The present invention relates to an apparatus for determining the concentration of at least one predetermined chemical species at a plurality of positions in a test region, and relates particularly, but not exclusively, to an apparatus for determining the concentration of sodium ions .

In biological monitoring applications, such as the observation of metabolic and signalling processes of in vitro cell cultures, various mechanisms of interest occur as a result of sodium ion activity. Valuable information can therefore be obtained from an indication of the concentration of sodium ions at various locations across the cell culture. This enables various effects, such as ion specific effects of drugs/toxins, to be studied, as well as observing the regulation and manipulation of extracellular concentrations by specific cells such as kidney cells, and spatiotemporal imaging of neural activity and communication processes by means of ionic pathways.

EP 1278064 discloses a high density microelectrode arrangement in which electrophysiological activity of in vitro cultured electrogenic cells is monitored by monitoring the voltage generated by the cells at a plurality of locations. The microelectrode device is manufactured using semiconductor manufacturing techniques. However, this arrangement suffers from the drawback that the voltage signals measured at the various locations are caused by a number of. factors, including sodium ion activity, and the measured voltage signals are therefore the signal caused by all of the electroactive species present. It is therefore not possible to monitor the concentration of a single chemical species.

Preferred embodiments of the present invention seek to overcome one or more of the above disadvantages of the prior art.

According to the present invention, there is provided an apparatus for determining the concentration of at least one predetermined chemical species at a plurality of locations in a test region, the apparatus comprising:

an array of sensors, wherein each said sensor comprises a respective membrane for allowing passage of a said predetermined chemical species, and a respective electrode for providing a respective first electrical signal dependent on the concentration of said chemical species in the vicinity of said membrane; and

signal processing means for processing a plurality of said first electrical signals to provide a plurality of second electrical signals dependent on the concentration of said chemical species in the vicinity of a plurality of said sensors.

By providing an array of sensors having membranes allowing passage of said chemical species, and signal processing means providing a plurality of second electrical signals dependent on the concentration of said chemical species in the vicinity of a plurality of said sensors, this provides the advantage of enabling the concentration of a predetermined chemical species to be monitored and therefore provides more detailed information of processing occurring in the entity being monitored, such as a cell culture. In addition, the use of membrane type sensors enables semiconductor manufacturing techniques to be used, as a result of which the process of manufacturing the apparatus can be simplified and its cost reduced. The further advantage is provided that the apparatus is under certain circumstances easier to use than conventional apparatus for this purpose, because chemical species in liquid form need not be permanently located In the apparatus .

A plurality of said membranes may include at least one ionophore.

A plurality of said sensors may include a respective conductive polymer layer arranged between the membrane and the electrode of said sensor.

This provides the advantage of improving electrical conductivity of the sensor, as a result of which the signal to noise ratio of the first electrical signals can be improved.

The array of first sensors may be provided on a substrate .

This provides the advantage of enabling semiconductor manufacturing techniques to be used, and to provide an apparatus which is easy to use. The signal processing means may comprise amplifier means for receiving a plurality of said first electrical signals .

This provides the advantage of reducing the effect of processing of the signals on the signals themselves as a result of the first electrical signals generating small amounts of current.

The signal processing means may include multiplexer means and analogue to digital converter means for receiving said first electrical signals and outputting said second electrical signals as serial data.

This provides the advantage of simplifying the construction of the apparatus by reducing the amount of electrical wiring or connections which needs to have access to the sensors, while at the same time enabling the apparatus to conveniently interface with a computer for receiving output data from the apparatus and/or supplying programming data to the apparatus.

The multiplexer means may be adapted to receive said first electrical signals from the sensors in adjustable sequences.

This provides the advantage of enabling the process of addressing the first electrical signals from the sensors to be adapted to the particular application, for example to enable the apparatus to automatically concentrate on regions of interest and/or increased activity. The signal processing means may be provided on said substrate as part of an integrated circuit device.

This provides the advantage of simplifying construction of the apparatus by enabling semiconductor manufacturing techniques to be used, thereby reducing the cost of manufacture of the apparatus, while also providing an apparatus which is easier to use.

The sensitivity of at least one said sensor may be adjustable.

This provides the advantage of enabling more sensitive calibration of the apparatus.

Preferred embodiments of the invention will now be described, by way of example only and not in any limitative sense, with reference to the accompanying drawings, in which: -

Figure 1 is a schematic view of a cell culture imaging and monitoring apparatus embodying the present invention;

Figure 2 is a schematic view of the sensor array and signal processing circuit of the apparatus shown in Figure 1;

Figure 3 is a plan view of a first embodiment of a sensor array of the apparatus of Figure 1;

Figure 4 is a cross sectional side view of the sensor array of Figure 3; Figure 5 is a detailed exploded view of a single sensor of the sensor array of Figures 3 and 4;

Figure 6 is a schematic view of the signal processing circuit of the apparatus of Figures 1 and 2;

Figure 7 is a circuit diagram of a buffer amplifier used in the signal processing circuit of Figure 6;

Figure 8 is a graph of the electrical output of the apparatus of Figures 1 and 2 using the sensor array of Figures 3 and 4 ;

Figure 9 is a graph of the variation of the electrical output of Figure 8 with concentration;

Figure 10 shows the time taken to detect an electrical output when a grain of sodium chloride is placed on the sensor array of Figures 3 and 4, when the sensor array is placed in water and sodium chloride solutions of various concentrations;

Figure 1OA is a schematic representation of the positioning of the sodium chloride grain providing the data of Figure 10;

Figure 11 is a graph of the variation of electrical output with time of the sensor array of Figures 3 and 4;

Figure 12 is a plan view of a sensor array of a second embodiment of the present invention; Figure 13 is a detailed view of the construction of a single sensor of the array of Figure 12; and

Figure 14 is a schematic cross sectional view of a single sensor of a sensor array of a third embodiment of the present invention showing an input transistor of the buffer amplifier of Figure 7.

Referring to Figure I ₁ . a cell culture imaging and monitoring apparatus 2 for providing an image of sodium concentration at various locations within an in vitro cell culture 4 comprises an array 6 of sodium concentration sensors embodying the present invention. Each sensor 8 (Figure 3) has a membrane 10 (Figure 5) sensitive to sodium ions, and a conventional CCD camera 12 and a microscope 14 for generating an image 16 of the cell culture 4, the outputs 18, 20 of which can be connected to a suitable computer 22 for providing a colour image 24 of sodium ion activity overlaid on the conventional CCD image 16 of the cell culture 4.

Referring to Figures 3 to 5, the array 6 comprises a linear arrangement of sodium ion selective sensors 8 arranged on a high electrical resistance quartz substrate 26. Each sensor 8 includes a gold/platinum electrode 28 bound to the substrate 26 by means of a titanium/chromium adhesion layer 30, and a sodium ion sensitive membrane 10 containing suitable ionophore mounted by means of a conductive polymer layer 32 to the gold/platinum electrode 28, the membrane 10 and conductive polymer layer 32 forming a stack located in an aperture 34 in an SU-8 photoresist layer 36, so that adjacent sensors 8 are electrically insulated from each other by the SU-8 layer 36 between adjacent membranes 10 and conductive polymer layers 32.

The membrane 10 can be formed from a variety of materials known to persons skilled in the art, such as 1.3% ionophore in the form of 4-tert-butylcalix [4 ] arene-o, o' , o' ' , o ^f ' ' -tetraacetic acid tetraethyl ester, 65.5% plasticiser in the form of 2-nitrophenyl octyl ether, 32.8% PVC polymer, and 0.5% ion exchanger in the form of potassium tetrakis (4-chlorophenyl borate) . Techniques for producing the membrane 10 are described in more detail in Bakker, E et al, Carrier-Based Ion-Selective Electrodes and Bulk Optodes. 1. General Characteristics. Chem. Rev. 1997, 97, 3083-3132. The sensor 8 operates such that a charge builds up in the membrane 10 which in turn induces via the conductive polymer layer 32 an electrical potential in the gold/platinum electrode 28 which is dependent upon the concentration of sodium ions in the vicinity of the membrane 10.

Referring to Figure 2, the voltages on the gold/platinum electrodes 28 of the array 6 are detected and are supplied to an array 38 (Figure 6) of buffer amplifiers 40 (Figure 7) which reproduce the voltage signals from the detector array 6 but with sufficient current to enable the voltage signals to be processed without significantly changing the voltage signals being detected, which would otherwise be the case because of the very small currents generated in the electrodes 28. The signals output from the electrodes 28 of the array 6 are fed via the array 38 of buffer amplifiers 40 to a multiplexer 42, which converts the parallel signals from the buffer amplifiers 40 to a serial signal, controlled by an array select circuit 44 and then fed to an analogue to digital converter 46 to output serial digital signals representing the concentration of sodium ions at the individual sensors 8. The signals are then output via output buffer amplifiers and bond pads 48 to the computer 22.

The array select circuit 44 can be controlled by means of a counter 50 sequentially scanning the array 6 of sensors 8, or a memory 52 can store a suitable scanning program which causes a state machine 54 to control the array selector 44 in a manner other than scanning the row of sensor sequentially, for example in such a way as to concentrate scanning in an area of interest or an area of increased activity. The state machine 54 can also be controlled from the computer 22, and a suitable software module 56 (Figure 6) can adjust the sensitivity of the individual sensors 8 to calibrate the array 6.

Referring to Figure 7, an individual buffer amplifier 40 of the buffer amplifier array 38 receives at an input terminal 58 of a current mirror amplifier the voltage signal from the gold/platinum electrode 28 from a sensor 8 and outputs at an output terminal 60 a voltage signal corresponding to the input signal but at higher current. The input signal input at input terminal 58 is fed to the gate of transistor Ml and modulates the channel current of transistor Ml. This in turn influences the gate voltage of transistor M8 because the gate of M8 is connected to the source of transistor M3. This in turn influences the channel current (and thereby the gate voltage) of transistor M9. Since the gate of transistor M9 is connected to that of transistor M7, variations in the gate voltage of transistor M9 also influence the gate voltage of M7, and the channel current of M7, which in turn influences voltage at the drain of Ml. As a result, the voltage signal at output terminal 60 follows that at input terminal 58, but has significantly higher current.

Figure 8 shows the output signals of the individual sensors 8 of the array 6 of Figures 3 and 4 when incorporated into the apparatus 2 of Figure 1 as the concentration of sodium ions in the solution being analysed is varied. The variation of the output signals with concentration is shown in Figure 9.

Referring to Figure 10, the behaviour of the apparatus 2 of Figure 1 is shown when a grain 62 of sodium chloride is placed on the sensor array 6 and allowed to dissolve. Initially, a signal is detected at sensor no. 4, i.e. the nearest sensor 8 to the grain 62 of sodium chloride. The signal is then subsequently detected at adjacent sensors nos. 3 and 5, i.e. the next nearest sensors 8 to the grain 62, and then subsequently at the sensors 8 located further away. The variation of the voltage detected at the electrodes 28 of the individual sensors 8 over time is shown in Figure 11.

Figure 12 shows a plan view of a two dimensional sensor array 70 of a second embodiment of the present invention. The sensors 72 of the array 70 are formed on a substrate 74 by means of photo-patterning and are arranged so that their active tips cover an array of locations in the area of interest. Each of the sensors 72 is connected by means of interconnect wires 76 separated by SU-8 photoresist 78 to bond pads 80 located at the edge 82 of the substrate 74. Various techniques for photo-patterning -lithe membrane of the sensors 72 are described in greater detail in Heng, L, Y et al. Assessing a photocured self- plasticised acrylic membrane recipe for Na+ and K+ ion selective electrodes. Analystica Chimica Acta 443 (2001) 25-40. One favoured material for the membrane is /i-butyl acrylate, 2-hexanedioldiacrylate, 2, 2-dimethoxy-2- phenylacetophenone, a sodium ionophore , sodium tetrakis (trifluoromethylphenyl borate) .

Referring to Figures 13 and 14, a sensor array 80 of a third embodiment of the invention is shown, in which the sensors 82 and signal processing circuit are formed as a single solid state device (ASIC) . Referring to Figure 14, an input transistor Ml of the buffer amplifier circuit 40 (Figure 7) has source 84 and drain 86 regions formed in a silicon substrate 88, and a gate electrode 90 separated from the source 84 and drain 86 by a suitable insulating layer 92.

By means of semiconductor fabrication techniques which will be familiar to persons skilled in the art, a conductive via 96 is formed on the gate 90 in a gap in an insulating layer 98. A metal layer is formed on the insulating layer 98 and then partly etched away to form an input conductor 100 connected to the source 84 of input transistor Ml and metal layers 102, 104 are connected to the gate 90 and drain 86.

A dielectric insulation layer 106 is formed over the other two metal layers 100, 102, 104, and then partly etched away to enable the conductive via 96 to be extended and a conductive via 108 to be connected to the metal layer 104 to enable connection to the drain 86 of the transistor Ml. A metal layer is then formed on top of the insulating layer 106, and then partly etched away to provide a metal layer 110 on top of the gate 90 and a metal layer 112 connected to the drain 86 forming interconnection connecting the transistor Ml to other parts of the amplifier circuit 40. A further layer of the conductive via 96 passes though a further insulating layer 114, and is covered by a top metal aluminium layer 116. A titanium/chromium seed layer 118 assists in bonding the gold/platinum electrode 120 of the sensor 82 to the top metal layer 116, and the device is sealed by means of passivation layers 122, 124. The conductive polymer layer 126 and ion selective membrane layer 128 are then provided on top of the gold/platinum electrode 120.

It will be appreciated by persons skilled in the art that the above embodiments have been described by way of example only, and not in any limitative sense, and that various alterations and modifications are possible without departure from the scope of the invention as defined by the appended claims. For example, although the present embodiments have been described with reference to the detection of sodium ions, the apparatus can be arranged to detect various chemical species and/or more than one chemical species.