US20110210016A1

US20110210016A1 - Electrochemical potentometric sensing

Info

Publication number: US20110210016A1
Application number: US13/061,091
Authority: US
Inventors: Matthias Merz
Original assignee: NXP BV
Current assignee: Morgan Stanley Senior Funding Inc
Priority date: 2008-08-25
Filing date: 2009-08-24
Publication date: 2011-09-01
Also published as: WO2010023611A1; US9006738B2; US20110156177A1; EP2321634A1; CN102132152A; CN102132153B; EP2335061B1; WO2010023610A1; EP2321635A1; US8801917B2; WO2010023569A1; EP2335061A1; CN102132154A; US20110208457A1; CN102132153A

Abstract

The invention relates to a method of determining a charged particle concentration in an analyte (100), the method comprising steps of: i) determining at least two measurement points of a surface-potential versus interface-temperature curve (c1, c2, c3, c4), wherein the interface temperature is defined as a temperature of the interface between a measurement electrode and the analyte (100), wherein the surface-potential is defined at the interface, and ii) calculating the charged particle concentration from locations of the at least two measurement points of said curve (c1, c2, c3, c4).This method, which still is a potentiometric electrochemical measurement, exploits the temperature dependency of a surface-potential of a measurement electrode. The invention further provides an electrochemical sensor and electrochemical sensor system for determining a charged particle concentration in an analyte. The invention also provides various sensors which can be used to determine the charged particle concentration, i.e. EGFET's and EIS capacitors.

Description

FIELD OF THE INVENTION

The invention relates to an electrochemical sensor for determining a charged particle concentration in an analyte, a semiconductor device comprising such sensor, an RF-ID tag comprising such sensor, and to an electrochemical sensor system for determining a charged particle concentration in an analyte. The invention further relates to a method of determining a charged particle concentration in an analyte.

BACKGROUND OF THE INVENTION

The pH-value is an integral parameter of every (aqueous) solution. It describes to which degree the solution is alkaline or acidic. Over a wide range it is well approximated by: pH=−log [H⁺], wherein [H⁺] denotes the hydrogen ion concentration of the solution in mol/L. Measuring a pH-value of an aqueous solution is a routine task in the industry and also in laboratories for process control and analysis. However, it could also become interesting for a wider range of applications if the pH-measurement units (sensor plus electronics) become sufficiently inexpensive. For example, there is a large potential for pH-measurement to monitor the quality of (liquid) perishables in the supply chain or even at the customer's himself. Experimental techniques for measuring ion concentrations (in particular pH) can be divided into two classes, non-electrochemical methods, e.g. optical (indicator dyes), catalytic, and swelling of polymers (gels), and electrochemical methods. The latter are widely used for many applications in industry and laboratories. Electrochemical ion concentration sensors rely on the potentiometric principle, i.e. they measure the electrical potential φ at a solid/liquid interface or across a membrane which is a function of the ion concentration to be determined. φ can be calculated from the Nernst equation: φ=kT/(nq) In(a₁/a₂), wherein k is the Boltzmann constant, T the absolute temperature in Kelvin, q the elementary charge, n the ionic charge (e.g., n=1 for H₃O⁺, Na⁺; n=2 for Ca²⁺), and a₁,a₂the respective activities at both sides of the membrane/interface.
Ion concentrations at both sides of the membrane/interface (1 and 2) are represented in terms of activities a_i=f_i*c_iwith f_ibeing the respective activity coefficient (f_i=1 for diluted electrolytes) and c_ithe respective ion concentration. According to the Nernst equation the electrode potential is a logarithmic function of the ion activity on one side of the membrane/interface if the activity on the other side is kept constant. Depending on the type of ion described by “a”, the sensor is sensitive to H₃O⁺-ions, Na⁺-ions, Ca²⁺-ions, etc.
All major pH (ion) measurement electrodes operate according to the principle described above, including the well-known glass electrodes (different glass compositions have been developed that are sensitive to pH, pNa, pK, etc., respectively), antimony electrodes, ISFET's (Ion Sensitive Field Effect Transistors) and EIS capacitors (Electrolyte Insulator Semiconductor capacitors; here the flat-band voltage is a function of the pH/pNa/pK/etc of the electrolyte).
In order to measure a potential difference, i.e. voltage, a reference electrode is needed; for the ISFETS and EIS devices the reference electrode also defines the electrolyte potential to set the operating point or do a capacitance voltage (C-V) measurement. The potential of the reference electrode with respect to the electrolyte potential must remain constant irrespective of the electrolyte composition. Besides the standard hydrogen electrode the Ag/AgCl electrode is the most well-known reference electrode. It consists of a chlorinated silver wire in contact with a well defined electrolyte (often 3 mol/L KCl). Galvanic contact between the analyte and the electrolyte is established via a diaphragm, such as a porous frit from glass or ceramics. During operation the electrolyte must continuously flow out of the reference electrode into the analyte. Other reference electrodes, e.g. calomel (based on mercury) or Tl/TlCl electrodes, are used for specific applications, e.g. at elevated temperatures. Their principle is the same as for the Ag/AgCl electrode, in particular with respect to the use of liquid electrolyte and contact via a diaphragm.
The problem with the known electrochemical sensors is that they require an accurate reference electrode with a reference analyte in order to determine the charged particle concentration from a measured potential (difference). Using reference electrodes, and in particular accurate reference electrodes, involves all kinds of difficulties such as the following:

- Electrolyte outflow in a reference electrode through the diaphragm is essential. That means the electrolyte needs to be refilled regularly. Moreover, the pressure conditions must be such that the outflow is guaranteed, i.e. the pressure in the analyte cannot be higher than in the reference electrode (otherwise the analyte enters the reference electrode and changes its potential, which is called reference electrode poisoning);
- Clogging of the diaphragm of the reference electrode causes measurement errors (depending on the application regular cleaning is needed);
- Most reference electrodes have rather large dimensions, which makes it difficult/impossible to integrate them into a miniaturized device. Some miniature reference electrodes exist but they have a limited lifetime (because reference electrolyte cannot be refilled);
- Reference electrodes have a limited temperature range, e.g., for high temperatures a Tl/TlCl electrode must be used; and
- Some reference electrodes may react to other environmental parameters, for example, the silver in Ag/AgCl electrodes is light sensitive.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an electrochemical sensor for determining a charged particle concentration, which does not require a conventional reference electrode which produces a known potential.
The invention is defined by the independent claims. The dependent claims define advantageous embodiments.
In a first aspect, the invention relates to an electrochemical sensor for determining a charged particle concentration in an analyte, the sensor comprising:

- a measurement electrode for measuring a surface-potential at an interface between the measurement electrode and the analyte in which the measurement electrode is immersed in operational use, and
- a control means for measuring the surface-potential at least two different temperatures of the interface to obtain at least two measurement points of a surface-potential versus interface-temperature curve.

The effect of the features of the electrochemical sensor in accordance with the invention is as follows. In all electrochemical sensors, the reactions of interest occur at the surface of the measurement electrode. It is of interest to measure the potential across the interface between the surface of the measurement electrode and the solution (i.e., the surface potential). However, it is impossible to control or measure this surface potential without placing another electrode in the solution. Thus, two potentials must be considered, neither of which can be measured independently. The reason why in the electrochemical sensors known from the prior art the reference electrode must produce a fairly accurate reference voltage is that otherwise the charged particle concentration cannot be determined from the Nernst equation, i.e. the absolute value of the surface-potential must be known.
The inventors have realized that the charged particle concentration may also be determined in a different manner, namely it may be determined from the surface-potential versus temperature curve, and in particular from the slope of this curve. In order to do so the electrochemical sensor comprises a measurement electrode for measuring a surface-potential at an interface of the measurement electrode and the analyte in which the measurement electrode is immersed in operational use. The electrochemical sensor further comprises a control means for measuring the surface-potential at at least two different temperatures of the interface between the measurement electrode and the analyte to obtain at least two measurement points of a surface-potential versus interface-temperature curve.
The electrochemical sensor enables determination of the charged particle concentration in the analyte as follows. First, the control means ensures that the temperature of the interface between the measurement electrode and the analyte reaches a first value. Subsequently, the measurement electrode can be “read-out” to give the surface-potential corresponding with the first temperature. These two steps are subsequently repeated for at least one other temperature different from the first temperature, which gives a total of at least two measurement points of a surface-potential versus temperature curve and which enables to determine a corresponding slope. The absolute values of the corresponding potential of the at least two measurement points in said curve are dependent on the absolute potential of the analyte as defined by the reference electrode. However, it is not required that the reference interface-potential is known and accurately determined, i.e. that it does not vary with the charged particle concentration, because the charged particle concentration is determined by the slope of said curve. Once the slope has been determined, the corresponding charged-particle concentration can be calculated from the slope. For this purpose a pseudo-reference electrode is sufficient. A pseudo-reference electrode is so named because it does not maintain a constant potential (potential depends on analyte composition); therefore, by definition, it is not a true/real reference electrode. However, its potential depends on conditions in a well-defined manner; if the conditions are known, the potential can be calculated and the electrode can be used as for reference potential.
In an embodiment of the sensor in accordance with the invention the control means comprises a temperature setting means arranged for setting a temperature of the interface at at least two different temperatures of the interface. Providing such temperature setting means is a first way of enabling to measure the surface-potential at at least two different temperatures of the interface between the measurement electrode and the analyte to obtain at least two measurement points of said curve.
In an embodiment of the sensor in accordance with the invention the temperature setting means comprises a heater and/or a cooler. Using at least one of these two components enables to set a temperature of the interface between the measurement electrode and the analyte. An example of a temperature settings means which can be used for heating and/or cooling is a peltier element.
In an embodiment of the sensor in accordance with the invention the temperature setting means comprises a resistive heater wherein the temperature is set by controlling a current through the heater. The advantage of the sensor in accordance with the invention is that the absolute value of the temperatures at which the surface-potential is measured need not be known. For obtaining slope information in said curve, it is only required to know the temperature shift. This embodiment is particularly advantageous because, in an environment, having a certain temperature and a constant heat loss, i.e. an environment in thermal equilibrium, each respective current value (or current duty cycle in case of pulsed current) through the resistive heater will correspond with a predetermined steady-state temperature of the analyte at the interface. Expressed differently, the current controls the interface temperature shift with respect to the environmental temperature, which gives the required information for determining the charged particle concentration. No additional temperature sensor for determining the absolute interface temperature is required.
In an embodiment of the sensor in accordance with the invention the temperature setting means further comprises means for determining the power dissipation of the resistive heater and thereby determining the temperature of the interface between the measurement electrode and the analyte.
In an embodiment of the sensor in accordance with the invention the control means comprises a controller, the controller being coupled to the measurement electrode and being arranged for initiating the measuring of the surface-potential with the measurement electrode at at least two different temperatures of the interface. Providing such controller, is a second way of enabling to measure the surface-potential at at least two different temperatures of the interface between the measurement electrode and the analyte to obtain at least two measurement points of said curve.
In an embodiment of the sensor in accordance with the invention the controller comprises a temperature sensor for measuring the temperature of the interface, and the controller is further arranged for initiating the measuring of the surface-potential at a desired interface temperature value. This embodiment is advantageous in case the temperature of the analyte is not constant over time, i.e. because of external influences. All what is required in that situation, is that the controller initiates the measurement of the surface-potential at two different temperature values measured by the temperature sensor.
In an embodiment of the sensor in accordance with the invention the controller comprises storage means for storing the respective measured values of the surface-potential and optionally the respective values of the temperature of the interface. Surface-potentials (optionally together with the respective temperature or temperature change) that have been stored in the storage means, can be read-out at any time in order to enable calculation of the charged particle concentration.
In an embodiment of the sensor in accordance with the invention the controller comprises a processor unit for calculating the charged particle concentration from the at least two measurement points of said curve. This embodiment conveniently provides the charged particle concentration when the measurement has been carried out. There is no need to do this manually anymore. The processor unit may be under software control or it may be a universal piece of hardware such as a gate array.
In an embodiment of the sensor in accordance with the invention the measurement electrode is selected from a group comprising: a glass-electrode, an ion-sensitive field-effect transistor, an ion-sensitive extended gate field-effect transistor, and an electrolyte semiconductor insulator structure. This list comprises the most convenient measurement electrodes.
In an embodiment of the sensor in accordance with the invention the measurement electrode comprises the ion-sensitive extended gate field-effect transistor which comprises i) a field-effect transistor, ii) a sensor electrode being electrically coupled to a gate of the field-effect transistor, and iii) an ion-sensitive sensor dielectric provided on the sensor electrode, the sensor electrode being arranged for contacting the analyte via the ion-sensitive sensor dielectric in operational use, and
the temperature setting means comprises a resistive heater which is arranged in thermal coupling with the sensor electrode for setting a temperature of the interface between the sensor dielectric and the analyte in operational use. In this embodiment the conventional extended-gate field effect transistor (EGFET) is modified. It has been provided with a temperature setting means, in the form of a resistive heater, which is arranged for setting the temperature of the interface in operational use. This embodiment of the sensor constitutes a relative simple but very effective EGFET-based sensor for carrying out the charged particle measurement in accordance with the invention. This embodiment of the sensor can be easily integrated in a semiconductor device. The fact that this sensor does not need an accurate conventional reference electrode further improves the miniaturization of the sensor. A simple pseudo-reference electrode (a simple electrode wire or metal contact in the analyte) is sufficient.
In a variation on last mentioned embodiment the sensor dielectric may be provided with or exchanged with an ion-exchange resin. Ion-exchange resins are based on special organic polymer membranes which contain a specific ion-exchange substance (resin). This is the most widespread type of ion-specific electrode. Usage of specific resins allows preparation of selective electrodes for tens of different ions, both single-atom or multi-atom. They are also the most widespread electrodes with anionic selectivity. However, such electrodes have low chemical and physical durability as well as “survival time”. An example is the potassium selective electrode, based on valinomycin as an ion-exchange agent.
In an embodiment of the sensor in accordance with the invention the measurement electrode comprises the electrolyte semiconductor insulator structure which comprises i) a conductive contact layer, ii) a semiconductor layer provided on the contact layer, iii) an ion-sensitive sensor dielectric provided on the semiconductor layer, the semiconductor layer being arranged for contacting the analyte via the ion-sensitive sensor dielectric in operational use, and the temperature setting means comprises a resistive heater which is arranged in thermal coupling with the electrolyte semiconductor insulator structure for setting a temperature of the interface between the sensor dielectric and the analyte in operational use. In this embodiment the conventional electrolyte semiconductor insulator
(EIS) capacitor is modified. It has been provided with a temperature setting means, in the form of a resistive heater, which is arranged for setting the temperature of the interface in operational use. This embodiment of the sensor constitutes a relative simple but very effective EIS-based sensor for carrying out the charged particle measurement in accordance with the invention. This embodiment of the sensor can be easily integrated in a semiconductor device. The fact that this sensor does not need an accurate conventional reference electrode further improves the miniaturization of the sensor. A simple pseudo-reference electrode (a simple electrode wire or metal contact in the analyte) is sufficient. In EIS-based sensors the flat-band voltage of the EIS capacitor yields information on the pH/ion concentration of the electrolyte. It is determined by C-V (capacitance-voltage) measurements or with a constant-capacitance method. Both methods require a reference electrode and an electrode to modulate the analyte potential for the capacitance measurements. If only small modulation currents are required, the modulation can also be done with the reference electrode itself. Otherwise a potentiostat and 3-electrode configuration are needed. Again the temperature at the dielectric/electrolyte interface is modulated with a heater underneath the EIS layer stack. Temperature changes affect the surface potential causing a shift in the flat-band voltage. Thus the surface potential is indirectly measured via the flat-band voltage. The semiconductor layer may comprises semiconductor materials, such as silicon, germanium, silicon-germanium, III-V compounds, II-VI compounds, etc.
In a variation on last mentioned embodiment the sensor dielectric may be provided with or exchanged with an ion-exchange resin. Ion-exchange resins are based on special organic polymer membranes which contain a specific ion-exchange substance (resin). This is the most widespread type of ion-specific electrode. Usage of specific resins allows preparation of selective electrodes for tens of different ions, both single-atom or multi-atom. They are also the most widespread electrodes with anionic selectivity. However, such electrodes have low chemical and physical durability as well as “survival time”. An example is the potassium selective electrode, based on valinomycin as an ion-exchange agent.
In an embodiment of the sensor in accordance with the invention the sensor is arranged for determining a hydrogen ion concentration and thereby a pH-value of the analyte.
In an embodiment of the sensor in accordance with the invention the sensor dielectric is further provided with a probe molecule layer comprising probe molecules, such as i) antibodies, and ii) DNA / RNA strands, the probe molecule layer being in direct contact with the analyte in operational use, the sensor dielectric thereby being-configured for binding charged target molecules for enabling to determine a charged target molecule concentration in the analyte. This embodiment of the sensor constitutes a molecule sensor, which makes use of the same principle as the charged particle sensor in accordance with the invention (measurement at two different temperatures). Such molecule sensor has a wide application area. In a variation on this embodiment the probe molecules are directly provided on the electrode. In that embodiment the sensor dielectric is not required.
In an embodiment of the sensor in accordance with the invention the charged target molecules are charged biomolecules.
There are various application areas for molecule or biosensors, for example: drug discovery, DNA sequencing, disease detection at the hospital/doctor (point of care), tumor marking, home use (e.g. glucose), security (biological warfare agents), forensic research. Corresponding biomolecules that may be of interest in these areas are: drugs, DNA, viruses and pathogens, tumor markers, glucose, antibodies, etc.
In an embodiment of the sensor in accordance with the invention the sensor further comprises a pseudo-reference electrode for providing a reference potential to the analyte, the reference potential being defined at a further interface of the pseudo-reference electrode and the analyte in which the pseudo-reference electrode is immersed in operational use. As the sensor in accordance with the invention only needs a pseudo-reference electrode, this reference electrode may be advantageously integrated with the measurement electrode.
In a second aspect, the invention relates to a semiconductor device comprising an electrochemical sensor in accordance with the invention. It is a great advantage of the invention that the electrochemical sensor can be integrated into a semiconductor device. All mentioned features in the embodiments can be integrated onto the same semiconductor device, including the temperature setting means, the control means, the controller, the temperature sensor, the pseudo-reference electrode, data processing means, memory etc.
In a third aspect, the invention relates to an RFID-tag comprising an electrochemical sensor in accordance with the invention. The invention is advantageously applied in this application area.
In a fourth aspect, the invention relates to electrochemical sensor system for determining a charged particle concentration in an analyte comprising:

- a measurement electrode for measuring a surface-potential at an interface between a measurement electrode and the analyte in which the measurement electrode is immersed in operational use;
- a temperature setting means arranged for setting a temperature of the interface at at least two different temperatures, and
- a controller coupled to the measurement electrode and being arranged for initiating the measuring of the surface-potential with the measurement electrode at at least two different temperatures of the interface.

The advantages and effects of the electrochemical sensor system in accordance with the invention follow that of the electrochemical sensor. All embodiments of the electrochemical sensor apply mutatis mutandis to the electrochemical sensor system.
In a fifth aspect, the invention relates to method of determining a charged particle concentration in an analyte, the method comprising steps of:

- determining at least two measurement points of a surface-potential versus interface-temperature curve, wherein the interface temperature is defined as a temperature of the interface between a measurement electrode and the analyte, wherein the surface-potential is defined at the interface;
- calculating the charged particle concentration from locations of the at least two measurement points of said curve.

The advantages and effects of the method in accordance with the invention follow that of corresponding embodiments of the electrochemical sensor. The inventors have realized that the particle concentration information is hidden in the slope of the surface-potential versus interface-temperature curve. A vertical shift of said curve does not have any influence on the slope. Thus any constant potential offset caused by an “inaccuracy” of the reference electrode does not have an effect on the measured slope and consequently the determined charged particle concentration. Expressed differently, if the reference electrode produces an output voltage, which is unknown upfront (i.e., because its output value depends on a yet-undetermined charged particle concentration), the invention still works. In the method of the prior art, the absolute value of the surface-potential has to be known for calculating the charged particle concentration. Therefore, in the prior art, a reference electrode is required which creates a potential, which is known and independent from the charged particle (ion) concentration of the analyte (that is, these electrodes ensure a constant analyte potential). The method of the invention does not require such reference electrode with a defined and independent output potential. A pseudo- or quasi reference electrode, which output depends on the charged particle concentration (and thus produces an output value which is unknown upfront) is sufficient. Any type of reference electrode (that is chemically inert to the analyte) will do. The only function the reference electrode has to perform is the establishment of a galvanic contact to the analyte such that the electric circuit is closed.
In an embodiment of the method in accordance with the invention the step of calculating the charged particle concentration comprises the following sub-steps: i) deriving the slope from the at least two measurement points, and ii) calculating the charged particle concentration in the analyte from the slope.
In an embodiment of the method in accordance with the invention, in the step of determining, at least three measurement points of said curve are determined, and wherein the step of calculating the charged particle concentration comprises the following sub-steps: i) determining a straight fitting line using the at least three measurement points of said curve, and ii) calculating the charged particle concentration from the straight fitting line. The advantage of this embodiment of the method is that measurement noise and measurement errors are reduced.
In an embodiment of the method in accordance with the invention the sub-step of calculating the charged particle concentration comprises: a) determining a slope of the straight fitting line, and b) calculating the charged-particle concentration from the slope.
In an embodiment of the method in accordance with the invention the step of determining of said curve comprises sub-steps of:

- setting the interface temperature to a first value;
- determining a first value of the surface-potential at the interface, wherein the first value of the interface temperature and the first value of the surface-potential together define a first respective one of the measurement points of said curve;
- setting the temperature of the interface to a second value different from the first value, and
- determining a second value of the surface-potential at the interface, wherein the second value of the interface temperature and the second value of the surface-potential together define a second respective one of the at least two measurement points of said curve.

This embodiment of the method constitutes a possible implementation of determining said curve.
In an embodiment of the method in accordance with the invention the difference between the first value of the temperature of the interface and the second value of the temperature of the interface is smaller than a predefined threshold, preferably smaller than or equal to 10K, and even more preferably smaller than or equal to 5K. Keeping the temperature difference between the first and second measurement within a certain threshold ensures that a measurement error, which is the result of a temperature dependency of a specific parameter of the sensor, is reduced. This applies especially in case of a dielectric sensor layer when the temperature dependence of the sensitivity parameter a is unknown. Keeping a small temperature range also reduces the power consumption. The pH of the analyte may itself be temperature dependent (e.g. buffers have temperature dependent buffer capacity). Thus if the pH of a solution must be known at a certain temperature the measurement process should not deviate too much from this temperature itself. One of those temperature dependent parameters is the sensitivity of the dielectric of the ISFET-based measurement electrode, which parameter is known to be temperature dependent. What is considered as an acceptable measurement error generally depends on the application. In case of a pH measurements an error of 0.1 pH is acceptable for most applications.
In an embodiment of the method in accordance with the invention the step of determining at least two measurement points of said curve is done by determining respective values of an output quantity that is indicative of the surface potential. Depending on the chosen type of transducer it may be that the output is a quantity that is representative of the surface-potential, i.e. a current through a transistor (for example an ISFET or EGFET).
It is important to note that, despite the fact that a real reference electrode is no longer needed in the electrochemical sensor in accordance with the invention, a real reference electrode may still be applied in the measurement principle in accordance with the invention (i.e. determining a charged-particle concentration from the slope of a potential-versus-temperature curve).
These and other aspects of the invention are apparent from and will be elucidated with reference to the embodiments described hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 shows some formula's for explaining the potentiometric measurement principle as known from the prior art;

FIGS. 2( a) to 2(c) show conventional electrodes and reference electrodes known from the prior art;

FIG. 3 shows some formula's for explaining the potentiometric measurement principle in accordance with the invention;

FIG. 4 shows a diagram with a couple of potential difference versus interface-temperature change curves for different charged particle concentrations;

FIGS. 5( a) and 5(b) show two embodiments of the electrochemical sensor in accordance with an embodiment of the invention;

FIGS. 6( a) to 6(d) show four different sensor-heater arrangements in accordance with other embodiments of the invention, and

FIGS. 7( a) to 7(d) show the manufacturing and operation principle of an electrochemical biosensor in accordance with yet another embodiment of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention provides a new method for determining a charged particle, i.e. ions and charged biomolecules, concentration in a liquid analyte. In an embodiment the method concerns determination of a hydrogen ion concentration and thereby the pH-value. It is based on measurements at different temperatures with a suitable electrochemical sensor. The electrochemical sensor may comprise measurement electrodes, such as glass electrodes, ISFETs, and EIS capacitors. The charged particle concentration is calculated from the measurement electrode readings and the respective temperatures. Because of the new measurement principle no accurate reference electrodes are needed any more. All problems and issues associated with these reference electrodes are thereby prevented (e.g. maintenance and refill of electrolyte, bulky device, limitations in temperature range, etc.). Moreover, the new method of measuring a charge particle concentration is a dynamic measurement type (the temperature is modulated), which minimizes drift and reduces the need for frequent calibration.
In view of the above the invention provides a method of determining a charged particle concentration, an electrochemical sensor for enabling to determine the charged particle concentration using such method, an electrochemical sensor system for enabling to determine the charged particle concentration.
In order to facilitate the discussion of the detailed embodiments a few expressions are defined hereinafter.
Throughout this description the term “interface temperature” should be interpreted as the temperature of a volume around the interface which includes volume with electrode material and a volume with analyte.
In electrochemistry, the Nernst equation is an equation which can be used (in conjunction with other information) to determine the equilibrium reduction potential of a half-cell in an electrochemical cell.
A half cell is a structure that contains a conductive electrode and a surrounding conductive electrolyte separated by a naturally-occurring Helmholtz double layer. Chemical reactions within this layer momentarily pump electric charges between the electrode and the electrolyte, resulting in a potential difference between the electrode and the electrolyte. The typical reaction involves a metal atom in the electrode being dissolved and transported as a positive ion across the double layer, causing the electrolyte to acquire a net positive charge while the electrode acquires a net negative charge. The growing potential difference creates an intense electric field within the double layer, and the potential rises in value until the field halts the net charge-pumping reactions. In a similar way the Nernst equation also describes the surface potential at the interface of a dielectric and an electrolyte or across a membrane with different ion concentrations in the electrolytes on either side.
Throughout this description the term “reference electrode” refers to an electrode which has a stable and well-known electrode potential. The high stability of the electrode potential is usually reached by employing a redox system with constant (buffered or saturated) concentrations of each participants of the redox reaction. Reference electrodes are used to build an electrochemical cell in conjunction with an electrode the potential of which is to be determined. Each electrode represents a half cell; both are required to complete the circuit and measure the unknown potential.
Throughout this description the term “pseudo-reference electrode” refers to an reference electrode which does not maintain a constant potential. By definition, a pseudo-reference electrode is not a true reference electrode. However, its potential depends on conditions in a well-defined manner; if the conditions are known, the potential can be calculated and the electrode can be used as for reference potential.
Throughout this description the term “measurement electrode” is considered either a glass electrode, ISFET or an EIS capacitor.
Throughout this description the term “charged particle” refers to ions and charged bio-molecules.
Throughout this description the term “interconnect layer” should be considered as synonym to “metallization layer” or “metal layer”. Both terms are used interchangeably and have to be interpreted as the layer comprising conductors (any conducting material), the insulating layer in which the conductors are embedded, and any vias (=contacts) to underlying layers. These terms are well-known to the person skilled in the art of semiconductor technology.
Throughout this description the term “substrate” should be interpreted broadly. The substrate may comprise an active layer with elements, such as transistors and diodes, which form the components of an electronic circuit. The substrate may further comprise interconnections between the elements which may be laid out in one or more interconnect layers and may further contain passive elements such as capacitors, resistors and inductors. In the figures, the elements have been left out in order to facilitate the understanding of the invention. The active layer in which the elements are formed may also be called a semiconductor body. The semiconductor body may comprise any one of the following semiconductor materials and compositions like silicon (Si), germanium (Ge), silicon germanium (SiGe), gallium-arsenide (GaAs) and other III-V compounds like indium-phosphide (InP), cadmium sulfide (CdS) and other II-VI compounds, or combinations of these materials and compositions as well as semiconducting polymers. The active elements together may form an electronic circuit. In any case, connection of the active elements is done via interconnect layers. These interconnect layers have parasitic capacitances which are defined by the dielectric constant of surrounding materials. The semiconductor body may even comprise contacts to lower layers (e.g. diffusion regions at the surface of an active region).
FIG. 1 shows some formula's for explaining the potentiometric measurement principle as known from the prior art. In the description of the figures the main principle will be explained with measurement of a concentration of hydrogen ions (pH-value). However, it must be stressed that the invention is also applicable to any other kind of charged particle concentration, i.e., Na⁺-ions, K⁺-ions, Ca²⁺-ions, etc.
The pH-value is an integral parameter of every (aqueous) solution. It describes to which degree the solution is alkaline or acidic. Over a wide range it is well approximated by: pH=−log [H⁺], wherein [H⁺] denotes the proton concentration of the solution in mol/L. pH-measurement is a routine task in industry and also in laboratories for process control and analysis. However, it could also become interesting for a wider application range if the pH-measurement units (sensor plus electronics) become sufficiently inexpensive. E.g., there is a large potential for pH-measurement to monitor the quality of (liquid) perishables in the supply chain or even at the customer himself. Experimental techniques for measuring ion concentrations (as is the case in pH-measurements) can be divided into two classes, non-electrochemical methods, e.g., optical (indicator dyes), catalytic, and swelling of polymers (gels), and electrochemical methods. The latter are widely used for many applications in industry and laboratories. Electrochemical ion concentration sensors rely on the potentiometric principle, i.e. they measure the electrical potential φ across a solid/liquid interface which is a function of the ion concentration to be determined. The potential φ can be calculated from the Nernst equation, given in formula (1) of FIG. 1. In this formula k is the Boltzmann constant, T the absolute temperature in Kelvin, q the elementary charge, and n the ionic charge (e.g. n=1 for H₃O⁺, Na⁺; n=2 for Ca²⁺). Ion concentrations at both sides of the membrane/interface (1 and 2) are represented in terms of activities a_i=f_i*c_iwith f_ibeing the activity coefficient (f_i=1 for diluted electrolytes) and c_ithe respective ion concentration in mol/L. According to the Nernst equation the electrode potential is a logarithmic function of the ion activity on one side of the membrane/interface if the activity on the other side is kept constant. Depending on the type of ion described by parameter “α” the sensor is sensitive to H₃O⁺-ions, Na⁺-ions, Ca²⁺-ions, etc.
FIGS. 2( a) to 2(c) show conventional electrodes and reference electrodes known from the prior art. All major pH-(ion)-measurement electrodes work according to the principle described above, including the well-known glass electrodes (different glass compositions sensitive to pH, pNa, pK etc. have been developed), antimony electrodes, ISFET's (Ion Sensitive Filed Effect Transistor) and EIS capacitors (Electrolyte Insulator Semiconductor capacitor; here the flat-band voltage is a function of the pH of the electrolyte). It is not possible to measure a potential; but it is possible to measure potential differences. In any case, in order to measure a potential difference with a measurement electrode a reference electrode is needed, wherein the potential difference is generated by a difference in the measurement electrode potential φ_mand the reference electrode potential φ_ref(see formula (2) in FIG. 1). In the case of ISFET and EIS devices as measurement electrode the reference electrode is also used to set the operating point and close the electric loop. In the prior art, the potential of the reference electrode φ_refwith respect to the electrolyte potential must remain constant irrespective of the analyte composition. Thus, in the prior art, what is measured is the potential difference Δφbetween the measurement electrode potential φ_mand the reference electrode potential φ_ref. This is given by formula (2) in FIG. 1.
In the case of a pH-measurement with a glass-electrode and a conventional reference electrode (with a reference liquid), the potential difference can be given by (3a) formula in FIG. 1. In formula (3a) pHin stands for the pH of the electrolyte in the glass-electrode and pHout stands for the pH of the analyte (which has to be determined). In fact formula (3) is the sum of two surface potentials at the inside and outside of the glass electrode as well as the contact potential of the wire inside the glass electrode with the electrolyte in the glass-electrode φ_contand the reference electrode potential φ_ref. However, in this configuration these terms cancel each other out when both electrodes have the same temperature. The derivation of formula (3a) and more information on reference electrodes can be found in the following publication:

- “Measuring, modeling, and controlling the PH-value and the dynamic chemical state.” By Jean-Peter Ylén, Helsinki University of Technology, Control Engineering Laboratory, Report 127, Espoo 2001 [REF1]. This document has been incorporated by reference in its entirety.

In the case of a pH-measurement with an ISFET-measurement electrode and a conventional reference electrode, the potential difference can be given by (3b) formula in FIG. 1. In formula (3b) pHpzc stands for the point of zero charge of the ISFET-measurement electrode (a material property defined by the dielectric sensor layer of the ISFET) and pHout stands for the pH of the analyte (which has to be determined). The derivation of formula (3b) and more information on ISFET electrodes can be found in the following publication:

- P. Bergveld, “Thirty years of ISFETOLOGY. What happened in the past 30 years and what may happen in the next 30 years.”, Sensors and Actuators B 88 (2003) 1-20 [REF2]. This document has been incorporated by reference in its entirety.

Besides the standard hydrogen electrode, the Ag/AgCl electrode is the most well-known reference electrode. This reference electrode RE is illustrated in FIG. 2( a). It consists of a chlorinated silver wire 10 (Ag/AgCl) in contact with a well-defined electrolyte 20 (often 3 mol/L KCl). Galvanic contact to the analyte is established via a diaphragm 30 (porous frit from glass or ceramics, etc.). During operation the electrolyte 20 must continuously flow out of the reference electrode RE into the analyte. Other reference electrodes, e.g. calomel electrodes (based on mercury) or Tl/TlCl electrodes are used for specific applications, e.g. at elevated temperatures. Their principle is the same as for the Ag/AgCl electrode, in particular the use of a liquid electrolyte 20 and contact via a diaphragm 30. The chlorinated silver wire 10 is connected to a contact cable 40.
FIG. 2( b) illustrates a measurement set-up in which the reference electrode RE is used in combination with a glass electrode GE. Both electrodes GE, RE are immersed into the analyte 100 in operational use. The glass electrode GE comprises a chlorinated silver wire 10 (Ag/AgCl) in contact with an electrolyte 20′ (buffer solution) with a well-defined pH_in-value. The electrolyte 20′ is provided in a pH-sensitive glass membrane 31, which is produced from a special glass. Its thickness is usually between 50-200 μm, but in the measurement of very aggressive solutions it can be even 1 mm. After immersion in water the glass electrode can measure the process solution 100 (analyte). A potential difference between the analyte 100 and the glass surface is created, and this difference is a function of the activity of H₃O⁺-ions and thus also a function of the pH-value of the analyte 100. The chlorinated silver wire 10′ is connected to a further contact cable 40′. The cable 40 and the further cable 40′ are both connect to the input of a voltmeter VM. The voltmeter gives the potential difference Δφ as given by formula (3a) in FIG. 1. More information about glass electrodes can be found in the first reference (REF1) given in this description.
FIG. 2( c) illustrates a measurement set-up in which the reference electrode RE is used in combination with an ISFET measurement electrode IE. Both electrodes IE, RE are immersed into the analyte in operational use. The ISFET measurement electrode IE comprises a transistor structure, which is very similar to a conventional field-effect transistor (FET). It comprises a p-type substrate 5 having an n-type source Src and an n-type drain Drn provided at a surface thereof defining a channel region in between. A gate dielectric 32 is provided on the substrate 5 covering source Src, drain Drn and channel. Alternative a p-type transistor can be used. A main difference is that the gate dielectric 32 is in direct contact with the analyte 100 instead of with a poly/metal gate contact. The gate dielectric 32 is the ion/pH sensitive layer (in an example embodiment it comprises SiO₂, but other dielectrics, such as Ta₂O₅can also be used). The transistor acts as transducer that converts the potential difference into a current between the source Src and drain Drn of the transistor. Above the channel region the dielectric may be thinner than elsewhere, in order to increase the sensitivity of the ISFET (better inversion of the channel in case of a predefined surface potential generation at the dielectric layer 32). More information about ISFET's can be found in the second reference (REF2) given in this description. A reference electrode RE is provided in the analyte 100 in order to establish a “working point” (reference potential) for the ISFET and define the analyte potential. A potential set by this reference electrode RE may be considered as the gate voltage V_Gof a conventional field-effect transistor. In the prior art pH-measurements it is of utmost importance that the potential of the reference electrode is independent of the composition of the analyte.
FIG. 3 shows some formula's for explaining the potentiometric measurement principle in accordance with the invention. An essential feature of the invention is to execute potentiometric pH/ion measurements at different temperatures in the (same) analyte. While temperature changes must be compensated or taken into account with the conventional potentiometric measurement principle of the prior art the invention exploits the temperature dependency of the sensor output to determine the quantity to be measured, e.g., the pH-value or ion concentration of a solution (or a charged biomolecule concentration as will be discussed in FIG. 7). The arguments described hereafter relate to pH-value but also apply to ion concentration or charged biomolecule concentration, then the pH needs to be replaced by pK and the charge number n must be taken into account).
The potential difference equation for a combination of a glass electrode and a conventional reference electrode (with reference liquid) is repeated in formula (4a) in FIG. 3, wherein pHout denotes the pH-value at the outside (analyte) and pHin denotes the pH-value of the electrolyte inside (ln10≈2.3). The inventors have realized that formula (4a) can be looked at differently. According to this formula Δφ shows a linear dependence on T with the slope of the straight line m given by formula (4b) in FIG. 3. It must be noted that all parameters of this formula are known or fixed, except for pHout which is the pH-value of the analyte to be measured. Following this approach the pH-value of an analyte can be obtained by recording the potential difference Δφ at different temperatures T, determining the slope m of the Δφ-T curve and subsequently calculating the pH-value using formula (4c) in FIG. 3.
FIG. 4 shows a diagram with a couple of potential difference (between measurement electrode and reference electrode) versus interface-temperature change curves for different charged particle concentrations. The diagram shows Δφ-ΔT-curves for various pHout-values in a temperature range 0K-10K (pHin=7). These curves have a direct relation with the surface-potential versus interface-temperature curves. The slopes of the curves allow a clear discrimination of the different pH-values. Four curves c1, c2, c3, c4 illustrate a pH-value equal to 5, 6, 7, and 8, respectively. Which curve runs horizontal depends on the value of parameter “pHin”. In principle parameter “pHout” can be calculated without calibrating the sensor since all parameters in formula (4a) in FIG. 3 are known. In reality calibration may still be advisory because of components of the system that do not behave ideally and may be temperature dependent (e.g. electrode contacting the reference electrolyte inside a glass electrode with pHin).
It should be noted that being a potential φ cannot be measured alone but only as a potential difference (voltage) to another potential i.e. the potential from the reference electrode. However, this does not imply any restriction if the reference potential (and all other potentials involved; e.g. potential between the inner electrode and reference electrolyte of a glass electrode) is independent of the analyte composition and only the surface potential of the measurement is modulated by a very localized change in the interface temperature (temperature at other interfaces e.g. between reference electrode and analyte are kept constant during the measurement).
Since the information about the pH-value (hydrogen-ion concentration) is conveyed in the slope of the Δφ-ΔT-curves rather than in the absolute value of φ (as is the case with conventional potentiometric measurements) any vertical shift of the curves has no effect on the measurement. Thus any potential offset caused by using a pseudo-reference electrode instead of a ‘real’ reference electrode is neglected. A pseudo-reference electrode consists of a simple metal wire (e.g. Pt or Ag) immersed in the analyte (sample solution). This pseudo-reference electrode provides a constant reference potential during the measurement, but depends on the analyte composition (e.g. its ion concentration). However, such a pseudo reference electrode is fully sufficient for our invention. For a precise measurement it must only be made sure that the potential of the pseudo-reference electrode remains constant during the measurement itself, i.e. during changing of the temperature T and recording of the respective values from the measurement electrode. For the method it is not even necessary to know the absolute temperature. The only value which must be known (in arbitrary units) is the interval ΔT between different measurements. For example the temperature T can be given as: T=T₀+a*U₂/R*t , wherein parameter “T₀” denotes the temperature at t=0s, parameter “R” denotes the ohmic resistance of a resistive heater, parameter “U” denotes the applied voltage and parameter “t” denotes the time the heater is activated. Parameter “a” comprises all other system parameters e.g. the volume of the heated liquid and its heat capacity. Substituting this formula for the temperature with formula (4a) in FIG. 3, gives a formula for the potential difference Δφ as a function of time t for which the heater is activated. The absolute value of the start temperature T₀does not need to be known, since it only causes a vertical shift of the curve, whereas the pH-value (pHout) is conveyed in the slope. A calibration of the system (i.e. measure the slope of a curve with a buffer of defined pHout) may be necessary in order to determine parameter “α”. Moreover, parameter “α” should preferably remain constant between calibration and real measurement since it directly affects the slope.
Potentiometric measurements as known from the prior art are static measurements, which rely on the thermodynamic equilibrium. Static measurements are often subject to drift which makes frequent calibration necessary. Besides the associated effort and cost, some systems are difficult to calibrate, e.g. because the sensor is fixed in a vessel/pipe and would need to be removed or because the system cannot be accessed at all (perishable monitoring, medical applications). Drift is a particular problem for ISFET sensors. Various algorithms and procedures have been developed to predict drift and correct the measurements Moreover, new sensors must equilibrate for a certain time before they can be used. An advantage of the measurement principle of the invention is that due to the dynamic measurement principle drift is considerably reduced increasing the calibration intervals and measurement accuracy. More information on drift and counter-measures can be found in the following publication:

- S. Jamsab, “An Analytical Technique for Counteracting drift in Ion-Selective Field effect Transistors (ISFETs)”, IEEE Sensors J., 4 (6), 795-801, 2004 [REF3]. This document has been incorporated by reference in its entirety.

Another advantage of the new measurement principle in accordance with the invention is the noise reduction. If the slope of a Δφ-ΔT-curve is determined by fitting a straight line to several φ values recorded at different temperatures, noise and statistical measurement errors are averaged out.
Until now, for the sake of clarity only the fundamental principles and equations have been shown and discussed. In real applications it might be slightly more complex. This also depends on the type of measurement electrode and reference electrodes chosen.
If a glass electrode is used for the measurements not only the membrane potential across the ion sensitive glass membrane has to be taken into account but also the potentials of the (pseudo) reference and measurement electrode (contacting the reference liquid with pHin). Unfortunately, the potential of the reference electrode may also be temperature dependent. However if the same electrode material and electrolyte are used in the measurement and ‘real’ reference electrode and both electrodes are kept at the same temperature they cancel out (φ_cont→φ_ref). This is not the case if a pseudo-reference electrode is used. In order to prevent errors with this system it is better to heat the analyte only locally near the ion sensitive glass membrane while the analyte at the pseudo-reference electrode remains at its initial temperature.
In the case of a pH-measurement with an ISFET-measurement electrode and a reference electrode, the potential difference can be given by (5a) formula in FIG. 3 wherein the first part describes the surface potential (which yields the information on the pH-value of the analyte) of the dielectric/analyte interface, wherein parameter pHpzc denotes the point-of-zero-charge, i.e. the pH-value of the analyte for which the oxide surface is electrically neutral, wherein parameter pHout denotes the actual pH-value of the analyte in contact with the dielectric, wherein parameter a denotes a temperature dependent sensitivity parameter which is characteristic for the specific ISFET sensor dielectric. Parameter α lies between 0 and 1 (in case of a sensitivity equal to 1 the sensor has the maximum sensitivity). Formula's (5b) and (5c) can be derived from formula (5a) in a way that is similar to that of formula's (4b) and (4c) in FIG. 3.
Parameter a for an ISFET is known to be defined as given in formula (6) in FIG. 3, wherein parameter C_Sdenotes the double layer capacitance (which depends on the ion concentration in the analyte), and wherein parameter β_Sdenotes the surface buffer capacity which is a material parameter of the sensor dielectric. Other parameters are already explained earlier in the description.
The temperature dependency of the sensor sensitivity α may complicate the measurement method a bit. It can be addressed in several ways (or combinations thereof).
1) Use a sensor dielectric material with high surface buffer capacity β_S. This measure minimizes the temperature dependence of the sensitivity α. The advantage of this approach is that the measurement principle described above can applied without modification. In a preferred embodiment the sensor dielectric material comprises tantalum oxide (Ta₂O₅) which has the advantage that it has a very high β_S.
2) Perform the different temperature measurements in a small temperature “window”, e.g. 5K. Within this temperature window the sensitivity α may be assumed to be constant. Consequently, a small change in the sensitivity α results in a relatively small error and can be neglected. This second approach requires that the calibration and “real” measurement are done at the same temperature. Otherwise the error will increase because of the earlier mentioned temperature dependency, which thus results in different slopes.
3) Determine C_Sand β_Sduring sensor calibration. A single calibration run with one reference solution is sufficient. However, the potential difference must be measured for several temperatures to allow fitting of the Δφ-T-curve in order to obtain C_Sand β_S. This is the most accurate approach but the absolute temperature must be known. A temperature sensor for determining the absolute temperature is thus required.
The method for measuring pH or ion concentrations can be realized by installing a small heater/cooler next to the sensor (glass electrode, ISFET). The heater/cooler heats/cools the analyte in close proximity to the sensor. The sensor readings (representing Δ_φ) at different temperatures (T measured with integrated sensor or determined from heating energy) are stored or plotted; the chemical parameter is then obtained from the slope of the curve according to the method described above. Instead of a close-by heater/cooler the analyte temperature can also be controlled by a remote device and applied to the sensor by a fluidic system (e.g. flush liquid onto sensor).
If no temperature sensor is used in the method, sufficient time must pass between subsequent heat pulses to allow cooling of the sensor to the initial (ambient) temperature. If only short heat pulses are used a heat wave will propagate towards the dielectric/analyte interface leading to a transient temperature increase. Continuous measurement of the surface potential (transducer output) will result in a maximum/minimum value, which value shall be used for further data extraction (when this value is reached the temperature at the interface is highest/lowest before it cools off / heats up again). To increase measurement accuracy a curve can be fitted to determine the extreme value (taking into account the temporal behavior of the temperature at the interface following a heat pulse). A simpler way is to average a few values in an interval around the extreme value.
Where in this specification the wording “obtaining of measurement points of a surface-potential versus temperature curve” is used, it is often meant that measurement points of a potential-difference (between the first electrode and a (pseudo)-reference electrode) versus temperature (of the interface at the measurement electrode) is meant. Nevertheless, as in the invention it is not required to know the absolute temperature, but only to determine the slope of the surface-potential versus temperature curve, the latter curve has a clear relation with the first curve and is sufficient to obtain the slope.
So far, the description of the figures mainly dealt with the method of determining a charged particle concentration in an analyte in accordance with the invention. However, the invention also relates to an electrochemical sensor, which can be used to carry out this method. It has already been discussed that such electrochemical sensor may comprise conventional measurement electrodes, such as glass electrodes, and conventional reference electrodes. So, in any case the electrochemical sensor in accordance with the invention must comprise a measurement electrode for measuring a surface-potential at an interface between the measurement electrode and the analyte in which the measurement electrode is immersed in operational use. Further the electrochemical sensor in accordance with the invention must also comprise at least a control means for enabling to measure the surface-potential at at least two different temperatures of the interface to obtain at least two measurement points of a surface-potential versus interface-temperature curve. Such control means can be a temperature setting means arranged for setting a temperature of the interface at at least two different temperatures of the interface. Alternatively, such control means can be a controller, wherein the controller is coupled to the measurement electrode and is arranged for initiating the measuring of the surface-potential with the measurement electrode at at least two different temperatures of the interface. A combination of both is also possible.
Miniaturized solutions for the electrochemical sensor are of particular interest as that opens up new application possibilities (due to small form factor and reduced cost). An example of such miniaturization is the ISFET measurement electrode. A disadvantage of the
ISFET is that with the measurement principle of the prior art still an accurate reference electrode (with reference electrolyte) is required, which electrode cannot be easily miniaturized. Miniaturized versions, which have been reported in the prior art so far, have a very limited life-time.
A major advantage of the invention is that this cumbersome reference electrode is no longer required. Instead a pseudo-reference electrode (which is basically a metal contact immersed in and in electrical contact with the analyte in operational use) can be used. This reference electrode can be easily integrated into the ISFET using the interconnect technology already present. Miniaturization has thus become very easy. Nevertheless, it is still possible to combine the electrochemical sensor of the invention with a conventional reference electrode. As already mentioned reference electrode allows to set a DC-potential of the analyte to a known value, which may be advantageous if the measurement method of the invention is combined with conventional measurement methods.
The main building blocks of an electrochemical sensor in accordance with an embodiment of the invention are:

- a sensor electrode covered with a suitable sensor material (depending on the application pH or ion sensitive);
- a heater/cooler in close proximity the sensor, and
- a transducer for transducing the sensor output into an electrical signal for further processing.

Moreover, the electrochemical sensor may include circuits for data processing and storage, power supply. The electrochemical sensor may further comprise circuit blocks such as AD/DA converters, digital signal processors, memory and RF units for wireless data transfer.
In a first embodiment of the electrochemical sensor the measurement electrode is an ISFET (as illustrated in FIG. 2( c)). The ISFET has been discussed earlier in this description. In order to use an ISFET according to our invention a small temperature setting means (i.e. a heater) is needed near the gate dielectric. This could be a resistive heater (thin lines of metal wire) processed next to or surrounding the gate area, e.g. by metal deposition and etch with a suitable mask. The heater may be covered by dielectric layers (e.g. be integrated into the metal interconnect) protecting it from direct contact with the electrolyte. The reference electrode can be used in the analyte to set the working point of the ISFET. If the ISFET is used according to our invention a simple pseudo-reference electrode can be used. A major disadvantage of ISFETs is the direct contact between analyte and the ion-sensitive gate dielectric. This makes CMOS process integration difficult because all layers above the sensor gate must be removed (e.g. by etch) to allow direct contact with the analyte. Moreover, because of the close contact between analyte and active layer (only thin dielectric layers for protection) there is a high risk that ions diffuse into the integrated circuit and shift the threshold voltage of transistors that are close to the opening and destroy the CMOS circuit.
FIGS. 5( a) and 5(b) show two embodiments of the electrochemical sensor in accordance with an embodiment of the invention. FIG. 5( a) shows a so-called Extended Gate Field-Effect-Transistor (EGFET). FIG. 5( b) shows a so-called Electrolyte Semiconductor
Insulator (EIS) structure.
Referring to FIG. 5( a), in this structure the issues, described in the last paragraph above, do not exist. It consists of a conventional transistor NM having a source Src, a drain Drn, and a gate Gt, e.g. an NMOS transistor. The gate Gt of the transistor NM is connected to a sensor electrode Snse via standard metal interconnect ‘wires’. On the sensor electrode Snse a sensor dielectric Snsd is provided that is sensitive to certain ions. The sensor has been exemplified in a simplified way to facilitate understanding of the invention. A heater Htr (temperature setting means) has been provided close, for example underneath, to the sensor electrode Snse and sensor dielectric Snsd. What is important is that the heater Htr is provided such that it is thermally coupled to the sensor part for setting its temperature.
Many variations are possible in this respect. Some of these variations are illustrated in FIG. 6. The transistor NM of the sensor has a floating gate, because the connection between gate Gt and sensor electrode Snse is not galvanically connected to any voltage source. Instead, it is surrounded by insulators such as the gate dielectric, sensor dielectric Snsd and interconnect dielectric. The working point of the sensor is controlled by a reference electrode, here a pseudo-reference electrode PR, in the analyte. The pseudo-reference electrode PR can be integrated with the EGFET, for example in the top metal layer.
The major advantage of the EGFET as compared to the ISFET is that the sensor electrode Snse is implemented in the top metal layer of the chip and thus ‘far away’ from the layer comprising the transistor NM. This reduces risk of contamination with, e.g.
alkaline ions, such as Na⁺. Moreover, it allows easy integration with standard CMOS processes.
Referring to FIG. 5( b), in this structure the issues, described in the before-last paragraph, also do not exist, because it can be manufactured in the upper layer(s) of a chip. The Electrolyte Semiconductor Insulator structure comprises a conductive contact layer Cl (e.g. metal pad, silicide) onto which a silicon layer S1 is provided. On the silicon layer S1 a sensor dielectric Snsd is provided. The stack is similar to a MOS (Metal Oxide Semiconductor) capacitor. It differs from there in that the dielectric/oxide is contacted by the analyte rather than by metal. The flat-band voltage of the EIS capacitor yields information on the pH-value/ion concentration of the analyte. It is determined by C-V (capacitance voltage) measurements or with a constant capacitance method. Both methods at least require a reference electrode to define the DC potential of the analyte and to modulate the analyte potential for the capacitance measurements. Again the temperature at the sensor dielectric/electrolyte interface is modulated with a heater Htr near, for example underneath, the EIS layer stack. Temperature changes affect the surface potential that subsequently causes a shift in the flat-band voltage. Thus the surface potential is indirectly measured via the flat-band voltage. The reference electrode can be a simple pseudo-reference electrode PR for the same reason discussed with FIG. 5( a).
More information on the electrolyte-insulator semiconductor structure can be found in the following document:

- Shoji Yoshida, Nobuyoshi Hara, and Katsuhisa Sugimoto, “Development of a Wide Range pH Sensor based on Electrolyte-Insulator Semiconductor Structure with Corrosion-Resistant Al₂O₃—Ta₂O₅and Al₂O₃—ZrO₂Double-Oxide Thin Films.”, Journal of The Electrochemical Society, 151 (3) H53-H58 (2004) [REF4]. This document has been incorporated by reference in its entirety.

More information on C-V measurements can be found in the following document:

- M. Klein, “CHARACTERIZATION OF ION-SENSITIVE LAYER SYSTEMS WITH A C(V) MEASUREMENT METHOD OPERATING AT CONSTANT CAPACITANCE.”, Sensors and Actuators B1 (1-6): p354-356, January 1990 [REFS]. This document has been incorporated by reference in its entirety.

Because of the special measurement principle of the invention the earlier described problems related to the reference electrode and calibration are no longer relevant (or at least to a much smaller degree) for the electrochemical sensor in accordance with the invention. In particular, the embodiments described here can be easily miniaturized and integrated into standard CMOS devices. Only minor additions to a standard processing scheme are necessary. Moreover, these modifications are after all conventional processing has been finished, and before dicing and packaging).
FIGS. 6( a) to 6(d) show four different sensor-heater arrangements in accordance with other embodiments of the invention. All figures are simplified, in particular for the sensor. For the sensor only the sensor electrodes are shown. In FIG. 6( a) the sensor Snsr is arranged as a large pad, whereas the heater Htr is arranged (in a same plane) around the periphery of the pad. In FIG. 6( b) the heater Htr is arranged under the sensor pad Snsr in the form of a meander. This configuration ensures a more uniform temperature of the sensor. In FIG. 6( c) the sensor Snsr is arranged as a meander structure, and the heater Htr is arranged, in a same plane, as a meander structure on both sides of the sensor Snsr in a river-routing fashion. In FIG. 6( d) the sensor Snsr is arranged as a meander structure. The heater Htr is arranged below the sensor Snsr as a meander structure in a 90°-rotated. The actual arrangement of heater Htr and sensor Snsr may considerably affect the temperature uniformity of the sensor. The person skilled in the art may easily come up with further variations of the sensor Snsr and heater Htr. In any case, what is important is that the heater Htr (temperature settings means) is thermally coupled to the sensor Snsr for enabling the setting of the sensor temperature.

Method of Manufacturing

Sensor manufacturing of the embodiments of FIGS. 5 and 6 follows standard CMOS processing schemes. This is the case for the transducers as well as for most parts of the sensor (and heater). If we consider a CMOS process with five metal layers for interconnect the heater can be implemented as a thin metal line (resistive heater) in Metal4 and the sensor electrode in Metal5 (for the geometries of FIGS. 6( a) and 6(c) both the heater Htr and sensor Snsr can be implemented in Metal5). Metal layers are separated by inter-layer-dielectric (ILD). Standard back-end-of-line processes are used for this, such as (dual)-damascene processing. Depending on the actual interconnect technology aluminum and copper are the most commonly used metals. The only non-standard steps are: i) deposition of the sensor dielectric on top of Metal5 and ii) opening of the bond pads. Both steps can be done as the final processing steps before dicing and packaging. Thus no changes are required for the standard processing part of the manufacturing method. The sensor dielectric can cover the entire device surface (including the heaters in arrangements in FIGS. 6( a) and 6(c)) thus acting as additional protective layer against the electrolyte. If a scratch protection/passivation stack is used the process steps may involve: opening of the scratch protection (lithography, etch) on top of sensor electrodes and bondpads, uniform deposition of sensor dielectric, removal of sensor dielectric on bond pads.
In order to improve protection, e.g. reduce pin holes, stacks of different dielectrics can be deposited. The actual sensitivity is determined by the final layer in contact with the electrolyte. For transducer/sensor configurations that use a pseudo-reference electrode the dielectric (and scratch protection) is also removed on a metal pad in an area later covered by electrolyte to establish a contact (processed together with bondpad opening). If needed, e.g. to improve corrosion resistance, other metals (silver, gold, platinum etc.) can be deposited on top of this pad by electrochemical or electroless deposition, PVD or CVD etc. with subsequent patterning or lift off.

Energy Consumption

Despite the use of a heater in some embodiment of the electrochemical sensor in accordance with the invention, the overall energy consumption is low, because the heated volume can be very small. If we consider a sensor having a surface area of 1000 μm2 the overall heat capacity is about 7.4*10-9 J/K. The following assumptions are made for this calculation:

- the heater is assumed in Metal4;
- the heat propagation to Metal3 and Metal5 are assumed to be identical;
- the total thickness of the heated stack is assumed to be around 3.5 μm;
- aluminum is used as metal;
- siliconoxide is used as intra-metal-dielectric and inter-layer dielectric, and;
- the electrolyte itself needs hardly to be heated only at the interface to the dielectric).

Doing ten measurements at ten different temperatures with 1K temperature difference and cool down in between (starting from the equilibrium temperature) requires an overall energy of 3.3*10⁻⁷J which corresponds to 92 pAh (at 1V). This energy is so small that it does not impose any restriction to the sensor, even not for miniature sensors in autonomous sensors tags powered with a small battery (capacity in the range of 1 mAh). The low-energy consumption is also beneficial for a rapid cool down. The heated volume is very small (3500 μm³). This means that less than 1 μL of analyte is sufficient to act as “reservoir” with constant temperature. This reservoir serves to cool down the sensor to the initial temperature after a heat pulse.
As already described earlier in this description, the invention may also be applied in different application areas, i.e. in de field of molecule sensors. FIGS. 7( a) to 7(d) show the manufacturing and operation principle of an electrochemical biosensor in accordance with yet another embodiment of the invention and its principle of operation. The electrochemical biosensor is to a large extent very similar to the already described embodiments of the sensor. Therefore, the biosensor will only be discussed in as far as it differs from the sensor already described. FIG. 7( a) shows such (plain) sensor that has already been described. In FIG. 7( b) the sensor is modified for turning the sensor into the biosensor. In order to do so the entire surface of the sensor dielectric Snsd is provided with a probe molecule layer Pml. In operational use the probe molecule layer Pml is in direct contact with the analyte. The probe molecule layer Pml is applied such that the sensor dielectric is configured for binding charged target molecules in the analyte. This enables to determine a charged target molecule concentration in the analyte. FIGS. 7( c) and 7(d) illustrate the operation principle of the biosensor. In FIG. 7( c) the sensor is applied in an analyte having biomolecules Bm in it. Biomolecules Bm that match with functional parts of the probe molecule layer Pml bind to the surface and change the surface potential of the sensor. In FIG. 7( d) the analyte is replaced by a measuring solution. This step is optional, which depends on which approach, as discussed below, is chosen. The measuring solution is an electrolyte that does not contain any biomolecules but closes the electrical circuit.
The surface of the sensor dielectric Snsd is functionalized with probe molecules capable of binding to target molecules that have to be detected in the analyte. The functionalized surface may comprise immobilized nucleic acids, e.g. probe-cDNA or mRNA. When the nucleic acid sequence of the (immobilized) probe-cDNA or mRNA is complementary to the nucleic acid sequence of the target DNA (in the analyte), the probe-cDNA or mRNA hybridizes to the DNA fragment and changes the sensor surface potential. Similarly proteins, hormones and various pathogens may be detected by immobilizing the respective antibodies on the sensor surface. Probe-DNA and antibodies may be immobilized using linkers, self assembled monolayer's (SAM), in situ nucleic acid synthesis, etc. In a variation on this embodiment the probe molecules are directly provided on the first electrode. In that embodiment the first ion-sensitive dielectric is not required.
The core of the measurement principle of the biosensor is the same as for pH/ion measurement, namely to vary a temperature of the analyte near the measurement electrode and measure any change in the potential difference, i.e. output from the transducer. However, due to the different “binding”-mechanism of the biomolecules (the binding is not automatically reversible as is the case for the pH/ion sensor), a slightly modified scheme must be followed. There are multiple approaches possible of which two are discussed below.

Approach 1:

As a first step, a calibration step is performed. In this step a measurement is done using a reference solution of which its content is known. The reference solution is an electrolyte with fixed pH and salt concentration to close the electrical circuit. With “measurement” is meant a measurement in accordance with the invention at at least two different temperatures (determining at least two different potential differences). The obtained data is saved or stored. This calibration step can be done in a manufacturing environment as part of the manufacturing process, if desired.
As a second step, the analyte 100 is applied to the sensor for a predetermined amount of time. During this time period target molecules are bound to the probe molecule layer Pml.
As a third step, the surface of the sensor is flushed. In this flushing step in principle any solution can do that does not contain target molecules nor remove bound target molecules during flushing.
As a fourth step, another measurement (measurement at at least two different temperatures) is performed on the reference solution. The result of this measurement is compared with the data from the reference measurement. If target molecules have bound to the probe molecule layer Pml they will stay there during flushing and the results from the second measurement will be different form the first. This difference is indicative of the concentration of the target molecules in the analyte 100.

Approach 2:

A more simple approach is to measure constantly during application of the analyte 100. With “measurement” is again meant measurement at at least two different temperatures. As the target molecules slowly bind to the sensor probe molecule layer Pml the sensor readings gradually change. It is important that the measurements at different temperatures are performed quickly so that the at least two measurements per “measurement” experience approximately the same biomolecule concentration. The difference between measurements right after analyte exposure and measurements after a certain exposure time is indicative of the original concentration of target molecules in the analyte 100.
The biosensor may comprise several sensors (e.g. in an array) functionalized with different probe molecules (deposited by ink jet spotting, etc.) to detect different target molecules in a single measurement run.
The thermo potentiometric principle in accordance with the invention only allows the detection of charged particles, such as ions, as these charged particles attach to the sensor surface and change the surface potential (Nernst equation only applies to ions, pH is a special ion: H₃O⁺, OH⁻). Therefore the biosensors in accordance with the invention are also applicable to charged target molecules. Charged target molecules of interest are DNA for example. DNA is known to be charged, although this charge may have many different values. Unlike normal ions, such as Nations, the charge on biomolecules heavily depends on the pH-value of the analyte in which they are dissolved, which makes these charged particles somewhat more special.
The invention thus provides a method of determining a charged particles concentration in an analyte. This method, which still is a potentiometric electrochemical measurement, exploits the temperature dependency of a surface-potential of a measurement electrode. The invention further provides an electrochemical sensor and electrochemical sensor system for enabling to determine a charged particle concentration in an analyte. The invention also provides various sensors which can be used to determine the charged particle concentration, i.e. EGFET's and EIS capacitors.
The invention may be applied in a wide variety of application areas, for example in ion concentration sensors, and in particular in pH-sensors. Further the invention may be applied in miniature sensors integrated into autonomous (RFID) tags. The invention may also be applied in potentiometric sensors with surface modifications, e.g. detection of biomolecules attaching to a sensor surface.
Various variations of the sensor and method in accordance with the invention are possible and do not depart from the scope of the invention as claimed. These variations for example relate to material choice, layer thickness, spatial arrangement of the sensor parts, etc. Also, in the method of determining a charged particle concentration in accordance with an embodiment of the method of the invention, many alterations are possible. Such alterations fall within the normal routine of the person skilled in the art and do not deviate from the inventive concept here disclosed. The most important variations are:

- Sensor dielectrics may include materials like: SiO₂, Ta₂O₅, SiN, TiO₂, HfO₂, Al₂O₃, and similar materials.
- Non-dielectric sensor materials can also be used, such as antimony and other metals, polymers, such as Polyaniline, Polypyrrole, Linear Polyethylenimine, Linear Polypropylenimine, and similar materials. These materials may be either in direct contact with the sensor electrode or with a dielectric in between.

A temperature sensor may be implemented near the sensor to accurately determine the temperature at the interface between sensor material (dielectric) and analyte.
For example, a thermistor can be realized by an additional thin metal wire surrounding the sensor pad (similar to the arrangement of the heater around the pad in FIG. 6( a)).
Several sensors which are configured for different analytes can be implemented on a single chip, e.g. pH-value and Na⁺-ion concentration.

- An inductive heater may be used instead of a resistive heater.
- The heater can be operated in constant-power mode (wherein activation time is adjusted) or in constant-activation-time mode (wherein the power is adjusted).
- The sensor capacitance forms a capacitive voltage divider together with the input capacitance of the transducer (e.g. gate capacitance of the transistor, input capacitance of operational amplifier). In order to improve the signal of the sensor, the sensor capacitance can be increased (it should be preferably larger than the transducer's input capacitance). The capacitance can be increased by making a the sensor area larger, or by making the sensor dielectric layer thinner
- A peltier element may be used as a cooler (temperature setting means).
- The sensor dielectric may be provided with or exchanged with an ion-exchange resin. Ion-exchange resins are based on special organic polymer membranes which contain a specific ion-exchange substance (resin). This is the most widespread type of ion-specific electrode. Usage of specific resins allows preparation of selective electrodes for tens of different ions, both single-atom or multi-atom. They are also the most widespread electrodes with anionic selectivity. However, such electrodes have low chemical and physical durability as well as “survival time”. An example is the potassium selective electrode, based on valinomycin as an ion-exchange agent.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb “comprise” and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article “a” or “an” preceding an element does not exclude the presence of a plurality of such elements. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Throughout the Figures, similar or corresponding features are indicated by same reference numerals or labels.

Claims

1. An electrochemical sensor for determining a charged particle concentration in an analyte, the sensor comprising:

a measurement electrode for measuring a surface-potential at an interface between the measurement electrode and the analyte in which the measurement electrode is immersed in operational use, and

a control means for measuring the surface-potential at at least two different temperatures of the interface to obtain at least two measurement points of a surface-potential versus interface-temperature curve.

2. The electrochemical sensor as claimed in claim 1, wherein the control means comprises a temperature setting means (Htr) arranged for setting a temperature of the interface at at least two different temperatures of the interface.

3. The electrochemical sensor as claimed in claim 1, wherein the control means comprises a controller, the controller being coupled to the measurement electrode and being arranged for initiating the measuring of the surface-potential with the measurement electrode at at least two different temperatures of the interface.

4. The electrochemical sensor as claimed in claim 3, wherein the controller comprises a temperature sensor for measuring the temperature of the interface, and wherein the controller is further arranged for initiating the measuring of the surface-potential at a desired interface temperature value.

5. The electrochemical sensor as claimed in claim 3, wherein the controller comprises storage means for storing the respective measured values of the surface-potential and optionally the respective values of the temperature of the interface.

6. The electrochemical sensor as claimed in claim 2, wherein the measurement electrode comprises an ion-sensitive extended gate field-effect transistor which comprises a field-effect transistor, a sensor electrode being electrically coupled to a gate of the field-effect transistor, and an ion-sensitive sensor dielectric provided on the sensor electrode, the sensor electrode being arranged for contacting the analyte via the ion-sensitive sensor dielectric in operational use, and wherein the temperature setting means comprises a resistive heater which is arranged in thermal coupling with the sensor electrode for setting a temperature of the interface between the sensor dielectric and the analyte in operational use.

7. The electrochemical sensor as claimed in claim 2, wherein the measurement electrode comprises an electrolyte semiconductor insulator structure which comprises a conductive contact layer, a semiconductor layer provided on the contact layer, an ion-sensitive sensor dielectric provided on the semiconductor layer, the semiconductor layer being arranged for contacting the analyte via the ion-sensitive sensor dielectric in operational use, and wherein the temperature setting means comprises a resistive heater which is arranged in thermal coupling with the electrolyte semiconductor insulator structure for setting a temperature of the interface between the sensor dielectric and the analyte in operational use.

8. The electrochemical sensor as claimed in claim 6, wherein the sensor dielectric is further provided with a probe molecule layer comprising probe molecules, such as antibodies, and DNA/RNA strands, the probe molecule layer being in direct contact with the analyte in operational use, the sensor dielectric thereby being configured for binding charged target molecules for enabling to determine a charged target molecule concentration in the analyte.

9. A semiconductor device comprising the electrochemical sensor as claimed in claim 1.

10. An RF-ID tag comprising the electrochemical sensor as claimed in claim 1.

11. An electrochemical sensor system for determining a charged particle concentration in an analyte, the system comprising:

a measurement electrode for measuring a surface-potential at an interface between a measurement electrode and the analyte in which the measurement electrode is immersed in operational use;

a temperature setting means arranged for setting a temperature of the interface at at least two different temperatures, and

a controller coupled to the measurement electrode and being arranged for initiating the measuring of the surface-potential with the measurement electrode at at least two different temperatures of the interface.

12. A method of determining a charged particle concentration in an analyte, the method comprising steps of:

determining at least two measurement points of a surface-potential versus interface-temperature curve, wherein the interface temperature is defined as a temperature of the interface between a measurement electrode and the analyte, wherein the surface-potential is defined at the interface;

calculating the charged particle concentration from locations of the at least two measurement points of said curve.

13. The method as claimed in claim 12, wherein the step of determining of said curve comprises sub-steps of:

setting the interface temperature to a first value;

determining a first value of the surface-potential at the interface, wherein the first value of the interface temperature and the first value of the surface-potential together define a first respective one of the measurement points of said curve;

setting the temperature of the interface to a second value different from the first value, and

determining a second value of the surface-potential at the interface, wherein the second value of the interface temperature and the second value of the surface-potential together define a second respective one of the at least two measurement points of said curve.

14. The method as claimed in claim 13, wherein the difference between the first value of the temperature of the interface and the second value of the temperature of the interface is smaller than a predefined threshold.