been used to detect microorganisms in biological fluids. A particular test involves detecting microbial growth on a culture plate after contacting the plate with the sample. However, optimal practice of this test requires significant knowledge about the microorganism. For example, it is generally helpful to know how well the microorganism grows on the culture plate. A long incubation time may be required to detect growth especially when the sample has few and/or slow growing microbes. Accordingly, it has been difficult to use many tests effectively.

More focused attention has been directed toward developing methods for detecting microorganisms in biological fluids obtained from patients. For O 00/57178 ,. . .

2 example, there has been a need to develop rapid and efficient methods for detecting potentially pathogenic microorganisms in patients suffering from or susceptible to infection, e.g., septicemia. Patients receiving antibiotic therapy have also demonstrated a need for the methods especially when continued therapy needs to be evaluated. Additionally, there has been an emerging need to develop methods for screening donated biological fluids such as blood or components thereof.

As an illustration, patient blood has been analyzed for bacteria by the following methods. In one approach, blood has been diluted into a sealable culture tube that includes a suitable growth medium. The tube is then monitored for bacterial growth by assaying for production of certain metabolic products. A particular example is carbon dioxide production. A specific system for detecting bacteria using this method is the BACTEC line of devices manufactured by Becton Dickinson. See Swenson, R.J., supra.

However, the methods for analyzing bacteria in blood have been associated with significant drawbacks. For example, optimal practice typically requires a substantial bacterial presence, usually from between about 10⁵ to 10⁶ cells per milliliter of blood. It is generally accepted that as little as 1 living bacterium per milliliter of blood can be life threatening. Accordingly, the methods must be conducted for an extended period of time of up to a week or more to obtain good results. More specific shortcomings of the BACTEC devices have been reported. See e.g., Swenson, R . supra.

Additional approaches have been used to detect microorganisms in blood and other biological fluids. For example, it has been reported that bacteria can be detected in blood by visual inspection. As a specific example, blood has been added to a culture tube and subsequently monitored for turbidity or eventual color changes. However, detection methods that rely on turbidity or color changes have been associated with substantial problems. As an example, when the methods are employed in an automated or semi-automated format, invasive manipulations are often involved. These manipulations have been reported to result in significant cross-contamination particularly between different culture tubes. It has been disclosed that the cross-contamination substantially decreases assay sensitivity and reliability.

Additional efforts have been directed toward detecting microorganisms in sealable containers and particularly to monitoring the containers for positive and/or negative pressure changes. However, the methods suffer from substantial shortcomings that include need for a substantial microorganism presence to conduct the test. Further, the pressure changes can be adversely affected by temperature fluctuations occuring in most laboratory environments.

There have been some attempts to remedy these problems. For example, one attempt has involved developing substantially non-invasive detection systems that use chemical sensors. In one approach, a chemical sensor is positioned inside a sealable tube comprising the biological fluid of interest along with a suitable growth medium. Specific chemical sensors have been reported to respond to changes in carbon dioxide levels by registering changes in the growth medium. See e.g., Swenson, F. J. (1990) Sensors and Actuators B, 1 1 , 315.

Additional methods for detecting microorganisms in biological fluids have been disclosed. For example, see Hobson, N.S. supra, Swenson, F.J, supra, Nakamura, N. et al. (1991) Biosensors & Bioelectronics, 6, 575; U.S. Patent Nos. 5,770,440 and 5,716,798; EPO 0751216 A1 , and PCT WO/97/10494. O 00/57178 ,. . .

4

These methods however also have drawbacks. For example, practice of the methods generally requires significant cell growth before optimal detection results can be achieved. Thus, the methods cannot always provide reliable detection results within a few hours or less time. This condition is especially problematic when the methods are employed to detect fluids with few microorganisms.

It has been reported that small volume elements (sometimes referenced as

SVEs) can be used to detect microorganisms such as bacteria. However, such methods have been disclosed as having shortcomings. In particular, use of electrical conductance to detect cells in SVEs has been plagued by severe problems. See e.g., Weaver, J.C. (1984) Ann. NYAcad. Science 434, 362.

In addition, U.S. patents 4,399,219 and 4,401 ,755 disclose specific methods for detecting cells in a microdroplet. However these methods are believed to involve approaches that are not amenable to continual, repeated or continuous monitoring. Other difficulties may be manifested when the methods are used to analyze especially with biological fluids such as blood.

It would be desirable to have a system for detecting cells in a fluid that is sensitive and can provide results within a few hours or less time. It would be particularly desirable to have a system for detecting living cells in a fluid that does not require significant cell growth for optimal detection. It would also be desirable to have a system and methods for detecting living microorganisms in a biological fluid.

SUMMARY OF THE INVENTION

The present invention features a system for detecting cells and particularly living cells in a fluid. The system is generally sensitive and can provide detection results within a few hours or less time. Significantly, the system is adapted to detect one or a few living cells in the fluid while avoiding the need to grow cells or handle large cell volumes. The system achieves these goals by concentrating living cells from the fluid and dispersing same into small volume elements adapted to detect cell metabolism. Preferred systems of this invention essentially ignore non-living cells, thereby providing a reliable indicator of viable cells in the fluid. A more preferred system is adapted to detect at least one living cell in at least one of the small volume elements. Also provided are methods for detecting the living cells especially in a biological fluid. The present invention has a variety of useful applications including the detection of living microbes in blood or related fluid obtained from a mammal such as a patient.

There is nearly universal recognition that living cells can change the media in which they grow. As an illustration, it has been acknowledged that microorganisms can alter certain properties in the media such as ionic character. Attempts to take advantage of this observation in prior detection strategies have nearly always been confined to analyzing large cell concentrations, e.g., millions of cells or more per milliliter of fluid. In some strategies large cell volumes up to a liter or more have been analyzed. However, it is believed that it is possible to relate media changes manifested by large cell numbers and fluid volumes to those made by much smaller cell numbers and fluid volumes. More particularly, it is believed that it is possible to detect media changes made by one or a few cells in a small fluid volume much less than about a milliliter.

A more specific illustration of this concept as it relates to the present invention is as follows. It is believed that about one living microorganism in a small fluid volume (about 10⁴ μ³ ) will have as much effect on its growth media as would a much larger of the cells in a large volume (e.g., about 10⁸ microbes per milliliter of fluid). The present invention recognizes and extends this concept, e.g., by providing a cell detection system that concentrates living cells from the fluid and disperses same over small volume elements adapted to detect the living cells. More particular small volume elements are specifically adapted to detect cell metabolism and to hold small fluid volumes within each element. Accordingly, in one aspect, the invention features a system for detecting cells and particularly living cells in a fluid. In one embodiment, the system includes a first implementation adapted to receive the fluid and to render at least a portion of any living cells in the fluid suitable for concentration. The system further includes a second implementation adapted to receive at least a portion of the fluid from the first implementation. In this embodiment, the second implementation includes an array of individual small volume elements adapted to concentrate the living cells from the fluid. Preferred small volume elements are specifically adapted to reduce diffusion of the fluid between each element. Sometimes the small volume elements will be referred to as "interrogation" elements to denote compatibility with various detection formats discussed below.

A more particular system of the invention is adapted to disperse (i.e., divide substantially evenly) the living cells into each of the individual small volume elements of the second implementation. The system preferably also includes a detector operably linked to each individual element in the array which detector is capable of registering small volume elements having at least one concentrated and living cell therein. Preferred is a detector adapted to detect metabolism in at least one of the small volume elements, collect data from the array, and output the data to a user of the system. In general, detection of the living cells by the system is indicative of the presence of the living cells in the fluid subjected to analysis by the system.

The system and methods of this invention can be used in a variety of assay formats including embodiments in which the fluid is known or suspected of having cells and particularly living cells. Living cells in the fluid can include one or a variety of cell species or genuses. As will be apparent from the discussion and examples that follow, the invention is capable of detecting cell metabolism in the array of small volume elements and is not constrained by a specific cell species or genus. Especially preferred for some applications are cells that include a cell wall.

In general, components of the present system (e.g., the first implementation, the second implementation and the detector) are operably linked to each other to optimize the function for which the system is intended, i.e., the detection of one or a few living cells in the fluid. Preferred components of the system are preferably associated with one another in a configuration suited to optimize that function. Thus the term "operably linked" as it is used herein particularly refers to the association of at least two system components in a functional relationship. Thus, for example, a first component of the system is "operably linked" to a second system component when it is placed into a mutual functional relationship which facilitates optimal function of the system.

As an illustration, the first implementation, the second implementation and the detector can each be directly linked (i.e. physically connected to each other in the mutual functional relationship) to support cell detection by the system. In this embodiment, operation of the system in a substantially continuous fluid flow format will be positively impacted. However, for some applications, it will be useful to link certain system components indirectly. For example, the first and second implementations can be linked indirectly (i.e., separated physically but linked functionally). Such a system is desirable to allow fluid provided by the first implementation to be collected and stored prior to analysis by the second implementation. In this embodiment, the fluid can be "pre-concentrated" if desired before contact with the second implementation as discussed below.

It will be apparent from the foregoing as well as the discussion and examples that follow that the present system is highly flexible and can be configured in a wide variety of ways to suit intended use. As an illustration, the system can be formatted as a directly linked stand-alone system preferably operated automatically. In another embodiment however, the system can be configured as a combination of indirectly linked system components. Choice of a specific system configuration will be guided by several parameters including the amount of fluid to be analyzed, the amount of living cells known or suspected to be in the fluid, the sensitivity of detection needed, and the resources available to conduct the analysis.

For some applications, analysis of fluid as a flow stream (sometimes called a fluid flow stream) will be preferred. In this embodiment, the system will usually include a pump implementation preferably adapted to move the flow stream at least through the first implementation and also through the second implementation or other system components as needed. In particular, the pump implementation can be a conventional pump operably linked to the system such as to the first implementation, the second implementation, or both implementations. Choice of a specific pump implementation will be guided e.g., by the amount of fluid to be analyzed. Preferred are pump implementations that move the fluid through the system under conditions of controllable fluid pressure.

As discussed, the system of this invention is adapted to concentrate the living cells from the fluid and to disperse those cells into each of the small volume elements of the array. A particular system of this invention is adapted to disperse the concentrated cells substantially evenly over the array as discussed below. A preferred array is adapted to detect at least one of the living cells in at least one of the small volume elements, with detection of between from about one to ten or more of the cells in about a corresponding number of small volume elements being preferred for many applications. Methods for making and using arrays having volume elements sufficient to hold the small fluid volumes are known in the field and are discussed below.

As noted, the first implementation is preferably adapted to concentrate living cells from the fluid. That concentration can be achieved by one or a combination of different strategies including the specific magnetic and filtration methods discussed below. The concentration can also be accomplished by tagging living cells with a standard moiety sufficient to facilitate concentration of those "tagged" cells by the second implementation. In some embodiments, the moiety may have potential to absorb or emit light under preferred assay conditions. Specific methods for tagging cells are known in the field and include recognized spectrophotometric approaches. More particular tagging methods involve well known immunological approaches using detectable and non- detectable antibodies. Preferred are tagging molecules rendered chromogenic, fluorescent, phosphorescent or chemiluminescent in the presence of the cell metabolism and light conditions suited to detect the tag.

As discussed, the present system is adapted to concentrate the living cells in the fluid and to disperse same substantially evenly over the array of individual small volume elements. Suitable dispersal of the living cells can be accomplished by one or more standard techniques. For example, preferred dispersal is accomplished by optimal positioning of first and second implementation sufficient to allow the fluid to flow substantially evenly over the array. In this embodiment, optional tubing and guiding structures can be used to further enhance optimal fluid flow and disposal. However for some applications it may be useful in certain instances to adapt the system so that the dispersal is facilitated by including an operably linked dispersing implementation. A particular dispersing implementation is adapted to spread cells over a flat surface (e.g., a culture plate or multi-well dish) using conventional cell culture manipulations. More specific examples of dispersing implementations compatible with the system include manual, automatic or semi-automatic pipetting devices.

Additionally, the present system can be readily adapted to include known devices and especially fluid dispersal devices such as baffles and the like to aid in the dispersal of cells over the array. As noted, substantially even dispersal of the fluid over each of the individual small volume elements of the array is generally preferred for most applications. The volume of fluid used will be guided by several parameters including the amount of sample to be analyzed, whether non-continuous, semi- continuous, or continuos flow is to be used, and the configuration of the second implementation, particularly the number of small volume elements in the array.

The first implementation is generally configured as a first chamber adapted to provide contact between the fluid and a composition capable of disrupting pre- selected cells in the fluid. As an illustration, the composition can be formulated to disrupt some or all-eukaryotic cells in the fluid, if present, while leaving cells with a cell wall substantially intact. A more particular first implementation is adapted so that the contact is sufficient to render any specific living cells in the fluid suitable for concentration by the second implementation. That is, the first implementation preferably has a configuration sufficient to allow at least about 90%, preferably up to about 100% of the living cells to be concentrated by the second implementation. Standard cell culture methods can be used to quantifying levels of the concentrated living cells and are discussed below.

In a more particular embodiment, the first chamber is especially adapted to provide efficient mixing between the sample fluid and the composition. Preferred is a composition that includes one or more components formulated to render the living cells susceptible to a magnetic field. More preferred are compositions specifically formulated to provide for distinction of microorganisms from cells without a cell wall, e.g., blood cells etc. In this embodiment, the first chamber is preferably composed of an essentially non-magnetic material such as plastic or glass although many other non-magnetic materials may be equally suitable. In particular, the contact provided by the first implementation is preferably sufficient to magnetize at least about 90%, preferably up to about 100% of the living cells in the fluid. Specific techniques for making and quantifying the magnetized living cells are discussed further below. For applications in which the first chamber is specifically adapted to magnetize the living cells, it will be helpful to further adapt the system to impress a magnetic field thereon, preferably with respect to the second implementation. Also in this embodiment, the system will further include a magnetic implementation such as a permanent magnet or electromagnet operably linked to the system and especially the second implementation. The magnetic implementation may be directly linked to the second implementation or other system component, or it may be configured as an indirectly linked stand-alone apparatus as needed.

Particular use of the system involves engaging the magnetic implementation and attracting the magnetized living cells toward the second implementation and particularly into the individual small volume elements of the array. The attraction of the magnetized cells into the array is especially facilitated by the substantially even dispersal of the fluid over the small volume elements as discussed above and in the discussion and examples that follow.

For some applications, it may be useful to pre-concentrate the fluid prior to the concentration by the second implementation. For example, pre-concentration may be desirable to facilitate cell detection in cases in which the fluid includes or is suspected to include only a few living cells. In this instance, the first implementation can be further adapted to pre-concentrate the living cells by operably linking a standard centrifugation or electrophoresis apparatus.

The second implementation of the present system is generally configured as a second chamber adapted to receive at least a portion of the fluid from the first implementation and particularly the first chamber and to position the array of individual small volume elements in operable linkage with respect to the first implementation. The second implementation particularly includes components adapted to facilitate concentration of the living cells into the individual small „_ .

12 volume elements. Examples of such components include the optional fluid dispersing components, magnetic implementation, and filters discussed previously.

The individual small volume elements discussed herein are more particularly referenced as "interrogation elements". Preferred interrogation elements are formatted to hold a small and discrete fluid sample and to reduce or eliminate fluid diffusion between individual elements. That reduction in fluid diffusion can be accomplished in several ways including configuring the elements with permeability barriers such as walls or zones through which fluid movement is significantly hindered or prevented.

Particular substrate surfaces are interrogation elements that include at least one surface for sensing cell metabolism and for communicating same to the detector. That surface will sometimes be referred to herein as a "sensing" surface that is preferably configured to register the metabolism directly or indirectly as discussed below.

As noted, the second implementation is operably linked to the detector preferably through the sensing surface. Preferred is a conventional detector adapted to register electronic, optic, electro-optic, or electrochemical signals communicated from the array via the sensing surface. A preferred detector further includes in an operably linked arrangement specific components needed to optimize cell detection provided by the system. The components will vary depending on the specific system configuration selected but typically will include electrodes and especially sensing or drive electrodes, light guides, photodiodes, and/or devices for registering and communicating output to a system user. Presence of a particular component or group of components in the detector is pre-determined and is generally guided by intended use. A more particular system of this invention will include one or more computational devices such as a PC or related device operably linked to the system and particularly the detector. In one embodiment, the computation device is formatted to receive output from the detector and to manipulate same using one or more pre-determined computer software algorithms. The computational system can be adapted to store the output or provide same to a user of the system in real-time. Preferred output can be provided by interfacing the system with a flat liquid display panel. This embodiment will often be preferred in embodiments where automated use of the system is desirable.

A more particular detector is operably linked to the sensing surface and includes a standard sensor array comprising a plurality of standard sensing pixels. In this embodiment, each of the sensing pixels is operably linked to the sensing surface of each individual interrogation element. A more particular detector is adapted to collect output from each of the individual sensing pixels in the sensor array and to provide output indicative of the presence (or absence) of living cells in the second implementation. Preferred sensing pixels are configured as well electrodes as will be explained in more detail below.

In a particular embodiment of the second implementation, the individual interrogation elements are configured in a well array and the sensing surface in each well is configured on one side of that well. In this example, the sensing surface is preferably positioned at the bottom of the well although for some applications other configurations are envisioned, e.g., positioning the sensing surface on the side of the well. Additionally, essentially the whole well surface may be a sensor, e.g., to increase sensitivity. In this embodiment, it is generally preferred that substantial diffusion between wells be minimized, i.e. each well is divided into multiple wells. Also, each well in the array can be configured to include a sensing surface built in to a portion of one side of the well. Each well preferably has the same sensing surface configuration although wells with different configurations are contemplated as part of this invention. Choice of a specific well configuration will be guided by several considerations including the amount of fluid to be analyzed, the concentration of living cells known or suspected of being present in the fluid, the level of detection sensitivity required, and the sensor array selected.

In a more particular embodiment of the second implementation, the sensing surface of each well in the array includes an electrically conductive polymer. Preferred is a polymer that has been formulated to function as a standard reporting electrode, in this embodiment, the second implementation further comprises a standard counter electrode or electrically inert surface that is preferably adapted to sealably engage the well array when the system is in a detection mode. In particular, the sealing engagement is typically provided by opposing the counter electrode (or the inert surface) and the well array sufficient to provide for a reduction and preferably an elimination of fluid contact or diffusion of component in the fluid between each of the wells in the system. Engagement of the counter electrode or the inert surface with the well array in this way can be achieved by manual, automatic or semi-automatic sealing movement of the counter electrode (or the inert surface) with respect to an essentially stationary well array. Alternatively, the engagement can be achieved by moving the well array toward an essentially stationary counter electrode or inert surface.

In another particular embodiment of the present system, the well array of the second implementation includes at least one filter, usually one or two filters, which filter is preferably positioned between the well array and the counter electrode or the inert surface. Suitable positioning of the filter with respect to the small volume well array and the counter electrode typically results in isolation. Preferred is a filter having a porosity sufficient to trap a wide variety of cells and especially microorganisms having a cell wall such as bacteria or yeast.

Preferred filtration results in deposition of cells on the filters which cells are then divided substantially evenly into the wells. A particularly preferred interrogation element is a well provided in a microchip ("chip") format. An especially preferred chip is configured as a disposable and sterile chip component of the system.

In embodiments in which the system is adapted to analyze a fluid flow stream, the opposition provided by the counter electrode or the inert surface and the well array preferably prohibits movement of the flow stream through the system and especially the second implementation. Preferred opposition achieves the sealing engagement between the counter electrode or the inert surface and the well array which engagement serves to further reduce or eliminate any diffusion between each of the wells in the array.

A variety of sensor arrays are compatible with the present system and particularly the second implementation. For example, a particular sensor array includes a plurality of individual electrodes in which each electrode is operably linked to the sensing surface in each of the wells. For many applications, a sensing surface configured as a conventional reporting electrode will be preferred. In this example, it is also preferred that the reporting electrode be capable of detecting a change in at least one fluid property in the well. In particular, that change will be manifested by metabolism of at least one living cell in at least one of the wells. Preferably the change is registered by the detector as an increase or decrease in the fluid property when compared to a suitable control well, i.e., a well having essentially the same type and volume of fluid in the well but without the living cell therein. Illustrative fluid properties are readily detectable by the reporting electrode and include conductivity, pH, ionic strength, redox potential, electrical impedance or other recognized indicators of cell metabolism. A reporting electrode capable of registering changes in electrical impedance will be preferred for many applications. In another embodiment of the present system, the linkage between the detector and each of the individual elements in the array is provided by an optically transparent surface. In this illustration of the invention, the array is a plurality of individual interrogation elements, preferably wells, in which each well is optically coupled through the surface to a sensor array. The sensor array typically includes a plurality of individual sensing pixels in which each sensing pixel is optically coupled to the optically transparent surface as discussed, a preferred sensing pixel is a well electrode. A particular detector for use with this embodiment is adapted to detect light emitted from the wells compared to a suitable control well.

Another particular detector for use with the present system is adapted to output light through the optically transparent surface and into each of the interrogation elements preferably configured as a well array. In this example, the detector is preferably adapted to detect any light absorbance in each of the wells compared to a suitable control well.

In embodiments in which the interrogation elements are configured as a well array, the position of the optically transparent surface in each well will be guided by intended use. For most applications, the surface is preferably positioned at the bottom of the well. Alternatively, each well in the array can be configured to include an optically transparent surface built into a part or an entire side of each well. Each well preferably has the same optically transparent surface configuration although wells with different configurations are contemplated.

In a particular embodiment of the system, the second implementation further includes the inert surface preferably adapted to sealably engage the well array when the system is in a detection mode. Preferred sealing engagement between the inert surface and the well array has already been discussed. A preferred sensory array adapted for optical cell detection is compatible with a wide variety of standard scanning devices. In one embodiment, the sensory array is operably linked to the scanning device and is capable of detecting a fluorescent, phosphorescent, chemiluminescent or chromogenic signal in each of the wells. A particular scanning device is adapted to collect the signal from the sensor array and output same to the system user. In a particular embodiment, the scanning device is operably linked to the computational system sufficient to provide delayed or real-time output to a user of the system as needed.

For example, the second implementation can be adapted to include a specific well array in which each well in the array includes at least one and typically one detectable reporter. The reporter can be added to the well array by a user of the system or it can be provided as one component of the system. Preferred is a reporter made to register presence of at least one living cell in the well. Alternatively, the detectable reporter can be added to the fluid prior to analysis by the system. More specific detectable reporters are rendered chromogenic, fluorescent, phosphorescent or chemiluminescent in the presence of cell metabolism and suitable light conditions. In the absence of that cell metabolism, the reporter is preferably not detectable or is ignored by the system. Preferred are reporter molecules rendered fluorescent, phosphorescent or chemiluminescent in the presence of the cell metabolism and light excitation conditions suited to release detectable photons by the reporter. Detection of the living cells by fluorescent, phosphorescent, chemiluminescent or chromogenic detection can be accomplished by choice of an appropriate reporter. Exemplary reporters and methods of use are disclosed below.

In another embodiment of the second implementation, the linkage between the detector and the array of individual elements is provided by a sensing surface which surface includes or consists of a standard biosensor. In this illustration of the invention, the detector comprises a sensor array comprising a plurality of standard sensing pixels in which each sensing pixel, preferably configured as a well electrode, is operably linked to the biosensor. Preferred in this embodiment are interrogation elements configured as a well array in which each well includes the biosensor. For most applications, each well in the array will include the same biosensor although at least one well comprising a different biosensor from another well is contemplated. Preferred embodiments of the second implementation include the biosensor and the inert surface adapted to sealably engage the well array as discussed previously.

Another particular system of this invention is especially adapted to receive a biological fluid such as blood or a blood related product such as plasma. In one embodiment, at least the first implementation and preferably the first implementation and the second implementation are each provided as sterile system components to facilitate handling of the biological fluid. A more specific system includes the first and second implementations as sterile and hermetically sealed components. In this embodiment, at least the first implementation is disposable and can be attached or detached from the system as needed. Additionally preferred is a system in which the second implementation and especially the detection head is configured as a sterile and disposable system component.

In another aspect, the present invention features methods for detecting presence of living cells in a fluid comprising or suspected of comprising the living cells. In one embodiment, the method includes the steps of:

a) receiving at least a portion of any living cells in the fluid in a first implementation adapted to render at least a portion of any living cells in the fluid suitable for concentration; b) concentrating the living cells in a second implementation adapted to receive at least a portion of the fluid from the first implementation, wherein the second implementation comprises an array of individual elements adapted to concentrate the living cells from the fluid and to reduce diffusion of the fluid between each element, the concentrating step further comprising dispersing the living cells substantially, evenly into each of the individual elements; and

c) detecting at least one living cell in at least one of the elements of the second implementation, wherein detection of cells by the system is indicative of the presence of the living cells in the fluid.

In another aspect, the invention features methods for detecting presence of cells in a fluid. In one embodiment, there is provided a method for detecting presence of living cells in a fluid, which said method includes the steps of:

a) concentrating any cells in the fluid in a first implementation adapted to receive the fluid and render the cells suitable for concentration;

b) dividing the cells into a second implementation and concentrating the living cells which second implementation includes a plurality of individual elements configured to individually communicate with a detector and to prevent diffusion between the elements; wherein the division is sufficient to detect at least one cell in at least one of the elements; and

c) detecting at least one cell in at least one element as being indicative of the presence of the living cells in the fluid.

The system and methods of this invention have advantages with respect to many prior cell detection strategies. For example, the present system generally provides for faster detection of positive fluid samples that have or are suspected of having small numbers of slow growing microorganisms. Significantly, living cells can be detected in accord with this invention even when present in minute amounts. In addition, prior detection schemes (e.g., cell culture techniques) usually require several hours or days to detect living cells in a fluid sample. In contrast, the present invention can usually provide test results quickly (ie.in a few hours or less time), thereby positively impacting cell detection in many regulatory and clinical settings. As a particular illustration, the present invention can be employed to detect microorganisms rapidly in a biological fluid taken from a patient. In this example, implementation of potentially expensive and agressive treatment protocols can be postponed without significant health risk until diagnosis of microbial contamination can be confirmed. In this instance, the invention can reduce or eliminate unecessary in-patient or out-patient care.

Additionally, the detection provided by the present invention is generally not dependent on a bulk property of the sample fluid but rather on the small volume present in each well of the interrogation array. That is, sensitivity and time of detection is usually independent of the concentration of microorganisms in the fluid to be analyzed. The system and methods of this invention are adapted to respond to interrogation elements that include actively metabolizing living cells. Thus it is believed that small numbers of living cells can be detected as readily as many millions of cells or more. In practice, a significant timesaving can result even if in some instances a small cell colony (microcolony) is required for optimal detection by the invention. Although not wishing to be bound to theory, the theoretical limit of sensitivity for this invention is affected by the concentration step, by statistical considerations impacting the possibility that a single living

(concentrated) microorganism may die or fail to metabolize (grow), and by background signals that may resemble metabolism (growth) in an array element.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic drawing showing one embodiment of the present invention. Illustrated is a detection system including a sample collection bottle comprising a first and second chamber, detection head, and vacuum connection attached to the second chamber. The detection system is preferably used to concentrate/detect bacteria by impedance.

Figure 2A and 2B are schematic drawings showing a detailed view of one embodiment of the detection head shown in Figure 1. In this example, the detection head is adapted to provide magnetic concentration of living cells (black ovals) into an array of small volume element (2A) and monitoring of electrical impedance of the cells in the array (2B). Additionally shown is the direction of fluid flow passing through the detection head. The direction of fluid flow in Figure 2A is shown by an arrow. Figure 2B more particularly shows engagement of the two components in a sealable engagement.

Figure 3A and 3B are schematic drawings showing a detailed view of another embodiment of the detection head shown in Figure 1. In this example, the detection head is adapted to concentrate living cells (black ovals) by filtration into an array of small volume elements (3A) and monitoring of electrical impedance of the cells in the array (3B). Also shown is the direction of fluid flow passing through the detection head. Figure 3B particularly shows the detection head in a sealed engagement.

Figure 4 is a schematic drawing showing an array of small volume elements in the detection head. Particularly shown is an array of interrogation elements configured as wells for detection of electrical impedance. The black oval represents a living cell whose metabolism is detectable by the array.

Figures 5A and 5B are schematic drawings showing well embodiments of the array shown in Figure 4. Figure 5A shows one element of a disposable small volume well array. The black oval represents a living cell whose metabolism is detectable by the well. Figure 5B illustrates one element of the detector array. The detector array embodiment shown in Figure 5B is sometimes referenced herein as a "chip".

DETAILED DESCRIPTION OF THE INVENTION As discussed, the present invention features a system and methods for detecting cells in a fluid and especially living cells in a biological fluid. The system is particularly adapted to concentrate living cells from the fluid and to disperse the living cells into small volume elements. The present invention has a variety of applications including detection of viable microbes (e.g., bacteria, yeast and fungi) in a biological fluid such as blood or a blood related product such as plasma.

In general, the first implementation of the present system is adapted to receive the fluid and to render any living cells in the fluid suitable for concentration by the second implementation. The second implementation is operably linked to the first implementation which second implementation is adapted to receive the fluid from the first implementation and to concentrate the living cells therefrom. The second implementation is operably linked to a suitable detector which detector is further operably linked to an array of small volume elements adapted to detect cell metabolism occuring in at least one of the elements. In a preferred embodiment, the detector is operably linked to a suitable computational implementation configured to manipulate output from the detector and particularly to analyze the output and provide same in real-time or as stored output to a user of the system.

By the term "small volume element" or like term as it is used herein is meant a substrate surface configured to hold an essentially unitary fluid sample. A preferred substrate surface is adapted to be provided with a plurality of other substrate surfaces such as in an array association. The substrate surface preferably includes a fluid-impermeable backing, a fluid-impermeable film formed on the backing, and a grid formed on the film. A particular grid of interest includes grid elements impervious to the fluid extending from the backing to positions typically raised above the film into a plurality of fluid-impervious interrogation elements. Preferred substrate surfaces are configured as standard well arrangements made from fluid-impervious material such as plastic or glass.

By the term "array of small volume elements" or like term is meant a linear or two-dimensional array of preferably discrete regions, each having a definable area formed on a suitable solid support surface.

A particular system of this invention is adapted to detect at least about one living cell in the biological fluid, preferably between from about one to about ten living cells or more in a fluid volume of from between about 10³μ³ to 100μl, preferably 10³μ³ to 1 μl and more preferrably about 10 μ³* The optimal amount of fluid needed for the detection will be guided by several parameters such as the type of fluid analyzed and the sensitivity of detection required.

As discussed, the system of this invention is well-suited to detect viable cells in a variety of fluids. In one embodiment, the system includes at least one and preferably all of the following components:

1 ) a first implementation adapted to receive the fluid and to output same, e.g., as a flow stream,

2) a second implementation adapted to receive the fluid from the first chamber, the second chamber preferably including the following components:

a) an inlet positioned at a first end of the second chamber for receiving the fluid, b) a detection head preferably made at least in part from an elastic material such as an electrically conductive polymer which detection head is adapted to receive the fluid from the inlet and including an array of small volume elements adapted to concentrate the living cells from the fluid and to dispense the living cells substantially evenly into each of the elements of the array, the detection head further including:

i) a detector positioned, e.g., between a counter electrode (or inert surface) in sealable engagement with the array of small volume elements which elements are preferably configured to individually communicate with the detector and to reduce and preferably prevent diffusion between the small volume elements, wherein the system is further adapted to divide the living cells into each of the wells sufficient to detect at least one living cell in each of the wells; and

c) an outlet positioned at a second end of the second chamber for outputting the flow stream from the detection head,

wherein detection of the cells by the system is indicative of the presence of the living cells in the fluid.

A schematic illustration of one embodiment of a present system is shown as detection system (10) in Figure 1. In this example, the first implemenation is preferably configured as a first chamber (20) adapted to hold the fluid of interest and to combine same efficiently with a pre-selected composition. As discussed, that composition preferably renders any living cells in the fluid suitable for concentration by the second implementation. The first chamber can be configured into a variety of shapes other than that specifically shown in Figure 1 with the proviso that the shape chosen facilitate the function for which the system was intended.

Referring to Figure 1 , the first chamber (20) includes a first chamber wall (21) within which is positioned at least one sealable orifice. The sealable orifice(s) of the first chamber are generally positioned to optimize fluid intake and to enhance contact between the fluid and the pre-selected composition. A more particular first chamber includes in the chamber wall (21) a first orifice (22) for receiving the fluid of interest and a second orifice into which a detection head (30) is sealably positioned preferably as a detachable system component. In Figure 1 , the second chamber orifice is essentially completely filled by the detection head (30). The second orifice is preferrably positioned in the first chamber wall (21) to maximize output of the fluid from the first chamber (20) and to provide for essentially even flow of the fluid inside the detection head (30). In operation, the fluid and the composition can be added to the first chamber (20) particularly through the first orifice (22) at the same time or at different times as required.

As shown in Figure 1 , the first chamber (20) includes in the first chamber wall (21) the first chamber orifice (22) positioned along a first axis substantially perpendicular to a second axis defined between centerpoints of the second orifice and the detection head (30). In an other embodiment of the system (10), the first chamber orifice (22) can be positioned along a third axis defined by centerpoints between the first chamber orifice (22), the second chamber orifice and the detection head (30).

The detection system (10) shown in Figure 1 further includes a second chamber (40) adapted to collect fluid outputted from the detection head (30). Although not always necessary to practice this invention, the second chamber (40) is especially useful when potentially hazardous biological fluids need to be collected, e.g., prior to sterilization and disposal. The second chamber (40) includes a second chamber wall (41) into which is positioned a second chamber O /57178 „, . .

26 output assembly (50) optionally connected to an exit tube (51) for disposing fluid or for providing an optional connection to a vacuum (or vacuum pump) adapted to move fluid through the system. In some embodiments, the exit tube (51) extends to a position near the bottom of the second chamber (52). The position between the second chamber output assembly (50) and the detection head (30) is typically optimized to provide for convenient collection of fluid from the detection head (30). The second chamber can be configured into a variety of shapes including that shown in Figure 1 with preferred shapes being chosen to facilitate function of the detection head (30), the first chamber (20), as well as collection and disposal of the fluid.

An illustrative detection head (30) compatible with the detection system (10) is shown in Figure 2A. In this example, the detection system (10) preferably employs magnetic concentration to attract living cells (104) in the fluid into an array of small volume elements (60). In Figure 2A, the array (60) is configured as a plurality of wells (61) although other interrogation elements may be used if desired. The array of small volume elements (60) is preferably positioned between: 1) a detection head inlet (110) configured to receive the fluid from the first chamber (20) and 2) a detection head outlet (120) adapted to output the fluid to the second chamber (40). The detection head (30) is preferably adapted to receive the fluid through a passage (111) adapted to communicate the first detection head inlet (110) and the second detection head outlet (120). Preferred movement of the fluid through the detection head (30) and particularly between the first detection head inlet (110) and the second detection head outlet (120) provides for an essentially even dispersal of the fluid over the array of small volume elements (60). In this embodiment, the living cells (104) have been rendered magnetic by contact with the pre-selected composition provided in the first chamber (20). Engagement of a magnet (80) positioned opposite to a counter electrode (90) impresses a magnetic field on the detection head (30) sufficient to draw the magnetized cells into the wells (61) of the array (60). In an exemplary embodiment of the second implementation, the array of individual elements (60) is a well array in which each well (61) has a total volume of from between about 10⁴ to about 10⁸ μ³, with about 10 μ³ being preferred for many applications. It is also preferred that in this embodiment each well (61) has a length of from between about 50 μ to 1 cm; a width from between about 50 μ to 1 cm; and a height of from between about 10 μ to about 100 μ.

As discussed, the detection head (30) is preferably adapted to detect metabolism of the living cells in each well (61) the array (60). That detection is accomplished by one or more of a combination of approaches including monitoring electrical impedance. Figure 2B shows the detection head (30) engaged to detect electical impedance in each of wells (61) of the array (60). In this example, the counter electrode (90) is in sealable engagement with a detector (70). That is, the detection head (30) is adapted to allow movement between the counter electrode (90) and the detector (70) sufficient to reduce and preferably stop fluid movement through the passage (111) between the detection head inlet (110) and the detection head outlet (120). In one embodiment, the counter electrode (90) can move in relation to the detector (70) which detector is stationary. Alternatively, the counter electrode (90) can be stationary and the detector (70) can move toward that counter electrode (90).

In Figure 2B, the detector (70) is shown about 90° from its preferred orientation about parallel to the wells (61) of the array (60) so as to show the plurality of well electrodes (140) of the detector (30) . In this example, each of the well electrodes (140) is operably linked to each of the wells (61) to facilitate the detection of the electrical impedance in each of the wells (61) of the array (60). In use, the detector (30) registers a signal indicative of the electrical impedance in each well (61), compares each of the signals, and outputs a combined signal representative of the array (60) and indicative of the presence or absence of a magnetized living cell (104) in one or more of the wells (61). Preferred detectors are capable of effectively discriminating between the electrical impedance in wells (61) that include viable living cells and those wells (61) that do not include any viable cells.

In some instances it will be useful to include in the array (60) a pre- determined number of wells (61) that have the fluid to be analyzed but without any living cells therein. For example, the array (60) shown in Figures 2A and 2B can be configured to include at least one of such wells as needed. Sometimes the fluid will be referred to as "control fluid" and the wells "control wells". However it will be appreciated that the detector (30) need not always include control wells particularly when the fluid of interest is one which is routinely analyzed. In this instance, the detector (30) can be adapted to compare the electrical impedance in each of the wells (61) of the array (60) to a pre-determined value representing the electrical impedance of the control fluid.

Referring now to Figures 3A and 3B, there is provided another embodiment of a detection head (31) suitable for use with the detection system (10). In this example, preferred use of the detection system (10) involves concentrating and detecting living cells in the fluid by filtration or related methods. In particular, Figure 3A shows the detection head (31) in which filtration is employed to concentrate the living cells (104) and to facilitate movement of same into the array (60). In this embodiment, the array (60) preferably includes wells (61) and a suitable cell trapping filter (100) positioned between the counter electrode (90) and the wells (61). In this example, the detection head (31) is adapted to receive the fluid through the passage (111). Preferred movement of the fluid through the detection head (31) particularly between the first detection head inlet (110) and the second detection head outlet (120) provides essentially even dispersal of the fluid over the filter (100). Living cells (104) are trapped on filter (100). Preferred capture of the living cells (104) by the detection head (31) is facilitated by maintaining essentially even fluid flow and selecting the filter (100) having a porosity less than the diameter of the living cell to be analyzed but not so small as to block or significantly impede fluid flow. An especially preferred filter for use in the detection head (31) is sufficient to trap microbes such as bacteria, fungii or yeast, or other suitable microbes. For applications involving use of potentially hazardous biological fluids, the detection head (31) can be operably linked to the second chamber (40) as discussed previously.

The detection head (31) is preferrably adapted to detect metabolism of the living cells positioned in array (60). In Figure 3B, living cells (104) captured by the filter (100) are positioned in each of the wells (61) of the array (60) by movement between the detector (70) and the counter electrode (90). Each or some of the wells, preferably some of the wells, are positioned so as to provide at least some of the wells without living cells to serve as control cells. In one embodiment, the detector (70) is essentially stationary and the counter electrode (90) is moved toward the wells (61) to facilitate positioning of the living cells (104) in the array (60). Alternatively, the counter electrode (90) can be essentially stationary and the detector (70) can be moved toward the counter electrode (90).

As discussed, the number of small volume elements in the array (60) will be guided by several parameters including intended use and especially by the sensitivity or rapidity of detection required. As an illustration, the well array can be adapted to include from between about 10² to about 10⁸ wells, e.g., about 10³ to 10⁴ wells. Figure 4 shows a portion of the array (60) showing sixteen small volume elements in a "4 x 4" configuration. In this illustration, the living cell (104) is shown positioned just above one element of the array (60). Particular arrays can be manufactured by a variety of well known techniques although molding or embossing on a suitable plastic support will be generally preferred for most applications.

As also discussed, it is generally preferred that the small volume elements be formatted as wells (61). In Figure 5A, a preferred embodiment of the well (61) is shown with well walls (101 , 102) enclosing a chamber wall (103) tapered toward a well bottom (130). The overall shape of the well (61) is not important so long as it can provide the function for which it was intended. For example, although the configuration of the chamber wall (103) is roughly pyramidal in shape, a variety of other chamber geometries are compatible with the detection head (30, 31) including those tapered to assume an essentially square or rectangular shape.

A preferred well bottom (130) includes or consists of a conductive material suited to provide electrical contact between the well (61) and a permanent electrode (62) including a well electrode (140) as shown in Figure 5B. A preferred conductive material is a polymer such as a standard conductive polymer such as an electrically conductive epoxy. In this example, the well bottom (130) is preferably adapted to provide electrical communication between the well (61) of the array (60) and the well electrode (140) of the permanent electrode (62). The well electrode (140) is further connected to wires (141 , 142, 143, and 144) which wires can be configured to communicate with at least one other permanent electrode in the array (60). More prefered wires (141 , 142, 143 and 144) communicate, either directly or indirectly, with a suitable computational implementation as described herein.

In a more particular embodiment of the array (60), each of the permanent electrodes is operably linked to provide an electrical matrix between each of the wells (61) in the array (60). In particular, a signal from the well electrode (140) is registered which electrode is operably linked to the permanent electrode (62) and preferably the well electrode (140) and the detector (70). In use, changes in electrical impedence arising from metabolism of the living cell (104) in the well (61) are registered by the well electrode (140) and outputed to the detector (70) for output to the user of the system.

As discussed, it is preferred that the fluid of interest be dispersed substantially evenly within the detection head (30, 31) and particularly over the array of small volume elements (60). That dispersal can be achieved by one or a combination of conventional strategies. For example, in one embodiment, the cell dispersal can be accomplished manually, e.g., by removing the detection head (30, 31) and dispersing the fluid with the aid of a standard fluid dispersal or tilting apparatus. In a more specific example, the manual dispersal of the cells can be achieved by use of a pipetting or spreading apparatus capable of dispersing (spreading) the cells substantially evenly over each of the elements. In these embodiments it is generally preferred to configure the detection head (30, 31 ) as a detachable system component to aid the cell dispersal.

However for most applications it will be preferred to provide substantially even fluid dispersal by employing automatic or semi-automatic techniques. For example, as has been discussed, the detection system (10) can be operably linked to a suitable pump sufficient to pressurize the flow stream and move same through the detection head (30, 31 ) as a substantially even and dispersed flow.

Conventional methods can be used for ensuring that flow can be implemented as needed, e.g., by operably linking the detection system (10) and especially the detection head (30, 31) and the first chamber (20) to facilitate substantially even fluid flow. If needed, the detection head can optionally include appropriate tubing and fluid baffles. In this embodiment significant amounts of fluid can be analyzed therefor by maintaining an essentially continuous and even flow stream through the detection system (10). A preferred pump is operably linked to the first chamber (10), e.g., through the first chamber inlet (21) or provide a vaccum through the second chamber (40) and outlet (51 ) although other configurations can be employed for certain applications.

Additional methods for subdividing biological samples into discrete zones have been described in U.S. Patent Nos. 5,716,798; 5,770,440 and EPO 751 393 A2, the disclosures of which are hereby incorporated by reference.

As discussed, a preferred system of this invention includes a detector that comprises conventional sensors typically configured in an array format. A variety of sensors are known in the field that are generally capable of converting one form of energy into another. One function of the sensor is to provide the user of the present system with interpretable output in response to a specific measurable input. See generally Fundamentals of Microfabrication, Chapter 10, CRC Press, 1997 and Hobson, N.S. et al. supra.

Further preferred systems of this invention are compatible with a wide variety of conventional sensors particularly those configured in the sensor array. Specific methods for making and using electrical sensor arrays such as those employed in conjunction with an electrical measuring implementation have been disclosed in U.S. Patent No. 5,571 ,401 to Lewis and Freund. See also Kagan, R.L. et al. (1977) J. of Clinical Microbiology 51-57; and Cady, P. et al. (1978; J. of Clinical Microbiology 265-272. Additional disclosure relating to sensor arrays can be found, e.g., in U.S. Patent Nos. 5,120,421 ; 5,672,256; 5,832,411 ; 5,776,791 and 5,830,343.

The use of specific sensors and sensor arrays to detect the electrical impedance and conductivity of cells has been reported in detail. See Hobson, N.S. et al. (1996) in Biosensors & Bioelectronics Vol. 11 , 455-477 and references cited therein.

Specific sensor configurations for fluorescence detection have also been reported. See e.g., WO 97/10494.

Additionally compatible with the present system are standard biosensors or related sensing devices. The term "biosensor" or like term is defined herein as a sensor configured to detect a desired biological material such as a cell or portion thereof such as protein, polypeptide, amino acid or nucleic acid. Alternatively, the term "biosensor" includes sensing implementations that incorporate use of biological molecules capable of specific binding activity such as antibodies, enzymes, or nucleic acids. Illustrative biosensors include those disclosed by Swenson, F.J. (1993) Sensors and Actuators, B, 11 315-321. Additional disclosure relating to biosensors provided in a chip format has been disclosed in U.S. Patent No. 5,545,531. See also U.S. Patent No. 5,135,852 and Spener, F. et al. (1997) in Frontiers in Biosensorics II, Practical Applications Birkhauser Verla Basel/ Switzerland; 27-44.

Additionally preferred systems of this invention includes a reporting electrode responsive to metal ion, hydrogen ion, hydroxide, carbon dioxide, carbonate, oxygen or a halide.

For many applications including those in which real-time output to a user of the system is desired, it will be useful to operably link the system of this invention and especially the detector (70) to a suitable computational system. A suitable computational system can be run in a variety of conventional formats. For example, the computational system can be run on analog or digital computer systems. Software implementing the system may be written in a high level language such as Fortran, C or C++ and run on the mini-computer or CPU. The software may be a stand-alone executable, a functional library an add-on to another application, or embedded in a specific purpose apparatus. The computational system may also be implemented using a high-level development or mathematical system such as spreadsheet or symbolic mathematical software. Specific data used and/or output by the present test systems may be stored in any form such as a database including relational and object-oriented databases stored on any apparatus including read-only memory (ROM) magnetic disk, CD Rom and erasable magnetic disks. The computational system may also be implemented in hardware, firmware, including special purpose designed integrated circuits (IC) such as ASICs (application specific integrated circuits), programmable logic arrays (PLAs), and gate arrays including field-programmable gate arrays (FPGAs). An especially preferred computational system is operably linked to a flat liquid crystal display for providing the output to the user of the system. As discussed, the first chamber is preferably adapted to receive the fluid of interest and to provide contact with a composition sufficient to render any living cells in the fluid suitable for concentration by the detection head. In one embodiment, the first chamber is especially adapted to provide a pre-determined amount of that composition. Alternatively, the composition can be added to the fluid prior to contact with the first chamber.

Suitable compositions in accord with this invention more particularly include one or a combination of components sufficient to disrupt pre-determined cells while leaving at least one other type of cell in the fluid substantially intact. A wide variety of compositions are suitable for such use. For example, an illustrative composition for rendering cells including a cell wall suitable for concentration by the present system includes at least one lysing component sufficient to disrupt cells without a cell wall in the fluid while leaving living cells with the cell wall essentially intact. Examples of cells including a cell wall include prokaryotic cells such as bacteria and certain primitive eukaryotic cells such as yeast. In one embodiment, the composition preferably further includes at least one polycation capable of binding negatively charged cells in the fluid. Preferably that binding is sufficient to form a binding pair between the polymer and the cells including the cell wall. Sometimes this complex will be referred to as a polycationic complex.

In other embodiments of the present invention, concentration of the living cells can be achieved without adding the components to the fluid such as those comprising the polycations. In particular, living cells in the fluid can be suitably concentrated by using filters, as discussed.

In embodiments of this invention in which concentration of living cells including a cell wall is desired it is contemplated that in some instances a particular fluid will not include appreciable levels of eukaryotic cells. In this instance, it may be useful for some applications to reduce the concentration of or eliminate the lysing component from the composition.

The polycationic complex so formed can be concentrated by the second implementation and particularly by the detection head by one or a combination of different strategies. In one approach, the composition further includes a ferrofluid sufficient to bind the polycationic complex. That ferrofluid can be added to the fluid as needed and in some instances may be provided as one component of the pre-determined amount of the composition added to the first chamber. The resulting complex formed between the polycationic complex and the ferrofluid is magnetic and can be manipulated by exposing the fluid to a suitable magnetic field. As discussed, that magnetic field can be provided in several ways including providing the detection head with a magnet.

Methods for making and using a variety of compositions to render living cells in the fluid suitable for concentration by the second implementation and particularly the detection head have been described. For example, compositions have been disclosed for rendering cells suitable for concentration. See U.S. Patent No. 4,935,147 to Ullman, F.F. et al., the disclosure of which is hereby incorporated by reference. Choice of a particular composition will be guided by several parameters including the type of cells known or suspected to reside in the fluid of interest and the sensitivity of detection required. See the Examples below for additional disclosure relating to the preferred compositions.

The present system is compatible with a wide variety of other methods for concentrating microorganisms. As an illustration, certain standard methods can be used to "pre-concentrate" living microorganisms such as bacteria prior to manipulation by the detection system (10) and particularly the first chamber (20). Examples include concentration of bacteria from blood by centrifugation. A commercially available device for performing the centrifugation is the Isolator™ system from Wampole Laboratories. The pellet from such a concentration could be introduced into the present system. Electrophoretic or dielectrophoretic approaches could also be envisioned.

Additional use of the present invention involves concentration, detection and enumeration of eukaryotic cells such as red blood cells and lymphocytes. A method for magnetic concentration of red blood cells from whole blood samples was previously described in a US patent 4934147 (Ullman et al). For concentration of blood cells other than red blood cells, it is desirable to lyse the red blood cells prior to concentration of the other cells of interest. Reagents for specific lysis of red blood cells in a blood sample were previously described (Tlinkainen M. I. 1995, Cytometry, 20, 341-348; Van Algthoven A, Eur. Patent Appl. EP 635707; Kim Y. R. et al, Can. Pat. Appl. CA 2070244; Ledis S. L. et al, PCT Int. Appl. WO 8505684). For concentration, detection and enumeration of lymphocytes using the present invention, the blood sample is added to the first chamber where it is contacted by reagents which specifically lyse the red blood cells. It is possible to include in the reagent tag antibodies which will specifically bind to defined lymphocyte cell types, for example T4 and T8 lymphocytes. Antibodies specific for binding to defined cell surface markers, are known in the art and are commonly used for flow cytometric analysis of lymphocytes. Following lysis of the red blood cells and binding of the tagged antibodies to the specific lymphocyte cell population, the sample is contacted with ferrofluid reagent and polycation reagent to form magnetic aggregates, comprising of the magnetic particles and the white blood cells. Additional use would include contact with a suitable magnetic reagent capable of reacting with the tag. Concentration and dispersal of the magnetic aggregates on the small volume well arrays of the detection head follows as described above. Detection of the individual lymphocytes in the individual small volume wells can be achieved either by detection of the presence of the tagged antibody or direct detection of living cells. When tagged antibodies are used for marking specific cell populations, it is possible to employ numerous tags for simultaneous analysis of numerous cell types in a single blood sample. The number of small volume wells of the well __ . .

37 array, containing lymphocytes of specific cell type, is directly related to the number of cells of the specific cell type in the sample, thus enabling enumeration of lymphocytes of specific cell type in the sample. The analysis of lymphocyte content and enumeration of specific cell types is of great clinical importance, e.g., in the monitoring of patient immune status.

For applications in which it is necessary to concentrate a specific living eukaryotic cell such as an immune cell, the composition can include a detectable antibody or suitable fragment thereof which antibody specifically binds that cell. Methods for concentrating cells to which an antibody has been specifically bound are known in the field and are provided below. It may be useful to detectably- label the antibody (or fragment thereof) to facilitate the concentration and optionally the detection of the eukaryotic cells in the fluid. Illustrative labels include standard immunological tags such as those described below.

Additionally, the system of this invention can be used to detect living cells in a variety of fluids including those specified below. Preferred are biological fluids obtained from a mammal and especially a primate such as a human patient. The biological fluid can be analyzed when taken from the mammal or it can be stored prior to analysis. Preferred is a biological fluid that has or is suspected of having the cells at a concentration of from between about 0.1 to 10⁷ cells/ml. As discussed the biological fluid may have or be suspected of having at least one up to about 10 or more viable microorganisms such as viable bacteria, yeast and/or fungi. Illustrative biological fluids include blood, blood products such as plasma and reconstituted blood preparations, fluids including purified or semi-purified preparations of blood cells such as erythrocytes and immune system cells, e.g., white blood cells and lymphocytes; cerebrospinal fluid, amniotic fluid, synovial fluid and ocular fluid. Also contemplated is use of this invention to detect viable microorganisms in fluids intended for administration to a mammal and especially a primate such as a human patient, e.g., vaccines and other medicinals. Solid compounds to be analyzed can be dissolved in a suitable solvent such as physiological saline or other suitable material to make the fluid.

For many applications it will be desirable to provide at least the detection head of the second chamber as a sterile and preferably detachable system component. Other specific components of the system including one or more interrogation elements (ie. chip) up to and including the entire system can be provided sterile prior to use. If desired, at least the detection head of the second chamber can be additionally provided as a hermetically sealed component.

As discussed, the present invention is useful for detecting a variety of live microorganisms in a fluid of interest including a biological fluid such as blood or plasma. In embodiments in which blood is assayed specific advantages will be apparent. For example, when human blood is assayed for bacteria and/or yeast, practice of the invention makes it possible to realize faster cell detection of positive samples when compared to prior methods. As discussed, microorganisms in blood are presently detected by diluting the blood into a liquid growth medium in a bottle and monitoring the bottle for growth. Most modern systems detect bacterial growth by carbon dioxide production and require the presence of 1x10⁵ to 1X10⁶ organisms/mL for detection. Since even 1 live microorganism/mL is clinically significant and must be detected, a long growth period is usually necessary before enough organisms are present to detect. This period averages about 16 hours for positive samples, and bottles must be held and observed for 5 days or longer to ensure that no growth has occurred. Septicemia (the clinical syndrome caused by infection of the blood) is often fatal and can progress rather rapidly. Consequently, when septicemia is suspected, blood samples are drawn and the patient is immediately treated aggressively with antibiotics. Since about 90% of blood cultures are ultimately negative, very rapid test results could make possible less expensive treatment or just observation of the patient until the test result is available. Any test that might prevent significant numbers of hospital admissions should be very cost-effective. Additional advantages will be apparent when the present invention is used to detect living bacteria in blood. For example, in this embodiment, there is little or no dependence on a bulk property of the medium (such as carbon dioxide concentration). Significantly, the time to detection is independent of the original concentration of microorganisms. Since the procedure counts the number of wells containing live organisms, 1 organism in the well can in theory be detected as rapidly as 1x10⁶ cells in a larger volume. In practice, a tremendous timesaving results even if a small microcolony is required for detection. The theoretical limit of sensitivity for this method is set by any inefficiencies in the concentration step, by the statistical possibility that a single captured organism may die or fail to grow, and by background signals resembling growth in an array element.

All documents mentioned herein are fully incorporated herein by reference in their entirety.

The following non-limiting examples are illustrative of the present invention.

Abbreviations used in the examples include: TW20 = Tween 20; FF = ferrofluid;

PB = polybrene; PE = polyethylenimine; PVM = poly(vinyl pyrrolidone- dimethylaminoethyl methylacrylate) ; PBS = phosphate buffer saline; SP = spermine; and EDTA = ethylenediamine tetraacetic acid.

Example 1- Concentration of Bacteria from Blood Using a Magnet

This example describes methods of concentrating bacteria from blood using compositions having broad cross reactivity. Preferred use of the methods facilitate nonspecific ionic interactions between the compositions and living bacteria. Those interactions can be used in accord with this invention to concentrate the bacteria especially in the second implementation of the present system. The method is broadly applicable for the concentration of many viable microorganisms from blood such as certain primitive eukaryotic cells like yeast and fungi. More particularly, the following methods provide for the selective lysis of blood followed by concentration of viable microorganisms and specifically bacteria. The blood lysis is believed to destroy red cells and most blood cells such as lymphocytes. In accord with the methods, a negatively charged ferrofluid is then added along with a composition that includes a suitable polycation. As described below, it was found that a combination of polycations is generally preferred to facilitate concentration of bacteria from blood. Without wishing to be bound to theory, the methods are believed to form co-aggregates between the bacteria and the ferrofluid. Further binding by the composition of polycations enhances co-aggregation. In specific methods that follow, concentration of the co-aggregates (magnetic pellet) is achieved by using a magnet to attract same toward a desired surface. General methods for concentrating magnet pellets of cells have been disclosed in the U.S. Patent No. 4,934, 147 to Ullman, F.F. et al., the disclosure of which is incorporated herein by reference.

A. Magnetic Capture of bacterial cells from blood samples Formation of the magnetic pellet was achieved by performing the co- aggregation step described in Example 1 B. In particular, about 80 μl per 1 ml blood sample of stock ferrofluid reagent (0.7x10¹⁵ particles/ml in water) and 50 μl of a stock solution of spermine and PVM in water (containing 154 mg/ml spermine and 2.8 mg/ml PVM) was added to the lysed blood sample to facilitate formation of that pellet.

Blood samples (from blood bank volunteers) were spiked with bacterial cells to a total of 500 to 1000 cells per ml. Blood lysis was carried out by mixing 200 μL of blood with 800 μL of lysis buffer consisting of Rhozyme (3.125 mg/ml) and Tween 20 (0.825% v/v). The mixture was incubated at 37°C for 1 .5 hrs. , 37.5 μL of stock ferrofluid reagent (0.7x10¹⁵ particles per ml) and 12.5 μL of stock solution of a mixture of spermine and PVM were added to the blood sample following lysis. Three mixtures of polycation were tested. The concentration of WO 00/57178 PCT/USOO/06408-

41 spermine and PVM in the stock solutions were as follows: spermine 153.6 mg/ml and PVM 2.8 mg/ml. The ferrofluid and polycation solution were prepared in water. Following further incubation for 15 minutes, the magnetic aggregates were concentrated and separated from the sample fluid using a neodymium-iron-boron magnet. The sample fluid was discarded and the magnetic pellet containing the captured bacteria was resuspended in 0.1 M citrate buffer. The suspension was then plated on nutrient agar. Bacterial colonies were observed following overnight incubation at 37°C.

Table I

Nonspecific Bacterial Capture From Lysed Blood With Spermine And PVM as polycations. % CAPTURE

BACTERIUM SAMPLE 1 SAMPLE 2 SAMPLE 3 SAMPLE 4

1 46 18 34 38

2 27 12 20 64

3 100 100 100 100

4 16 10 19 15

5 32 54 47 78

6 89 89 85 55

7 26 18 33 41

8 65 88 96 95

9 70 87 90 95

10 95 97 100 99

11 37 28 37 54 rial sp: 1. C. albicans; 2. S. aureus; 3. Strep. pyoqenes

4. P. vulqaris; 5. Ps^ aeruginosa 9021 ; 6. Ps. aeruqinosa 27853; 7. S. marcescens 8. E. coli K1 ; 9. E. coli K2; 10. B coli Kδ; 11 E. coli K30.

Table I, above shows efficient capture of 11 different species of microorganisms which have been spiked in low numbers into 4 different lysed blood samples, then separated magnetically according to the method described above. The percentage of bacteria captured was determined by plating the supernatant and pellet according to the method described above. Capture of most species was good, and all species gave some capture from all samples.

B. Detection Of Captured Bacteria

1. Cell culture detection

The method used for the detection of captured bacteria was plating of the resuspended magnetic pellet on nutrient broth agar. In all cases colonies were apparent within 24 hrs following plating. Thus the bacteria making up the pellet are viable. This result indicates the removal of blood related components capable of inhibiting bacterial growth. Further investigation of the effective removal of potential growth inhibitors can also be carried out with patient samples. The analysis in this example was carried out with normal blood samples collected from a blood bank. The normal growth rate of the bacteria is expected to be the rate limiting step for the detection of captured bacteria when using a detection procedure based on growth parameters. Bacterial detection based on colony formation is slow and would serve only as an indication of the potential of the capture method for separation of the bacteria from sample depended interference.

Example 2- Detection of Bacterial Metabolism Using Small Volume Elements

The detection system (10) illustrated in Figure 1 can be used to detect living bacteria in blood or other suitable biological fluid. In particular, it is envisioned that the disposable array could be incorporated into a sealed system, allowing both bacterial concentration and measurement in an automated instrument. Schematic drawings of preferred systems of this invention are shown in Figures 1-5. For example, Figure 1 shows a detection system (bottle) that is provided with a first chamber (20) and a second chamber (40). Blood is collected into the first chamber (20), where it is lysed and reacted with a ferrofluid, if necessary, as described above in Example 1. The solution is then passed through the detection head (30) preferably constructed of elastic material into the second chamber (40). Bacteria are collected on a disposable array in this head, either by a magnet applied to the outside or by passage through a filter. The array is then contacted with the counter electrode (90), the filter (100) if present, and the permanent measuring array by compressing the surface of the head against the permanent electrode array. This assembly is monitored long enough to determine presence or absence of living cells (metabolism) in any of the wells. If not (90% of cases), the bottle is simply removed from the apparatus and discarded. If living cells are detected in any well, the bottle may be further processed to recover the live organism and determine its identity and antibiotic susceptibility in the usual manner. A preferred detection system (10) is automated although other embodiments have been discussed above.

1. Magnetic Method

About 1 to 10mls of blood from a human patient or other suitable mammal can be collected and then lysed as described in Example 1A. Preferred use of the detection system (10) involves performing that lysis in the first chamber (20) although for some applications it may be useful to conduct the lysis outside the detection system (10). Additionally preferred is use of a pump attached to the first chamber inlet (22) to facilitate movement of the lysed blood as a flow stream through the detection system (10).

Formation of the magnetic bacterial pellet is achieved by performing the co-aggregation step described in Example 1 , above. In particular, a FF, spermine and PVM composition as described in Example 1 can be added to the lysed blood sample to facilitate formation of that pellet. After about 15 min. at room temperature, magnetic separation is performed in the detection head (30) which includes the array of small volume elements (60) preferably configured as the wells (61). The magnet (80) preferably provided as the neodymium-iron-boron magnet is contacted to or brought in close opposition to the array of small volume elements (60) for about 15 to 20 minutes to facilitate attraction of the magnetized and living bacteria into the wells (61) or until all the lysed blood flowed from the first chamber to the second chamber.

As discussed, detection of bacterial growth by electrical impedance changes is well known in the literature. In effect, bacterial metabolism changes the ionic character of the medium and thus its electrical resistance and the capacitance of electrode surfaces in contact with the medium. Studies with different species indicate that most common bacteria can be detected rapidly (ca. 30 min) at concentrations of 3x10⁷ bacteria/mL. This concentration corresponds to 1 bacterium in a volume of 3x10⁴ μ³. Thus, 1 bacterium confined to a volume this small should have as much effect on the medium as do 3x10⁷ bacteria /mL. Measurement of impedance of the medium in this volume as a function of time should show detectable changes in impedance.

The concept is further illustrated in Figure 4 in which a bacterium has been concentrated from the lysed blood onto the surface of a "chip" which is further subdivided into the wells (61). The impedance of all of these wells is then continually monitored. The well containing the bacterium will show a change in impedance with time resulting from metabolism. The other wells should show no change and in fact serve as negative controls to compensate for drift in temperature or electrode properties that may affect impedance. Actual time to detection of single microorganisms may be longer than that extrapolated from bulk culture because of occurrence of a lag phase sometimes seen in bacterial growth when transferred to a new medium. For example, Weaver reports a lag phase of 10-60 minutes for E. Coli in a small-volume application (U. S. 4,401 ,755).

Preferred detection of the concentrated bacteria on the chip would preferably be carried out using a disposable array. One approach for construction of such an array has been discussed previously and is shown in Figures 5A-B. At the top of the figure is one well (61) of the array (60), consisting of an inverted pyramidal well molded or embossed into a plastic sheet. In this case, the dimensions of the well are 100 μ square and 10 μ deep, giving approximately the required volume; a well the thickness of 1 mil Mylar (25 μ) would have to be about 60 μ square. In the bottom of this well (130) is an orifice filled with a conducting material, preferably a polymer such as conducting epoxy, which serves as an electrode in contact with the medium in the well. This material also contacts a permanent electrode on the surface of the chip, which serves as the measurement device. This chip includes one unit of an active matrix array (61 and 62) , where each electrode is in contact with a corresponding electrode in the disposable array and can monitor continually the impedance in one well. In this example, the counter electrode (90) shown in Figs. 2A-B, 3A-B would serve as a "lid" covering all of the wells and preventing diffusion of medium components between wells.

As discussed, fluid passing through the detection head (30) would move to the second chamber (40) for collection and eventual disposal as needed. See the U.S. Patent No. 5,536,644 for additional disclosure relating to separation methods using magnetic and non-magnetic particles.

2. Filtration Method

It has been shown that lysed blood can be filtered easily through either Millipore or Nucleopore filters with very effective capture of suspended bacteria. See e.g., Zierdt, C. et al. (1977) J. of Clinical Microbiology 46-50. In most instances, the captured bacteria are still alive and can be grown from the filter. In this method, blood from a human patient or other suitable mammal can be lysed as described above. After about 15 min. at room temperature, cell filtration is performed using the detection head (31) described above and illustrated in Figures 3A-B. For some applications such as those in which the fluid of interest includes large numbers of cells it is possible that clogging of the detection head (31) may be problematic. However, if clogging does occur, optimization of lysis reagent may help to reduce or eliminate potential clogging.

Example 3- Additional Embodiments of the Invention

1 . Detection of Fluid Properties-

(a) Chemical or Electrical Methods of Detection - Electrical properties of the medium other than impedance could be measured. It is also possible that electrical properties of the microorganism itself could be detected (for example, contact with the electrode might alter the behavior of the electrode). The presence of the organism or its metabolites could also be detected electrochemically, following either the presence of natural metabolites or redox reactions of synthetic nutrients. Optical detection systems could also be considered, measuring for example the appearance of fluorescent or colored metabolites in wells containing live organisms. The same principle of rapid detection by confining changes to the medium in a small volume still applies. Sensors could also be used, measuring, for example, carbon dioxide, oxygen, pH, or specific ion changes.

(b) Biological Methods of Detection- Sensors incorporating a biological element such as an enzyme or antibody could even detect specific metabolites. Detection systems in which the organism is labeled and the label is detected are also included. For example, an antibody-enzyme conjugate might be bound to the organism and the product of this enzyme rapidly detected in a small volume. Sensitive detection of fluorescent or magnetic labels might also be possible. (c) Detection of immune cells

A similar device might be imagined for enumeration of lymphocyte subtypes. In this case, immunomagnetic concentration might be used to bring cells to a surface where cell types would be distinguished based on a second label, perhaps different conjugates producing different products which could be differentiated electrochemically.

Similarly, the ability to count microorganisms can be used for microbial identification and susceptibility testing. Thus one can imagine a matrix in which various parts of the array contain different antibiotics or growth media. After growth to provide a statistically significant number of organisms, a bacterial sample could be spread over this matrix and should show further growth in some areas but not others.

References

1 . Reagents and method for rapid detection of bacteria in clinical samples. (Becton, Dickinson and Co., USA). Japan. Kokai Tokyo Koho 9 pp. CODEN:JKXXAF JP 60164497 A2 850827 Showa. Patent written in Japanese. Application: JP 85-5622 8501 16. Priority: US 84-571001 840116. CAN 103:175008 AN 1985:575008

2. Detection of bacterial concentration by luminescence. Plakas, Chris J. (Vitatect Corp., USA). U.S. 7pp. CODEN: USXXAM US 4144134 790313 Patent written in English. Application: US 77-764180 770131. CAN 91 :2177 AN 1979:402177

3. Development of a lysis filtration blood culture technique. Zierdt C. N. et al., J Clinical Microbiology. 1977, 8, 46-50 4. Evaluation of lysis filtration as an adjunct to conventional blood culture. Chan R, Munro R, Tomlinson P. J. Clin Pathol 1986 Jan; 39(1 ):89- 92

5. Citation for Rhozyme use for lysis: An improved technique for the isolation of higher plant chromosomes. Griesbach, R.J.; Malmberg, R.L.; Carlson, P.S. Dep. Hortic, Michigan State University, East Lansing, Ml, USA. Plant Sci. Lett. (1982), 24(1), 55-60

All documents disclosed herein are incorporated by reference. The invention has been described with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention.

Claims

What is claimed is:

1. A system for detecting living cells in a fluid comprising or suspected of comprising the living cells, the system comprising:

a) a first implementation adapted to receive the fluid and to render at least a portion of any living cells in the fluid suitable for concentration;

b) a second implementation adapted to receive at least a portion of the fluid from the first implementation, wherein the second implementation comprises an array of individual elements adapted to concentrate the living cells from the fluid and to reduce diffusion of the fluid between each element, the system being further adapted to disperse the living cells from the first implementation into each of the individual elements; and

c) a detector operably linked to each individual element in the array, the detector being capable of registering elements having at least one living cell therein,

wherein, detection of cells by the system is indicative of the presence of the living cells in the fluid.

2. The system of claim 1 , wherein the system further comprises a pump implementation adapted to move the fluid as a flow stream through the second implementation

3. The system of claim 2, wherein the pump implementation is operably linked to the first or second implementation.

4. The system of claim 3, wherein the concentration and division of the living cells by the system is facilitated by filtration. O 00/57178 _.- .

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5. The system of claim 4, wherein the division of the living cells is facilitated by a manually operated dispersing implementation operably linked to the second implementation.

6. The system of claim 5, wherein the manually operated dispersing implementation facilitates spreading of the living cells substantially evenly over each of the individual elements in the array.

7. The system of claim 1 , wherein the first implementation is further adapted to provide contact between the fluid and a composition capable of disrupting any eukaryotic cells in the fluid, the contact being sufficient to render living cells comprising a cell wall suitable for concentration by the second implementation.

8. The system of claim 7, wherein the composition is further capable of magnetizing living cells comprising a cell wall, the contact being sufficient to render the magnetized living cells suitable for concentration by the second implementation.

9. The system of claim 8, wherein the first implementation is further adapted to pre-concentrate the living cells using centrifugation or electrophoresis prior to the concentration by the second implementation.

10. The system of claim 8, wherein the system is further adapted to impress a magnetic field on at least the second implementation.

11. The system of claim 1 , wherein the system further comprises a magnetic implementation operably linked to the second implementation.

12. The system of claim 11 , wherein the concentration and division of the cells into each of the elements is facilitated by engaging the magnetic implementation and attracting the living cells toward the array.

13. The system of claim 12, wherein the linkage between the detector and each of the individual elements in the array is provided by a sensing surface.

14. The system of claim 13, wherein the detector comprises a sensor array comprising a plurality of individual sensing pixels, each sensing pixel being operably linked to the sensing surface of each individual element.

15. The system of claim 14, wherein the array of individual elements is a well array.

16. The system of claim 15, wherein the sensing surface comprises a conductive polymer and the second implementation further comprises a counter electrode adapted to sealably engage the well array, the counter electrode being operably linked to each well.

17. The system of claim 16, wherein a filter is positioned between the well array and the counter electrode, the filter being sufficient to concentrate the living cells in the second implementation and to position at least one concentrated living cell into at least one well.

18. The system of claim 16, wherein the sensor array comprises an array of individual electrodes, each electrode being operably linked to the sensing surface in each of the wells.

19. The system of claim 18, wherein the sensing surface is configured as a reporting electrode. O 0 5717 „_ .

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20. The system of claim 19, wherein the reporting electrode is adapted to detect cell metabolism in the well.

21. The system of claim 20, wherein the cell metabolism is detected as a change in at least one property of the fluid.

22. The system of claim 21 , wherein the fluid property detected by the reporting electrode is conductivity, pH, ionic strength, redox potential, or electrical impedance.

23. The system of claim 19, wherein the reporting electrode is responsive to metal ion, hydrogen ion, hydroxide, carbon dioxide, carbonate, oxygen or a halide.

24. The system of claim 19, wherein the reporting electrode comprises an epoxy resin sensitive to electrical impedance.

25. The system of claim 1 , wherein the linkage between the detector and each of the individual elements in the array is provided by an optically transparent surface and each of the individual elements is optically coupled through the surface to a sensor array, the sensor array comprising a plurality of individual sensing pixels, each sensing pixel being optically coupled to the surface.

26. The system of claim 25, wherein each of the individual elements in the array is a well and each well is optically monitored through the optically transparent surface by the sensor array.

27. The system of claim 26, wherein the second implementation further comprises a counter electrode or inert surface adapted to sealably engage each well.

28. The system of claim 27, wherein the sensor array is monitored by a scanning device operably linked to the second implementation.

29. The system of claim 27, wherein the fluid in each of the individual elements is monitored by a scanning device operably linked to the second implementation.

30. The system of claim 29, wherein the scanning device is capable of detecting a fluorescent, phosphorescent, chemiluminescent, or chromogenic substance in each of the wells.

31. The system of claim 28, wherein the sensor array is capable of detecting a fluorescent, phosphorescent, chemiluminescent or chromagenic substance in each of the wells, the scanning device being capable of registering a signal from the sensor array.

32. The system of claim 13, wherein the sensing surface comprises a biosensor.

33. The system of claim 32, wherein the detector comprises a sensor array comprising a plurality of individual sensing pixels, wherein each sensing pixel is operably linked to the biosensor.

34. The system of claim 33, wherein each of the individual elements is a well.

35. The system of claim 33, wherein the second implementation further comprises a counter electrode adapted to sealably engage each well, the counter electrode being operably linked to each well.

36. The system of claim 35, wherein the biosensor comprises a cell, nucleic acid or a protein.

37. The system of claim 36, wherein the protein is an enzyme, receptor, antibody; or functional fragment thereof.

38. The system of claim 1 , wherein the array of individual elements in the second implementation is a well array comprising from between about 10² to about 10 ⁸ wells.

39. The system of claim 34, wherein the well array is molded or embossed on a plastic sheet.

40. The system of claim 38, wherein each of the wells in the array has a total volume of from between about 10⁴ to about 10¹⁰ μ³.

41. The system of claim 38, wherein each well has a length of from between about 50 μ to 1 cm; a width from between about 50 μ to 1 cm; and a height of from between about 10μ to about 100μ.

42. The system of claim 41 , wherein the first implementation is further adapted to receive a biological fluid.

43. The system of claim 42, wherein the biological fluid is blood and the living cell is a blood cell or microorganism.

44. The system of claim 43, wherein the blood cell is an immune cell and the microorganism is yeast or bacteria. O 00/57178 ... .

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45. The system of claim 44, wherein the microorganism is present in the biological fluid at a concentration of from between about 0.1 to 10⁷ cells/ml.

46. The system of claim 1 , wherein the system further comprises a flat panel liquid crystal display for providing output from the detector to a user of the system.

47. The system of claim 38, wherein the well array is molded or embossed on a plastic sheet.

48. The system of claim 39, wherein each of the wells in the array has a total volume of from between about 10⁴ to about 10¹⁰ μ³.

49. A method for detecting presence of living cells in a fluid comprising or suspected of comprising the living cells, the method comprising:

a) receiving at least a portion of any living cells in the fluid in a first implementation adapted to render at least a portion of any living cells in the fluid suitable for concentration;

b) concentrating the living cells in a second implementation adapted to receive at least a portion of the fluid from the first implementation, wherein the second implementation comprises an array of individual elements adapted to concentrate the living cells from the fluid and to reduce diffusion of the fluid between each element, the concentrating step further comprising dividing the living cells into each of the individual elements; and

50. The method of claim 49, wherein the living cells in the fluid are rendered suitable for concentration by contacting the fluid with a composition comprising a solution comprising spermine (96 mg/ml), and PVM (7mg/ml).