GB2436145A

GB2436145A - A system for monitoring properties of a single cell

Info

Publication number: GB2436145A
Application number: GB0605243A
Authority: GB
Inventors: Christer Spegel; Simon Pedersen; Ras Kaas Vestergaard; Rafael Taboryski
Original assignee: Sophion Bioscience AS
Current assignee: Sophion Bioscience AS
Priority date: 2006-03-16
Filing date: 2006-03-16
Publication date: 2007-09-19
Also published as: GB0605243D0; WO2007104565A1

Abstract

A system for determining and/or monitoring properties in a single particle, the system comprising an assembly comprising a front side structure, a rear side structure and a substrate, the substrate comprising: ```a first upper surface part and a first lower surface part for holding the single particle, a first site comprising a passage in the substrate, a first end of the passage being in contact with a first domain at the first upper surface part of the substrate and a second end of the passage being in contact with a second domain at the first lower surface of the substrate, the first end of the passage defining an aperture adapted to receive the single particle; ```at least one electrode positioned on the first upper surface part of the substrate; ```a reference electrode in electrical contact with the first domain; ```at least one working electrode, situated on a surface of the substrate, and in ```electrical contact with the first domain; ```the first end of the passage being adapted to locate the single particle held at the first site, the particle thereby separating the first domain of the site from the second domain; ```the at least one electrode on the upper surface part being in close proximity to the first end of the passage; ```the system further comprising: ```locating means for locating the single particle at the aperture; ```and at least one interface for interfacing the substrate with an analysing system.

Description

A SYSTEM

This invention relates to a system for determining and/or monitoring properties in a smgle particle, and particularly to a system for determining and/or monitoring properties in a single biological cell.

The charactensation of single cells is important since all cells differ in their composition and function. The understanding of these differences in composition and the consequent difference in function is very important in clinical science.

There is an increasing interest in the study of single cells, i.e. single cell characterisation. To be able to study biology on the smgle cell level, delicate handling systems are required.

Previously it has not been possible to study and make use of processes related to single cells, due to the lack of proper tools. However, the recent breakthrough in micro-and nano-technology has made single cell analysis and manipulation possible.

A typical example of the advantages gained from single cell analysis is regular blood testing, where thousands of cells are homogenized to provide sufficient amounts of analytes for quantification. At early stages of e.g. carcinogenesis, it is possible that only a few cells might carry the specific marker for the disease.

These cells may therefore be masked by the complex sample matrix. If cells are examined individually, the chance of recognizing abnormalities is much higher.

Single cell analysis can also be utilized for testing and/or screening of pharmaceutical agents and toxins, increasing the throughput compared to classical multi cellular assays. The development phase of new drugs is highly associated with expensive and ethically controversial animal experiments. Single cell screening systems and methods can, in the future, to at least some extent replace animal experiments.

During the last decade several techniques have been developed and optimized for single cell studies. Among these techniques are patch clamp techniques, utilized for the investigation of ion-channels [0. P. Harnill, et al., Pflcugers Arch, 391, 1981, 85-100]; optical techniques such as laser confocal microscopy, utilized to study and localize bioactive components in single cells [S. Maiti, et al., Science, 275, 1 997, 530-532]; and microelectrodes, for amperometric determination of single cell catecholamine exocytosis [R. H. Chow, et al., Nature, 356, 1992, 60-63].

Huang and Rubinsky have developed a flow through micro-electroporation chip for single cell electroporation [Y. Huang and B. Ruhinsky, Sensors and Actuators A, 2003, 104 (3), 205-2 12]. The cells are captured on a micro-hole by a pressure difference and can then be individually electroporated. However, the system is not constructed to perform electrochernical studies on the captured cell.

The most common method, however, for single cell manipulation is the utilization of patch-clamp pipettes or other types of capillanes, which operate in a similar manner as the presented invention.

Recent progresses in the development of microchip based high-throughput patch clamp systems has lead to new cell positioning devices, which is an inherent part of the patch clamp methodology.

WO 02/29402, Sophion Bioscience A/S, discloses an apparatus for a high throughput system for determining and/or monitoring electrophysiological properties of ion channels of ion channel-containing membranes. The substrate contains no electrodes for amperometry, dielectric spectroscopy, electrorotation, electroporation, etc., thus, measurements are limited to those of ion-channels and ion-channel containing membranes.

US 6,315,940 BI, Nisch et al. discloses a microelement device composed from microelectrodes adapted for making contact to cells. The cells are directed onto the cells by hydrodynamic force or negatl\Je pressure. The method is thus limited to those where the electrodes arc in contact with the cells and measurements of potentials associated with the whole cell, i.e not electrochemical potentials and currents.

US 5,187,096, Giaever et al., discloses an apparatus and method for the monitoring of cell-substrate impedance. However, the apparatus contains no means for smgl e parti ci c/cell positioning.

WO 2001005511, Lock and Pething, discloses a system composed of electrodes for the study of AC electrokinetic Dielectrophoresis (DEP, travelling wave dielectrophoresis (TWD) or electrorotation. The system does not, however, contain any feature for single particle positioning.

WO 9316383, Parton et al., discloses a method for the detection of various substances upon their interaction with surface modified particles. There is no feature concerning the positioning of the particle, described in the application.

WO 2002031171, Haas et al., discloses an electroporation method composed of a container. The container is placed in close proximity to the cell, thus, no method for the precise positioning of the cell exists.

WO 9924110, Orwar et al., discloses a method for the pernieabilization of single cells utilizing individually controlled microelectrodes. However, no system for the industrially feasible positioning of the single cell is described.

The analysis of single cell content is fundamental for the understanding of several functions such as; cell conmiunication, chemical storage, protein expression, responses to external stimuli, etc. According to a first aspect of the present invention there is provided a system for determining and/or monitoring properties in a single particle, the system comprising an assembly comprising a front side structure, a rear side structure and a substrate, the substrate comprising: a first upper surface part and a first lower surface part for holding the single particles, a first site comprising a passage in the substrate, a first end of the s passage bemg in contact with a first dornam at the first upper surface part of the substrate and a second end of the passage being in contact with a second domain at the first lower surface of the substrate, the first end of the passage defining an aperture adapted to receive the single particle at least one electrode positioned on the first upper surface part of the o substrate; a reference electrode in electrical contact with the first domain; at least one working electrode, situated on a surface of the substrate, and in electrical contact with the first domain; the first end of the passage being adapted to locate the single particle held at the first site, the particle thereby separating the first domain from the second domain; the at least one electrode on the first upper surface part being in close proximity to the first end of the passage; the system further comprising: locating means for locating the single particle at the aperture; and at least one interface for interfacing the substrate with an analysing system.

Most known methods utilizing electrodes for the characterization of single cells are associated with high demands on equipment for the positioning of both the cell and the electrodes. By combining both the positioning of the cell and the electrodes for stimulation/recording on a single substrate, these high demands can be reduced.

ln a preferred embodiment, the at least one electrode on the first upper surface part is less than 100 im from the first end of the passage.

Advantageously, the at least one electrode on the first upper surface part is less than 100 irn from the first end of the passage.

Preferably, the at least one electrode on the first upper surface part is less than 10 S tm from the first end of the passage.

Preferably, the particle is any oh1ect with dimensions within the range 0.5 to 500 tm. More preferably the particle comprises a lipid containing vesicle such as a biological cell.

Advantageously, the interface comprises an electrical contact for forming an electrical connection between the system and the analysing system.

Advantageously, the assembly comprises a contact receiving portion, and the electrical contact comprises a contact portion that is positioned within the contact receiving portion, extends from the system, and is in electrical contact with the Advantageously, the system comprises a plurality of analysing systems.

An important feature of the present invention as claimed is that the system allows the measurement of properties such electrochemical and electrical properties, of an object such as a biological cell to be carried out, and then to be analysed using one or more different analysing systems. The one or more different analysing systems can conveniently interface with the system through the at least one interface which allows the one or more analysing systems to interface with the substrate on which the particle is located. Any number of different types of analyse may be carried out on the particle once it has been accurately positioned in the system according to the present invention. Some of the various analytical methods that can be used on a particle positioned on a system according to the present invention are set out below.

All the methods fall within the general definition of electro-chernical methods.

Electrochemistry is the study of electromc and electrical aspects of chemical reactions.

The system according to the present invention allows the measurement of properties of a cell by means of electro-chemical methods, which differ from patch clamping techniques in relation to the measurement of the ion content of a cell, in patch clamping techniques, the ion content of a cell is measured through the membrane whereas with electro-chemical methods, electronic and electrical properties of a cell are determined by analysing chemical excretion from the cell.

A great variety of electrochernical methods exist. However, only a few have been utilised for the study of single cells, the most common being amperometry and cyclic voltarnmetiy.

The most common method, based on the application of DC voltage and the observation of a generated current, is amperometry. The use of constant voltage amperometry for single cell characterization has mainly been used for the detection of neurotransrnitter release and for the general characterization of cell functionality, utilizing the scanning electrochemical microscope and carbon fibre microelectrodes. Processes associated with single biological cells, that have been studied using amperometric and biosensor methods are, among others, exocytosis of neurotransmitters [R. H. Chow, et al., Nature, 356, 1992, 60-63], drug efflux [H. Lu and M. Gratzl, Analytical Chemistry, 71, 1999, 2821-2830], oxygen consumption [T. Kaya, et al., Biosensors and Bioelectronics, 18 (11), 2003, 13791383], secretion of insulin [R. T. Kennedy, J. Am. Chem. Soc., 118 (7), 1996, 1795-1796], nitric oxide [T. Malinski, Z. Taha, Nature, 358, 1992, 676-678], superoxide radicals [K. Tanaka, el al., Bioelectrochemistry and Bioenergetics, 41 (2), 1996, 201-203] and cellular reduction of various electrochemically active mediators [W. Feng, et al., Analytical Chemistry, 75(16), 2003, 4148-4154]. etc. Another technique suitable for single cell electrochemical studies is cyclic voltammetry (CV). In CV the applied potential is swept in cycles between two voltages, resultmg in a current versus potential trace.

Cyclic voltammetry (CV) has been investigated as a tool for the characterization of secretory events from single biological cells, e.g. Wightman et al. utilized fast scan CV (FSCV) to distinguish between adrenaline and noradrenaline at the single cell level [D. J. Michael and R. M. Wightrnan, Journal of Pharmaceutical and Biomedical Analysis, 19(1-2), 1999, 33-46].

The above descnbed methods are, however, not associated with any industrially feasible system that incorporates single cell positioning.

In potentiometric experiments, the current is kept at zero and the potential is determined as a function of the concentrations of analytes. Potentiornetric methods can be used to study the perturbation of a redox system, that is, changes in the ratio of oxidized and reduced species. Changes in the concentration ratio will result in a change in the measured potential according to Nern's equation: E = E nF cR Where E is the determined potential, which is related to the standard potential, E , of the redox couple and the concentration of the oxidized species, C0, and the reduced species, CR. R is the gas constant, 8.3 1441 J moF' K', T is the temperature in Kelvin, n is the number of electrons participating in the studied reaction and F is the faraday constant, 9.64846 x I O C/equivalent.

A few potentiometric techniques are available, such as open circuit potential versus time techniques, chronopotentiometry and double potential step chronopotentiometry.

Rahmowitz ci a] p. D. Rahmowitz, ci al., J. Am. Chern. Soc., 1998, 120, 2464- 2473] have utilized the lipophilic redox couple menadione/menadiol to probe intracellular redox enzyme activity in living cells by potentiometric measurements. The intracellular enzyme activity was monitored as changes in the redoxpotential, E, of the redox couple ferri-/ferrocyanide, which accepted the electrons from menadiol. The experiments where conducted Ofl approximately I 0 L-929 cells in a modified microphysiometer.

An electrochen-ucal biosensor is a self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochernical transduction element.

{D. R. Thévenot et al., Electrochernical Biosensors: Recommended Defimtions and Classification (Technical Report). Pure and Applied Chemistry, 1999, 71(12), 2333-2348]. The biological recognition element is, in most cases, an enzyme, but can also be a whole living cell or a part of a cell, i.e. macromolecules present in their original biological environment, isolated macromolecules or engineered macromolecules.

Enzyme based biosensors are classified into the 1st, 2nd and 3rd generations. The function of biosensors of the 1st generation is based on detection of either the consumption of a cosubstrate (e.g. oxygen) [S. J. Updike et al., Nature, 1967, 214 (92), 986-988] or the enzymatic generation of a product (e.g. H202, as in the case of many oxidases) [G.G. Guilbault and G.J. Lubrano, Analytica Chimica Acta,.

1972, 60 (1), 254-256]. In the 2nd generation, a mediator (e.g. fenocene and quinones), able to transport electrons between the enzyme and the electrochemical transducer, is introduced (i.e. mediated electron transfer, MET) [A.R.

Vijayakurnar et al., Analytica Chimica Acta, 1996, 327 (3), 223-234]. Biosensors of the 3rd generation do not require any co- substrate or mediator, since the electrons are transferred directly from the active site of the enzyme to the electrode (direct electron transfer, DET) [L. Gorton et al., Analytica Chirnica Acta, 1999, 400 (1-3), 91-108].

JP 96-214946, Niwa et al., discloses an online hiosensor for single cell studies.

The sensor system comprises a sampling capillary, positioned near a single cell and a pump to deliver solution, captured near the cell, to a flow cell hiosensor, The sampling capillary is positioned near a single cell, present in a cell culture, hence, no method for single cell captunng is presented Also, dynamic measurements on the single cell are not possible, due to the distance between the cell and the sensing element.

Capacitive hiosensors can be formed by immobilizing recognition elements on an o electrode. When an analyte binds to the recognition element, or in some way changes the characteristics of the recognition element, changes in the dielectric properties of the sensing layer are detected. One common application of capacitive biosensors IS the detection and quantification of heavy metals, which, when they bind to an immobilized protein, change three dimensional conformation of the protein, causing a change in capacitance.

Dielectric properties relate to the ability of matenals to polarise under the influence of an electromagnetic field. The degree to which the material is polarized depends on the structure and molecular properties. Dielectric measurements can thus provide information of these characteristics. Dielectric properties are determined utilizing a technique known as dielectric spectroscopy.

Dielectric measurements are based on the application of an AC current or voltage to a sample of material, and the measurement of the amplitude and phase shift of the resulting voltage or current. Impedance and admittance can then be calculated from the ratio of the current to the voltage and the ratio of the voltage to the current, respectively. From the impedance and the admittance, various parameters can be calculated, characterizing the studied material, e.g. penrnttivlty, susceptibility, resistivity and conductivity.

Electrorotation is a type of dielectric spectroscopy that can be utilized for particle characterization, based on the electric polarizahility of the particles.

A non-uniform field can induce a torque on a particle, causing it to rotate The first time this principle was utilized for the induction of cellular spin was in 1982 [W. M. Arnold and U Zimmerman, Z. Naturforsch. 1982, 37c, 908-915] and {M.

Mischel et al., J. Biol. Phys. 1982, 10, 223-226]. A generated electrical field giving a 900 phase difference between adiacent electrodes, causes a particle to rotate. The particle can rotate with or against the rotating current, depending on the properties of the particle.

Electrorotation can be used to study dynamic changes in the dielectric properties and the morphology of small objects, e.g. cells, since it provides a sensitive method for monitoring the physiological state of cells and determining their sensitivity to exposure to chemicals and other agents.

Electrorotation can also be utilized as a method for quantification of selected analytes. For example, receptor-modified particles will change their electrorotation properties upon interaction with a target substance. Examples of interaction that can be studied are; antibody-antigen interaction, covalent and non- covalent interactions, adsorption interactions, absorption interactions, surface-active interactions, etc. Electroporation is a process in which a current pulse is passed through a cell. The process results in the formation of pores in the membrane of the cell, making it possible for extracellular, water soluble, compounds to enter the cytosolic compartment of the cell. Electroporation is very important in biotechnology and medicine, where it is important to have the tools for the introduction of, membrane impermeable compounds, into living cells.

In 1978 Neher, Sakmann and Steinbeck realized that the ion-channel activity in single cells could be monitored through the measurement of current flow between an electrode situated inside a solution filled capillary, pressed against the surface of the cell, and an electrode present in a cell bath solution. The quality of these expenments was, however, not fully satisfactory, due to a high level of noise in a registered signal. The noise was ascribed to the low resistance of the cell-pipette seal.

A few years later it was discovered that a higher resistance could be obtained by fire polishing the pipette, and assisting the cell-pipette seal by applying a negative pressure to the intenor of the pipette. This method increased the resistance of the cell-pipette seal from below 5() MQ to more than 1 GQ. Since the noise is inversely related to the seal resistance, a huge increase in the signal to noise ratio was achieved.

A vanety of microscopes provide the required sensitivity for single micro-particle characterization. The most common approaches for the study of single cells are fluorescence microscopy and classical bright-field imaging techniques.

A few endogenous cellular molecules, inheriting natural fluorescence, such as NAD(P)H, can be directly observed utilizing a fluorescence microscope.

However, the most common approach for the study of dynamic cellular processes is by green fluorescence protein (GFP) technology [J. Lippincott-Schwarz and G. H. Patterson, Science, 300, 2003, 87-91] as well as specific molecular probes, which can he inserted into cells by microinjection, diffusion or electroporation. A deeper understanding of optical techniques for single cell monitoring can be found in "Light Microscopy Techniques for Live Cell imaging" {D. J. Stephens and V. J. Allan, Science, 300, 2003, 82-86].

Techniques based on bright-field imaging are mostly utilized for studies concerning drastic changes in cell morphology, such as apoptosis (programmed cell death) and mitosis (cell division).

By means of the present invention, it is possible to combine two or more of the techniques described hereinabove when analysing a single cell that has been positioned using the system according to the present invention. By combining two or more techniques, the amount of information gained from single cell studies can he significantly increased.

By combining electrophysiological methods, i.e., patch clamp techniques, and electrochernical methods, detailed information about the secretory processes in living cells can he revealed. 2'

Electrophysiological methods can detect changes in the surface area of the cell and, electrochemical methods can reveal the amplitude and time resolution of the secretory events.

By means of the present invention, therefore, a substrate is provided Ofl which a single particle can he accurately located. The invention also allows for the substrate on which the particle is positioned to interface with one or more analysing systems in order that the single particle can be accurately analysed.

In addition to the above mentioned applications for amperometric sensing in relation to single cells, several other analytical applications exist for the detection of, among others, oxygen, nitric oxide, etc. The present invention is adapted to all types of amperometric recordings, that is methods in which the current is proportional to the concentration of the species generating the current.

Advantageously, the first domain comprises a first canal and the second domain comprises a second canal.

Preferably the first canal has a first input and a first output, and the second canal has a second input and second output.

The first and second canals may be formed integrally with the substrate, or may be formed as separate structures. If the canals are formed as separate structures they are then interfaced with the substrate.

If the canals are formed on an external structure which is then interfaced and aligned with the substrate, a top section of the canals preferably comprises a transparent or semi-transparent material. The transparent or semi-transparent rnatenal allows for visual inspection of the positiomng of the particle on the substrate, it also allows for visual inspection of the interfacing of the substrate with optical manipulation, observation and analytical tools such as microscopes and photosensitive detectors.

Advantageously, the locating means comprises pressure means for applying a pressure gradient along the passage such that the pressure at the first end of the passage is greater than the pressure at the second end of the passage.

A particle is thus positioned accurately at the aperture using hydrodynamic forces.

Advantageously, the locating means comprises a pump which pump is connectable to the second canal Advantageously, the system further compnses a detector for detecting when the single particle is positioned at the aperture.

The detector may comprises a transparent part formed in the front side structure positioned above the substrate. The transparent part allows visual inspection and monitoring of the positioning of the single particle.

Alternatively, the detector comprises a system for measuring electrical resistance through the passage. This is achieved by applying a potential difference across the substrate passage, and simultaneously monitonng the cunent flow through the passage.

if the particle is appropriately positioned at the aperture, resistance is increased leading to a lower cunent flow The at least one electrode on the first upper surface part of the substrate may be either a conducting electrode or a semi-conducting electrode.

By introducing conducting or semiconducting electrodes for single particle charactenzation on a substrate comprismg means for single particle positioning.

several analytical techniques can be employed, among others; classical electrochemical methods such as (i) amperometry and (ii) potentiometry, as well as methods based on modified electrodes such as (iii) biosensors. Also methods for the determination of electrical properties of particles such as (iv) dielectric spectroscopy and (v) electrorotation are possible on the proposed substrate.

Mampulative/analytical techniques such as (vi) electroporation and electrophysiological techniques such as (vii) patch clamp are also possible.

io Finally, the platform can easily be interfaced with external analytical tools such as (viii) microscopes.

The invention will now be further described by way of example only with reference to the accompanying drawings in which: Figure 1 is cross sectional representation of a first embodiment of a system according to the present invention; Figures 2A to 2J are schematic representations of embodiments of substrates suitable for inclusion in the system of Figure 1; Figures 3A to 3C are schematic representations of reference electrode configurations suitable for forming part of the system of Figure 1; Figures 4A to 4F are optical images of various configurations of working electrode suitable for forming part of the system of Figure 1; Figure 5 is a schematic representation showing the system of Figure 1 coimected to analysis systems; Figure 6A is an optical image of a single HEK cell positioned on the an electrode configuration presented in Figure 4C.

Figure 6B is a graphical representation of the electrode configuration in Figure 6A.

Figure 6C is a graphical representation showing the increase in resistance over a front to back passage when the single HEK cell is positioned over an aperture forming part of the system of Figure 1; Figures 7A and 7B are graphical representations showing an ampcrometnc set up forming part of a two and three electrode configuration respectively forming part of the system of Figure 1; Figure 8A is a graphical representation of an amperometric response curved for ferricyanide, obtained using the electrode configuration of Figure 8B; Figure 8B is an optical image of the working electrode configuration utilized for the experiments presented in Figure 8A.

Figure 9A is a graphical representation of a cyclic voltamm.ogram for pure buffer and 10 mM ferricynanide, obtamed using the electrode configuration shown in Figure 9B; Figure 9B is an optical image of the working electrode configuration utilized for the experiments presented in Figure 9A.

F]gure I OA is a graphical representation of a cyclic voltammogram for the hexacyanoferrate complex prussian blue immobilised on the electrode configuration shown in Figure lOB; Figure I OB is an optical image of the working electrode configuration utilized for the experiments presented in I OA.

Figure II is a schematic representation illustrating the principle of amperometric analysis of exocytosis from a single cell using the system ofFigure 1; Figures l2A to 12C are schematic representations illustrating three different types of electrochernical communication within living cells; Figure 13 is a schematic representation illustrating the functionality of capacitive biosensor; Figure 14 is a schematic representation showing the principle of dielectric measurements; Figure 1 5A is a schematic representation showing the principle of electrorotati on; and Figure 1 5B is an optical image of an electrode configuration suitable for electrorotation expenments.

Referring to Figure 1, a system for determining and/or monitoring properties in a single particle is illustrated schematically and is designated generally by the reference numeral 2 The system comprises an assembly 4. which assembly comprises a substrate 6. The assembly further comprises a front side structure 8 and a rear side structure 10 which together sandwich the substrate 6 and hold the substrate in place.

The front side structure S can be produced fiom any material. In the embodiment shown in Figure 1 the front side structure 8 comprises a portion 1 2 which is positioned over the substrate 6 which portion 12 is formed from a transparent or translucent material. The transparency or translucency of portion 12 facilitates the interfacing of the system 2 with optical analysis systems.

The rear side structure 10 may be constructed from any material, preferably plastic, glass or metal.

The assembly further comprises canal 14 associated with the front side structure 8, and canal 16 associated with the rear side structure 10. The canals 14, 16 allow liquid to he introduced into the system 2.

The system 2 further comprises a passage 28 extending between a first domain and a second domain, the canals 14, 16 defining the first domain and second domain respectively. The passage 28 comprises a front side 30 and a back side 32 in contact with the first domain 14 and the second domain 16 respectively. The passage 28 forms an aperture 34 on a front side of the substrate which allows for the positiomng of a smgle particle. The passage 28 can be formed using an isotropic and/or anisotropic etching technique.

The system 2 further comprises a first reference electrode 18 associated with the front side structure 8, and a second reference electrode 20 associated with the rear side structure 1 0. In a preferred embodiment, the first and second reference electrodes 18,20 are positioned inside the front and rear side structures 8,10 respectively.

The first and second electrodes may however be placed at different locations relative to the front side structure 8 and the rear side structure 10 For example, a first reference electrode 18 and a second reference electrode 20 may be positioned proximate the entrance of the respective canals 14,16. Alternatively a first reference electrode 1 8?! and a second reference electrode 20" may be placed directly on the front and backside 30,32 of the substrate 6 respectively.

The system 2 further comprises working electrodes 54 and electrical connections 22 positioned on the substrate 6. If the system 2 is operated in a three-electrode setup, the system 2 further comprises counter electrodes positioned on the substrate 6.

The working electrodes 54 are positioned close to the passage 28, preferably less than 1 mm from the passage 28. Moreover, the working electrodes 54 are manufactured from a material suitable for electrical and electrochernical measurements on a positioned particle.

Electrical and electrochemical measurements in respect of a positioned particle take place between two of the electrodes on the front side structure of the system, one of which is a working electrode 54.

The coimection part of the substrate 6 is composed of a material enabling connection of the substrate 6 to an external measurement equipment.

The substrate 6 further compnses an insulating polymer layer 24 formed from, for example, photoresist, silicon oxide or silicon nitride. The electrical connections 22 provide a connection between a substrate 6 and the external environment, and are composed of any conductive material preferably a metal. The electrical connectors 22 further comprise a probe 26 preferably a spring probe or other electn cal low-resistance connector.

Due to the configuration of the system 2. it is only possible for electric current to flow through the passage 28 from the front side 30 of the passage 28 to the hack side 32 of the passage S This ensures that current flowing through the passage 28 from the front side to the hack side of the passage is influenced by a particle captured and located at the aperture 34 of the passage.

Instead of using connectors 22, 26, the system 2 could use commercially available IC sockets.

In addition, the substrate 6 could he cornhmed into an array system by using, for example, standardised 96, 384 or 1536 well plate format.

Further although the embodiment illustrated in Figure 1 has a single passage 28, in other embodiments, there may be a plurality of passages 28.

The portion of the substrate in which the passage is formed can have a number of different configurations which are illustrate schematically in Figures 2A to 2J.

Parts of the system shown in Figures 2A to 2J that correspond to parts shown in Figure 1 have been given corresponding reference numerals for ease of reference.

Figures 2A and 2B are views of a portion of the substrate 6 from above, and Figures 2C to 2J are cross-sectional representations of a portion of the substrate 6.

Referring to Figure 2A, the aperture 34 formed on a front side of the passage 28 is formed on a thin membrane 36. The membrane 36 is positioned in a cavity 38 having a substantially pyramidal shape. A top surface 40 of the cavity is substantially rectangular or square shaped, and a bottom surface 42 of the cavity 38 is also substantially square or rectangular. The cavity comprises side walls 44, and in this embodiment each side wall has an angle of approximately 54.7 to a plane substantially peiendicular to the surface of the substrate 6.

Referring now to Figure 2B, the aperture 34 of the passage 28 is also formed on the thin membrane 36 As can be seen from Figures 2C to 2J, there are number of different configurations that are possible for the relative positions of the membrane 36.

The passage 28 typically has a diameter of 0.5 to 1 0 jim, and the thin membrane 36 has a thickness in the range of 0.1 to 30 jim.

The membrane 36 can he formed from silicon, silicon mtnde, silicon oxide, i 0 polymers or various combinations of these materials.

Working electrodes 54 are positioned at least partially on the thin membrane 36.

The electrodes 54 are positioned up to 100 jim away from the passage 28, preferably less than 1 0 jim from the passage 28, and have submicrometer to sub-millimetre dimensions.

The electrodes 54 are utilized to sense the presence of a particle cell at the aperture 34.

A substrate may be manufactured in several different ways.

The substrate fabrication may be divided into two major parts, the formation of the particle positioning structure and the deposition and formation of electrode on the particle positi onmg structure.

Details of a possible fabrication process when the passage is formed from a silicon particle positioning structure are set out below.

Passage forming process * Define and structure the front to backside passage by photornaskmg and advanced silicon etch (ASE) the silicon.

* Deposit silicon nitride onto both sides of the substrate.

* Define windows in the nitride on the rear side of the substrate by photornaskmg followed by reactive ion etch (RIE) * Etch pyramidal cavities through the rear side window by anisotropic etching of the silicon. This process results in the formation of pyrarnidial side walls with a slope of 54.7 0 The etching is stopped when a membrane with the desired thickness, 0.5 to 30 trn has been fhrmed.

* Strip the mtride in hot phosphoric acid.

* Deposit silicon oxide as an insulating layer onto both sides of the silicon substrate.

First electrode forming process * Define the front side electrodes by photomasking and evaporate metal(s) onto the substrate. L] ft off the photomasking material and the excess metal in aceton.

* Define the active electrode area and contact pads by depositing an insulating polymeric, e.g. SU8, layer and develop the structure.

The reference electrodes may he formed by evaporating a conducting material, preferably a metal onto the substrate 6.

To form metal/metal salt reference electrode, for example, Ag/AgCI, chemical or electrochemical oxidation of the appropriate metal is performed.

The reference electrode may be positioned either on the substrate 6 or in one or more of the canals.

The layer of polymeric coating 24 prevents leakage of reference electrode material into the surrounding solution.

Referring to Figures 3A to 3B, an embodiment of the system of Figure 1 is shown in which a reference electrode 46 is formed in a canal 14 or 16.

In Figure 3A the reference electrode 46 is situated within a compartment 48 which is separated from the canal 1 4, 16 by a conducting bndge 50 In the embodiment shown in Figure 3B, the reference electrode 46 is positioned directly in a canal 14, 1 6, and in the Figure 3C embodiment the reference electrode 46 is positioned s directly in a canal 14, 16 hut is shielded from the solution in the canal by a polymeric coating 52.

The working electrodes 54 can have a number of different configurations. Figures 4A to 4F show several different possible configurations of the working electrode.

The working electrode 54 shown in the embodiments shown in Figures 4A to 4F is positioned close to the aperture 34.

Further electrodes 56, may be positioned on the substrate 6 further away from the aperture 34 as shown in Figure 4E. The electrodes 56 may be used as counter electrodes and/or pseudo reference electrodes as well as working electrodes.

For arnperometnc measurement, a voltage is applied on the working electrode 54 against the reference electrode 46. Current will flow upon the application of a voltage, in a two-electrode system, the current will flow between the working electrode 54 and reference electrode 46. In a three-electrode system, the voltage is applied between the working electrode 54 and the reference electrode 46.

However, a high impedance circuit is positioned between the working electrode 54 and reference electrode 46 with the consequence that the current will flow between the working electrode 54 and the counter electrode 56.

The canals 14, 16 may he fhrmed directly in the substrate 6 or in a second structure. When the canals 14, 1 6 are fbrmed in a second structure they may have a number of different functions such as: introduction of cells onto the front side of the substrate 6; 2. application of a negative pressure for particle locatmg and positioning; 3. introduction of solutions for stimulation and/or modification of the positioned particle; and 4. introduction of solutions for modification and/or washing of the solution canals, particle, substrate and electrodes etc. To allow rapid exchange of solutions in the canals 14, 16, the dimensions of the canals are chosen so that the solution flow in the canals will be laminar. The laminar characteristics of the fluid in the canals 14, 16 make it possible to test and/or deliver to the particle several different compounds within a short timescale.

The material used to form the canals may be hydrophobic and/or hydrophilic in order to achieve a specific solution flow characteristic.

An advantage of the system described particularly with reference to Figure 1 is that the canals 14, 16 are positioned to allow for easy access to all liquid and electrical connections.

Turning now to Figure 5, an embodiment of a system according to the present invention is designated generally by the reference numeral 60. The system 60 is particularly suitable for the electric charactensation of a single particle such as a cell. The system comprises a substrate 62 sandwiched between a front side structure 64 and a rear side structure 66. The front side structure 64 defines a front canal (not shown), and the rear side structure 66 defines a rear canal (not shown). The front canal is formed from a transparent material which aids in the optical inspection analysis of a particle positioned at an aperture (not shown) defining a front side of a passage (not shown) formed in the substrate 62. Particles and liquids can be injected into the front canal, and liquids can he injected into the rear canal. The system 60 further comprises a rack 72 comprising, for example, a compound library to be tested on the positioned particle. The rack 72 is connected to the front canal. The rear canal is connected to a pump 74 which can generate poSltJ\'e pressure for the priming of the passage, and negative pressure for the positioning of a particle on the aperture.

External equipment 76 such as voltmeters, potentiostats, frequency generators, amperometers, impedance analysers etc are connected to the substrate 62 via electrical connections of the type shown in Figure 1. A common feature of all the external equipment, is that each device responds to local changes in the liquid composition close to the electrode surface, and/or to physical changes in the interface between the electrode and the liquid. These local changes originate from the positioned particle. The connections are appropriate for the application of the external equipment is met. For example the system may include connectors for working, reference and counter electrodes for amperometric recordings in a three-electrode setup; for working and reference electrode for amperometric recordings in a two-electrode setup; for working, counter and reference electrode for dielectric measurements in a three-electrode setup; for working and reference electrode or working and counter electrode for dielectric measurements in a two-electrode setup. Pumps and other external equipment are preferably controlled by a computer 78.

A central task of the present invention is to capture and position a single particle in a close proximity to the working electrodes, which preferably are positioned on the substrate 6.

In the embodiments where the working electrodes are positioned on the substrate, the working electrodes are utilized to sense the presence of the particle.

Figure 6A shows an optical microscopy image of a cell 80 captured on the substrate 6 of the system of Figure 1. In Figure 6B a graphic representation of the working electrode configuration utilized in 6A is presented. The cell 80 is positioned on top of the passage 28 at the aperture 34, in close proximity to the working electrodes 54 (hidden by the cell 80 in Figure 6A), which are defined as an opening in the insulating resist 82.

The positioning of the cell 80 can both he monitored optically as shown in Figure 6A, or electrically When the cell is positioned over the aperture there will he an increase in the electrical resistance along passage 28.

In Figure 6C, the resistance over a front to hack passage prior to positioning cell (AA) and the resistance with a positioned cell (BA) are presented. The resistance is determined between an electrode positioned in a front side canal and an electrode positioned in a rear-side canal. The Error bar indicates a 95 % confidence interval for a population of 4.

The present invention relates to a platform employed for a variety of single particle characterization and manipulation techniques, particularly el ectrochemi cal techniques and methods. Below, the different techniques, and their associated substrate designs are discussed.

The present invention possesses features necessary for electrochemical experiments on single particles. In Figure 7A-B, two different circuits 84, 86, for electrochemical experiments are depicted. The circuits 84, 86 are utilized for experiments in a two-and three-electrode configuration, respectively.

In circuit 84 a voltage is applied from a potentiostat 88, between a reference electrode 90 and working electrode 92 and the current at 94 is measured with an ammeter. In the three-electrode circuit 86, the current is passed between a working electrode 96 and a counter electrode 98. The potential of the working electrode 96 is monitored relative to a reference electrode 100, situated in a circuit with high input impedance causing a negligible current to be drawn through the reference electrode 100.

The counter electrode 98 can he of any material, since its properties do not influence the behaviour of the sensing working electrode.

Preferably. the counter electrode is composed of a matenal that does not produce any substances that can reach, and interfere with the processes taking place at the working electrode, e.g platinum or gold.

Several electrolytic techniques (i.e. electrochernical techniques involving the oxidation or reduction of an analyte at the surface of an electrode as the result of an applied potential) are suitable for the characterization of single particles, especially biological cells. These techniques involve, cyclic voltammetry, linear sweep voltammetry, chronoamperometry, DC potential amperornetry (referred to as amperometry) chronoeou]ometry, sampled current polarography, normal pulse voltammetry, differential pulse voltanrn-ietry, square wave voltammetry and stripping voltammetry.

A few of these techniques have been utilized for single cell characterization, the most common being cyclic voltaninietry and amperometry.

Figure 8A illustrates graphically a current versus time trace from an amperometric recording, utihzmg a working electrode 1 02 shown in more detail in Figure 8B with an applied potential of -l 00 mV vs Ag(AgCl) pseudo reference electrode.

Before a time indicated by 1 04, the substrate solution canals only contained phosphate buffer. At home 1 04, the phosphate buffer is instantly exchanged with I mM of ferricyanide, leading to an instant increase in reduction current. At time 106, the solution is exchanged again with fresh 1 mM ferncyanide. The current trace is brought back to its initial value by the exchange of the ferricyanide with phosphate buffer at time 1 08. The same procedure is repeated with 100 tM ferricyamde at time 11 0 to time 114, i.e. injection of 100!M ferricyanide at times I IC) and 112, followed by an injection of phosphate buffer at time 114. The substrate 116 shown in Figure 8B contains a passage 11 8 utilized for single particle positioning, and a working electrode 120 connected to the external environment through connector 122. The working electrode dimension is defined by an opening 123 in a photoresist.

Referring now to Figures 9A and 9W Figure 9A illustrates graphically a cyclic voltammogram in phosphate buffer (AB) and in 1 0 mM ferricyanide (BB), utilizing a working electrcde 132. The substrate 128 in Figure 9B contains a passage 1 30 for particle positioning, and two gold electrodes 132 and 1 34 with dimensions in the im range. The working electrode area is defined in a photoresist 136 and is connected to the external environment through connector 138.

According to the present invention, the electrodes on the substrate can be further modified to perform various analytical tasks. In Figure 1OA, a cyclic o voltammogram obtained from a Prussian blue modified gold working electrode 142 with the geometry shown in Figure 1 OB, defined in photoresist 144. Prussian blue is a hexacyanoferrate complex with catalytical activity towards, among others, hydrogen peroxide [A. Karyakin and Lo Gorton, Talanta, 43, 1996, 1 597- 1605]. The anodic peak (AC) is almost on top of the cathodic peak (BC), which is characteristic for adsorbed redox species.

Amperometric recordings from single particles, particularly cells, are highly dependent on the particle to electrode distance.

For amperometric recordings on single particles the reference electrode should be positioned as close as possible to the working electrode, although not closer than a distance corresponding to twice the reference electrode diameter, to decrease uncompensated solution resistance.

A single granule, containing about 3 million molecules of electroactive neurotransmitters, e.g. adrenaline, noradrenaline and doparnine, can release its content in about 10 ms. if all molecules are completely oxidized at the electrode, delivering two electrons each, the total recorded current will be 100 pA, calculated from: O=$idi=Men.

Where 0 is the total charge transferred, / is the current. 1 is time, M is the number of molecules, c is the elementary charge I 6x 1 0- 19 and 11 is the number of moles of electrons transferred per mole of neurotransmitter.

s In practice, not all neurotransrnitters will he oxidized smce many of them will diffuse out into the surrounding solution and the registered current will therefore be smaller. The amount of non-detected neurotransmitters is highly dependent on the cell to electrode distance.

The recording of such small currents requires careftil shielding of the device from all interfering powerimes and other equipment. In addition all metal parts need to be carefully grounded.

Refemng to Figure 11, a system according to the invention and used to record single cell secretory events is depicted. A cell 150 is positioned onto an aperture 152 formed by passage 154 in close relation to working electrode 156, in order to achieve as high collection efficiency as possible. When the cell 150 is properly positioned, as observed either visually through transparent front side structure 1 64, or as determined from increased resistance between electrodes positioned on opposite sides of the passage 154, stimuli can be added, either as compounds in solution canal 166 or as a voltage pulse over the passage 154. Depending on the type of stimuli, initiation, decrease or disruption of the secretory characteristic of the cell will be monitored. The secretory event is initiated with the movement and fusion of a neurotransmitter-containing vesicle 1 58 with the cell membrane and consequent diffusion of the neurotransrnitters 1 60 into the extracellular space. A voltage is applied between the reference electrode 168 and the working electrode 156. The extracellular neurotransmitters 160 are electrochemically converted to reduced or oxidised counterparts 1 62 on the working electrode 156, resulting in the flow of electrons between the working 1 56 and reference electrode 1 68. The flow of electrons is measured with an ammeter, and the resulting current is proportional to the amount of neurotransmitters released by the cell.

Amperometnc biosensors are based on the measurements of an electrode current, which is generated when an enzyme-modified electrode is exposed to a solution containing the anal yte of interest The hiosensor produces a current proportional to the analyte concentration Enzymatic hiosensor can he divided into the 1, 2' and 3rd generation.

The function of biosensors of the generation is based on detection of either the consumption of a cosubstrate (e.g. oxygen) or the enzymatic generation of a product (e.g. i2O2, in the case of many oxidises, for example glutamate oxidise, GlOx). By modifying the electrode surface, the detection of the product can be catalysed, e.g. Prussian blue modified surface for H202.

The 7nd generation biosensor is based on a mediator (Med), able to transport electrons between an enzyme and the electrode.

Biosensors of the 3rd generation do not require any co-substrate or mediator, since the electrons are transferred directly from the active site of the enzyme to the electrode (direct electron transfer, DET). Some enzymes able to directly communicate with the electrode are, among others, laccase, hydrogenase, Oxidoreductase enzymes present in living cells can communicate with electrodes through electron transfer mediators in the process of MET. The characteristics of the mediator will determine the location of the enzyme or group of enzymes that can he probed, i.e. hydrophilic mediators, e.g. ferricyanide, will only be able to communicate with enzymes situated in the cytoplasmic membrane [J. Tkac, et al., Biosensors and Bioelectronics, 2003, 18(9), 1125-1134], whereas lipophilic mediators, e.g. quinones, are able to penetrate the cytoplasmic membrane, thus facilitating the comm un i cation with intracellular oxidored uctase enzymes {A.

Heiskanen, Ct al., Electrochemistry Communications, 2004, 6(2), 219-224].

Electrochemical methods utilizmg lipophilic redox mediators have been shown to be able to communicate with intracellular enzymes in living yeast cells [A.

1-leiskanen, et al., Electrochemistry Communications, 2004, 6(2), 219-224], mammalian cells [J. D. V. Rabmowitz, et al., Journal of the American Chemical Society. 1 998, 120(1 0), 2464-2473] and various strains of bacteria [T. K. Ikeda, et al., Journal of Electroanalytical Chemistry, 1997, 430(1-2), 197-204]. Moreover, the method has been used to study various energy generatmg pathways in living, genetically engmecred, yeast cells [C. Spégel, et. al. Manuscript]. Mediators s immobilized on the electrode surface, e.g. Prussian blue, Nile blue and Meldola blue [R. Garjonyte, et al., Sensors and Actuators B. Chemical, 2003, 96(3), 509-515] have also been shown to mediate enzyme catalysed electron transfer reactions with whole living cells By choosing or designing a proper mediator, selective determination of the activity of a specific redox enzyme or class of redox enzymes is possible. Development of selective electrochemical enzyme assays in living cells can become a useful complement to classical in vitro spectophotometric enzyme assays, since it is possible to sense the enzyme activity in its natural environment.

Figures 1 2A.-C illustrate the principles of electron transfer with intracellular enzymes (Figure 12A) and membrane bound enzymes (Figure 12A). Direct electron transfer with cellular is shown in Figure 1 2C.

In Figure 12A, electrochemical communication with intracellular enzymes is presented. A membrane soluble mediater 200 diffuses through the plasma membrane and is reduced by intracellular enzymes 202. The oxidized enzyme is re-reduced by intracellular cofactors 204, which are consequently oxidized to their oxidized counterparts 206. The reduced mediator 208 can then diffuse out from the cell and deliver its electrons 210 to the electrode, resulting in the formation of a current proportional to the intracellular enzyme activity.

In Figure 12B, membrane hound enzymes 202 are probed using water soluble mediator redox couple 212, 214, resulting in a current proportional to the membrane hound enzyme activity.

Direct, mediatorless, electron transfer is presented in Figure 12C. In this case the enzyme 202 directly delivers the electrons 210 from the intracellular cofactor redox couple 204, 206 to the electrode, also resulting in a current proportional to the activity of the probed enzyme.

The working electiode 54 in Figure 1, can he modified with various sensing s elements to form capacitive biosensors.

A capacitive biosensor 222 is represented in Figure 13. The working electrode 54, e.g gold, can he modified with a layer 220, e.g. thiols, promoting adsorption of the sensing element 222. e.g. a protein or antibody. When the analyte 224 of interest binds or interacts with thesensing element 222, a perturbation of the interface equilibrium takes place, resulting in a detectable signal.

The substrate according to the present invention can he utilized for dielectric measurements on single particles, particularly el ectrochemi cal impedance spectroscopic studies on single cells. Dielectric characterization of single particles can reveal information on the interaction of a particle or single cell with a substrate (base platform), as well as give information about the functional and morpho-physiological characteristic of the cell/particle. The dielectric response of the cell can be presented by a number of equivalent schemes consisting of interconnected capacitance and resistance elements [Joachirn Wegener, Sigrid Zmk, Peter Rosen, Hans-Joachirn Galla. Use of electrochemical impedance measurements to monitor b-adrenergic stimulation of bovine aortic endothelial cells. Eur. J. Physiol, 1999, 437, 925-934. Koji Asarni. Characterization of heterogeneous systems by dielectric spectroscopy. Prog. Polyrn. Sci. 2002, 27, 1617-1659.]. Usually, impedance/dielectric measurements are performed in the I to 108 Hz frequency window.

An example of an impedance scheme modelling the impedance properties of a cell is presented in Figure 14 A constant phase element describes the interaction (contact) of a cell with an opening on a platform. Rm and Cm are the resistance and capacitance of a cell membrane, Z,1 is an impedance of cytosol, which can include dielectnc characteristics of nucleus and other organelles in the cell.

represents the ion transfer characteristics of a solution surrounding the cell. More precise modelling of a single cell in microsystems by equivalent circuit elements can also he sensitive to the geometry of canals and electrodes surrounding the cell due to a heterogeneous electrical field [Shady gawad, Karen Cheung, Urban Seger, Amaud Bertsch and Philippe Renaud. Dielectric spectroscopy in a micromachmed flow cytometer: theoretical and practical considerations. Lag Chip, 2004, 4,241-251.] The present in\/ention provides a platform for studies of dielectric properties of single cells and enables monitoring of functional and morphological characteristics of cells including viability.

The substrate according to the invention is useful for electrorotation experiments on single particles, particularly cells.

A substrate configuration suitable for electrorotation experiments is presented in Figure I 5A. The electrodes 250 are given a 90 phase difference, causing the particle 252 to rotate in the generated electric field.

A microscopy image of a possible substrate 254 for electrorotational studies is presented in Figure 1 5B. A passage 256 is utilized for particle capture and electrodes 258 generate the electric field utilized for particle characterization.

The substrate according to the invention can also he utilized as a cell manipulation tool, performing electroporation. By applying short voltage pulses to a captured cell, pores in a cell are created allowing, otherwise membrane impermeable, water-soluble molecules to enter the intracellular compartment via a combination of diffusion, electrophoresis and electroosmosis. Such molecules include DNA, dyes, drugs, proteins, peptides, amino acids etc. Optical techniques are easily interfaced to the present invention by manufacturing the front side structure from an optically transparent material.

Claims

CLAIMS

1. A system for deterrnimng and/or monitoring properties in a single particle, the system comprising an assembly comprising a front side structure, a rear side structure and a substrate, the substrate comprising a first upper surface part and a first lower surface part for holding the single particle, a first site comprising a passage in thy substrate, a first end of the passage being in contact with a first domain at the/first upper surface part of the substrate and a second end of the passage being ji contact with a second domain at the first lower surface of the substrate, the first end of the passage defining an aperture adapted to receive the single particle; at least one electrode positioned on the first upper surface part of the substrate; a reference e]ectrode in electrical contact with the first dornam; at least one working electrode, situated on a surface of the substrate, and in electrical contact with the first domain; the first end of the passage being adapted to locate the single particle held at the first site, the particle thereby separating the first domain from the second domain; the at least one electrode on the first upper surface part being in close proximity to the first end of the passage; the system further comprising: locating means for locating the single particle at the aperture; and at least one interface for interfacing the substrate with an analysing system.

2. A system according to Claim I wherein the at least one electrode on the first upper surface part is less than I mm from the first end of the passage.

3. A system according to Claim 1 or Claim 2 wherein the at least one electrode on the first upper surface part is less than 100 im from the first end of the passage.

4. A system according to any one of the preceding claims wherein the at least one electrode on the first upper surface part is less than 1 0 p.m from the first end of the passage.

5. A system according to any one of the preceding claims wherein the particle comprises a biological cell.

6. A system according to any one of the precedmg claims, wherein the interface comprises an electrical contact for forming an electrical connection between the system and the analysing system.

7. A system according to Claim 6, wherein the assembly comprises a contact receiving portion, and the electrical contact comprises a contact portion that is positioned in the contact receiving portion, extends from the system and is in electncal contact with the working electrode.

8. A system according to any one of the preceding claims, wherein the first domain compnses a first canal and the second domain compnses a second canal.

9. A system according to Claim 8, wherein the first canal has a first input and a first output, and the second canal has a second input and a second output.

10. A system according to any one of the preceding claims, wherein the locating means comprises pressure means for applying a pressure gradient along the passage such that the pressure at the first end of the passage is greater than the pressure at the second end of the passage.

11. A system according to Claim 6 when dependent on Claim 9, wherein the locating means comprises pump, which pump is connectable to the second canal.

12. A system according to any one of the preceding claims, further comprising a detector for detecting when the single particle is positioned at the aperture.

13. A system according to Claim 12, wherein the detector comprises a transparent part in the front side structure positioned above the substrate.

1 4 A system according to Claim 1 2, wherein the detector comprises a system fhr measuring electrical resistance through the passage.

15. .A system according to any one of the preceding claims, wherein the at least one electrode on the first upper surface part is a conducting electrode.

16. A system according to any one of Claims 1 to 14, wherein the at least one electrode on the first upper surface part is a semi-conducting electrode.

17. A system as generally hereinbefore described with reference to and/or illustrated in the accompanying drawings.