EP0730760A1 - Biocapteurs potentiometriques, systeme de commande de ces derniers et leurs applications - Google Patents

Biocapteurs potentiometriques, systeme de commande de ces derniers et leurs applications

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Publication number
EP0730760A1
EP0730760A1 EP95901567A EP95901567A EP0730760A1 EP 0730760 A1 EP0730760 A1 EP 0730760A1 EP 95901567 A EP95901567 A EP 95901567A EP 95901567 A EP95901567 A EP 95901567A EP 0730760 A1 EP0730760 A1 EP 0730760A1
Authority
EP
European Patent Office
Prior art keywords
transducer
sensor
biosensor
redox potential
variation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95901567A
Other languages
German (de)
English (en)
Inventor
Marco Sartore
Manuela Adami
Claudio Nicolini
Antonio Fanigliulo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TECHNOBIOCHIP
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TECHNOBIOCHIP
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB939324258A external-priority patent/GB9324258D0/en
Priority claimed from IT93RM000846A external-priority patent/IT1266466B1/it
Priority claimed from GB9411072A external-priority patent/GB9411072D0/en
Priority claimed from GB9411059A external-priority patent/GB9411059D0/en
Priority claimed from GB9414189A external-priority patent/GB9414189D0/en
Application filed by TECHNOBIOCHIP filed Critical TECHNOBIOCHIP
Publication of EP0730760A1 publication Critical patent/EP0730760A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • 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
    • G—PHYSICS
    • G05—CONTROLLING; REGULATING
    • G05D—SYSTEMS FOR CONTROLLING OR REGULATING NON-ELECTRIC VARIABLES
    • G05D23/00—Control of temperature
    • G05D23/19—Control of temperature characterised by the use of electric means
    • G05D23/1919—Control of temperature characterised by the use of electric means characterised by the type of controller
    • G—PHYSICS
    • G05—CONTROLLING; REGULATING
    • G05D—SYSTEMS FOR CONTROLLING OR REGULATING NON-ELECTRIC VARIABLES
    • G05D23/00—Control of temperature
    • G05D23/19—Control of temperature characterised by the use of electric means
    • G05D23/20—Control of temperature characterised by the use of electric means with sensing elements having variation of electric or magnetic properties with change of temperature

Definitions

  • Temperature control is very important in many systems, mainly when a temperature sensitive device is being utilized.
  • a silicon-based potentiometric sensor able to detect the H* ions concentration (i.e. the pH) of an electrolyte in contact with it.
  • a cover slip with attached living cells is put closely in front of the sensitive surface of the silicon sensor, in order to obtain a micro-volume nearby the sensor; cells are in contact with fresh medium, by means of a flow mechanism; when the flow is stopped, or some drug is added to the medium, cells acidificate the micro-environment more quickly; monitoring of the acidification rate allows us to understand the "status" of the cells under different conditions, and to predict the effects of drugs on the considered cell system.
  • the sensor used is silicon-based, hence it is extremely sensitive to temperature variations; if operating in a non-controlled system, the output signal cannot be unequivocally related to any physical event.
  • cells behaviour depends on temperature; the whole population must be at the same temperature if quantitative monitoring is performed. extracellular acidification is strongly affected by the temperature at which cells are kept; usually 37°C is a suitable value for optimal cells conditions.
  • Temperature control is needed in very precise measurements. If one just wants to perform basic experiments on the device, acquisitions at room temperature in a normal laboratory are more than satisfactory, and no temperature control system is needed.
  • temperature control is essential .
  • usual temperature control is obtained by means of setups based on a warm fluid flowing around a considered region.
  • a (big) thermostat is kept outside the measuring chamber, and even outside the measuring desk; this system produces a warm fluid circulation in a tube coil, placed just around the measuring chamber.
  • a temperature control device comprises a heating/cooling device in the form of a Peltier cell; a temperature sensor for sensing the temperature in the vicinity of the cell; and control means responsive to the sensed temperature for controlling the heating and cooling activity of the Peltier cell so as to maintain the temperature in the vicinity of the cell at or near a predetermined value.
  • This temperature control device or thermostat is effective, cheap and easy to use. It can be applied in a variety of situations, when a precision temperature control is needed in a relatively small environment.
  • a biosensor including a temperature control device for controlling the temperature in a sample region, the device comprising a heating/cooling device; a temperature sensor for sensing the temperature in the vicinity of the device and control means responsive to the sensed temperature for controlling the heating and cooling activity of the heating/cooling device so as to maintain the temperature in the vicinity of the device at or near a predetermined value.
  • the temperature control device used with this biosensor could take a variety of forms, preferably the heating/cooling device is in the form of a Peltier cell. It has been found that in applications where the temperature of a small environment must be controlled, the use of Peltier elements guarantees high efficiency and uniform temperature distribution. Furthermore, a Peltier-based device enables temperatures to be controlled at target or predetermined values higher or lower than room temperature. Furthermore, the Peltier cell can easily and rapidly be used as a heating or cooling element. Finally, this system is very compact and the driving electronics can be installed in a small box.
  • a number of biosensors are known to employ light-addressable potentiometric sensors (LAPS) .
  • LAPS light-addressable potentiometric sensors
  • the principle of operation of a light-addressable potentiometric sensor is described by Hafeman et al, Science (1988) 240:1182 and by Sartore et al, Biosensors & Bioelectronics (1992) 7:57-64.
  • Use of such sensors has allowed the design of biosensors that are capable of measuring either pH variations (if the sensor's surface is coated with a H* sensitive insulator, such as Si 3 N 4 or Ta 2 0 5 ) or redox potential variations (if metal spots are evaporated onto the sensor's surface) .
  • Light-addressable potentiometric sensors have also been used to monitor extracellular acidification inmicro environments, again through pH variation. Many important metabolic processes of the cells (namely the catabolism of sugars, amino acids and fatty acids) produce H* ions, which are excreted through the cytoplasmic membrane and out of the cell, thereby causing an extracellular pH variation.
  • the use of light-addressable potentiometric sensors to measure this pH variation is described by Owicki et al, Biosensors & Bioelectronics, (1992) 7:255-272; Owicki et al, Proc. Natl. Acad. Sci . (1990) 87:4007-4011; and Wada et al, Journal of Cell Biology, (1991) 115:A2455.
  • the cell medium usually contains a buffer whose capacity masks any pH change due to extracellular acidification, which therefore cannot be measured.
  • the present invention relates to a biosensor, and in particular to a biosensor for measuring pH variations and/or redox potential variations in enzymic reactions, either taking place within the cell and causing extracellular variations, or in isolation.
  • a method of measuring a pH variation or a redox potential variation in an enzymic reaction that generates ions that cause, or whose generation causes, such a variation comprises monitoring the reaction over a period of time using a light-addressable potentiometric sensor that generates a current on the binding of the respective ions thereto; converting current measured over that time to voltage; if desired, calculating the pH variation or the redox potential variation, or the number of the respective ions generated, as a function of the voltage; and comparing the voltage, pH variation or redox potential variation, or number of ions with a precalibrated standard.
  • the method of the invention allows the monitoring of range of enzymic reactions through a variation in pH and/or in redox potential occurring in those reactions.
  • the method of the invention can be applied to determine enzyme and substrate concentrations to low orders of magnitude, for example in the 10" 10 M range.
  • a sensor that is sensitive to redox potential variations, the problem related to the measurement of small pH variations in buffered solutions is overcome. In this case the measured quantity is a surface potential, and therefore buffers, even with high buffer capacity, do not affect the sensitivity of the method.
  • a biosensor can be programmed to carry out either or both of the above methods, and to display the results thereof in a suitable manner. This allows the user to carry out a wide variety of experiments on the same or different biological samples in a simple and efficient manner.
  • a biosensor suitable for carrying out the method of the invention comprises a reaction chamber in which the reaction is to proceed and into which at least one sensor can be immersed.
  • the reaction chamber can be separate from at least one measuring chamber, each having a sensor associated therewith.
  • the biosensor further comprises specific electronic cards and related software programs to acquire the necessary data quickly, and present it in a user-friendly manner.
  • the biosensor is preferably completely automated and driven by a personal computer.
  • the electronics is designed in order to obtain a good general signal-to-noise ratio, hence ensuring reproducible results.
  • the software package typically consists of low-level programs (written, for example, both in C and Assembler) , to interact with the data acquisition and controlling cards, and of high- level programs (for example written in C) , to obtain, display, save and print data files, and to present a suitable user interface.
  • a very important peculiarity of the biosensor of the invention is the ability to connect different sensors or a plurality of measuring chambers each associated with a different sensor, to the same device; this allows the user to perform a wide variety of experiments, utilizing the same or different biological elements, by simply changing the measuring chamber under consideration and selecting the appropriate acquisition software. This can be done by simply substituting electrical connections and does not require physical movement of the measuring chamber per se.
  • sensors examples include a sensor sensitive to pH variation by the binding thereto of H" ions, and a sensor sensitive to redox potential variation by the binding thereto of ions generated in a redox reaction.
  • An example of the latter kind of sensor is one which has had gold metal evaporated onto its surface.
  • Sensors that are sensitive to inorganic ions are known, and can also be used in the biosensor of the invention.
  • sensors that are sensitive to more than one of the above types of ion can be used.
  • the biosensor of the invention utilizes the fast information recovery from the sensor output signal, and can acquire quantitative data at fraction of a second intervals over period of time; it can therefore be utilized ' to monitor fast acidification or redox processes.
  • PAB Patentiometric Alternating Biosensor
  • LAPS Light Addressable Potentiometric Sensor, Hafeman et al, 1988
  • the system has been tested with cells (Gavazzo et al .
  • YADH Alcohol Dehydrogenase from Yeast, Adami et al, 1994b
  • GST Glutathione-S-Transferase, Antolini et al, 1994
  • a potentiometric sensor based on a silicon chip, able to detect redox potential changes in solution is produced and some of its possible applications are investigated.
  • the redox potential of a solution in contact with the surface of a metal layer deposited on the chip affects the amplitude of a photocurrent signal generated in the silicon by means of a modulated light source.
  • HRP is linked to the widespread use of this enzyme as label in immunoassays, therefore giving the possibility to use this system as an immunosensor.
  • other enzymes can be used and another type of assay is proposed, using Diaphorase together with Alcohol Dehydrogenase.
  • a biosensor capable of 2D acquisition and/or multisensing; the device is potentiometric and is based on a silicon transducer with signal generation caused by light excitation.
  • the system can recover the local spatial information of pH, redox and other significant quantities on a sensing area of about 1cm 2 , which is usually contained in the particular measuring flow chamber specifically designed for the given biosensing application.
  • the bidimensional PAB system allows a fast potentiometric measurement in the 2D space of the transducer surface. Spatial information recovery is achieved by means of local light stimulation of the silicon transducer. In this way the surface potential of the system is locally measured; the surface charge, giving rise to the potential, can be originated in different ways (Delia Ciana et al, 1991) , allowing multisensing operations onto a single chip; for instance, the transducer surface can be partially covered by Si 3 N 4 for pH detection, or by gold, for redox measurements, or by different chemically sensitive sites, such as LaF 3 for F" detection; moreover, specially functionalized sites can be created onto the sensing surface, for instance by chemical interactions between gold spots and specific compounds (i.e. thiols) .
  • the system produces a 2D map of the solution pH under investigation; instead, when additional sensitive sites are used, the system can be considered both bidimensional and multisensitive; in other words the local surface potential can still be recovered preserving the spatial information, so obtaining a 2D multisensor.
  • PAB One of the most relevant characteristics of PAB is the versatility of the system.
  • a fifth aspect of the invention provides for the application of PAB system for the investigation of the effects of antineoplastic drugs on two stabilized cell lines (normal mouse fibroblasts 3T6 and transformed HEPG2 hepatomas) and on primary cultures (normal rat hepatocytes) ; this work shows the feasibility to utilize the PAB biosensor as an innovative test with respect to conventional ones for the estimation of drug efficacy and toxicity.
  • the liver is the major site for the uptake of drugs and chemicals, converting them topharmacologically inactive, active or even toxic metabolites.
  • Biotransformation of xenobiotics and other specific liver functions, mostly performed by parenchimal liver cells (hepatocytes) are difficult to study in the whole organism, because of the influence of other organs, tissues and exogenous/endogenous factors; consequently, in vitro hepatocytes represent a valuable tool in pharmoco-toxicology, since it was demonstrated that they preserve, when in culture, the functional drug metabolising enzymes in culture (Engelmann et al 1985; Dich et al, 1988; Martelli et al, 1988) .
  • a possible application of the PAB system as an im unosensor can imply the usage of a modified Langmuir-Blodgett (LB) technique for the production of antibody monolayer.
  • LB Langmuir-Blodgett
  • urease an enzyme
  • the immobilized monolayer was characterised by means of ellipsometric and nanogravimetric techniques in order to evaluate the main physical parameters such as thickness and surface density. Together with the enzymatic activity we also investigated the operative lifetime of the monolayer. The performance of the PAB system utilizing the thin film technology was analyzed.
  • the aim of our work is to study the enzymatic activity of the urease immobilized onto a support (glass or silicon nitride (Si 3 N practical) ) inserted into a microvolume reaction chamber in order to evaluate the application of the LB technology to immunosensors based on PAB.
  • the enzyme used in this experiment was urease, which catalyzes the hydrolysis of the urea.
  • FIG. 1 Schematic view of a typical measuring flow chamber utilizing the thermostat here described.
  • An aluminum plate is in contact with the heating element, a Peltier cell; the temperature sensor is placed just under a micro-volume area (the region to thermostat) . Liquid flows in and out by means of two channels, and the measuring area is just nearby the sensitive layer (Si 3 N 4 ) of a silicon-based sensor placed in front of a cover slip.
  • FIG. 2 Control loop of the thermostat. User can set a target temperature T d , continuously compared with the measured temperature to generate an error signal e(t) .
  • Non linear control is accomplished by means of a "Bang-Bang" system which drives the Peltier unit either with full power heating or with full power cooling.
  • a temperature sensor closes the loop, and gives the actual temperature value.
  • Figure 3 Theoretical temperature signal vs. time, as derived from equations 7, 10 and 11 (see text) .
  • the figure also contains a plot of the input square wave u(t) and of h(t), as expressed in the mentioned formulas. All values are in arbitrary units, and normalized between +1 and -1. Input sequence wave [u(t)], output signal [y(t)] and its deviation from input [h(t)] is shown.
  • FIGS 4A and 4B Hardware circuitry of the thermostat. Terminals referred to as V p should be connected to an external constant-current power supply, regulated for an output voltage as specified in the particular Peltier cell data sheets. The connection to a milli-voltmeter can be also used to interface the circuit to an A/D converter, in order to monitor temperature changes in time by a computer.
  • Figure 5 Computer acquired temperature signal as a function of time.
  • the acquisition interval corresponds to a complete semi- period of the (square-wave) control signal.
  • Target temperature was set to +37°C, and the system kept this value in about 3 minutes after system switch on.
  • a fit with the theoretical expression of y(t) is also shown; data and fitted curve are in considerably good agreement.
  • the control signal frequency was about 5.5 KHz and the resulting value of ⁇ was 22.1 ⁇ s . Variation of measured temperature vs. time for a Peltier cell, and fitting with theoretical expression is shown.
  • Figure 7 Relationship between produced H + moles and pH variations: a region is shown with a direct proportionality between n and ⁇ pH, where a fitting straight line is also shown.
  • Figure 9 A typical experiment for the determination of enzyme concentration. Three different zones are visible: a first constant region, a small step and an exponential-like curve; they all are due to the particular configuration of the measuring system, as described in the text.
  • Figure 10 Output signal slope vs. YADH concentration; each data is calculated on the respective third (exponential) portion of the data presented in Figure 9, and the tangent is calculated at the first valid point.
  • FIG. 11 Sensor output signal variation during the enzymatic reaction: the figure shows a typical result.
  • Figure 12 Output signal variation vs. EtOH concentrations; each point in the plot is measured as shown ' in Figure 11.
  • FIG. 13 schematic representation of the measuring system.
  • Cell medium flow is obtained by means of a peristaltic pump, which is connected to a measuring chamber, containing the transducer and a coverslip with cells; the cell medium flows in a very small region between transducer and coverslip.
  • the figure also shows a control electronic block, which provides the system with all the necessary driving signals.
  • Figure 14 output signal during ON-OFF acquisitions.
  • the pump is ON the pH in the measuring chamber is maintained constant, while switching OFF the pump yields to a net pH variation, due to the presence of acidic products in the micro environment.
  • Figure 15 a similar signal as in Figure 9 is here shown, relative to a single OFF time interval.
  • the signal is approximately linear in the considered region, and can be fitted with a straight line, whose slope represents the acidification rate.
  • Figure 17 two images of the 3T6 cell monolayer as usually appears before (A) and after (B) an acquisition session with the presented system. Images have been acquired by a CCD camera with an optical microscope interfaced with a Personal Computer.
  • Fig. 18 Schematic representation of the PAB system.
  • a central control unit drives and controls the solution flowing system and the measuring chambers containing the transducer; the control unit also dislogs with a Personal Computer, utilized to bias the device and acquire and process the experimental data, via appropriate AD/DA interfaces .
  • Fig. 19 Block diagram of the main electronics.
  • the LAPS is biased via a potentiostat and the current signal is converted to voltage, filtered and synchronously demodulated, before being digitized and computer acquired.
  • the figure also shows a digital output interface suitable to drive the flow circuit through an interface; in addition a temperature control scheme is provided.
  • Fig. 20 Schematic circuit of the synchronous demodulation technique utilized, me input signal, after a current to voltage conversion and a filtering, is multiplied by the reference signal coming from the LED driver; the output of these blocks are then filtered and amplified before being fed into the circuit for calculation of the modulus.
  • Fig. 21 Software organization of the PAB system.
  • a shell offers a suitable user interface, and allows parameters input, data display, saving and printing; the usual procedure consists in the acquisition of a complete characteristic curve (current versus bias potential) and in the consequent determination of the inflection point; our software then bias the device and continuously acquires the digitized signals.
  • Fig. 22 Schematic of a very simple measuring chamber; a well in the upper part confines the measuring solution in a region delimited by an 0-ring, directly pressed against the LAPS surface. Counter and Reference electrodes are dipped in the measuring solution from the top, while a LED confined in the lower part just near the back of the chip provides the light excitation.
  • Fig. 23 Measuring flow chamber suitable for enzymic experiments.
  • the lower side is quite similar to that of Fig. 22; the upper part contains an inlet channel towards a well able to contain a membrane; usually some biological specie is entrapped in the membrane at this level; the reaction products flow then in a small volume region near the sensitive surface, through a channel as tiny as possible, in order to reduce the delay time of the sensor response.
  • the solution flows away through an outlet channel, contacting the reference electrode.
  • Part A shows the immunoreaction scheme, where the antibody is fixed onto a substrate and the antigen with.enzyme is injected in the solution to compete with the free antigen; the binding of enzyme-linked molecules is detectable after the production of electrons.
  • Part B shows a typical acquisition curve (I/V characteristic) obtained utilizing Horse Radish Peroxidase as the labelling enzyme, 2,4 dichlorophenossyacetic acid (2,4-D) as antigen and a monoclonal antibody against the pesticide 2,4-D; the right-band curve is before binding, the leftmost after binding.
  • Fig. 25 Measuring flow chamber for cellular acidification on experiments.
  • the counter electrode is inserted inside the inlet channel, while the reference electrode is in the outlet.
  • the LAPS is separated from the cells immobilized onto a glass coverslip by a teflon spacer, whose thickness defines the micro volume of the measuring chamber.
  • Thermal control is achieved by a Peltier cell connected to an appropriate circuitry and fixed on the top of the chamber; heating is obtained near the cells by a metal cylinder (usually aluminum) .
  • the right part of the figure shows the top view of the gasket, utilized to prevent medium leakage, of the coverslip where cells are grown and of the teflon spacer, giving a indication of the flow circuit inside the "sandwich".
  • Fig. 26 Two-chips measuring chamber suitable for differential measurements.
  • the flowing solution appears at the first chip, then passes through a removable reaction chamber, before reaching a second chip.
  • both chip should be affected by the same noise, as the particular design ensures for both the same environmental conditions, hence a differential acquisition scheme detects only the true reaction signal.
  • Fig. 27 Schematic representations of the PAB system in a redox configuration.
  • the measuring chamber is shown in detail. Biasing of the transducer is achieved by a potentiosta , while signal measurements are performed by a synchronous demodulation technique.
  • Fig. 28 Different curves of chips with and without metal layer to a solution containing ImM Fe(II) , ImM Fe(III) and lOOmM sodium citrate buffer pH 6.0
  • Fig. 29a Biphasic response curves of a chip with a gold layer covering part of the sensing surface; buffered solution containing the redox pair in different ratios were used. From left to right the curves correspond to solutions of Fe(II) : Fe(III) ratios ranging from 1:100 to 100:1.
  • Fig. 29b Biphasic response curves of a chip with partial gold covering of its exposed surface to solutions at two different pH values : at left pH 6; at right pH 9.
  • Fig. 30 Bias potential values corresponding to the inflection points at different redox pair ratios plotted versus the logarithm of the ratios themselves for chips with gold and chromium layer.
  • Fig. 31 Variation of the logarithm of the redox pair concentration ratio when Fe(II) is oxidized to Fe(III) .
  • the experimental conditions are: reaction chamber volume 10 , 0.8mM initial Fe(II) concentration. 0.4mM initial Fe(III) concentration.
  • the curve can be approximated to a straight line in the y-interval between +0.3 and -0.3.
  • Fig. 32 Calibration curve for the HRP enzyme in solution.
  • the enzymatic activity units are here defined as moles of TMB oxidized per minute, corresponding, in turn, to half of the moles of Fe(II) converted to Fe(III) .
  • Fig. 33 Calibration curve for the HRP enzyme immobilized on activated membrane.
  • Fig. 34 Monitoring of Alcohol Dehydrogenase activity in redox configuration by the use of Diaphorase as auxiliary enzyme.
  • Fig. 35 Block diagram of the PAB system.
  • the transducer is biased by a potentiostat, and the alternating current photogenerated by LEDs is converter into voltage and amplified; after filtering, the RMS values of the signal are extracted by synchronous demodulation, and acquired by a DAC card into a Personal Computer.
  • a temperature control system based on a Peltier cell is provided, in order to stabilize the environmental conditions of the measurement.
  • Fig. 36 Schematic structure of the transducer-optical fibres system. The distance of adjacent light spots is related to the wafer thickness, because the main process in the signal generation is the diffusion of charge carries.
  • Part A represents a simple equivalent circuit of the three-electrode system; Zrc is the impedance between counter and reference electrodes, and Zwr is that between working and reference electrodes; changes in these impedances cause signal amplitude variations, as depicted in Part B.
  • the normalization procedure described in the text avoids the eventual artefacts due to the monitoring of such impedance variations.
  • Fig. 38 Schematic representation of a measuring flow chamber of the 2D PAB system. Part A is connected to B after the eventual insertion of some biological element (i.e. membrane) into the small volume region F. Medium flows from inlet C, passes through the sensing spots H and G, and exits through outlet E, after filing the electrodes region D. An array of optical fibres I is fixed at a given distance from the chip backside by the holder L.
  • some biological element i.e. membrane
  • Fig. 39 Hardware circuit for the optical fibres array selection.
  • the driving signal (usually a sine- or a square- wave) is fed to a single selected LED at a time, passing through the operational amplifier and the switching transistor.
  • the remaining blocks are used to digitally connect a desired LED to the drive electronics; in particular two 4-bit words are the row-column addressed for the CD 4028 multiplexers, which activate corresponding digital switches. (Drawing missing) .
  • Fig. 40 Schematic representation of the transducer sensing surface utilized in the presented experiments. Two gold spots have been evaporated onto the silicon nitride surface, and four light spots are created in order to obtain two redox sensitive and two pH sensitive sites.
  • Fig. 41 Typical acquisition results after the insertion of a Potassium Ferrocianide-Ferricianide solution into the measuring chamber. Data corresponding to coordinates (1,1) and (2,2) are relative to redox, while those corresponding to coordinates (1,2) and (2,1) refer to pH. (Drawing missing) .
  • Fig. 42 Series of measurements as the one shown in Fig. 41. Data from A to E show redox pair concentration variations range from 1:100 to 100;1 at pH 7; data from F to L range back from 100:1 to 1:100 at pH 9. It is evident that variations in the redox concentrations selectively affect the only redox-specific spots, while the pH variation is specifically sensed by the silicon nitride regions. (Drawing missing) .
  • Fig. 43 shows the dose/effect trend of ara-C on S-phase 3T6 cells acquired with PAB system
  • Fig. 44 shows a comparison between PAB results and results of the Trypan Blue Test
  • Fig. 45 shows rat hepatocyte acidification curves acquired with PAB.
  • Fig. 46 shows monolayer surface density v. surface pressure obtained by means of nanogravimetry
  • Fig. 47 shows pH variation due to urease activity monitored by means of PAB system: the enzyme was immobilized on silanized glass: the regression line helps in evaluating the initial alcalinization rate of the reaction; and
  • Fig. 48 shows activity of urease monolayer immobilized of silanized glass during repeated assays (Deposition Pressure 20mN/m) .
  • the system utilizes a flow chamber 1, as depicted in Figure 1; a silicon chip 2 is put in front of a cover slip 3, and the medium flows in between.
  • the cover slip (containing cells) occupies a well 4 in an aluminium plate 5, which is in contact with the heating element (the Peltier cell) 6.
  • a temperature sensor 7 (AD 590, by Analog Devices) is placed just under the cover slip 3.
  • the other side of the Peltier cell 6 is connected to a convection heat sink 8.
  • Another typical control strategy utilizes a PID (Proportional Integral Derivative) algorithm; in the latter case the control signal is generated by adding three terms, one proportional, the second related to the first derivative and the last related to the integral of the error signal.
  • PID Proportional Integral Derivative
  • the Bang-Bang control technique is much easier and immediate than the PID one, as it is not necessary to compute (either by software or by hardware) the proportionality factors at each control step.
  • an hardware solution for the control system (as the one here presented) only requires a comparator- based stage in the circuit.
  • the performance here requested thermal stability within 0.1°C around the desired temperature, and the latter ranging between +20°C and +45°C) can be fully obtained without a PID control.
  • the desired temperature T d can be manually set; it, is continuously subtracted from the actual measured temperature y(t) by a subtracter 10, hence generating an error signal e(t) used to set the desired temperature, while the two lateral 500 ⁇ variable resistors are trimmers used to set the minimum and maximum allowed temperature; in order to set these two values one should turn the potentiometer first completely to the left, then adjusting the left trimmer until the desired minimum value appears on the connected digital display; then one should turn the potentiometer completely to the right, and adjust the right trimmer for the maximum value.
  • the second signal to the comparator comes from the Operational Amplifier CA313024, used as a driver for the temperature sensor (AD 590) .
  • the 10K ⁇ trimmer on the right allows to adjust the driver in order to sense the correct temperature value; to set the temperature measuring section it is necessary to utilize at least a 0.1°C precision thermometer and to perform several steps of temperature monitoring near the two desired extremes with both methods, then adjusting the trimmer in order to read the true value on the milli-voltmeter.
  • a switch 25 connects either the pre-set desired temperature signal, or the actual measured temperature signal to a milli-voltmeter (digital display) ; usually one sets the switch to the T d position, selects the desired temperature, and then switch to the monitoring position in order to read the actual temperature while thermostating process is on.
  • the signal to the display can be directly connected (with no additional circuitry) to an A/D converter input, if computer monitoring of the temperature is needed.
  • the error e(t) is greater than 0 if the actual temperature is lower than the desired one, lower in the opposite case:
  • the signal e(t) is used as input to a non linear stage 11, which produces a control signal u(t), which, power-amplified 12, corresponds to the driving signal of the Peltier cell 6.
  • a control signal u(t) When the control signal u(t) is low, one side of the Peltier element 6 is heating, the opposite is cooling (and vice-versa when the control signal is high) ; fast transitions of the control signal allows the temperature control around the target value.
  • the feedback system is completed by a temperature measuring block 7. Temperature sensing is accomplished by using a temperature probe connected to an appropriate driving system, whose output is y(t) .
  • a typical set up as the one here described takes some time to reach a stable state corresponding to the desired temperature; the elapsed interval from the system power-up to the stable situation depends upon several factors: the selected current in the Peltier power supplying circuit, the temperature probe location with respect to the heating element, the type and shape of the heat sink, the system to thermostat.
  • the driving control signal is a square wave with a 50% duty cycle.
  • a is the semi-period of the square wave ranging between - 1 and +1
  • the first term depends on time through the exponential factor exp(-t/ ⁇ ) , and represents the initial transient response following the system power-on; this term expires with the time constant T.
  • the second term represents the steady-state portion of the response to the square wave, as its dependence on t is expressed by exponential terms like exp[- (t- na)/ ⁇ ] , related to the number of sweeps before the actual one.
  • the circuit implementing the Bang-Bang control here described is quite simple ( Figure 4) .
  • the heating-cooling technique is achieved by feeding the Peltier element 6 with a given current flowing in one sense or in the opposite one; this can be easily obtained by applying a fixed voltage V p to the unit in order to heat, and the opposite -V p in order to cool.
  • the circuitry of the control system is depicted in Figure 4.
  • the Peltier element 6 connected to the external power supply generating V p by means of power transistors pairs (2N3055) 13 used as switching elements; if the right pair or the left pair is on, current flows into the Peltier cells in one sense or in the opposite one, yielding the heating or cooling of a given cell side.
  • transistors 17,18 are used to drive red and green LEDs 19,20 indicating to the user when the system is heating and when it is cooling.
  • the non-linear control signal is generated by an AD 845 (Analog Devices) Operational Amplifier 21, here utilized as a zero- hysteresis comparator.
  • AD 845 Analog Devices Operational Amplifier 21
  • This integrated circuit is a suitable comparator because its slew-rate is quite high (100 V/ ⁇ s) .
  • the operational amplifier 21 continuously compares the two signals fed into its inverting and non-inverting input pins, hence generating a +V sat /-V sat output if the actual temperature is lower/higher than the desired one.
  • the central variable resistors 23 is a multi-turn potentiometer
  • the described thermostat has been realized and tested with a 3x3 cm, 65 W, Peltier cell.
  • the system was supplied by a commercial +.12 V power supply; in addition a selectable constan -current power supply was connected to the terminals V p in Figure 4, i.e. to the Peltier element through the transistors pairs.
  • this external power supply was 8.5 V, and 1.5 A.
  • the measuring chamber to thermostat is a plexiglass-aluminum cylinder of about 5 cm base diameter, and 5 cm high.
  • the Peltier cell 6 as connected to the aluminum part 5 depicted in Figure 1, and the system was tested at room temperature for target temperatures ranging between +20°C and +45°C. After switching on the thermostat, the output of the comparator is at high/low level depending upon the desired temperature as compared to the actual one; then a heating/cooling process starts, and no commutation arises until the temperature becomes higher/lower than the desired one. At this point a transition in the control system causes an inverse cooling/heating process with respect to the prior one (the heated side is now cooled and vice versa) .
  • the system reaches and keeps the target temperature.
  • the actual temperature has been computer- acquired, after the initial transient expired and the target temperature was kept; values have been fit with the theoretical expression of y(t) presented before.
  • Figure 5 shows the result; for a sake of clarity, the figure corresponds exactly to a low- level semi-period of the control signal square wave when the target temperature was set at 37°C.
  • the total semi-period is 90 ⁇ s, which implies a commutation frequency of the Bang-Bang control system of about 5.5 KHz, as can also be easily observed by connecting an oscilloscope to the comparator outpu ; the behaviour of the actual temperature vs. time has an exponential nature, as predicted by the expression of y(t) . Moreover, fitting with y(t) gives an estimation of T, which results to be about 22 ⁇ s.
  • the described thermostat can be easily applied in different research areas, when a precision temperature control is needed in a small environment; the application here reported makes use of a flow chamber, and the "sensitive region" to control is about 8x8 cm; by using bigger Peltier elements it is possible to control, by means of the very same circuitry, a bigger area and a different system.
  • PAB Potentiometric Alternating Biosensor
  • the enzymic reaction under consideration is that of YADH on NAD + and C 2 H 5 0H.
  • the transducer output signal is a photocurrent, converted to a voltage by a current to voltage converter with a usual gain of 10 6 ; this voltage is directly proportional to the actual pH of the solution in contact with the sensing area, see Figure 6.
  • the sensitivity ⁇ of the transducer corresponds to the bias voltage shift to apply to the system in order to get the same ⁇ after a pH unit step; to this sensitivity directly corresponds a y-axis sensitivity, which also depends upon the maximum curve slope ⁇ ; a variation ⁇ of the output signal can be expressed in terms of ⁇ pH, as follows:
  • the first portion of the curve can be approximated very well with a straight line, by means of a least squares fitting; the fitting line is also shown in Figure 7.
  • the portion utilized for the fitting corresponds to a (pH-pK a ) ranging from +0.3 to -0.5.
  • the total pH variation caused by YADH is comprised in the mentioned interval.
  • a buffer is preferably chosen with a pK a value very close to the optimal enzyme pH range, and with a molarity which is a good compromise between the need for a pH-controlled environment and the need for detection of small pH variations.
  • the enzyme reaction is monitored by the system depicted in Figure 8. It is divided into two main sections: a reaction chamber and a measuring chamber, with a working volume of about 10 ⁇ l, which contains the silicon transducer, its driving light source and two electrodes (a Pt counter electrode and a Calomel reference electrode (AMEL 303/scg/6J) ) connected to a potentiostat. The whole system is thermostated at 25°C.
  • the system is biased through a Personal Computer AT bus 80486 processor, by means of a digital-to-analog card directly connected to the potentiostat.
  • the photocurrent signal is derived from an ohmic contact on the chip back side, and it is converted into a voltage by a current- to-voltage amplifier; then it is fed into a commercial Lock-In Amplifier (EG&G mod. 5210) .
  • the output signal (previously referred to as T) is then computer acquired by means of a 16 bit analog-to-digital conversion card (National Instruments Mod. AT-MIO-16X) . Appropriate software written under National Instruments LabWindows ambient allows fast signal acquisition and processing.
  • ⁇ vs. bias potential curves (as depicted in Figure 6) have been acquired, in order to determine both the sensitivity of the actual chip and its maximum slope, necessary to define a system sensitivity in terms of ⁇ , as already stated.
  • the signal ⁇ is recorded vs. time at a fixed bias potential; the total acquisition time interval can be set by the user: for the experiments here reported it was always set to 22 min.
  • the YADH (Sigma, cat.n.A7011) concentration was varied between 1.4xlO “8 M and 7. lxlO "10 M, while the concentrations of substrates were kept constan .
  • formula (10) can be used to fit our experimental data, as shown in the previous Figure 9.
  • Figure 10 shows a set of experimental data at different YADH concentrations; each point is the slope of the tangent line to the exponential fitting function of the signal; fitting is performed only on the third (exponential) portion of the data, and the tangent is calculated at the first valid point.
  • the EtOH concentration was varied between 0.8 and 352 mM, and the same kind of acquisition (output signal vs. time) has been performed as previously described.
  • Figure 12 represents data values at different EtOH concentrations; each point in the plot is measured as described above, and as shown in Figure 11.
  • the biosensor here described has been tested to monitor enzyme reactions in order to detect both enzyme and substrate concentrations.
  • the sensor seems to be more accurate and useful for enzyme determination than for substrate detection, as appears from the presented results.
  • Figure 13 shows a schematic arrangement of the experimental set ⁇ up.
  • a coverslip with living cells is placed in close proximity to the transducer, in order to obtain a microvolume chamber: in this tiny environment it is possible to detect even the pH variation related to the cellular activity.
  • the experiments here reported have been carried out with a volume of about 16 ⁇ l, obtained by interposing a thin spacer (typically 50-250 ⁇ m thick) between coverslip and transducer.
  • a suitable hydraulic circuit based on an electronic-controlled pump, continuously refreshes the cell medium into the microvolume chamber. To ensure an optimal condition for the biological sample, both the nutrient medium and the measuring chamber have been thermostated at 37°C.
  • a light addressable sensor should be biased by an external voltage source a counter electrode.
  • a modulated light source shining on the silicon chip causes an alternating current (photocurrent) to flow in the circuit; the current is measured, usually after a conversion to voltage, a filtration and a rectification.
  • Output voltage (V out ) versus bias potential (V bias ) characteristics curves have a sigmoidal shape, as V out varies from a high level (corresponding to silicon inversion condition) , through a falling edge (corresponding to the depletion of majority carriers in the silicon) , to a low level (corresponding to silicon accumulation condition) .
  • V out versus V bias characteristics shift along the V bias axis accordingly with the pH variations of the solution in contact with the insulator surface and with the chip sensitivity.
  • Monitoring of the relative position, on the V bias axis, of the curve inflection point is a good method to follow pH variations.
  • Our transducer is biased by means of two electrodes (an Ag/AgCl reference electrode and a Pt counter electrode) connected to a potentiostat circuit; the modulation of light is obtained by driving an infrared LED with a sinusoidal waveform.
  • the output photocurrent is converted into voltage (with an usual gain of 106) and filtered.
  • a computer interfaced AD/DA acquisition card (National Instruments AT-MI0-16X) allows both to set the bias voltage and to acquire the output signal, by means of specific software programs.
  • the mouse fibroblast line 3T6 has been utilized in our study. All culturing was under standard conditions; 3T6 cells were in fact grown routinely in monolayer cultures at 37°C and 5% C0 2 ; culture medium consisted of RPMI 1640 (SIGMA, catalog number R- 6504) supplemented of antibiotic and 10% Fetal Calf Serum (Boheringher Mannhein, catalog number 210463) .
  • the transducer is usually prepared by cleaning the sensitive surface (Si 3 N 4 layer) with isopropanol and double distilled water to remove impurities. Then the wafer is placed into the measuring chamber together with the spacer and a coverslip with cells. After these preliminary steps, the medium starts to flow into the chamber, feeding the cells. At this stage it is necessary to acquire the I/O sigmoidal characteristic (V out versus V biaa ) in order to evaluate its inflection point and its slope ct; these parameters will be used to define the proportionality factor between the read voltage and the actual ⁇ pH, as will be shown.
  • a software program allows the user to select one of several acquisition procedures; in every acquisition session the system is biased at a value corresponding to the inflection point, and the V out signal is continuously recorded.
  • the peristaltic pump is automatically switched ON and OFF at desired fixed time intervals .
  • the acidification rate can be expressed as: pH _ ⁇ J ⁇ [pH units/sec] di)
  • ⁇ V out (expressed in mV) is the variation of V out during the considered time interval and ⁇ t is the considered time interval (expressed in sec) .
  • Acidification data are presented as plots of V out as a function of time; by means of the above formula, V out values can be easily converted into pH units.
  • the device finalized to detect the pH variations of a microenvironment containing living cells, represents a valid tool to monitor the metabolic activity and eventually the physiological and pathological alterations of the cells . Because this potentiometric system detects pH changes and not directly the number of H" effectively excreted, it is important to define the mathematical expression representing the relationship between the protons produced by the cells and the resulting pH variation in the given environment; the link between pH variations and number of protons is as follows:
  • ⁇ v is the buffer capacity of the cell medium
  • R is the proton generation rate in mol/sec
  • V is the volume in litres.
  • the informative portions of the plot of Figure 14 correspond to the OFF intervals.
  • the pH signal decreases linearly, and this variation, of the order of 10" 2 -10 "3 V/min (depending on the total acquisition time) , represents the change in the acidification rate during the experiments.
  • the decreasing in the acidification rate may be due to a slowing down of the metabolic activities.
  • the PAB system integrates mechanics, hardware and software aspects, and consists in a complete stand-alone device for biosensing purposes; the block diagram is visible in Fig.18. As it clearly appears, the system is driven by a computer; this choice allows continuous measurements in time, and the automatic recording of data. As the interfacing electronics is standard and based on a 16 bit AT-BUS, a normal Personal Computer can be utilized, without any particular hardware modification.
  • the computer "talks" with the electronics through a digital interface, opportunely studied to guarantee high speed and fast acquisitions; in particular an analog-to-digital board converts the measuring signal, while a digital-to-analog board gives the proper supply to bias the transducer; in addition a digital output card provides eight digital signals to be used as switches for external controls.
  • the main block is the central Control Unit, consisting of several cards devoted to a precise signal amplification and conditioning, to the rectification and finally to the control of several parts of the system.
  • This unit interfaces directly with the solution pumping system and with the measuring chambers.
  • the latter always contain one or more transducers, directly biased and interfaced to the control unit.
  • FIG. 19 A schematic block diagram of the electronics is shown in Fig. 19. Several circuits are necessary to drive the LAPS transducer, and to measure the output signal; in this respect, the amount of circuitry is bigger than in the case of ISFETs, where a few operational amplifiers can fully drive the transducer.
  • the picture shows the main blocks used to drive the transducer, but also some additional circuit devoted to the control of a pump and of the thermal regulation of the measuring environment.
  • the transducer is connected to a current-to-voltage converter; in fact, the measuring signal is an alternating current of the order of several micro amperes, usually generated by means of modulated light impinging the silicon surface.
  • the voltage-converted signal is then filtered and measured as RMS values; as the driving signal is available, our system utilizes a synchronous demodulation technique to recover the information.
  • a schematic diagram of this circuit is shown in Fig. 20.
  • both the filtered input and the reference signals are fed in two multipliers; the first output consists of the product of the input signal with the reference component in phase with it, while the second output is derived by the 90° shifted reference component.
  • the output of this block is then a DC signal corresponding to the RMS of the actual (alternating) current signal, in a suitable form to be computer acquired after digitalization.
  • the generally used procedure to detect these fluctuations of the signal with respect to the applied potential involves the determination of the bias voltage corresponding to the inflection point of the characteristic; this value can be successfully used as an indicator of the actual surface potential.
  • a suitable electronics must be provided in order to achieve rather fast voltage scans, and the corresponding signal acquisitions; then the inflection point can be computed either by hardware circuitry or by software.
  • the information can be retrieved only after a complete voltage scan, the global signal acquisition and the relative signal processing (usually involving the second derivative computation) ; the computational time needed to perform all the steps is the limiting factor in the acquisition velocity.
  • the first circuit allows a proper biasing and control of the electrolyte- transducer system; the input consists of the digitally- converted bias potential, coming from the computer settings; a controlling electrode (Pt wire) and a reference electrode (SCE) are connected to this block.
  • the functional feature of the potentiostat is to maintain a controlled potential among the two electrodes and the transducer, which is considered as the working electrode, regardless any impedance fluctuation among them.
  • Thermal control is a must for bi ⁇ sensing purposes; it is definitely necessary when a biological sample is utilized, as in the case of cells (to maintain the population in living conditions) or in the case of enzymes (to guarantee the good working temperature) .
  • temperature control is necessary because of the use of a silicon transducer, extremely sensitive to thermal variations.
  • a good thermostat system is necessary to obtain true reproducible results.
  • Our system is equipped with a temperature control system, consisting of a circuit implementing a non-linear control algorithm and whose heating/cooling element is a Peltier cell; of course, the setup depends upon the application and varies accordingly to the actual measuring chamber; in fact the Peltier cell is always fixed inside a chamber, together with a temperature sensor utilized in the feedback circuit.
  • a temperature control system consisting of a circuit implementing a non-linear control algorithm and whose heating/cooling element is a Peltier cell; of course, the setup depends upon the application and varies accordingly to the actual measuring chamber; in fact the Peltier cell is always fixed inside a chamber, together with a temperature sensor utilized in the feedback circuit.
  • the last block is the interface with the flowing circuit; a flow of a measuring solution, of a given compound (i.e. enzyme substrates) , or of cell medium is usually utilized in the standard experiments.
  • a flow of a measuring solution, of a given compound (i.e. enzyme substrates) , or of cell medium is usually utilized in the standard experiments.
  • Our system makes use of a peristaltic pump which allows the solutions to flow from a beaker to the measuring chamber; the pump is completely automated, in the sense that start and stop commands come from the computer. This allows a very precise flow control in time, which is for example very useful in acidification experiments.
  • the interface with the motor pump has been designed by means of opto-couplers, which allows a good controlling technique and avoid interference problems, as the motor and driving circuit grounds are physically separated.
  • the complete hardware system described above practically consists of an interface board to plug into a PC bus, a connection cable, and an external circuit connected to the pump, to the electrodes and to the measuring chambers.
  • FIG. 21 Another important portion of the system is the set of program to drive the circuitry, to acquire the data and to perform the necessary processing.
  • the block diagram of software is shown in Fig. 21; it represents the main procedures for a typical data acquisition session.
  • a shell provides a user-friendly interface, and allows both parameters input and a complete data acquisition control.
  • the acquisition procedure depends upon the particular experiment, but in general a surface potential measurement is requested versus time, either pH or redox; basically, one can even acquire simple characteristic curves, for instance to investigate some surface property or to test a particular sensitive layer; in this case the user connects a standard (static) measuring chamber, biases the transducer and records the corresponding current values. This procedure can be easily performed, as it is the basis for further and more complicate acquisitions.
  • the pH or redox detection method implies the acquisition of a characteristic curve (current signal versus bias voltage) ; then the program automatically computes the bias potential corresponding to the inflection point, which s a good indication of the actual surface potential.
  • V b ⁇ as the system is biased at the inflection point corresponding to the middle of a characteristic region which can be approximated by a straight line, as we have already proved (Adami et al. , 1992b) . Due to this fact, if a small surface potential variation occurs, the characteristic curve shift can be regarded as a line shift; then the current value corresponding to the previous bias voltage can be linearly related to the surface potential change, as it can be, first of all, linearly related to the transducer sensitivity.
  • the procedure is time consuming, but in most of cases it is applied only once; in fact the system is biased at the value just found, and the current signal is then acquired continuously, and related to the surface potential as previously stated.
  • This procedure allows an information detection every fraction of a second; hence data acquisition can be performed to monitor fast pH or redox changes.
  • the described method can be applied only when no impedance variation among the electrodes arises during the measurement; this can happen, for instance, if a membrane is pressed against the transducer.
  • an interrupt-based structure guarantees faster communication from/to the circuits, and allows a fast data acquisition.
  • an interrupt occurs every time a data conversion is performed, the control then passes to a specific routine which acquires the data and resets the program.
  • the actual temperature monitoring is also possible, by an additional AD related software.
  • the system can be equipped with a variety of measuring chambers; the purpose is to allow different experiments with the same device.
  • a standard static cell able to contain a solution in contact with the transducer; the system is depicted in Fig. 22.
  • the solution is confined by an O-ring pressed against the chip, and the two electrodes are dipped from the top.
  • This chamber is the simplest example, and it is useful for standard measurements; in addition it can be utilized for experiments on the transducer, as it can be thermostated and generally guarantees the maintaining of conditions constant in time.
  • Fig. 23 shows a flow cell suitable for enzymic applications; the solution flows through a reaction chamber where a membrane can be easily located; for instance, the membrane could be a PALL ImmunodyneTM for the immobilization of proteins (enzymes or antibodies) .
  • the picture also shows the transducer and the related light emitting diode; again the measuring volume is delimited by a small O-ring, and a Pt wire is placed just in the proximity of the sensitive surface; the reference electrode can be located in the outlet channel.
  • the membrane can also be placed in the small volume near the chip, but in this case the eventual remotion implies opening the entire chamber.
  • Fig. 9 shows a typical acquisition for the determination of enzyme concentration; the signal decay is due to the H* production after the reaction took place, yielding to an acidification of the solution in contact with the transducer. Curves as the previous one allow the calculation of calibration curves, as the one shown in Fig. 10, which evidence the good sensitivity of the system, since it was possible to discriminate enzyme concentrations of up to 10" 10 M.
  • FIG. 25 Another exciting application available to this system concerns measurements of extracellular acidification; hence, a specific micro-volume flow chamber able to contain a cell population has been designed and realised and is visible in Fig. 25.
  • One important feature of the chamber is the possibility to insert glass coverslips, routinely utilized by biologists; other methods imply fixing the cells inside a porous membrane, but this method is not as standardized as the usage of coverslips.
  • Another flexibility offered by our measuring chamber is the possibility to vary the microvolume, i.e. the distance between the transducer and the cells; this is accomplished by using different teflon spacers in between.
  • the set-up mechanism is very easy: the user should simply take the coverslip containing living cells and insert it into the appropriate guides visible in the figure; then one should insert the desired spacer (usually ranging between 50 and 400 ⁇ m) and close the chamber. It is quite necessary to thermostat the system a few minutes before starting this set-up procedure, in order to offer a good thermal condition to the cell population, and to start the medium flow immediately after the set-up is complete. In usual applications the entire procedure takes a few seconds to be carried out, hence no particular perturbation is caused to the cells, and quantitative measurements are possible.
  • a more recent application makes use of microorganisms; in this case and in many other applications it is preferable to have a quantitative method to take into account eventual noise or possible artefacts deriving from the particular biological setup; tins can be accomplished using a differential scheme.
  • the solution must utilize the above described electronics for both signals, in order to ensure the same electrical characteristics and the same gains for the two measurements.
  • a good solution consists of a digital-driven switch to alternatively select the two signals.
  • a related chamber has been designed and is shown in Fig.
  • Different measuring chambers can be connected to the same device rendering the system suitable for numerous applications: some chambers for standard solution measurements, a mini-volume chamber for enzymic applications, a micro-volume flow-through chamber for cellular experiments.
  • the latter has a completely new flow circuit which allows the realization of controlled and variable micro volumes, simply by inserting different Teflon spacers.
  • the PAB system can be thermostated by means of a purpose- designed temperature controlling system, based on a Peltier cell, which has an elevated precision and reliability.
  • a transducer whose surface is sensitive to many types of ion, and not only to hydrogen as in the case of silicon nitride transducers, may be used, for example, a gallium arsenide- based transducer; in addition to the GaAs property of a very high charge mobility, in fact, it is possible to build extremely sensitive layers by LB technology.
  • the transducer is essentially a heterostructure made of silicon ("n"- or » p » - type), silicon dioxide and silicon nitride (Sartore et al, 1992a; Sartore et al 1992b; Bousse et al. 1994) .
  • the insulator is pH-sensitive , due to the proton binding capacity of its groups (essentially Si-O and Si-NH 2 ) over a large pH range (2-12) , with an theoretical Nernstian response (if hysteresis and drift phenomena are not considered) .
  • Redox potential measurements can be obtained by the deposition of metal layers over the silicon nitride surface; when the solution in contact with the silicon structure contains a redox pair, the metal changes its potential toward a value stated by the Nerst equation and determined by the ratio of the concentrations of the solution species (Bard et al, 1980) .
  • This process is due to the initial differences in the electron affinities of the two phases that are put in contact, the meal layer and the electrolyte solution; from this interfacial discontinuity an exchange of electrons between metal and solution originates and continues until the resulting potential change reaches the equilibrium point, i.e. the electron affinities of the two conductive phases are equal. So the metal, in some way, adequates its characteristics to the solution and reflects the peculiarity of the liquid phase. By our configuration we measure a signal that responds to changes in redox potential of the electrolyte.
  • the sinusoidally-modulated LED illuminates the back-side of this modified structure, it produces an alternating photocurrent: its shape depends on the ratio between the metal layer size and the light spot size. If the light source illuminate both a silicon nitride region and a metal region, a biphasic response is obtained, due to the different surface potentials for the two zones (see Fig. 28, curve B) . In this case, an infrared LED illuminates an area that is, approximately, in an equal percent, silicon nitride and metal layer in contact with the electrolyte solution.
  • the liquid phase contains ImM Potassium Ferricyanide, ImM Potassium Ferrocyanide and lOOmM sodium citrate buffer, pH 6.
  • the second reaction is spontaneously occurring in the presence of the redox pair.
  • the pFe depends, as defined, on the conversion of Fe(II) into Fe(III); a theoretical prediction of the pFe behaviour can be estimated taking into account the concentrations and the buffering capacity of the solutions; the theoretical approach already published in the case of ADH experiments by Adami et al 1994a can be full applied also in this case.
  • Fig. 31 The pFe dependence on the conversation of Fe(II) into Fe(III) is shown in Fig. 31; in the interval between +0.3 and -0.3 of the pFe axis the curve can be well approximated with a straight line: its slope is the proportionality factor between the pFe variations and n, the number of nanomoles of Fe transformed.
  • the enzymatic activity can be expressed in units defined as micromoles of TMB oxidized per minute, equivalent to half of the micromoles of Fe converted. So, it is possible to correlate the output signal of the sensor with the enzymatic concentration, if the specific enzymatic activity is known (Bousse et al, 1992; Adami et al 1994b) .
  • the calibration curve for the HRP enzyme in solution has been obtained, as shown in Fig. 32: the lowest detection limit in enzyme concentration is 5X10 "11 M.
  • the enzymatic activity determined with the procedure above described is in good agreement with the value coming from usual spectrophotometric assays.
  • it is essential to determine the activity of the immobilized enzyme.
  • the same configuration as depicted in Fig. 27 has been used, by simply inserting in the measuring chamber a membrane (Pall Biodyne B) of about 4x4 mm on which the enzyme has been immobilized.
  • the relative calibration curve has been obtained, as shown in Fig. 33: the lowest detection limit, in this case, is of about 2.5xl0 "8 M.
  • the loss of activity due to the immobilization is considerable but does not prevent from the applicability of this system to an immunoenzymatic assay.
  • Diaphorase an auxiliary enzyme, Diaphorase, which can reduce Fe(III) in the presence of NADH
  • Fig. 34 shows the transducer output signal vs. time; this is the basic data by which calibration curves are determined, and shows the signal variation during the enzymatic reaction.
  • the detection of the ADH activity in a redox configuration presents several advantages with respect to a pH sensitive configuration, namely: i) the membrane buffer capacity does not interfere with the redox measurements; ii) a high buffer concentration can be used, stabilizing the enzyme activity; iii) thermodynamic equilibrium is not reached because of coenzyme recycling. 2 Dimensional PAB System
  • FIG. 1 A block diagram of a bidimensional PAB system is shown in Figure 1.
  • PAB Patentiometric Alternating Biosensor
  • the transducer consists of a light-addressable silicon chip, which provides regions properly modified and functionalized to yield sensitivity to either pH or redox potentials.
  • the system is electronically controlled and driven, and it is connected to a common personal Computer; ad hoc software drives an array of light sources, allowing 2D signal acquisition and processing up to the production of response images or 2D histograms.
  • the system is here utilized with a specific measuring chamber to monitor biological events related to in vivo cell metabolism. Several other chambers have been designed to monitor different phenomena such as enzymatic activity (Y-ADH, Urease, HRP) and antigen-antibody binding.
  • the system can provide multiparameter information related to the specific distinct local modifications made on the sensing surface of the transducer.
  • the quantity which strongly affects the choice of dimensions in the design of this 2D system is the minority carriers diffusion length, defined as:
  • D is the diffusion coefficient and ⁇ is the minority carriers lifetime; in particular depends upon the chip used and strongly varies from wafer to wafer (Bousse et al 1994 and Sartore et al 1993) .
  • the charge carriers can diffuse rapidly and can reach positions quite far from the generation area; this effect is undesired, as it affects the spatial resolution.
  • the signal acquired in a certain area is inevitably conditioned by the surface potential of adjacent zones; in the case of a multisensor, this can mean for example that a pH measurement can be affected by the redox potential of an area close to the pH sensitive one.
  • a preliminary setup was based on a single optical fibre scanned along the chip backside; at each step position a new value was acquired; this setup has the advantage of a uniform optical excitation of the chip, as the light source maintains the same features even if moved across the transducer; every acquired signal can be directly related to the other ones because all of them derive from the very same light excitation.
  • This setup has the big disadvantage to need an XY mover, which can cause displacement errors and increase the total cost of the device; this solution also requires specific hardware and software to drive at least two movers.
  • the final version of the system is based on an optical fibre array fixed in the close proximity of the transducer backside; each fibre is addressed at a given time interval and the corresponding surface potential is measured.
  • any "true” data is the ratio between the inflection point value and the maximum one; this represents an easy way to overcome the above mentioned underived effects.
  • the design of a suitable measuring chamber for the 2D system combines the need of a relatively wide sensing area (with different sensing spots) and the corresponding backside positioning of the optical fibres array; in this sense a big effort should be spent in order to obtain a perfect alignment between the sensing surface spots and the corresponding fibres.
  • the chamber design allows the positioning of membranes in the close proximity of the sensing surface, and a cell population can either be grown directly on the chip or on a cover slip fixed in front of the transducer, at a very small distance (usually 50-200 ⁇ m) . With these peculiarities the chamber directly allows biosensing applications, such as enzymatic or cellular measurements.
  • FIG. 38 A schematic of the flow chamber is presented Fig. 38.
  • the chamber is made of two parts (indicated A and B) to be connected after the eventual insertion of a biological layer in the microvolume region F.
  • a medium flow comes from inlet C and fills the measuring chamber F, allowing a multidetection through the sensing sites H, formed onto the transducer surface G.
  • the medium flow enters the area indicated with D, where both a counter and a reference electrode can be placed (alternatively the counter electrode can be accommodated even within the inlet connection) .
  • An array of optical fibres I is fixed at a predetermined distance from the transducer backside by an attachment L; this array is connected on the opposite side to a corresponding array of infrared LEDs (Light Emitting Diodes), driven by a multiplexing hardware.
  • infrared LEDs Light Emitting Diodes
  • the driving electronics of the 2D system requires a single light modulating source and two 4-bit (or one 8-bit) digital ports.
  • each fibre is connected to a LED which is addressed digitally.
  • the modulating signal is fed into a switching transistor through an operational amplifier, in order to ensure the proper current to the LEDs without affecting the local oscillator.
  • the driving transistor is connected to a series of digitally-controlled switches, in order to address a single LED at a time.
  • the selection of a given light emitting diode is performed by sending the corresponding digital address to a couple of multiplexers, namely the CD 4028.
  • a couple of multiplexers namely the CD 4028.
  • Up to 10 rows and 10 columns can be addressed by the proposed scheme, which can be connected either to a serial port (by an appropriate interface) or to a DIO (Digital Input Output) card of a common Personal Computer.
  • DIO Digital Input Output
  • the driving software can be easily integrated within the single-channel acquisition program already described (Adami et al, 1994B) ; in fact it is necessary to repeat acquisitions at the different sensing locations.
  • the program Before acquiring the signal, the program sends two 4-bit words to the hardware circuitry in order to select a single LED, so enabling a given sensing spot; then the corresponding data is acquired, and the process re-starts.
  • a bidimensional pattern has been acquired at different measuring conditions.
  • a Si/Si0 2 /Si 3 N 4 chip has been partially covered with gold in two distinct areas; the gold was evaporated onto the chip by means of a Balzer M10 metal evaporator; for a better results, a thin layer of Chromium was previously deposited onto the silicon nitride.
  • the chip has been addressed by 4 optical fibres focused on the transducer backside at locations corresponding to the front side sensing regions; the surfacial pattern of the chip is visible in Fig. 40.
  • the chip was inserted into the measuring chamber, and then the measuring solutions were injected. Solutions containing different ratios of Potassium Ferrocyanide and Potassium Ferricyanide (namely 1:100, 1:10, 1:1, 10:1, 100:1) at pH 7 and 9 have been prepared.
  • Fig. 41 shows a typical acquisition when a solution containing the redox pair in a ratio of 1:10 in concentration of pH 7 is injected into the measuring chamber; the "bar chart" directly corresponds to the chip shape shown in the schematics of Fig. 40, i.e. the higher bars, whose coordinates are (1,1) and (2,2), are relative to the gold spots (redox measurements) and the lower bars, whose coordinates are (1,2) and (2,1), refer to the Si 3 N 4 sites (pH measurements) .
  • Fig. 41 shows the possibility to obtain bidimensional patterns relative to different sensitivities; one can very either the pH or the redox compounds ratio, causing a variation only of the specific local signal.
  • Fig. 42 is a collection of 2D patterns of the same type of the previous Fig. 41, varying both pH and redox pair concentrations; in particular ten 2D patterns are shown and indicated with letters A through L; patterns A to E refer to pH 7 solutions at redox pair ratios range from 1:100 to 100:1, while patterns F to L refer to pH 9 solutions at the same redox pair ratios, precisely ranging back from 100:1 to 1:100; it is possible to observe a very specific variation corresponding to a change in the measuring solution.
  • Biosensor for In Vitro Drug Screening Toxicitv Testing in Cancerous Hepatocytes
  • the Potentiometric Alternating Biosensor (PAB) system has been utilized to monitor the effects of two antineoplastic drugs, Cytosine arabinoside and Mitoxantrone, on two distinct cell lines, namely on established cell line (3T6 mouse fibroblast) in different phases of cell cycle and primary culture (rat hepatocytes) in resting GO cells.
  • PAB Potentiometric Alternating Biosensor
  • An ad hoc microvolume flow chamber has been designed and produced; the chamber is equipped with inlet and outlet circuits and with a fixed transducer (Si/Si0 2 /Si 3 N 4 chip) , facing a cover slip on which cells grow; the transducer allows monitoring of pH in the microenvironment where the cells are placed; the system is used in the pH-sensitive configuration and a single measuring spot has been used for the experiments, warranting an accurate determination of the change in the extracellular acidification rate resulting from drug administration.
  • a fixed transducer Si/Si0 2 /Si 3 N 4 chip
  • New insights into normal vs. abnormal cell growth and differentiation and into in vitro drug toxicity and efficacy can be obtained with the presented system, towards effective human cancer treatment.
  • the effect of increasing drug concentrations on cellular metabolism is here compared with the results coming from conventional tests (optical microscopy, Neutral Red and Trypan Blue assays) .
  • hepatocarcinomas which includes drugs as mitoxantrone, adriamycin and cis-platinum.
  • the cellular line chosen for experiment with Ara-C is 3T6 (Swiss albino mouse embryo, fibroblast) purchased from ATCC (American Type Culture Collection) . Conventional culture procedures have been followed using culture medium RPMI 1640 (Sigma) .
  • the cells have been plated onto a glass support.
  • the glass supports have been previously treated with collagen (Sigma C3511) .
  • rat hepatocytes The mitoxantrone toxicity tests, we have chosen primary cultures of rat hepatocytes because they are easy to obtain and, like all hepatocytes, they maintain, in the first hours in vitro, their metabolic skills practically unchanged with respect to the in vivo situation. Hepatocytes were isolated from liver of Sprague-Dawley albino rats (200-250g) by in situ collagenase perfusion according to Williams (1977) .
  • Isolated cells were suspended in Williams E. Medium (WME) , supplemented with 10% fetal bovine serum and genamicin (50 g/ml) at the concentration at 5xl0 5 hepatocytes/ml. Aliquots of this suspension were plated as follows: a) lxlO 5 cells were plated on the glass support (coated with collagen) for the measurement with PAB; b) 6x10 s cells were plated on 35 mm dishes, coated with collagen, for conventional toxicity tests.
  • Ara-C is a synthetic nucleotide, which differs from the natural ones (cytidine and deoxycytidine) for substitution of ribose and deoxyribose with arabinose.
  • Ara-C is principally used to induce regression of the acute proliferation hemopaties of the granulocytic series in the adult.
  • the secondary application is in the other proliferative forms of the leucocytes series in both the adult and child.
  • Mitoxantrone is a potent inhibitor of RNA and DNA synthesis. It intercalates on DNA, inducing cross links intra- and inter-strand, especially on GC base pairs. It interacts with the cellular membranes changing their functions.
  • the recommended administration route is the intravenous one. According to the autopic observations, the largest residual amount of mitoxantrone can be found in liver. In fact, in addition to renal excretion, the hepatobiliar pathway is largely involved in drug removal. There are evidences that the drug undergoes hepatic metabolisation; in fact four different metabolites have been isolated from urine.
  • the assay times are the following:
  • the tests have been carried out on primary culture of rat hepatocytes and on HEPG2 cells, with exposure times of 30 minutes and of 30 minutes + 20 hours of incubation in absence of drug, with doses of 2.5 - 5 - 10 - 20 - 40 - 80 g/ml for hepatocytes and 5 - 10 - 20 g/ml for HEPG2 cells (dosage selected after the tests on hepatocytes) .
  • Exposure time 30 minutes, cell line: rat hepatocytes; the cells showed a continuous distribution and a polygonal form similar to the one of the controls at doses 2.5 - 5 - 10 and 20 g/ml, even if a certain detachment of cells was observed with increasing doses.
  • the cells were losing the polygonal form and looked damaged, while at 80 g/ml the appearance of the cells was better: the phenomenon could be attributed to an edema of the cells caused by loss of normal permeability.
  • Exposure time 30 minutes, cell line: HEPG2; cells did not reach confluence, not even in checking dishes; cell morphology was comparable at all doses even if, increasing the dose (especially at 20 g/ml) , we could observe several detached cells, especially among the piled groups (major exposure to chemotherapeutic) .
  • Exposure time 30 minutes + 20 hours, cell line: HEPG2; all the cultures appeared suffering compared to the control; we have observed a maximum cell detachment at a dose of 10 g/ml.
  • the cells presented several cytoplasmic vacuoles and extended cytoplasm.
  • the still viable hepatocytes showed, at all doses, to have incorporated mitoxantrone, in order to metabolise it.
  • the measurements with PAB are based on the monitoring of extracellular acidification; the relationship between production of acidic metabolites and rate of extracellular acidification is the following: dn dt
  • dn/dt is the generation rate of H + ions, due to acidic dissociation of excreted metabolites
  • ⁇ is the buffering capacity of the examined solution
  • V is the volume of the chamber; in order to improve the sensitivity of the method, we can lower both volume and buffering capacity of the solution.
  • d out dpH dt dt S o.
  • S is the sensitivity (50mV/pH, in our case) and a is the slope of the I-V characteristic curve of the transducer.
  • Figure 43 shows the relationship between extracellular acidification of 3T6 and drug (Ara-C) dose: low doses give no evident effect on the cell population while, at doses greater than 40 ⁇ g/ml, the action of the antimetabolite Ara-C on the cells synchronized is evident and consists of a linear decrease in extracellular acidification rates.
  • Figure 45 shows some of the acidification data acquired with PAB system on rat hepatocytes (30 min + 20 h treatment) .
  • a remarkable feature of PAB system is the possibility to monitor in continuum the effect of a drug on a cell population, while other tests measure only the end point of an experiment (i.e. the death of cells) .
  • Table III Trypan Blue exclusion test of HEPG2 hepatoma cell line exposed to mitoxantrone.
  • the scheme of the PAB system is shown in figures 1, 8, 27 and 35.
  • a sinusoidally-modulated IR radiation lights the back side of the sensor chip a sinusoidal photocurrent is obtained, and its profile mainly depends on the chemical reaction which takes place in the measuring chamber.
  • the urease (Urea aminohydrolase, 61000 unit per gr.) from jack beans was purchased from Sigma and the 3- glycidoxypropyltriethoxysilane (GOPTS) from Aldrich.
  • the monolayer of urease was formed spreading 0.2 ml of 1 mg/ml enzyme solution at the water-air interface of the Langmuir- Blodgett trough (MDT Corp- Russia) whose dimensions are 120x240x30 mm. Carbonate buffer (pH 8.6) was used as subphase.
  • the vacuum silanization of substrate with GOPTS is based on the method developed by M. Malmquist et al, 1989. We activated both the glass and then the silicon nitride surface of the transducer in order to choose the best measuring configuration.
  • the enzyme monolayer was transferred onto the activated support by Langmuir-Schaefer technique. After the incubation at temperature of 4°C for 4 hours the monolayer was washed by water flow and dried with nitrogen. The obtained enzyme monolayer was characterized by measuring the thickness and the surface density by means of ellipsometric and nanograviometric technique respectively. Characterization of monolayer
  • the thickness of the monolayer measured was about 45 A, that also corresponds, in order of magnitude, to the dimensions of the urease molecule. Therefore, both these measurements confirmed that the obtained monolayer was densely packed.
  • Figure 47 shows one of the curves acquired.
  • the pump When the pump is ON the products of the enzymic reaction flow away from the chamber and the dynamic equilibrium condition is reached: in this way the pH is nearly constant (first part of the curve) .
  • the pump When the pump is OFF, the flow stops and the products of the reaction accumulate in a standing volume: this causes an increase of the pH (second part of the curve) .
  • the "initial alcalinization rates" as the slope of the regression line in the first relevant points of the enzymatic reaction curve (see Figure 47) . By repeating several times the measurements, always in the same conditions, it was possible to evaluate the stability of the monolayer.
  • LAPS light-addressable potentiometric sensor
  • PAB a newly designed potentiometric alternating biosensor system, Biosensors and Bioelectronics, in press.
  • Nicolini, C. (1975) The discrete phases of the cell cycle: autoradiographic, physical and chemical evidences, Journal National Cancer Inst., 55, 821.
  • Nicolini, C. (1976) The principles and methods of cell synchronization in cancer chemotherapy. Biochemica Biophysica Acta, 458, 243.
  • Gavazzo P. Paddeu S., Sartore M. and Nicolini C. "Study of the relationship between extra cellular acidification and cell viability by a silicon based sensor” . Sensors and Actuators (1993, in press) .
  • Nicolini C. Adami, M. Zunino M. and Sartore, M. "A newly designed silicon-based cell biosensor for in vivo drug screening. Part 1: a 2D system for simultaneous pH and redox determination" .
  • LAPS light addressable potentiometric sensor

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Abstract

La présente invention se rapporte généralement à un système de biocapteur potentiométrique alternatif (PAB) utilisant un transducteur sur puce de silicium adressable par stimulation lumineuse pour mesurer des paramètres tels que le pH et le potentiel redox. Dans un premier aspect, l'invention se rapporte à la régulation de température d'un système PAB. Dans un second aspect, elle se rapporte à un biocapteur permettant de mesurer les variations du pH et/ou du potentiel redox au cours de réactions enzymatiques associées à des cellules. Dans un troisième aspect, l'invention concerne un biocapteur de potentiel redox à transducteur de silicium, sur au moins une partie de la surface duquel a été déposée une couche métallique, ainsi qu'à son utilisation dans un système PAB afin de surveiller des enzymes catalysant les réactions redox. Dans un quatrième aspect, un système PAB à deux dimensions apte à mesurer simultanément le pH et le potentiel redox est décrit. Dans un cinquième aspect, l'invention porte sur l'utilisation d'un système PAB pour mesurer l'acidification extracellulaire en vue de surveiller le métabolisme cellulaire, ainsi que pour mettre à l'essai la toxicité/l'activité anticancéreuse de composés par rapport à des cellules. Dans un sixième aspect, l'invention concerne un système PAB susceptible d'agir comme un immunocapteur au cours d'un dosage immunologique en vertu de l'application d'une monocouche immobilisée d'une enzyme sur le transducteur.
EP95901567A 1993-11-25 1994-11-25 Biocapteurs potentiometriques, systeme de commande de ces derniers et leurs applications Withdrawn EP0730760A1 (fr)

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GB9324258 1993-11-25
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ITRM930846 1993-12-22
IT93RM000846A IT1266466B1 (it) 1993-12-22 1993-12-22 Dispositivo per il controllo automatico della temperatura.
GB9411072 1994-06-02
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GB9411072A GB9411072D0 (en) 1994-06-02 1994-06-02 Biosensor and its use
GB9411059A GB9411059D0 (en) 1994-06-02 1994-06-02 Biosensor and its use
GB9414189 1994-07-14
GB9414189A GB9414189D0 (en) 1994-07-14 1994-07-14 Sensor
PCT/IB1994/000408 WO1995014962A1 (fr) 1993-11-25 1994-11-25 Biocapteurs potentiometriques, systeme de commande de ces derniers et leurs applications

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US5972715A (en) * 1996-12-23 1999-10-26 Bayer Corporation Use of thermochromic liquid crystals in reflectometry based diagnostic methods
US6780296B1 (en) 1999-12-23 2004-08-24 Roche Diagnostics Corporation Thermally conductive sensor
DE10004642C2 (de) * 2000-02-03 2003-11-20 Danfoss Analytical As Sonderbo Biologischer Mikrosensor
DE10052670A1 (de) * 2000-10-24 2002-05-08 Forschungszentrum Juelich Gmbh Meßanordnung zum Nachweis einer ein- oder mehrdimensionalen Verteilung einer chemischen oder biochemischen Komponente
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US7947223B2 (en) 2005-10-31 2011-05-24 Senzime Ab Biosensor apparatus for detection of thermal flow
DE102009010639B4 (de) * 2009-02-26 2020-07-02 Boehringer Ingelheim Vetmedica Gmbh Verfahren und Anordnung zur Inhibierung einer chemischen Reaktion von Substanzen in einer Flüssigkeit vor einer Messung
CN108344714B (zh) * 2018-01-16 2020-07-31 东南大学 基于有序多孔纳米结构薄膜干涉效应的生物检测仪及其进行生物分子检测的方法
IT202100015608A1 (it) * 2021-06-15 2021-09-15 Claudio Gianotti Dispositivo termoelettrico per il raggiungimento della normotermia umana

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US4020830A (en) * 1975-03-12 1977-05-03 The University Of Utah Selective chemical sensitive FET transducers
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