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• • H—ELECTRICITY
  • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
  • H05B—ELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
  • H05B3/00—Ohmic-resistance heating
  • H05B3/20—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater
  • H05B3/22—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater non-flexible
  • H05B3/26—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater non-flexible heating conductor mounted on insulating base
  • H05B3/265—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater non-flexible heating conductor mounted on insulating base the insulating base being an inorganic material, e.g. ceramic
• • C—CHEMISTRY; METALLURGY
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
  • C12Q1/004—Enzyme electrodes mediator-assisted
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
  • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
  • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
  • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/403—Cells and electrode assemblies
  • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
  • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
• • H—ELECTRICITY
  • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
  • H05B—ELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
  • H05B3/00—Ohmic-resistance heating
  • H05B3/20—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater
  • H05B3/22—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater non-flexible
  • H05B3/26—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater non-flexible heating conductor mounted on insulating base
  • H05B3/262—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater non-flexible heating conductor mounted on insulating base the insulating base being an insulated metal plate
• • H—ELECTRICITY
  • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
  • H05B—ELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
  • H05B3/00—Ohmic-resistance heating
  • H05B3/20—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater
  • H05B3/22—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater non-flexible
  • H05B3/28—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater non-flexible heating conductor embedded in insulating material
  • H05B3/30—Heating elements having extended surface area substantially in a two-dimensional plane, e.g. plate-heater non-flexible heating conductor embedded in insulating material on or between metallic plates
• • H—ELECTRICITY
  • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
  • H05B—ELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
  • H05B3/00—Ohmic-resistance heating
  • H05B3/78—Heating arrangements specially adapted for immersion heating
  • H05B3/82—Fixedly-mounted immersion heaters
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/28—Electrolytic cell components
  • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
  • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
• • H—ELECTRICITY
  • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
  • H05B—ELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
  • H05B2203/00—Aspects relating to Ohmic resistive heating covered by group H05B3/00
  • H05B2203/013—Heaters using resistive films or coatings
• • H—ELECTRICITY
  • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
  • H05B—ELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
  • H05B2203/00—Aspects relating to Ohmic resistive heating covered by group H05B3/00
  • H05B2203/017—Manufacturing methods or apparatus for heaters

Definitions

• This invention relates to sensor devices, and more particularly to improved sensor devices useful in analytical methods involving the detection or measurement of pH changes, especially in enzyme-based biosensor systems, and to analytical methods using them.
• an electrode is employed to provide output signals by which the presence or absence of an analyte in a sample can be determined.
• an analyte or a species derived from it which is electro-active generates a detectable signal at an electrode, and this signal can be used as the basis for detection or measurement of the presence and/or amount of the analyte in a sample .
• Bio-sensors have been found to be very successful in use for such purposes, especially when the bio-component is an enzyme .
• An enzyme has the advantage that it can be more specific to the analyte sought and also, when the analyte itself is not sufficiently electro-active, can be used to interact with the analyte to generate another species which is electro-active and to which the electrode can respond to produce the desired output signals.
• the classic example of such a sensor is the glucose oxidase enzyme-electrode, in which an immobilised glucose oxidase enzyme catalyses the oxidation of glucose to form hydrogen peroxide, which is then detected and determined by amperometric measurement of the effect it produces (increase in electrical current) at a polarised electrode (typically platinum) . Measurements can be made conveniently in the usual manner, polarising the platinum electrode at 650 mV against an Ag/AgCl reference.
• amperometric sensors and their use suffer from the limitation that other electro-active species present in a sample under examination (additional to the analyte sought) can cause interference, and also that enzymes do not always produce -electro-active products. Consequently some alternative to amperometric transduction is desirable.
• Such buffering components may be present in the sample itself or may be deliberately added, for example as a stabiliser or a preservative for the sample.
• a persistent problem encountered with pH-sensitive sensors is derived from the presence of any buffering salts in the sample matrix.
• samples of high buffering capacity the pH change induced by the enzyme is not detected by the sensing surface, hence no change in signal is observed and a false interpretation of the sample's composition is drawn.
• samples that have a variable buffering capacity will result in a significant "drift" in the sensor signals, again leading to practical limitations in their use.
• This coating of DLC serves as a non-ionic coating layer which is not used as part of the ion-sensitive electrode (ISE) itself, and so is not put there to function as the ion-sensitive element or one of the construction components making up the ISE, for example as a necessary insulating component or layer in it. It is there to serve as a partial diffusion barrier to modify the manner in which components of media behave in the vicinity of the ISE.
• ISE ion-sensitive electrode
• the senor which is responsive to ionic changes in the media in contact with it, and especially the pH, is associated with an adjacent enzyme, so that interaction of the enzyme with the selected analyte can produce a change in the ionic content, especially the pH, at the sensor surface.
• the DLC coating not only has the properties of diffusion control indicated above, but also that it can be applied to an enzyme-containing system without causing the enzyme, which is a sensitive material, to lose its activity.
• a sample containing a selected analyte is brought into contact with a sensor which is responsive to changes in ionic content, especially pH, of media in contact with it and the change (especially in pH) induced by the interaction of the enzyme and the analyte is measured and used to provide a measure of the amount of the analyte present in the sample, characterised in that the said sensor has a coating comprising a carbonaceous material having structural characteristics comparable with that of diamond.
• a form of carbonaceous material known as "diamond-like carbon" (DLC) .
• DLC is a form of amorphous carbon or a hydrocarbon polymer with properties approaching those of diamond rather than those of other hydrocarbon polymers .
• Various names have been used for it, for example “diamond-like hydrocarbon” (DLHC) and “diamond-like carbon” (DLC) , but the term “DLC” appears to be the most common. It possesses properties attributable to a tetrahedral molecular structure of the carbon atoms in it, similar to that of diamond but with some hydrogen atoms attached. It has been described in the art as being a designation for "dense amorphous hydrocarbon polymers with properties that differ markedly from those of other hydrocarbon polymers, but which in many respects resemble diamond" [J.C. Angus, EMRS Symposia Proc . , 17, 179 (1987) ] .
• DLC diamond-like carbon
• hydrocarbon precursor gases e.g. propane, butane or acetylene
• glow-discharge deposition by laser-induced chemical vapour decomposition, by a dual-ion beam technique, or by introduction of the hydrocarbon gases directly into a saddle-field source.
• a saddle-field source is a source of ions produced by a collision between gas atoms excited by thermionic emission, and this method is preferred because it allows heat- sensitive materials to be coated by a beam that is uncharged -- so facilitating the coating of insulating or non- conductive materials.
• Its properties can vary according to the particular raw materials used and its mode of formation. It can also be made in other ways, for example by sputtering solid carbon, as an alternative to dissociating hydrocarbon gases.
• the convenient source of the DLC carbon is a hydrocarbon gas or vapour, especially one which is readily decomposed by an electric field or discharge.
• a very convenient source gas is acetylene, though others may be used if desired.
• Individual hydrocarbons or mixtures thereof may be used, and diluent gases may be added if desired, and the decomposition/deposition procedure may be carried out at pressures at atmospheric or above or below atmospheric, as may be found most suitable for particular instances and the sensitivity of the enzyme used.
• the sensor which is responsive to ionic changes of media in contact with it, and especially to changes in pH, may be any of those known in the art .
• responsive sensors are field effect transistor devices, which are commonly made of solid semiconductor materials, for example silicon - though other materials or mixtures of materials may be used.
• a preferred example of this type of sensor is that known as an enzyme field effect transistor ("ENFET”) .
• EFET enzyme field effect transistor
• Such devices are well known.
• an enzyme is immobilised on the surface of a semiconductor material (which is typically made of silicon, though other materials may be used) carrying inter-digitating electrodes with a bridging conducting polymer film.
• the enzyme is one which acts on a substrate to form a product which is then detected by its effect on the medium in its vicinity, especially its pH.
• the change in pH resulting from enzymic activity alters the conductivity of this material -- which is detected by monitoring the extent of electric current flow through it (i.e. the "drain current”) .
• ENFET devices have advantages over devices used for amperometric detection -- notably a lower susceptibility to electro-active interference and also usefulness with enzymes which do not form electro-active products. They have so far been based primarily on silicon semiconductor technology, but alternatives to silicon may be used if desired, for example organic conducting polymer films.
• Some conducting polymers have properties similar to semiconductors, in that it is possible to "switch" them repeatedly between conducting and insulating states . This switch can be induced by change in the polymer redox state and/or their degree of protonation (pH) . It is this latter property which has been the basis for the polymers used for ion-sensitive devices, especially in ENFET type devices. In this respect, when a redox moderator has ionisable groups in its structure then its properties will be ion-dependent, for example pH-dependent .
• An especially useful conducting polymer is poly (pyrrole) . This is made is known manner, for example by electro-polymerisation or oxidation of pyrrole, usually in aqueous solution - the polymer precipitating as it is formed. Other known conducting polymers can be used if desired.
• Another example of its use is in a sensor sensitive to penicillin, based on pol (pyrrole) with an overlaying layer of immobilised penicillinase. Upon exposure to penicillin, the polymer becomes more conductive (through protonation) which is detected by an increase in drain current passing through the conducting polymer film.
• impedance (resistance) measurements can be used as the measure of polymer conductivity as, in principle, both drain current and impedance provide the same information regarding the film conductivity, though the latter is recognised as being a more sensitive technique.
• the thickness of the DLC coating may vary according to the particular requirements desired for the performance of the sensor and the system to be analysed.
• the thickness of the DLC coating or deposit may be in the range 0.01 to 1.0 nm, but thicker or thinner coatings may be used if desired.
• a typical and convenient coating deposit is one approximately 0.5 nm thick, but this is not necessarily the optimum for all purposes.
• the optimum thickness in any particular case will depend upon such factors as the nature (physical and chemical) of the material upon which the DLC is deposited, and its porosity or permeability, and the particular enzyme and substrate concerned, and the characteristics appropriate to the intended use of the sensor.
• the coating with DLC is conveniently carried out at a rate which allows the deposit to adhere to the membrane material and form a coating of the desired thickness - preferably also evenly coated so as to cover substantially all the surface without leaving any areas too thinly covered or even un-*covered.
• the deposition may be carried out at a rate of up to 0.5 nm per hour, though higher or lower rates may be used if desired.
• the invention is applicable to a variety of enzyme systems which act to form acidic or basic products which can change the pH of the media in which they act .
• the principal example is an enzyme which hydrolyses a substrate
• ammonia is strongly basic, but can also be applied to systems in which other pH- altering compounds can be formed, for example amines.
• the commonest enzyme we find useful is urease, which hydrolyses urea to carbon dioxide and ammonia.
• the improved sensors according to the present invention have the advantage that their range of linear response (i.e. the relationship between the sensor output signal and the amount of the substrate analyte) is extended, in comparison with known sensors . This makes them much more useful in practical clinical or laboratory conditions; by their use, the ease, speed and reliability of measurement or detection can be extended considerably.
• this range of linear response is increased from 2 mM to 6 mM of urea.
• the levels of urea analyte present may be in a region (e.g. 20 mM urea) which too high for sensors to be used satisfactorily.
• the present invention does not eliminate entirely the need for some dilution of a sample, nevertheless it does reduce it considerably because the DLC-coated sensors of our invention have an increased range of linear response. The result of this is that sample dilution becomes far less critical and one can simplify considerably the previous need to carry out multiple tests (by several dilution steps) to determine the optimum degree of .dilution for the particular sample under test .
• the electrode of our invention can be used to carry out the method of our invention by simple immersion in a predetermined volume of a buffer solution to which the sample to be analysed has been added, and applying an alternating voltage (AC) , with a superimposed bias voltage, so that measurements of the impedance can be made.
• AC alternating voltage
• This measurement procedure may be done in conventional manner, using conventional equipment. Measurements of the impedance (resistance) may be taken and the measurements taken and recorded as desired, intermittently or continuously. For this, conventional apparatus may be used. Samples of the media for examination may be obtained by standard methods . The quantity of sample should be sufficient to cover the sensor and the current measured at a fixed time or after a stable response has been achieved. Likewise, samples of other media may be obtained in any convenient manner and brought into contact with a sensor of the present invention for the purpose of component detection. If desired, the procedure may also be calibrated by use of solutions containing known amounts of the substances sought, and its accuracy this checked and confirmed.
• the procedure may be carried out using known amounts of compounds which are considered to be potentially troublesome by their expected ability to interfere with the measurement, so that the degree of interference (if any) can be established.
• Conventional apparatus may be used, for the cell, electrodes and the measurement and recording of the impedance relationships for the samples under test. Measurements may be made continuously or intermittently, as desired.
• the most suitable bias voltage is +0.4V against an Ag/AgCl reference .
• the pH of the sample/buffer mixture being examined may vary in the pH range 5 to 8 within which lies the pH optimum of many enzymes.
• the selected pH for an assay mixture may be chosen to be close to 7.4, which is the physiological pH of blood plasma.
• the sample under examination may be stirred or not, as desired or convenien .
• the measurement procedure for use of the sensors of our invention may be carried out over a considerable range of temperatures, for example in the range 20 to 40 degrees C..
• the medium comprising the sample examined is commonly aqueous, but need not necessarily be so, and an organic solvent may be used if desired (as such, or in admixture with each other and/or water) provided it is an electrolyte and dissolves any desired reagents, but is not medically relevant to the assay carried out .
• Diamond-like carbon coatings have the advantages of a high degree of inertness and also a high degree of bio- compatibility.
• the DLC coating also serves as a mechanical barrier against abrasion of the enzyme layer; an ultra-thin, low permeability layer that controls the permeation of the analyte without adversely extending response times; reduces access to the electrode by buffer interferents; reduces protein and cell bio-fouling of the electrode and so increases bio-compatibility.
• the invention is illustrated but not limited by the following Examples .
• An inter-digitating electrode (“IDE”) is used as the starting device.
• This comprises a base of an insulating material carrying on its surface two electrically separate conducting arrays which intermesh with each other so that they form, over the surface of the base, a pattern combining the two conducting elements which do not make contact with each other.
• This pattern area forms a "sensing area" which can respond to the conductivity of any media spread across it and contact both conducting element arrays .
• the two conducting elements are connected electrically to simple terminals by which they can be connected into an electrical circuit, for example for measurement of the electrical impedance (resistance) between the two element arrays. It was made as follows : -
• Gold inter-digitated electrodes were fabricated by photo-lithography and consisted of a gold layer (500 nm thick) deposited on to an insulating silicon substrate which comprised a 1 nm thermal oxide layer on which a 0.16 nm layer of silicon nitride had been deposited. A 30 nm chromium layer assured adhesion of the gold on to this underlying substrate.
• the IDA consisted of 50 digits, each 10 um wide and 500 urn long, separated by a 15 um gap.
• Poly (pyrrole) films were made on the surface of the IDE by deposition from a de-oxygenated aqueous solution containing pyrrole (100 mM) sodium dodecyl sulphate (2 mM) and urease (1350 U) .
• the pH of this solution was adjusted to 7, using 0.1 M sodium hydroxide, and polymerisation was achieved by potential cycling between -0.25 V and +0.9 V against an Ag/AgCl reference at a scan rate of 50 mV/s.
• the formed poly (pyrrole) precipitates from the aqueous solution on to the electrode surface and eventually forms a smooth film bridging the inter-digitating electrodes .
• the urease enzyme layer becomes entrapped and thereby immobilised. It was found that the growth of conducting polymer across the electrode gap could be more readily achieved by using a three-electrode system with the IDE as the working electrode, a separate counter (platinum flag, 2 x 3 cm) and an Ag/AgCl reference.
• the coated electrode was washed with a saline buffer (50 mM, at pH 7.4, containing 50 mM potassium chloride) to avoid enzyme de-naturation and to remove excess reagents .
• the monomer is oxidised, which makes it reactive and able to combine with further oxidisable monomers.
• the polymer becomes insoluble and is deposited on to the surface of the electrode and its support.
• the polymer deposit grows across the digits and thereby provides an electrical connection between them.
• it is the ammonia formed by the action of the enzyme on urea that de-protonates the poly (pyrrole) polymer -- thereby resulting in an increase in its resistance to current flow between adjacent electrodes which is detected by impedance measurement.
• the resulting sensor device was coated with a layer of diamond-like carbon 0.5 nm thick by deposition of the DLC by striking a plasma in an atmosphere containing hydrocarbon vapour (methane) .
• the DLC was deposited at a rate of l Angstrom a second, and the resulting DLC was in an insulating, inert, flexible and bio-compatible thin film.
• Urease catalyses the hydrolysis of urea to form ammonia and carbon dioxide.
• the ammonia thus produced de-protonates the supporting poly (pyrrole) film and, as a result, produces a decrease in the polymer conductivity (an increase in its resistance), which can be detected by impedance measurement.
• a sample solution containing urea the solution permeates through the polymer and the urea in it interacts with the urease, and the ammonia so formed then alters the impedance as described above.
• the IDE coated with urease/poly (pyrrole) prepared as described above is connected electrically, by its terminal connections, to a conventional apparatus for measuring the impedance (resistance) of the bridging conducting polymer film over the inter-meshed array of conducting elements, and then dipped into an aqueous solution containing urea as analyte, and the impedance (resistance) of the coated IDE is then measured, the impedance measurements provide a measure of the urea content .
• the range of urea concentrations used for calibration was 1 to 20 mM urea; for test purposes, a concentration of 5 mM urea was convenient for use.

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