WO2003087293A1 - Apparatus and method for biochemical analysis - Google Patents

Abstract

An apparatus and method for monitoring how a plurality of biological samples responds to one or more test agents. The apparatus comprises a plate having a plurality of wells, each of which is associated with a working electrode which is exposed to the well's interior. For one or more (e.g. each) of the wells, the relative positions of a biological sample within the well, and its respective working electrode, may be controlled. This provides for assay procedures that are optimized according to the nature of the biological sample (s), the test agent(s), and the purposes of the assays.

Description

APPARATUS AND METHOD FOR BIOCHEMICAL ANALYSIS

The present invention relates to apparatus, methods and means for biochemical analysis, more particularly for analysis which monitors the response of one or more biological samples to one or more test agents. The invention may be useful for e.g. studying the effects of prospective pharmaceuticals or other chemicals on biological systems, assaying environmental samples for the presence of pollutants, and clinical analysis of biological specimens for e.g. toxins and pathogens.

There is increasing public awareness of environmental issues such as quality of air, water and food, an ever expanding amount of mandatory safety legislation, and an increasing tendency to litigation in the event of negligence in the areas of industrial, pharmaceutical, consumer and environmental safety. This inevitably leads to increasing demand for the testing of compounds, in particular for their effects on biological systems. Here a conflict of interest arises: consumers wish for greater protection involving increased safety testing, but ethical concerns demand reducing, replacing or refining the use of laboratory animals for in vivo assays. In recent years, a number of in vitro assays have been developed, and several have been incorporated into the legislation.

EP 0 242 225 A2 describes a prior art system for detecting pollution in a continuous stream of aqueous liquid. An electron transfer mediator is added to the liquid, and the resulting mixture is passed to a sensor chamber containing bacteria. An activity of the bacteria is stimulated, and the level of that activity is measured at an electrode by means of electron transfer from the bacteria, to the electrode, by the mediator. There is no disclosure of any device which monitors, in parallel, the response of a plurality of biological samples to a particular test agent. There is also no disclosure of a method of monitoring a test agent having a finite volume, i.e. a test agent which is not a continuous stream.

A more general limitation of the prior art is in respect of the provision of apparatus, methods and means for monitoring biological cells, or components thereof, in states of organisation that are conducive to their proper functioning. For example, it may be necessary for the proper metabolic functioning of certain types of cell for the cells to be grown on a particular substrate. Furthermore, the mode of responsiveness of a population of a first variety of developmentally_'differentiated cells to an external stimulus, e.g. to a pollutant, may be conditional on the positioning of the cells in proximity to a population of a second variety of developmentally-differentiated cells. This situation is a simple manifestation of, or an analogy to, the more complex interdependency of cell varieties in a biological tissue, and/or the further interdependencies introduced by the arrangement of several biological tissues within a biological organ. Consequently, depending on the purpose for which the interaction of a biological sample with a test agent is to be monitored, and the particular biological sample and test agent concerned, it may be necessary to control the spatial arrangement and relative positioning of one or more populations of cells within a biological sample, with respect to one or more components of the test apparatus and, in the case of multiple populations of cells within the sample, with respect to one another. It may be necessary to study samples of a complete tissue or a complete organ. In relation to electrochemical methods of monitoring the function of cells, tissues and organs, e.g. methods employing an electrode and an electron transfer mediator, the control of the spatial arrangement and relative positioning of cell populations, tissues or complete organs, or indeed of sub-cellular organelles, may have further advantages. First, the biological sample may be positioned sufficiently far away from the electrode that all the components of the sample are held at a similar distance from the electrode, and effectively interact equivalently with the electrode via the electron transfer mediator. This may improve the consistency of the results obtained, and the correlation of those results with data that have been obtained from in vivo assays. Second, apparatus and methods may be devised in which the biological sample may be removed for a time, once or repeatedly, from the monitoring environment. This may be beneficial for the reasons that are described elsewhere herein. Third, the region around the electrode need not come into direct physical contact with the biological sample, which may enable the electrode and its environment to be regenerated effectively for subsequent reuse. Fourth, the performance of the monitoring system as a whole may be optimized by varying the relative positioning of, optionally including the distance between, the biological sample and the electrode: operational parameters including the fluid dynamics and the rate of electron transfer by the mediator may be adjusted in this way.

In a first aspect, the present invention provides an apparatus for monitoring how a plurality of biological samples responds to one or more test agents, the apparatus comprising a plate having a plurality of wells, each of which is associated with a working electrode which is exposed to its interior. The plurality of wells may be arranged in the plate in a regular pattern, e.g. as a series or array. It may comprise any number of wells, each of which may be of any convenient size and shape.

By means of the electrode which is present in each well, the apparatus is adapted for use in electrochemistry, e.g. for use in cyclic voltammetry, potentiometry and/or amperometry, e.g. for use in chemically mediated amperometry. In this latter technique, a chemical mediator (typically a redox coupling agent in its oxidised form) interacts with a biological sample and is thereby reduced by accepting electrons generated by biological (e.g. cellular) processes conducted by the sample. The mediator then transfers the electrons to a working electrode poised at a sufficient oxidising potential with respect to a reference electrode, and the resulting "current" is monitored. The chemical mediator, now re- oxidised, returns to the sample and the process is repeated. In this way, a biological activity of the sample is monitored over time, and any change in the activity, e.g. one which results from the addition of a test agent, causes a change in the observed current. Chemically mediated amperometry has never before been used in a high throughput bioassay format.

The apparatus of the invention provides for an assay system for the simultaneous analysis of a plurality of biological samples. The system therefore has a high throughput, relative to the disclosures of the prior art. It is also very versatile. A user can investigate, in parallel, the responses of a range of biological samples (e.g. a range of different bacterial, plant and/or animal cells) to any given test agent. Moreover, the different biological samples may be chosen so as to be relevant to the investigation in question. By way of illustration, if a given test chemical is suspected of being toxic to humans, but the target organ is unknown, then a range of human cells from e.g. the liver, kidney, gut, lung, muscle, skin, blood etc. can be tested simultaneously, in order to determine which organ is (most) affected. Alternatively, an environmentally relevant chemical may be tested against a range of micro-organisms, in order to determine which (if any) species is at most risk, should a release of the chemical into the environment occur (or indeed to determine which organism might be the most sensitive at detecting such a release).

The apparatus of the invention likewise provides for the simultaneous investigation of a broad spectrum of test agents on any given type of biological sample: a different test agent (e.g. solution or other medium) is contacted with each of a plurality of identical samples in the assay. The spectrum of different test agents may even include the same material at a range of different concentrations, thereby permitting a concentration-response curve to be established. By virtue of the multi-well format, it is even possible to monitor, in parallel, a plurality of different biological samples exposed to a plurality of different test agents.

One relevant use for the apparatus of the invention is in the high throughput screening of chemicals, e.g. to fill gaps in toxicity data (e.g. for the risk assessment of high production volume chemicals [HPVs]), e.g. in the development of drugs and pharmaceuticals. Historical data indicate that about 10,000-25,000 novel molecules are synthesised in order to produce a single compound which passes all the hurdles to reach the market place. A significant number of compounds are withdrawn for toxicological and/or (in the case of agrochemicals especially) ecotoxicological reasons. Carefully targeted short-term parallel screening tests can significantly improve the possibility of obtaining successful candidates, by winnowing out those with unacceptable structural and/or toxicological properties early on. By preventing such "false starts", the number of laboratory animals required for subsequent in vivo testing may also be reduced. In addition, the apparatus of the invention may be used for range-finding, i.e. determining a suitable dilution range at which a particular test compound should be administered to a higher organism, again reducing the necessity for repeat testing in animals. It may also be used for prioritising a group of test agents for subsequent analysis, e.g. in vivo.

The apparatus of the invention is adapted for high throughput screening by comprising inter alia a plate having a plurality of wells. The plate may comprise any convenient number of wells. It may have at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more wells. It may have at least 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more wells. The plate may have from 5-150, 5-125, 5-100, 10-150, 10-125, 10-100, 15-150, 15-125, 15-100, 20-150, 20-125, 20-100, 25-150, 25-125, 25-100, 30-150, 30-125 or 30-100 wells.

The wells may be arranged in a regular pattern. They may take any convenient size and shape. They may be rectangular (e.g. square) or circular in cross-section. They may define a substantially cylindrical or substantially hemispherical space. The wells in the plate may be identical e.g. as to shape and/or volume, but that is not essential. The apparatus may for example be adapted for simultaneously analysing a plurality of different test agents having different volumes. Two or more different shapes and/or sizes of wells may therefore be provided.

Any given well may have any convenient volume, as long as it is sufficient to hold a biological sample, e.g. at least one cell. It may have a volume of e.g. at least about 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 3000, 4000 or 5000μl. It may have a volume of from about 5 to about lOOμl, about 5 to about 200μl, about 5 to about 300μl, about 5 to about 400μl, about 5 to about 500μl, about 5 to about lOOOμl, about 5 to about 1500μl or about 5 to about 2000μl.

In preferred embodiments of the present invention, the apparatus may comprise 96 wells in an e.g. 8 12 array. Depending on the size and separation of the wells, the apparatus may conform to the standard 96-well format conventionally used in the art, e.g. i the fields of microbiology and biochemistry.

The use of the 96-well format may lead to significant advantages. By way of example, the apparatus may be capable of interfacing with other devices employing the 96-well format. It may be capable of interfacing with e.g. an automatic pipetting device for simultaneously loading the 96 wells with one or more samples or reagents. It may be capable of interfacing with e.g. a machine for analysing the contents of each well by an assay technique other than one based on electrochemistry, as discussed elsewhere herein.

In the apparatus of the invention, a plurality of wells is provided, each of which has a working electrode which is exposed to its interior. The presence of other wells, which are not associated with an electrode, is not however excluded. These 'non-electrode' wells may be used for analytical techniques other than chemically mediated amperometry. By way of example, optical analysis of the well contents may be used, e.g. by measurement of absorbance, luminescence or fluorescence.

Moreover, even if a biological sample is to be monitored in a well which does include an electrode, a form of analysis other than chemically mediated amperometry may be used, either as an addition, or as an alternative to that technique. In this event, the electrode will be configured, and the plate constructed, such that the alternative form of analysis is enabled. Electrode configuration and plate design are discussed elsewhere herein. By using multiple analytical techniques, a much greater amount of information, e.g. as to toxicity, can be obtained for any given test run, further improving the rate of sample/test agent throughput.

Besides the high throughput and optional interfacing with the standard 96-well format, a further advantage of the present invention's capacity for simultaneously testing a large number of agents/samples in parallel, is that each individual assay may be conducted under substantially the same conditions, e.g. as to temperature, pressure, humidity, oxygen and carbon dioxide levels, and the like. When the results of the various assays are later compared, these potential variables will hence be eliminated, and the effect of using a particular test agent and/or a particular biological sample will be easier to evaluate. The plate of the apparatus may comprise a substantially solid block, into which the plurality of wells is sunk. Alternatively, the plate may comprise a support, e.g. a substantially planar support, from which a wall or walls of each well may extend. In this latter case, the support may provide a common base to the plurality of wells, the remaining wall(s) of each well being configured as a tube, e.g. of circular or rectangular cross-section, that is upstanding from the support. In alternative embodiments, the support may comprise a web including a plurality of holes. The holes provide the mouths of the wells, a wall or walls of each well depending from the support, from around a respective hole. Depending wall(s) may be mounted to, or integral with, the support. They may terminate in a closed base that is individual to each well, or may again take the form of a tube, with a connected or integral substrate providing a common base. The support connects the wells together, whether or not it provides their base.

Any suitable material may be used to form the plate, or one or more of its components, e.g. the walls of the wells and/or any support and/or any base substrate. A ceramic material may be used. In certain embodiments of the invention, the plate, or one or more of its components, e.g. abase of the wells, maybe transparent. This will permit optical analysis of the well contents. Suitable materials include acetate, polystyrene and polycarbonate. The material of the plate, or at least that which provides the walls (including the base) of the wells, will preferably be chemically and/or biologically inert. In particular, it may be non- toxic to one or more of the types of biological sample described elsewhere herein.

Bonding substances may be used to secure one or more components of the apparatus together, e.g. the walls of the wells to a common base. A bonding substance should preferably be chemically and/or biologically inert. Alternatively or in addition, welding techniques (e.g. heat sealing or radio frequency welding) may be employed to bond components together.

Each well is associated with a working electrode for use in electrochemistry, e.g. for use in chemically mediated amperometry. At least a part of the electrode is exposed to the interior of the well. The electrode may be entirely contained within the well, and the apparatus may comprise conduction means, e.g. a wire or printed conductive (e.g. metallic) track, for providing a path of electrical communication from the outside the well, to the electrode. Alternatively, the electrode may extend into the interior of the well by passing through its mouth. The electrode may extend through a wall of the well, e.g. through a base of the well, such that its proximal end is exposed to and/or protrudes into the well interior. The distal end of the electrode is exposed to and/or located outside of the well.

Where an electrode or conduction means extends through a wall of the well, a substantially water-tight seal may be provided between the electrode/conduction means and the wall. A seal may be formed by intimate contact between the electrode or conduction means and the wall. A welded seal may be used. Alternatively, a separate sealing means may be provided, e.g. a cured resin, a gasket etc.. Any sealing component exposed to the interior of the well may be chemically and/or biologically inert. In certain embodiments of the invention, a plurality of working electrodes is provided on a support or substrate forming a common base to the plurality of wells. The remaining wall(s) of each well, which may be circular or rectangular in cross-section, will project from the common base. The walls of the wells are mounted on the base such that at least one working electrode is exposed to the interior of each well. A perforated web support may connect together, e.g. be integral with, the ends of the tubes which define well mouths, the interior of each tube being located in register with a respective hole in the web.

Electrodes may be provided on a common base by e.g. screen-printing, the base optionally being pre-scored. The base may be made of ceramic, glass or a polymeric material. Any suitable ink or other material may be used for the printing, e.g. gold, copper, a silver/silver chloride ink or a high conductivity carbon-based ink. The non-working areas of the base may be covered with an electrically insulating layer, e.g. a dielectric layer, which may be printed using a non-conducting polymer. In alternative embodiments, the electrodes may be electrically conductive pins or lugs which are mounted to and/or extend through the base.

In addition to the working electrodes, the apparatus of the present invention may comprise one or more reference electrodes, against which the potential of one or more associated working electrodes can be measured. Reference electrodes may be provided as combined reference/counter electrodes as is known in the art, but in certain embodiments of the present invention, one or more dedicated counter electrodes, separate from the reference electrode (s), may alternatively be provided (thereby forming a three electrode system).

In preferred embodiments of the invention, each well is associated with both a working electrode and a reference electrode, each electrode being exposed to the well's interior. A reference electrode may be associated with a well in the same way as a working electrode. It may for example extend through a wall of the well, or it may be provided on a common base of the plate, e.g. by screen-printing with an electrically conductive ink or other material, e.g. with a silver/silver chloride or high conductivity carbon-based ink. Any dedicated counter electrode (s) may also be so provided.

In certain embodiments of the present invention, working and reference electrodes are screen-printed onto a common base.

Electrode layouts, e.g. for screen-printing, may be designed on computer, e.g. using CAD or Gerber software.

Working and/or reference and/or counter electrodes may take any suitable configuration. In embodiments of the invention designed for optical analysis of the well contents, the electrodes will be structured appropriately. By way of example, if a working electrode is mounted on a transparent common base, then an O- or C-shaped electrode may be appropriate. This will permit a central, unobstructed portion of the base, through which optical analysis may be conducted. If a reference electrode (or other electrode) is to be included in the same well, then it may be provided with a similar O- or C-shaped configuration, optionally of different diameter. The working and reference electrodes may then be located concentrically, again providing a central, unobstructed portion of the base.

It will be clear from the foregoing discussion, that the working electrodes of different wells should ideally be maintained in electrical isolation, both from one another, and also from any reference electrode (s) . The individual responses of biological samples contained within the different wells can then be monitored. By way of contrast, if the apparatus comprises a plurality of reference electrodes, e.g. one associated with each well that has a working electrode, then these reference electrodes (or groups of them) maybe connected together. They may then be poised at the same potential. Electrical connection is achieved using conduction means. Unconnected electrodes may have different electrical potentials applied thereto.

It has also been noted that where an electrode is entirely contained within a well, the apparatus may comprise conduction means for providing a path of electrical communication from the outside the well to the electrode.

Further, the apparatus may contain one or more electrical terminals for connecting the electrodes to one or more electrical devices for controlling, monitoring, and optionally recording, their potential. The electrical terminals maybe mounted on the plate, e.g. at an edge thereof. Conduction means may be provided for establishing a path of electrical communication from the electrodes to the terminals. Such paths may be provided with electrical switches.

For any of the above connections, any conduction means well known to those skilled in the art may be used. A metallic or carbon-based conductor, e.g. in the form of a wire, may be suitable. Alternatively, a conductive track, screen-printed onto the plate, e.g. onto a support or base substrate, may be employed.

In certain embodiments of the invention, e.g. those involving a large number of wells, the use of copper conductors mounted on e.g. the support or base substrate (where present), may be advantageous. This is because of the length of electrical connections between the electrodes and/or terminals: carbon and/or silver-silver chloride inks, under certain circumstances, may not conduct low currents effectively over long distances.

Production of copper tracks may be performed using known methods of manufacture of printed circuit boards (PCBs).

In certain embodiments of the present invention, a high speed lithographic printing technique may be used to provide conductive tracks on e.g. a support or base substrate. Rather than etching away unwanted copper from a laminated substrate (as in PCB manufacture) , the conductive tracks are printed directly onto the substrate/support in the desired configuration. The process uses specialised inks or other materials, which can be used as conductors themselves, or as a base for subsequent plating to form the conductive tracks. The technology may achieve finer line width and spacing than etch technology, and the circuits can be printed on any substrate, including a range of plastics. Lithographic technology may increase the speed of production of the apparatus of the invention, whilst dramatically reducing the amount of copper or other metal required, and the overall cost of producing the apparatus. Furthermore, transparent materials may be used for the support/base substrate, thereby permitting optical analysis of the well contents. The risk of chemical contamination may also be reduced, as compared to etch technology.

Where conduction means are provided on a base component which is common to the wells, e.g. a support or additional base substrate, then those means may be carried, e.g. printed, on either side of the base. An electrical connection between any two conduction means on opposite sides of the base may be achieved using e.g. drilled holes (through-holes or vias) which are plated. By using both sides of the base, an increased amount of space is available for optimum configuration of the conduction means, thereby ameliorating the problem of how fit a large number of connections in the relatively narrow spaces between wells.

The apparatus may comprise positioning means for determining (and optionally adjusting) the relative positions of (including e.g. the distance between) a biological sample and its respective working electrode within at least one (and preferably each) of the wells. In certain embodiments of the invention, in order to effect such control of position, the apparatus may comprise, in addition to the plurality of wells, at least one insert for removably mounting a biological sample within a respective well. The apparatus may in fact comprise, in addition to the plurality of wells, a corresponding plurality of inserts for removably mounting a biological sample (and optionally adjusting its position) within each of said wells.

For those embodiments in which it is possible to control the position of a biological sample, relative to its respective working electrode, in a plurality (e.g. in all) of the wells, the control may be performed either uniformly across the wells, individually for each well, or otherwise non-uniformly, e.g. differently for each biological sample, differently for each test substance, or in a deliberately graduated fashion for a given biological sample or a given test substance, or otherwise. Greater flexibility may be achieved by means of, e.g. the permanent modification of preformed inserts, the use of alternative inserts having fixed dimensions, and/or the use of inserts having adjustable dimensions.

An insert may be constructed so as to permit the passage of a chemical mediator, e.g. a redox coupling reagent, between the biological sample and the working electrode. It is nevertheless capable of retaining the biological sample, thereby to permit its removal from the well at the end of the analysis. The exact configuration of any inserts may depend on the size and shape of the corresponding wells.

A portion or component of the insert may be porous, the holes being sufficiently large so as to permit the passage of a chemical mediator, but not the biological sample. A porous portion or component of the insert may comprise or consist of a membrane.

The exact configuration of an insert may depend on e.g. the volume of the well for which it is intended, and the nature of the biological sample which it is designed to carry. The insert may be configured for example as a cup, into which the biological sample, one or more test agents and a chemical mediator may be placed in use. The cup may be adapted so it can be mounted in the well such that its lower most portion is inside the well, but spaced a predetermined distance from the well base. This may be achieved using a shoulder or lip of the cup which is capable of resting on a portion of the plate which defines the mouth of the well. This portion of the plate may be e.g. an end of a wall of the well or a section of the support. Alternatively, or in addition, the cup may comprise one or more legs which in use contact the base of the well. Such a shoulder, lip, leg or other means of predetermining distance from the well base may be adapted to enable adjustment of that distance, for example by permanent modification, interposition of an additional component, or a built-in telescopic or ratchet mechanism

The configuration of an insert is preferably chosen so as not to obstruct the operation of any electrode (s) in the well. By way of example, if the cup is provided with legs, then these should be arranged and/or configured so as not to contact the electrode.

In certain embodiments of the invention, the inserts may be suitable for complete immersion within the contents of a well, whilst still preventing an escape of the biological sample. By way of example, a membranous bag may be used. The inserts may be beads.

The inserts may be made of any appropriate material, but that material is preferably electrically non-conductive and chemically and/or biologically inert. The materials described elsewhere herein for manufacture of the plate may be used. Where the apparatus is designed for optical analysis of the well contents, the inserts maybe made of a transparent material, e.g. of acetate, polystyrene or polycarbonate.

The use of inserts greatly enhances the versatility of the apparatus of the present invention. By way of example, a biological material e.g. cells, or viruses on a cell lawn, may be directly grown on the inserts or on a component thereof. Steps involved in the transfer of cells from an established culture to the sensory apparatus can therefore be avoided. Even where the culture is a liquid, such transfer may be detrimental to the cells. It may involve steps such as centrifugation, which may damage and/or alter the behaviour of the cells.

In particular, the provision of inserts may facilitate the use of eukaryotic cells, especially animal cells, as the biological sample. Animal cells are typically grown as a monolayer anchored to a culture dish; the use of an insert (or component thereof) on which the cells are grown, will obviate the need for removing the cells from that dish. Since such removal may comprise harmful and/or behaviour-altering steps, e.g. treating the cells with enzymes (e.g. trypsinisation), the integrity of the cells will be substantially preserved. Exposure of the cells to enzymes may also be time consuming.

The use of an insert further permits a biological sample to be returned to culture, after its , analysis, and optionally re-examined. This allows recovery studies to be conducted. It also provides for an investigation as to whether a given test agent (or concentration thereof) causes sensitisation or desensitisation of the biological sample: repeat exposure and monitoring of the same biological sample may be conducted periodically. The inserts of the plurality may be discrete from one another such that any given insert may be removed from its respective well, without disturbing the others. Alternatively, at least some of the inserts may be mechanically coupled or fused together, thereby forming a set of inserts to facilitate their co-ordinated manipulation. In embodiments of the invention in which the plurality of wells is arranged in an array, inserts may be joined or fused together to reflect the structure of a regular part thereof, e.g. a row or column.

As discussed elsewhere herein, the apparatus of the invention may be provided with contacts or terminals for connecting the electrodes to electrical devices. Such devices may be used for e.g. monitoring and optionally recording the potentials of the working electrodes. They may be used for e.g. maintaining the potential of the reference electrode (s) . The apparatus may comprise a multi-channelled potentiostat for controlling the potential difference applied to the electrodes and collating the signals from each individual electrode on a computer. A potentiostat will have a number of channels which ideally corresponds to (or exceeds) the total number of electrodes (or groups of electrodes) in electrical isolation from one another. By way of example, if e.g. amperometry is to be conducted in each well of a 96-well plate (using the standard 96-well format known in the art) , then a potentiostat having at least 96 channels for 96 working electrodes, and at least one channel for each reference or counter electrode (or group of such electrodes) is required. Each channel is in electrical communication with one of the electrodes. Potentiostats may be obtained from e.g. Whistonbrook Technologies Limited.

The apparatus of the invention may comprise data processing means, e.g. a computer and appropriate software, for collecting, storing and optionally recording the potential of each working electrode relative to an associated reference electrode. The computer may further permit data display and/or analysis. Connection between a computer and a potentiostat may employ any conventional means.

The apparatus of the invention, optionally including the potentiostat and/or computer, may be sufficiently small so as to be easily transportable between laboratories and/or useable in the field, e.g. where on-site analysis of environmental or clinical samples is required. A mobile power source, e.g. a battery pack, may be provided.

In certain embodiments of the invention, a potentiostat, a data processing means, a data display means, or one or more components of such devices, may be incorporated into, e.g. mounted on, the plate.

One or more components of the apparatus of the invention, e.g. the plate and/or the inserts (where present) which may comprise adjustment means for adjusting the location of a biological sample relative to a respective working electrode, may be disposable. Alternatively or in addition, the component (s) may be capable of being sterilised by one or more methods, without impairment of function.

Effective sterilisation techniques are prerequisites for the art of cell culture and cell-based assays. Cell culture provides an ideal environment for the growth of micro-organisms including viruses, and it is only by excluding such agents that cells can be successfully cultured and meaningful experimentation can be carried out (Roberts, P. L: Sterilisation. Basic Cell Culture - a Practical Approach, (ed. Davis, J M) pp. 27-55. Oxford University Press, Oxford [1994]). When using apparatus according to certain embodiments of the present invention, cells are located in inserts which are deposited into wells of e.g. a microtitre plate. If the cells are to be monitored electrochemically for more than a few hours (or if they are to be returned to culture after the test) then the presence of microorganisms becomes an issue. Such contamination could result in artefacts arising during the tests. Of even greater concern is the possibility that the return of a contaminated plate or insert to the incubator could result in the contamination of other cultures which are being handled within the laboratory. It may therefore be important that the apparatus of the invention, or at least the plate component and/or inserts thereof, is capable of being sterilised.

There are many methods by which laboratory items may be sterilised. High temperature sterilisation techniques using hot air or steam (e.g. hot air ovens and autoclaves) are generally favoured, but under certain circumstances may damage screen-printing inks used in the production of electrode working areas and their surrounding connections. In such circumstances, irradiation and/or appropriate liquid disinfectants may be of use. UV light inactivates micro-organisms through DNA damage but may require a long exposure time (e.g. overnight). In addition, some bacterial spores are resistant. A range of liquid disinfectants are available, with ranging¹ effectiveness against contamination. Mention may be made of alcohol, aldehydes, hypochlorite and phenolics. Ethanol is effective against bacteria and some viruses but not against fungi or endospores. The most effective concentration is approximately 70%.

The apparatus of the invention, or one or more of its component parts, may be capable of being sterilised by any of the above .methods, or a combination thereof, without impairment of function. The desire for a sterilisable apparatus should be taken into account when selecting the materials for its manufacture, including any screen-printing inks or adhesive. Such selection is well within the knowledge of the person of skill in the art.

To further facilitate the manipulation and analysis of biological samples under sterile conditions, the apparatus may comprise a lid or other covering means for overlying the mouth of at least one well, (preferably all wells) thereby to prevent (or at least inhibit) the entry of micro-organisms. The covering means may be provided with securing means for mounting it on the plate. Any conventional securing means may be employed, whether mechanical, e.g. a friction fitting, or chemical, e.g. an adhesive.

In a further aspect, the present invention provides a method of monitoring how a plurality of biological samples responds to one or more test agents, the method comprising the steps of contacting each sample with an electron transfer mediator and a test agent in the presence of an electrode, and monitoring in parallel the transfer of electrons from the samples to their respective electrodes by the mediators. The transfer may be monitored by measuring the electrical potential of each electrode relative to a respective (or common) reference electrode. In certain embodiments of the method of the invention, the positions of the biological samples relative to their respective working electrodes maybe controlled. This may be achieved using positioning means as described elsewhere herein. Different biological samples may be positioned at different distances from their respective working electrodes.

The method of the invention may be based on chemically mediated amperometry, as discussed elsewhere herein. Examples of appropriate chemical mediators and electrolytes are well known to those skilled in the art. Reference is made to e.g. Evans, M. R. et ah Pesticide Science 54, pp.447-452 [1988] . As to the chemical mediators, p-benzoquinone and 2,6-dimethyl-benzoquinone may be mentioned.

In order to properly evaluate the effect of a particular test agent on a particular biological sample under a certain set of conditions (e.g. a specific chemical mediator and a selected type of electrode), the method may further comprise a step of contacting an identical biological sample with an identical electron transfer mediator in the absence of the test agent. The transfer of electrons from the sample to an identical electrode is monitored, and the transfer is compared to the transfer recorded in the presence of the test agent. Monitoring of identical samples in the presence and absence of test agent may be conducted simultaneously, i.e. in parallel, e.g. in adjacent chambers of the same apparatus. This may help to eliminate variables such as temperature, oxygen and carbon dioxide levels. Alternatively, the transfer of electrons observed in the presence of test agent may be compared to pre-recorded data showing the transfer of electrons from the sample in the absence of test agent.

In certain embodiments of the invention, the effect of a given test agent on a particular biological sample may be determined by: (i) contacting the sample with an electron transfer mediator in the presence of an electrode; (ii) monitoring the transfer of electrons from the sample to the electrode by said mediator, thereby to establish a basal activity for the sample; (iii) contacting the sample with a test agent; and (iv) monitoring the transfer of electrons from the sample to the electrode by the mediator in the presence of the test agent, thereby to determine a test activity for the sample. The basal and test activities of the sample may then be compared.

The method of the invention may comprise such steps for two or more of the plurality of biological samples being monitored. It may comprise those steps for all of said samples.

Notwithstanding the above, a basal activity need not always be determined. By way of example, if the effect of concentration of a given substance on a particular biological sample is to be determined (e.g. for the production of a concentration-response curve), then a plurality of identical biological samples may be exposed to a respective plurality of test agents comprising the same substance at a range of different concentrations. The zero concentration activity may be determined by extrapolation.

Unlike most existing toxicity tests, the electrochemical component of the assay is designed for continuous monitoring. The rate of action of a given test compound, e.g. its rate of toxic action, can therefore also be determined.

The method of the invention may thus comprise a time period during which the transfer of electrons from the biological samples to their respective electrodes is continually, or periodically, monitored. The test agents are contacted with the samples at a particular point in the test period, and the change in electron transfer (if any) which results from the test agents is recorded. The rate of change of the measured 'current' is determined and optionally analysed.

In certain embodiments of the invention, the method may be used to determine the recovery of a particular biological sample, after its exposure to one or more test agents. By way of example, the method may comprise the steps of: (i) contacting at least one of said biological samples with an electron transfer mediator and a test agent in the presence of an electrode; (ii) monitoring the transfer of electrons from the sample to the electrode by the mediator, thereby to determine a test activity for the sample; (iii) removing the test agent from the sample; and (iv) periodically or continuously monitoring the transfer of electrons from the sample to the electrode by the mediator, after the test agent has been removed. Removal of test agent may occur by any conventional technique known to those skilled in the art, e.g. by aspiration, filtration, etc.

Prior to step (i), the method may comprise the steps of contacting the sample with the electron mediator in the presence of the electrode, and monitoring the transfer of electrons from the sample to the electrode by said mediator, thereby to establish a basal activity for the sample. In that way, the extent of recovery of the sample may be more easily determined, e.g. by comparison of the transfer of electrons monitored in step (iv), with the previously measured basal activity.

If the sample is to be monitored periodically (rather than continuously) in step (iv), then in between the measurements of electron transfer, the sample may be removed from the apparatus in which the electrode is located, and returned to conditions for cell culture, e.g. to an incubator. To facilitate such removal, the apparatus may comprise removable inserts or carriers on which, or within which, the sample is retained. In that way, the sample does not need to be subjected to treatments which might cause damage and/or a disturbance in behaviour, e.g. treatments for releasing anchored cells from a culture plate. The use of removable inserts, in association with electrochemistry, e.g. in association with chemically mediated amperometry, is discussed elsewhere herein, within the context of the present invention, more particularly the apparatus of first aspect of the invention.

Removable inserts may also facilitate methods of the invention in which an investigation is conducted as to whether a given test agent (or concentration thereof) causes sensitisation or desensitisation of a biological sample: repeat exposure and monitoring of the biological sample may be conducted periodically, with the sample being returned to culture conditions in between times.

Irrespective of the purpose of the method of the invention, the method involves monitoring a plurality of biological samples in parallel by electrochemistry, e.g. by chemically mediated amperometry. Accordingly, a sensory apparatus comprising a plurality of chambers, each of which is associated with a working electrode may be used. If a respective plurality of removable, discrete inserts is associated with the plurality of chambers, then it becomes easier to manipulate any particular assay without disturbing the others. By way of example, a first set of chambers in the sensory apparatus may be used to monitor how a first plurality of different biological samples reacts to a particular test agent. A second set of chambers in the same apparatus may be used to monitor how the same spectrum of samples reacts to a different test agent (e.g. the same test substance at a different concentration). A third set of chambers may be used to monitor recovery of a third plurality of biological samples, following their exposure to a further test agent. Whilst electron transfer is continually monitored in the first and second set of chambers, the third plurality of samples may be loaded into the third set of chambers on inserts, their electron transfer to respective working electrodes monitored, and the samples returned to culture.

To facilitate independence between different sets of chambers, the apparatus employed in the method of the invention may comprise a series of electrical switches for reversibly completing paths of electrical communication between electrodes and terminals.

Any method of the present invention may involve the use of any apparatus according to the first aspect of the invention. Various features of such apparatus are described elsewhere herein, and will not be repeated here.

Having said that, an apparatus employing the established 96-well format may particularly be mentioned. Such an apparatus may be based on a modified microtitre plate. In that way, only small sample volumes are required. This may be important where the quantity of the test agent(s) available for analysis is limited (e.g. in drug development).

In certain embodiments of the method of the invention, a sensory apparatus is used which comprises a plate having a plurality of sampling areas, each of which is associated with a working electrode. A biological sample and a test agent are located onto each sampling area, over the electrode. The sample and agent may be spotted onto the sampling area and held in place by surface tension. Such an apparatus is particularly suitable for use with small sample volumes of sample, e.g. in the field of proteomics. The apparatus may include any of the features (or combinations of features) that may be found in the apparatus according to the first aspect of the invention, unless the context requires otherwise. For example, the control of the relative positions of a sample and its respective working electrode may be achieved by floating a suitable membrane or other surface carrying the sample, on a spot of fluid retained over the electrode by surface tension. The fluid may contain the test agent and/or mediator. Alternatively, such a membrane or surface maybe distributed across the surface of the plate either as a continuous layer (itself being so composed and fabricated as to prevent mixing between the fluid volumes in contact with the working electrodes) or as discrete units, one for each location of a working electrode. Fluid or fluids containing, e.g. the test agent and/or the mediator may then be spotted onto the membrane or surface. In these various embodiments, the positioning of the membrane or surface relative to its respective working electrode (s) may be respectively adjusted by varying the volume of the retained spot of fluid, or by shaping the membrane or surface so as to determine its clearance in relation to each electrode that it covers. The membrane or surface may contain the biological sample, thereby preventing any direct physical contact between the sample and the electrode, or the biological sample may adhere to one or both sides of the membrane or surface.

The method of the invention may involve, in addition to analysis by electrochemistry, e.g. chemically mediated amperometry, which may be conducted using the apparatus of the first aspect of the invention, one or more alternative analytical techniques, e.g. assays based on optical, e.g. UV, visible, fluorescent or luminescent endpoints, e.g. where a compound undergoes a colour change due to its reduction oxidation by the sample (or by a mediator previously reduced/oxidised by the sample) . The assays may be conducted at the same time as the amperometry, either on biological samples which are simultaneously being monitored by amperometry, or on parallel samples, e.g. samples contained within separate wells of the same apparatus, or on both types of sample. Through the use of multiple parallel analytical techniques, an increased amount of information can be collected in the same test run. The throughput efficiency of the analytical system is therefore improved.

Examples of alternative assays include the fluorescein leakage test (which employs cells with tight junctions in order to detect subtle junctional defects) , the resazurin test (a cell viability test), and the Neutral Red and Alamar Blue assays. One alternative analytical technique employs biological cells which have been engineered to contain reporter genes. A reporter gene may provide for an additional and unrelated activity of a biological sample, which may be modified in response to a test agent.

Reporter genes may code for biological molecules possessing unique properties which are easily distinguishable from endogenous cellular functions. They can generate, e.g. colorimetric, fluorescent, luminescent, • chemiluminescent or electrochemical signals, which may be proportional to the concentration of the test agent to which their transgenic hosts are exposed.

To take just one example, luminescence may be achieved using the lux genes encoding luciferase enzymes. Such enzymes catalyse light-emitting reactions, and are commonly found in a range of bioluminescent (light-emitting) organisms. There are five lux genes responsible for the light-emitting reaction. If all five genes of the lux cassette are incorporated into a test cell, then a completely independent light producing system is created, requiring no additional substrate to be added, and no excitation by an external light source. Luminescent reporter genes may be used to determine the metabolic status of the cells, following chemical challenge. Disturbances in luminescence illustrate e.g. toxic effects of test agents which may result from e.g. transcriptional inhibition.

Reporter genes can provide a robust, cost-effective, quantitative method for the rapid and selective detection and monitoring of chemical and biological test agents. Similar to . chemically mediated amperometry, reporter technology can often be implemented in realtime, on-line bioassays, with intact, living cell systems, thereby providing a unique and revolutionary perspective on bacterial, plant and mammalian physiology, including cellular interactions. The technology may be used in the drug discovery process to study e.g. transcriptional regulation in response to a test agent. It may be used for secondary pharmacological and receptor binding assays.

The methods and apparatus of the present invention may be used in a diverse array of detection processes and behavioural studies, e.g. in methods of medical diagnostics, precision agriculture, environmental monitoring, food safety, process monitoring and control, and potential pharmaceutical analysis. Such uses in fact provide yet further aspects of the present invention. Particular examples of such uses are described elsewhere herein. The following provides yet further illustration, by specifying examples of the biological samples and/or test agents that may be employed. In general, the invention is useful where it is desired to establish whether (and optionally to what extent) a given test agent affects a particular biological sample.

In any method of the present invention, the plurality of biological samples maybe identical to one another, e.g. in the event that a user wishes to determine how a plurality of different test agents affects one particular cell type or tissue. The spectrum of test agents may even include the same material at a range of different concentrations, thereby permitting a concentration-response curve to be established.

Any given test agent may be a pure or substantially pure compound, e.g. in embodiments where the high throughput screening of individual chemicals is to be conducted. Such methods may be used in the risk assessment of high production volume chemicals (HPVs) , or in the development of drugs and pharmaceuticals, as discussed elsewhere herein. By way of example, the method may be used for the rapid testing of a large number of potentially therapeutic compounds generated by combinatorial chemistry.

In alternative embodiments, the test agent may comprise a mixture of substances. By way of example, the test agent may include a material taken from the environment, e.g. when the method of the invention is used to detect pollution and/or to monitor dispersion and/or degradation of one or more pollutants. An environmental material may be taken from e.g. soil, rock, sand, water (e.g. from a river, lake, ocean or underground water source). It may be taken from biological matter, e.g. it may comprise an extract or homogenate from a plant or animal, e.g. a specimen of blood, plasma, lymph, bile, gastric fluid, tissue fluid, urine, faeces, or the like. Biological materials may also used as test agents when the method of the invention is used in clinical analysis, e.g. where one or more isolated specimens (from the same or different patients) is used for a method of in vitro diagnosis. Any of the above-mentioned biomaterials may be used. Such materials, e.g. blood, may be pre-treated, prior to their use in the method of the invention, e.g. by the removal of cellular components, e.g. by centrifugation and/or filtration. A test agent may be a foodstuff, e.g. an aliquot of allegedly purified drinking water. A test agent may include a material obtained by or derived from an industrial process. It may include an industrial waste material (e.g. waste water) which may be destined for release into the environment.

Any given test agent may be solid or liquid in nature. Liquid test agents may be aqueous or non-aqueous, i.e. organic. A liquid agent may be a solution, suspension, or emulsion. The use of a monitoring apparatus, e.g. one affording control of the positioning of one or more (e.g. all) of the biological samples relative to their respective working electrodes, and in particular such an apparatus comprising a plurality of chambers, e.g. wells, each containing an electrode, and a respective plurality of removable carriers, e.g. inserts, may facilitate the use of test agents which are either: (i) solids of poor solubility; or (ii) non- aqueous liquids. By way of illustration, water-insoluble chemicals may be dissolved in non- aqueous solution (e.g. mineral oil) and placed in an insert which comprises a membrane and is loaded with a biological sample. The chemical mediator and an electrolyte are placed in a respective chamber of the apparatus, and the insert is located within the chamber. The membrane of the insert separates the two liquid phases but permits the passage of the chemical mediator. Where confluent, e.g. animal cells are grown, on the membrane, the cell layer will also participate in separating the two liquid phases.

In certain embodiments of the method of the invention, a single test agent may be contacted with a plurality of different biological samples. As discussed elsewhere herein, this may be appropriate where a given test chemical is suspected of being toxic to a particular animal, e.g. to a human, but the organ which is most affected is unknown: a range of different cells from the animal may be tested simultaneously. Alternatively, it may be appropriate where an environmentally relevant chemical is to be tested against a range of different micro-organisms, in order to determine which (if any) species is at most risk, should a release of the chemical into the' environment occur.

In the context of toxicity testing, it might be imagined that a biological organism is so complex that the monitoring of isolated cell-types provides little valuable information. To the contrary, the concept of basal cytotoxicity teaches that most toxic chemicals exert their ultimate action by interference with basic cellular functions. These functions are fundamental to the life of cells, and are expressed in a similar way in all cells, regardless of whether the cells are located in vitro or in vivo. Chemical compounds can therefore be tested for their acute toxicities in relatively simple culture systems. Intra-laboratory assessments during the early 1980s indicated that good correlations were obtained between rat LD₅₀ values and cytotoxicity test results with various isolated cell lines. Different cell types may nevertheless show different sensitivities to the same toxic chemical, as discussed elsewhere herein. Moreover, cell-cell interaction may modify a toxic (or pharmacological) response, e.g. where a given cell type, which is relatively insensitive to the direct effect of a particular test agent, is more severely compromised by the lack of physiological signals received from a second cell type, which is directly affected.

The one or more biological samples may be selected from the group consisting of eukaryotic cells, prokaryotic cells, cellular organelles, membranes and mixtures thereof. Eukaryotic cells include plant cells, animal cells and fungal cells. Algae or higher plant cells may be used. Animal cells may be taken from mammals, e.g. from humans or other primates (e.g. from chimpanzees). They may be taken from agricultural animals such as horses, cows, goats, sheep, pigs, or birds (e.g. from chickens or geese) , or they may be taken from domestic animals such as dogs, cats, rabbits, and the like. Prokaryotic cells may be taken from any species of bacteria, e.g. from photosynthetic bacteria or from cyanobacteria. Suitable organelles and membranes may include those which comprise, or are associated with, one or more components of an electron transport chain, e.g. chloroplasts and mitochondria and membranes therefrom. In certain embodiments, isolated proteins or mixtures of proteins may be used as a biological sample, e.g. cytochromes, photosynthetic pigments, and the like.

Biological samples may comprise transgenic cells, i.e. cells containing a heterologous nucleic acid sequence such as a reporter gene. The transgene may provide for an additional and unrelated activity of the biological sample which may be modified in response to the presence of a test agent.

Biological samples may take any suitable form known to those skilled in the art, e.g. cell suspensions or cell layers grown on removable carriers or inserts of the sensory apparatus, or on components thereof, e.g. on membranes. Multiple cell layers and back-to-back cultures may be used, as discussed elsewhere herein.

Notwithstanding the concept of basal cytotoxicity, it may be desirable to examine the effect of a particular test agent on an in vitro biological system comprising more than one cell type. Although the investigation of cytotoxicity is an important aspect of the present invention, the methods and apparatus described herein are not so limited. Moreover, (and as explained previously) , cell-cell interaction may influence the toxic effect of a compound in vivo, although it may ultimately act on a basic cellular metabolic function. Researchers have noted that many cells rely on the presence of other cell types in order to function correctly.

A range of cell-cell interaction factors (e.g. cytokines and growth factors) are known to regulate a wide array of cellular functions, including cellular defence and repair systems, which can affect the toxic or pharmacological response in an organism, subsequent to its exposure to one or more chemicals.

The influence of intercellular signalling cannot be investigated in conventional cell monocultures or suspensions, and due to the short life time and local range of the signalling factors, is difficult to study in vivo (Maier, P: Developments in Animal and Veterinary Sciences, 31 A: Progress in the Reduction, Refinement and Replacement of Animal Experimentation (eds. Balls, M. et l) pp.249-256. Elsevier, Oxford [2000]). Moreover, as noted elsewhere herein, there are also certain ethical concerns surrounding experimentation on animals.

In light of these facts, it may be of value to culture several cell- types in close proximity to one other, in order to permit intercellular interactions to occur. The situation in a tissue, or the interactions between organs, may therefore be mimicked. Cells may be co-cultured either as a mixture, or as separate cell layers within the same medium. In any of the methods of the present invention, co-cultures of two or more different cell types may be used.

Cell culture inserts (as used in certain embodiments of the apparatus and methods of the present invention) may be used to grow back-to-back cultures. Different cell types may be grown on each side of a micro-porous insert membrane, enabling cell-cell contacts or contact through soluble factors (Van Gompel, J : Developments in Animal and Veterinary Sciences, 31 A: Progress in the Reduction, Refinement and Replacement of Animal Experimentation (eds. Balls, M. et al) pp. 231-236. Elsevier, Oxford [2000]). Using chemically mediated amperometry, optionally in conjunction with optical endpoints, it is possible to monitor the effects of intercellular interactions, in particular by comparison with the results of assays on individual cell types.

The ability to monitor co-cultured cells has implications in a range of research topics which span across medical and pharmaceutical research. The assessment of liver toxicity, and the identification of metabolic pathways in the liver, are important aspects in the characterisation of the toxicity of chemicals in humans. By way of example, Kupffer cells in the liver are known to release a number of intercellularly-acting mediators which are suspected of modulating the effects of hepatotoxic compounds, by intercellular signalling directed towards hepatocyte cells (Maier, P: Developments in Animal and Veterinary Sciences, 31 A: Progress in the Reduction, Refinement and Replacement of Animal Experimentation, supra).

The identification of potentially neurotoxic activity may also be an important aspect of toxicity assessment. The neurons in the brain which receive, integrate and transmit information only account for 10% of brain cells. Glia (the majority of brain cells) provide mechanical support, regulation of the extracellular environment, production of cytokines and neurotrophins, and regeneration, and are thought to exacerbate neurotoxicity. Co- cultures could be used to bring cell types such as cortical neurons, astrocytes and microglia together. The results may be compared with pure cultures to determine cell relationships, e.g. to determine whether there is an increase or reduction in toxicity when the cells are cultured together (Kirkpatrick, C. J: Developments in Animal and Veterinary Sciences, 31 A: Progress in the Reduction, Refinement and Replacement of Animal Experimentation (eds. Balls, M. et al) pp. 329-337. Elsevier, Oxford [2000]). An interaction between the cells of the brain and those of the liver is also possible via the blood circulation. Co- cultures of brain cells and hepatocytes grown on culture inserts may be exposed to test compounds, and hepatocyte metabolites assessed for their toxicity to brain cells.

In addition to using the methods and apparatus of the invention to monitor co-cultures, it is also possible to directly examine the effects of compounds or chemicals on tissue explants, e.g. liver slices. Liver slices have been used in recent investigations of drug metabolism and liver toxicity because all the cells are present in their natural state, with their original cell-cell contacts intact. It is therefore believed that they may be more discriminating for compounds possessing intricate mechanisms of toxicity. They may be useful for the examination of chronic or long term toxicity.

Under chronic exposure conditions, where a small number of cells might die and repair/detoxification systems are active, the actual complexity of the responses is much higher than can be accurately predicted in standard cytotoxicity models (notwithstanding the fact that basal cytotoxicity models using isolated cell lines will still provide valuable information). From the cell-cycle point of view, hepatocytes in a liver slice are closer to hepatocytes in the liver than are isolated hepatocytes. Precision-cut liver slices are now commercially available. These are highly reproducible and stable (lasting up to 5 days). 75 slices may be obtained from a single rat liver and 20,000 from a human liver. These slices are each approximately 8- 10mm diameter, which is sufficient volume for a detailed study (Bach, P. H. et a The use of tissue slices for pharmacotoxicology studies. The report and recommendations of ECVAM workshop 20. ATLA pp. 893-923 [1996]). In accordance with the present invention, a plurality of tissue slices may be placed in a plurality of culture inserts, or otherwise positioned relative to respective electrodes for monitoring a plurality of biological samples, and immersed in culture media or perfusion solution, in the presence of a mediator for amperometric monitoring.

As will be apparent to those skilled in the art, the use of assay systems in which control is provided over the distribution of biological sample components and their position relative to the working electrode, e.g. by using inserts, greatly facilitates the examination of co- cultures and more complex in vitro models.

Various further aspects and embodiments of the present inverition will be apparent to those skilled in the art in view of the present disclosure. Certain aspects and embodiments will now be described by way of example only and with reference to the Figures described below.

All recited documents are incorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows examples of electrode arrays screen printed onto a ceramic base.

Figure 1(a) shows an 8 well linear arrangement as used in Experiment 1. The working area is shown in more detail in Figure 1 (b) , where the black area is the working electrode and the grey area is a silver/silver chloride reference electrode. Figure 1 (c) shows the design as applied to the 96-well format.

Figure 2 shows a comparison of the amperometric signal resulting from bacteria placed directly in a well, with the signal produced by bacteria contained in an insert. Bacteria were present in the insert at time 0, whereas bacteria were added to the well at time 180s. A control containing no bacteria is also shown.

Figure 3 shows a comparison of the amperometric signal resulting from bacteria placed directly in a well, with the signal produced by bacteria contained in an insert. Bacteria were present in the insert at time 0 whereas bacteria were added to the well at time 180s. A high concentration of the toxicant ZnS0₄ was added to the insert at time 1840s, resulting in a reduction of the bacterial signal to baseline. Figure 4 shows the amperometric signals obtained from bacteria exposed to different concentrations of the toxicant 3,5-dichloroρhenol.

Figure 5 shows a plot of percentage inhibition of bacteria vs. concentration of 3,5- dichlorophenol.

EXAMPLES

EXAMPLE l. AMPEROMETRIC MONITORING OF BACTERIAL RESPONSE

Introduction

As described elsewhere herein, the apparatus and methods of the present invention maybe used to monitor cultured mammalian cells in a plurality of wells of a 96-well microtitre plate, for high throughput screening applications. The present experiment demonstrates that the use of culture inserts has no problematic effect on the technique of chemically mediated amperometry, e.g. because the chemical mediator may be required to pass through a porous component of the insert, e.g. a membrane, in order to interact with the cells and thereafter pass back to the working electrode.

Because bacteria are simpler, quicker, and more cost effective to culture than mammalian cells, the response from bacteria held fn a culture insert was used as indicator of the approximate amperometric signal amplitude and profile which might be expected from mammalian cells cultured on the membrane of the insert.

The maximum speed of response and signal amplitude was also determined for a suspension of bacteria placed directly into a well, without the use of an culture insert (this would not be possible using an anchorage-dependent mammalian cell line).

The parameters with and without culture inserts were compared, in order to determine to what extent the flow dynamics and mixing within the wells are affected by the inserts. A plate shaker was used to promote mediator transfer.

Preparation of electrodelmicrotitre plate constructs

96-well microtitre plates with no well bases were provided by Life Technologies Ltd (Paisley, UK) . These plates were carefully machined into 2 x 8-well sections to which two screen-printed ceramic electrode strips (Figure 1) were fixed using either a cyanoacrylate "superglue" (Loctite) or an epoxy-based (Araldite Rapid) adhesive. The constructs were left overnight to ensure total setting of the adhesives.

Preparation of bacterial cultures

Nutrient broth for bacterial culture was prepared by dissolving 5 g of nutrient broth no. 2 (Oxoid Ltd., Basingstoke, UK) in 200 ml of ultra-pure water. This was divided into 4 ml aliquots in McCartney bottles (The Microbiological Supply Company, Toddington, UK) and autoclaved at 121 °C for 15 minutes in order to ensure sterilisation. A glycerol suspension of a non-pathogenic K12 strain of Esc ericlr a coli bacteria was obtained from the Sensor and Cryobiology Unit at Luton University. 50μl of this stock was added to a 4 ml aliquot of nutrient broth and placed in a shaking incubator at 37 ° C overnight together with another 4 ml aliquot (no bacteria). The resulting bacterial suspension was then sub-cultured by adding 200μl of the overnight culture to the 4 ml aliquot of nutrient broth and returning it to the 37 °C shaking incubator for 4 hours. This was performed in order to obtain a log phase bacterial population which would be expected to have the highest metabolic activity for testing.

Physiological saline solution (0.85% w/v) was prepared by dissolving one saline tablet (Oxoid Ltd., Basingstoke, UK) in 500 ml of ultra-pure water..

A vial of freeze-dried bacterial substrate solution (Cellsense Ltd., Cambridge, UK) was reconstituted by injecting 10 ml of the saline into the vial and agitating until all substrate was dissolved. Working strength saline substrate medium (SSm) was prepared by making the solution up to 500 ml with saline.

After 4 hours, the sub-cultured bacterial suspension was adjusted to obtain an optical density of 1.6 at 430 ran using a spectrophotometer which had been zeroed using nutrient broth as a blank. A 1 ml aliquot of the suspension was then centrifuged at 6000 rpm for 4 min to obtain a bacterial pellet. The supernatant was removed and the pellet reconstituted in 1 ml of saline. Centrifugation and resuspension was performed again twice, with the final reconstitution being in 250 μl of SSm/mediator solution.

A stock mediator solution had been prepared on the same afternoon by dissolving 5.4 mg of p rα-benzoquinone (pBQ) (Sigma-Aldrich Company Ltd., Poole, UK) in 10 ml of SSm. The SSm/mediator solution was then prepared immediately before the final bacterial resuspension by adding 100 μl of the fαrα-benzoquinone to 750 μl of SSm.

Mediated amperometric monitoring of bacterial responses

Amperometric monitoring of the E. coli bacterial cells was conducted in an electrode/microplate construct (secured on a microplate shaker at 600 rpm) coupled to an Autolab PGSTAT10 single channel electrochemical workstation (Windsor Scientific Ltd., Slough, UK). For the experiment using the culture insert, 15μl of bacterial solution was placed in an insert having a 0.2 μm anapore insert membrane, and blotted to remove the solution, leaving a layer of bacteria on the insert membrane. 15μl of SSm/mediator was added to the insert (without disrupting the bacterial layer) in order to protect the cells from dehydration. It is also important that the insert membrane is saturated in order to prevent the production of air bubbles below the insert. The insert was lowered into a well which contained 50 μl of SSm/mediator solution and amperometric monitoring initiated. Where no insert was used (i.e. a free solution of bacteria was tested), 15μl of the bacterial solution was added to 50 μl of SSm/mediator solution in the well after 180 seconds of amperometric monitoring. A control was also run using 65 μl of SSm/mediator solution (no bacteria). The results are shown in Figure 2. This experiment was repeated in order to determine the ability of the system to detect the effect of a high concentration of a known toxic chemical on the signal, directly reflecting the effect of the toxicant on the bacterial cells. The results are shown in Figure 3.

Results

The results of both experiments demonstrate that the signal profile for bacteria contained in an insert and bacteria free in solution are different. For bacteria which are free in solution in the well, a peak is seen immediately on addition of the bacteria. As these bacteria had been incubated with mediator prior to addition, there was probably a sufficient time period for the bacteria to reduce all of this mediator. Therefore, this peak is likely to be caused by re-oxidation of the mediator by the electrode. The signal then continues to climb as the remaining mediator in the well comes in contact with the bacteria and is reduced. The signal then deteriorates slowly.

The signal profiles for bacteria contained in inserts are different. These begin with a small drop in signal followed by a gradual climb. This suggests that reduced mediator is taking longer to reach the electrode. This is understandable, since mediator which had already been reduced by the bacteria through contact in the insert takes time to diffuse through the insert membrane and reach the electrode. Additionally, mediator present in the bulk solution below the insert will take even longer to generate a signal, since it must pass through the membrane to reach the bacteria, undergo reduction, and then diffuse back through the membrane for re-oxidation at the electrode surface.

Although the timescales for the assays were different, it can be seen that the final signal amplitudes for cells in bulk solution and those in inserts are not dissimilar, being in the region of 0.4-0.6 μA.

The second experiment demonstrates that the effects of a high concentration of the toxicant zinc sulphate on the assay can be clearly observed, even at these low currents. Extrapolation of the baseline (the signal produced when no bacteria were present in the well) provides almost an exact correlation with the bacterial response after several minutes of exposure to the toxicant. This suggests that the cells are no longer able to reduce the mediator, either because their electrochemical activity has been impaired or because they have ben killed by the exposure.

Conclusion

The results of Experiment 1 indicate that it is possible to perform chemically mediated amperometric monitoring of whole cells in electrode containing wells of a modified microtitre plate, and that the strength of the signal is dependent on the number of viable bacteria that are present. EXAMPLE 2: BACTERIAL RESPONSE TO INCREASING CONCENTRATION OF TOXICANT

Introduction

Example 1 demonstrates that a mediated amperometric signal maybe obtained from living bacterial cells contained in inserts suspended in electrode containing wells of a modified microtitre plate, and that the strength of the signal is dependent on the number of viable cells that are present. The present experiment demonstrates that the strength of the signal derived from a fixed concentration of bacteria is reduced in response to an increasing concentration of a toxic chemical.

Because the response of the bacteria was found to be related to the concentration of the toxicant chemical, and because different chemicals are known to have different toxicities to any given cell type, the experiment demonstrates that the amperometric assay may be used to compare the toxicities of different test agents. It also shows that the assay may also be utilised to determine the toxicity of an environmental sample compared to a corresponding unpolluted control sample.

Within this experiment, the bacteria were again contained within culture inserts as this gives an indication of the speed of mediator transfer between cells and electrodes and thus gives an indication of the toxic response time for both bacterial cells and anchorage- dependent cells (e.g. mammalian cells) which may be grown on the insert, or a component thereof, e.g on a membrane.

Preparation of electrodelmi rotitre plate constructs

96-well microtitre plates with no well bases were provided by Life Technologies Ltd (Paisley, UK) . These plates were carefully machined into 2 x 8 -well sections to which two screen-printed ceramic electrode strips were fixed using either a cyanoacrylate "superglue" (Loctite) or an epoxy-based (Araldite Rapid) adhesive. The constructs were left overnight to ensure total setting of the adhesives.

Preparation of bacterial cultures

Nutrient broth for bacterial culture was prepared by dissolving 5 g of nutrient broth no. 2 (Oxoid Ltd., Basingstoke, UK) in 200 ml of ultra-pure water. This was divided into 20 ml aliquots in 100 ml conical flasks and autoclaved at 121⁹C for 15 minutes in order to ensure sterilisation. A glycerol suspension of a non-pathogenic K12 strain of Escherichiα coli bacteria was obtained from the Sensor and Cryobiology Unit at Luton University. 50μl of the glycerol stock was added to a 20 ml aliquot of nutrient broth and placed in a shaking incubator (200 rpm) at 37⁹C overnight. The resulting bacterial suspension was then sub- cultured by adding 500μl of this overnight culture to another 20 ml aliquot of nutrient broth, and returning the resultant culture to the 37°C shaking incubator for 4 hours. This was performed in order to obtain a log phase bacterial population which would be expected to have the highest metabolic activity for testing. Physiological saline solution (0.85% w/v) was prepared by dissolving a saline tablet (Oxoid Ltd., Basingstoke, UK) in 500 ml of ultra-pure water.

A vial of freeze-dried bacterial substrate solution (Cellsense Ltd., Cambridge, UK) was reconstituted by injecting 10 ml of the saline into the vial and agitating until all substrate was dissolved. Working strength saline substrate medium (SSm) was prepared by making the solution up to 500 ml with saline.

After 4 hours, the sub-cultured bacterial suspension was adjusted to obtain an optical density of 1.6 at 430 n using a spectrophσtometer which had been zeroed using nutrient broth as a blank. 10 x 1 ml aliquots of the suspension were then centrifuged at 6000 rpm for 4 min to obtain bacterial pellets. The supematants were removed and the pellets each reconstituted in 1 ml of saline. Centrifugation and resuspension were performed again twice, with the final reconstitution of all pellets being in a total of 500 μl SSm.

A stock mediator solution of 50 mM potassium ferricyanide (Sigma- Aldrich Company Ltd., Poole, UK) in SSm was prepared on the same day. A 5 mM working mediator solution was then prepared immediately before preparing the bacterial resuspension by preparing a xlO dilution of the stock mediator solution in SSm.

A 1 g/L stock solution of the toxicant 3,5-DCP (3,5-dichlorophenol, Sigma- Aldrich Company Ltd., Poole, UK) was prepared in working mediator solution on the day of testing. This was used to prepare 25, 50 and 100 mg/L solutions of the toxicant in working mediator solution. A 0 mg/L control (working mediator solution only) was also set aside for testing.

Mediated amperometric monitoring of bacterial responses

Amperometric monitoring of E. coli bacterial cells was conducted in an electrode/microplate construct (secured on a microplate shaker at 500 rpm) coupled to a 16 channel potentiostat (Whistonbrook Technologies Ltd, Luton, UK) . The potentiostat was connected to a PC loaded with appropriate software for use with the potentiostat.

Each of the 16 wells of the electrode/microplate construct was loaded with 50 μl of 0, 25, 50 or 100 mg L of 3,5-DCP in working mediator solution (four wells of each concentration) . The inserts were then lowered into the wells so that the insert membranes were saturated. This ensured that no air was contained in the membranes which otherwise might have prevented mediator transfer. After 10 minutes, 30 μl of bacterial solution was placed in two of the inserts within each group of wells of a particular toxicant concentration.30 μl of SSm only (no bacteria) was placed in the two remaining inserts of each group. The microplate stirrer was turned on (500 rpm) and amperometric monitoring initiated. The assay was run for approximately 90 minutes. Results

Mean results of amperometric signal (current) vs. time were calculated for each pair of duplicate experiments, both for bacteria exposed to each concentration of 3,5-DCP, and for blanks (SSm only) exposed to each concentration of 3,5-DCP. The blanks had been assayed in order to account for any effect that the toxicant alone might have on the mediator signal. Mean results for bacteria exposed to each of the four concentrations of 3,5-DCP are shown in Figure 4.

Percentage inhibition of bacteria resulting from each concentration of toxicant was calculated as follows: the slope of the amperometric signal vs. time results between t=600s and the end of recording was calculated for each of the eight mean data sets (four concentrations of 3,5-DCP with bacteria [test slopes], and the same four concentrations without bacteria [blank slopes]). Each blank slope was subtracted from its corresponding test slope to give a "corrected slope". Percentage inhibition was then calculated using the following equation:

Inhibition (%) = ((1-corrected slope) /corrected slope) x 100

The results (as a plot of percentage inhibition vs. concentration of 3,5-DCP) are shown in Figure 5.

Conclusion

The results of Experiment 2 indicate that increasing the 3,5-DCP concentration resulted in an inhibition of bacterial response as measured by mediated amperometry. This demonstrates that mediated amperometry can be used to determine the effect of different toxicant concentrations on bacterial cellular response in a microplate format. Greater numbers of replicates (e.g. an entire 96 well plate) will improve the statistical significance of the results.

Claims

1. An appar tus for monitoring how a plurality of biological samples responds to one or more test agents, the apparatus comprising a plate having a plurality of wells, each of which is associated with a working electrode exposed to the well's interior, the apparatus further comprising positioning means which in respect of at least one well is capable of locating a biological sample within said well at a predetermined location relative to the well's electrode.

2. The apparatus according to Claim 1 , wherein each well is further associated with a reference electrode against which the potential of the working electrode can be measured, said reference electrode being exposed to the well's interior.

3. The apparatus according to Claim 2, wherein the reference electrodes, or one or more groups thereof, are connected together by electrical conduction means.

4. The apparatus according to any preceding claim, wherein the wells have a common base, and the electrodes are screen-printed onto said base.

5. The apparatus according to any preceding claim, wherein the plate, or at least the base of each well, is transparent.

6. The apparatus according to any preceding claim, wherein the apparatus comprises at least one insert for removably mounting a biological sample within a respective well.

7. The apparatus according to Claim 6, wherein the insert comprises a membrane on which eukaryotic cells can be grown.

8. The apparatus according to Claim 6 or Claim 7, wherein the apparatus comprises a plurality of said inserts, one for each well.

9. The apparatus according to any preceding claim, wherein the plate is a 96-well microtitre plate.

10. The apparatus according to any preceding claim, wherein the apparatus further comprises a multi-channel potentiostat for controlling the potential applied to the electrodes and/or collating the signals obtained therefrom.

11. A method of monitoring how a plurality of biological samples responds to one or more test agents, the method comprising contacting each sample with an electron transfer mediator and a test agent in the presence of a working electrode, and monitoring in parallel the transfer of electrons from the samples to their respective electrodes by the mediators, the method further comprising positioning each biological sample at a predetermined location with respect to its respective working electrode.

12. The metho ^r according to Claim 11, wherein the method further comprises contacting at least one additional biological sample with an electron transfer mediator in the presence of a working electrode but in the absence of test agent, monitoring the transfer of electrons from said sample to its respective electrode by the mediator, and comparing that transfer with the transfer recorded with an identical biological sample in the presence of a test agent.

13. The method according to Claim 11 or Claim 12, which comprises the steps of: (i) contacting at least one biological sample with an electron transfer mediator and a test agent in the presence of a workihg electrode; (ii) monitoring the transfer of electrons from the sample to the electrode by the mediator, thereby to deteπnine a test activity of the sample; (iii) removing the test agent from the sample; and (iv) monitoring the transfer of electrons from the sample to the electrode by the mediator after the test agent has been removed.

14. The method according to Claim 13, wherein the sample is monitored periodically in step (iv), and the biological sample is removed from the apparatus in which the ilectrode is located, and returned to an incubator, in between measurements of electron ϊansfer.

15. The method according to any one of Claims 11-14, wherein the biological samples in said plurality are the same, and the test agents are different from one another.

16. The method according to Claim 11-15, wherein the plurality of test agents comprises the same substance at different concentrations.

17. The method according to any one of Claims 11-16, wherein the test agents are selected from the group consisting of compounds generated by combinatorial chemistry, environmental samples, biological samples, and waste materials derived from an industrial process.

18. The method according to any one of Claims 11-17, wherein the biological samples are selected from the group consisting of eukaryotic cells, prokaryotic cells, cellular organelles, cellular membranes, and mixtures thereof.

19. The method according to any one of Claims 11-18, wherein the method is carried out using an apparatus according to any one of Claims 1-10.

20. The method according to any one of Claims 11-18, wherein the method is carried out using an apparatus comprising a plate having a plurality of sampling areas, each of which is associated with a working electrode, wherein a biological sample and a test agent are located onto each sampling area, over the electrode, and held in place by surface tension.

21. An apparatus or method substantially as herein described and/or as shown in any one of the accompanying drawings.