FI20175623A1

FI20175623A1 - A method and devices for adaptive measuring of electrochemiluminescence

Info

Publication number: FI20175623A1
Application number: FI20175623A
Authority: FI
Inventors: Andrei Pavlov; Timo Korpela; Tony Wahlroos
Original assignee: Labmaster Oy
Priority date: 2017-06-29
Filing date: 2017-06-29
Publication date: 2018-12-30
Also published as: WO2019002697A1; FI20175623A

Abstract

The present invention relates to a device that can generate highly energetic or hot electrons into solutions. The device comprises an electrical pulse generator, a reaction cavity having a solution into which hot electrons can be injected as a result of electrical pulses via anode and cathode electrodes by the electrical pulse generator, at least one sensor in the reaction cavity, and an adaptive system making decisions based on previous data obtained before the decision, predictive model, an algorithm and instant data for adjusting and controlling the electrical pulse generator to attain amplitude, duration and frequency values of the pulses which are the most favorable for producing hot electron flow into the solution in the reaction cavity. The present invention also relates to a method to provide and control a flux of highly energetic or hot electrons into solutions, and a computer program product.

Description

A METHOD AND DEVICES FOR ADAPTIVE MEASURING OF ELECTROCHEMILUMINESCENCE

FIELD OF INVENTION

The present invention relates to adaptive methods and systems in general, and particularly to use of adaptive methods in electro-chemistry. The invention is related to instruments and methods to produce highly energetic hot electrons into solutions to carry out unique chemical reactions. More specifically, the invention is related to optimization of chemical reactions producing light in aqueous solutions.

BACKGROUND OF THE INVENTION

There is a universal need for fast, sensitive, quantitative diagnostic technologies suitable for public health, veterinary medicine, environmental monitoring, etc. Among the various technologies used for diagnostics, the assays based on chemiluminesence are prominent. They include anodic as well as hot-electron induced cathodic electrochemiluminescence (ECL). The production of light with intensive electrical pulses is a specific, easily measurable, way of detection of chemical reactions.

For example, US20020081749 (Kulmala, S., et.al.) discloses that various longlife-time luminescent compounds can be excited in water or in aqueous-organic solutions with injecting cathodic pulses into the solutions through conductive cathodes coated with a thin flawless insulating film. The electronic circuitry to achieve the excitation effect can be any signal generator with adjustable pulsed voltage. Basically, the present disclosure is based on making very specific electrically assisted chemical reactions, part of which can be applied to generate light emission. When using highly energetic electrons, such reactions decompose molecules and create other reactive molecules. Although the present invention is illustrated with the examples of generation of light from specific fluorophores, the process is only an example how a variety of chemical reactions can be achieved by electrical pulses. The main significance of the present disclosure is to generate unique chemical reactions by electrical pulses. ECL can be considered as an easily detectable and measurable tool for a wide variety of consequence of electricity according to the present disclosure. Therefore, the term ECL may be considered in this context more widely than only producing light. A serious drawback, in ECL technologies is that even if an optimal voltage can be found to a specific sample for maximum light output signal (or recovery of a chemical reaction product), the output voltage is not optimal for every sample or condition and it also changes along with the time. There exists variation in the conductor materials, temperature, viscosity, coating films, their interactions, and the mixture of reactive compounds in the liquid phase. The main application of the devices described in US200220081749 (Kulmala, S., et al.) was bioaffinity assays including a specific biofilm above the insulating film. This biofilm has complex structure and it likely reacts in unknown ways itself with unspecific reactions by hot electrons causing interferences in the integrated emission output and consumes part of the electron flow. Since the luminescence labels are spatially fixed and a minor part of labels can diffuse from the bulk solution to this fixed volume, the time for integration of the light pulses is one of the limiting factors for obtaining the maximum signal. This and the signal-to-noise problems are the limiting factors for obtaining high sensitivity of the system described by Kulmala, S. et al (US 200220081749). The specificity of the excitation of a certain label molecule is dependent on average energy of the excitation reaction that is related in voltammetry as the half-wave potential.

There is a need to develop methods and devices where maximal integrated specific light output from the ECL system can be obtained, or in more general expression, when a specific chemical reaction will have the maximum yield.

There are various chemical and physical parameters which, in theory, could be targets of improvement of optimizing electrically assisted chemical reactions. So far it has been assumed that the improvements can be made only in the chemistry, materials, or in production technology of the electronic system. Here, however, a completely different approach was taken to apply adaptive methods to improve instrumental devices which enable significantly better injection of charged particles into the reaction medium of chemicals.

Adaptive methods have been applied to well-known physical phenomena like in optics, electronics, and tele-communications, which can be completely expressed in mathematical format. Byrne et al. in US 8,837,066 BI applied a least mean squares algorithm for reducing noise in signals using a set of filters in data storage devices. Colavolpe et al. (U S7,444,082 B2) suggested a method for the adaptive adjustment of a polarization mode dispersion compensator in optical fiber communication. Adriaan J. De Lind Van Wijngaarden et al. (US 8,122,330 B2) suggested a rate-adaptive forward error correction for optical transport. Aviv Ronen et al. proposed adaptive modifications in micro-opto-electro-mechanical systems (US 8,939,025 B2) for a rotation rate sensor. Bryant E. Sorensen et al. suggested methods for use of adaptive secondary path estimate to control equalization in an audio device (US 9,478,212 BI). Stephen Whitlow et al. proposed a user interface device (US 9,547,929 BI) that can be used for air traffic management.

The technologies, to which adaptive methods have been applied, have so far been well known physical phenomena that can be completely expressed in mathematical format. ECL phenomenon and technology, however, is a more complex cooperation between chemistry, physics and photonics. Both, chemical and physical parameters work/are valid, during very short periods of time only. Second, adaptive methods require mathematical methods and algorithms that are used in control electronics and software. For effective use, the adaptive methods require very fast response; calculations are to be made during microseconds. Because of these challenges, adaptive methods have not been applied to ECL technology (or to control electrically assisted chemical reactions) and adaptive methods have not been regarded as a possible solution to improve the performance of ECL devices and accuracy/ efficiency of diagnostic systems using ECL.

It was shown in the present disclosure that adaptive methods can be successfully applied to significantly more complex systems including unpredictable biological functions.

SUMMARY OF THE INVENTION

In this disclosure therefore, it is provided improvements in injecting hot electrons into a medium including sensing of the injection of hot electrons and/or time-dependent light output and adjustment of the electronic properties accordingly.

It is an object of this invention to control the injection of hot electrons into a reaction medium to generate specific chemical reactions.

It is another object of this invention to target hot electrons into a certain distance from the electrode.

It is still another object of this invention to regulate the energy of hot electrons to cause certain chemical reactions while preventing non-wanted chemical reactions.

It is another object of this invention to monitor the flow of hot electrons online after each electrical pulse over the electrodes and obtain feedback signals.

It is yet another object of this invention to apply optimal strength and duration of electrical pulses to produce hot electrons into the reaction medium.

It is an object of this invention to apply an optimal electrical pulse train to obtain maximum amount of reaction products in a specified period of time.

It is still another object of this invention to apply optimal electrical pulse or pulse train to achieve maximum amount of light emission from hot-electron excitable fluorophores in a specified period of time.

It is an object of this invention to produce a reproducible electrochemical system for diagnostic purposes.

It is yet another object of this invention to provide a system to avoid incidental errors due to chemical part of the diagnostic system.

It is an object of this invention to provide improved diagnostic systems to improve patient safety.

It is an object of this invention to provide a device capable of generating highly energetic or hot electrons into solutions, wherein the device comprises: i) an electrical pulse generator, ii) a reaction cavity having a solution into which hot electrons can be injected as a result of electrical pulses via anode and cathode electrodes by the electrical pulse generator; iii) the reaction cavity including at least one of the following sensors: a photon emission sensor, a sensor measuring electron flow to the cavity over tunneling and solution, and a sensor measuring electron flow at a distance of 50-500 nm from the electrode surface; and iv) an adaptive system making decisions for adjusting and controlling the pulse generator attaining the adjustment of the pulse generator to amplitude, duration and frequency values of the pulses which are the most favorable producing hot electron flow into the solution in the reaction cavity.

It is an object of the invention to provide a method to generate a flux of highly energetic or hot electrons into solutions in a device involving more than two different processes, wherein the method comprises an adaptive system making decisions for adjusting and controlling electric pulses generated by a pulse generator to attain the most favorable amplitude, duration, and frequency of the pulses to produce hot electron flow into the solution in the reaction cavity .

It is yet another object of the invention to provide a computer program product embodied on a computer readable storage medium, the computer program product comprising instructions executable by one or more processors to control production of a flux of highly energetic hot electrons into solutions by an adaptive system in a device involving at least photonic, electronic and chemical processes, wherein the adaptive system makes decisions based on previous data obtained before the decision, a predictive model, instant data and an external function.

BRIEF DESCRIPTION OF THE DRAWINGS FIG.l illustrates known art showing the luminescence in an immunoassay using organic Terbium chelates which produce typical delayed luminescence. The voltages of the excitation pulses vary from 7 to 25 volts. The ECL decays are shown with some delay time after the excitation pulse. FIG. 2 A and B. 2A illustrates a basic adaptive model of the present disclosure. Excitation voltage is increased gradually according to a mathematical model until to a forecasted voltage. The excitation time intervals are variable and the optimal voltage information is obtained from feedback signals. Several measurable parameters can be optionally subjected to the analysis process including the signals from separate tunnelling sensors. There are three main stages of operation: A, B. and C. Stage A operates mainly with electronic phenomena while the ECL is below a threshold value, tunnelling currents are main parameters in the feedback in stage A. Stage B operates mainly with measuring photon energy. A time-resolved spectroscopy is used to measure ECL decay times. Stage C is a relaxation process. After the ECL is decayed to a very low value, again the tunnelling currents are used to estimate relaxation times indicating that the system is returned to its basic electro-chemical state. Figure 2B shows similar graphs as Fig 2 A comprising typical process times. Stages A, B and C are not completely separated but are partially overlapping.

Fig. 3 is a schematic diagram of a variant of the ECL measuring instrument of the present disclosure. The tunnelling sensors probe 1 and probe 2 are shown in the figure. The part marked as “ECL device” represents the electrochemical chip. The basic parts of the chip are marked in the figure.

Fig. 4 illustrates ECL decays for different number of excited molecules. The curves with longer life-times correspond to higher number of excited molecules.

Fig. 5 illustrates means to express the present ECL diagrams. An ECL decay is shown as a spiral which are formed by rotation of the ECL light intensity vector. The length of the vector corresponds to the light intensity. Rotation angle corresponds to decay time. The diagram shows the ECL decay up to 0.1 ms. The angle is slightly bigger than 90 degrees.

Fig. 6 illustrates further ECL diagrams. An ECL decay is shown as a spiral formed by rotation of an ECL light intensity vector. The length of the vector corresponds to the light intensity. Rotation angle corresponds to decay time. The diagram shows ECL decay until 2.9 ms. There are several circles number of which is equal to 2.9/0.360 or about 8 circles.

Fig. 7 illustrates ECL diagrams. An ECL decay is shown as a spiral which is formed by rotation of a ECL light intensity vector. The length of the vector corresponds to the light intensity. The diagram shows ECL decay up to 2.9 ms. There are 8 circles. When the ECL vector intersects the x-axis, the life-time is obtained at this point. For longer decays, as in this case, there are a number of lifetime values corresponding to the decay times of 0.360 ms, 0.720 ms, ... i*360, ...8*0.360 ms. These lifetimes characterize the ECL process at different stages. These diagrams are convenient to automatically measure lifetimes of different paths of the decays. The feedback adjusts cathode voltage to obtain maximum τΝ. Here τ is the life time and N is the concentration of excited molecules.

Fig. 8 shows dependencies τΝ/τοΝο vs. time. The curves show electro-photo dynamics of the ECL process. A first dashed curve a) represent the ECL process characterized with a small number of excited molecules and quick ECL decay. Hence, the function Ντ is continuously decreasing with time. The second dotted curve b) represents the ECL process characterized by the same number of excited molecules as in the case a) but with longer lifetime. The curves c) and d) show the ECL process characterized with a higher number of excited molecules and longer lifetime due to the feedback. The threshold of the ECL process, curve e), corresponds to Nthith =C1= constant for initial stage which is obtained during first stage (see Fig. 2 A) of the feedback. The amplitude of the pulsed voltage is calculated using a special shaped function. The special shape function incorporates theoretical curve and experimental data for specific molecules used in ECL process. During the second stage (see Fig. 2A) of the feedback when ECL intensity is the main feedback parameter, the integrated value \Ντάί has its maximum. In practice, good results are obtained within 10% of variation from the maximum value. FIG. 9A is a schematic diagram of the adaptive mechanism related with the electronic feedback. The diagram illustrates how the adaptive mechanism works. There are initial control parameters and initial measured data. The control parameters for the voltage supply are voltage pulse, pulse duration, frequency, amplitude and DC offset. The measurement data include the tunneling current 1, duration, frequency, light intensity and tunneling current 2. These initial parameters and measured data as well as the initial value of the function Ντ are connected on the left side of the TUNNELING FEEDBACK loop. This loop is organized so that new values calculated using the adaptive coefficients and measured data are re-entered as the new initial condition in the loop. The new measurements are performed and new input values are calculated again using new adaptive coefficients. The procedure is iterated until the threshold condition is fulfilled. Then the new condition and the electronic adaptive coefficients are sent to the PHOTONIC FEEDBACK procedure shown in Fig. 9B. FIG. 9B is a schematic diagram of the adaptive mechanism related with the photonic feedback. The diagram reveals how the adaptive mechanism works. There are initial control parameters and initial measured data received from the TUNNELING FEEDBACK part. The control parameters for the voltage supply are voltage pulse, pulse duration, frequency, amplitude and DC offset. The measurement data include the tunneling current 1, duration, frequency, light intensity, tunneling current 2 and Ντ. These initial parameters and measured data as well as the initial value of the function Ντ are connected on the left side of the PHOTONIC FEEDBACK loop. This loop is organized so that new values calculated using the adaptive coefficients and measured data are reentered as the new initial condition in the loop. The new measurements are performed and new input values are calculated again using new adaptive coefficients. The procedure is repeated until the threshold condition is fulfilled. FIG. 9 C is a block diagram of the adaptive system comprises previous data that are used for initial condition for an algorithm; a predictive model which is another input for the algorithm; parameters that are produced by the algorithm; instant data that are obtained during measurements using the above parameters; another algorithm which analyses the instant data and makes a decision with the help of an external function. The external function does not depend on measurements or instant data. The algorithm makes a decision that is used to modify the predictive model. The adaptive system produces new data. It repeats all the steps described above until the decision stops the loop. FIG. 10 shows the ECL intensity vs. concentration of terbium chelates measured at different pulse voltages. FIG. 11 shows the ECL intensity vs. pulse voltage measured for different concentration of terbium chelates.

DETAILED DESCRIPTION OF THE INVENTION

Definitions in the present disclosure

Sample device is a cassette or more or less complex lab-on- chip which is often a single-to-use-device and where to a sample to be analysed is placed.

Measuring instrument is an electronic device according to the present disclosure which provides electricity to the sample device and measures the emitted light from it and which integrates all these parts as a functional system communicating internally to get maximum output. ECL. Electro-chemiluminesence (ECL) is the electro-chemical process in which certain molecules get additional energy as a result of chemical reactions, and molecules are thereby transferred into excited state from which they emit photons. Most of chemical reactions produce radiation during chemical reactions in certain energy area of the electronic spectrum.

Feedback is a method to improve performance of an instrument, method or model in a system characterised with a certain function where an output data is used as an input data to repeat the function. The function can be a measurement, calculation, or a sequence of such measurements or calculations.

Hot carrier is an electron having an exceptionally high kinetic energy. The hot carriers can tunnel through structures that have energy barrier for other type of carriers. In order to the hot carriers to tunnel through the barrier, their energies have to be high. They receive high kinetic energies through acceleration in high electrostatic fields produced by the excitation voltage pulse.

Excitation pulse is an electric voltage pulse applied over anode and cathode that is characterised with the amplitude, duration, and shape. In the case of a number or train of excitation pulses, they are also characterised by frequency. The excitation pulse is a source of energy for production of hot carriers.

Light emission is a flow of photons radiated by the excited molecules. The light emission is described by quantum mechanical principle including energy conservation and transferring the excited molecules into its basic energy state. The light emission can occur from a number of excited states characterised by different energy levels.

Adaptive excitation pulse refers in the present disclosure to an individual excitation pulse which yields optimum amount of hot electrons and/or photon counts in a train of excitation pulses.

Micro-electronic devices that use chemically active structures with variable electronic characteristics, including photon emission, cannot be described by standard methods and tools used in micro-electronics. Particularly, there are micro-electronic devices including bio-active materials which can change electronic and photonic characteristics of the device. Control of such devices requires non-standard systems which can adjust voltages and currents in order to obtain optimal photonic parameters. It is of utmost importance since biological materials are sensitive to electrostatic fields, charges, electrical currents, and electro-chemical reactions. Often all these factors are present and mutually interdependent.

Devices to transfer electron energy to photons in solutions in electro-chemiluminesence (ECL) system are well-known in the art. In the simplest form such devices comprise two electrodes, cathode and anode and an active layer capable of producing photons. If the cathode is covered additionally by a thin insulating film barrier, highly energetic or hot electrons can be injected into solutions through the insulating barrier. This insulating layer may have been covered also with another molecular layer, e.g. coated with antibodies or nucleic acids, and/or their luminescence labeled counterparts. Certain reactive chemicals can also locate in bulk solution and diffuse to the reaction phase. Such a system generating hot electrons enables special analytical, synthetic and/or destructive reactions to be performed which reactions may not be possible in conventional electrochemical systems described in textbooks. While certain details of the chemistry of hot electrons in solutions are known, the novel inclination in the present disclosure was to develop more effective controllable means to inject hot electrons into solutions to carry out specific chemical reactions. While the present disclosure is not limited to producing light emission by hot electrons the experimental method was focused to cathodic ECL systems used in clinical diagnostics since this field is of high immediate practical importance and standard methods exist to measure hot electron via chemical reactions of certain luminescent label compounds. Such commonly used methods are exemplified by LUCIA-technology (Labmaster, Finland; www.Labmaster.fi). The DELFIA-technology (Perkinelmer-Wallac) exploits similar label compounds but they are excited with UV-light pulses and not with hot electron pulses like in the present disclosure. Whereas the experimental studies of this disclosure were mainly conducted in electrolytic water solutions, the term solution as used in this disclosure includes also any aqueous solutions, including micellar solutions and water-organic solvent mixtures. As used here, the term solution can also mean steam phase allowing hot electrons to be stabilized with water molecules, i.e. to make hydrated and/or stabilized electrons. While the subject of the present disclosure is the construction and function of the source of the electrical pulses causing generation of hot electrons, the place and nature of the reactions caused by hot electrons affect the details of the construction and applied electrical parameters. The basic construction of the present disclosure, however, can be readily modified by a skillful artisan to fit it to different applications.

The barrier layer or insulating film on cathode is relatively thin (having a thickness of 0.5 to 6 nm) but provides hot-electron tunneling through it by electrical pulses (frequency being in a range of mHz-lOOkHz). Those hot electrons that travel/move into the solution phase or bio-active layer can interact with the active molecules, exemplified by label compounds, resulting in a change of their chemical composition. The basic electronic circuitry, according to the present disclosure, provides voltage pulses applied over the electrodes.

The whole electronic unit also contains means to measure electric currents through electrodes, and an optical system that measures light intensities. The generally used instruments have the standard excitation systems known from literature. Commercial pulse generators can be used as available from various commercial manufacturers. The light measuring unit generally used is a photodetector and photon counter. That shows that there has been no attempt to integrate the units functionally and therefore the improvements disclosed here have not been possible.

The electro-chemical-optical process is specific in the context of biological materials. They are neither solid state materials nor stable liquids with constant viscosity, density, and chemical composition. Moreover, biological systems depend largely on the temperature. All these above-mentioned characteristics are changing during hot electron flow and electro-chemical-optical process. Thus, logically the process requires a strict and unique control.

There are two clear functional limits in the operation of pulse generators in generating hot electrons: 1) the voltage pulse is too weak, it has small voltage amplitude, and/or short duration so that hot electrons are not generated because the energies of hot electrons are below the energy barrier defined by its properties and thickness, and 2) the amplitude of the voltage pulse is high enough enabling tunneling hot electrons through the resistive layer, but, duration is too long, resulting in existence of the electro-static field in the active layer after the hot electrons have been generated in the active layer. The electrostatic field in the active layer accelerates further hot electrons towards the anode and also attracts positively charged molecules in opposite direction towards the interface with the resistive layer. This can result in changing chemical reactions because the molecules of the active layer can interact with the surface molecules of the resistive layer. This current causes increase of temperature in the active layer which may cause secondary unwanted effects.

In a standard feedback scheme, one can use light intensity and anodic current to adjust the amplitude of the pulsed voltage and its duration, but that would not be enough because in an ideal case the electrical current should be zero as all hot electrons are involved in the electro-chemical reaction and stay in the active layer. Hence, there is a situation when the feedback parameters are unclear. Therefore, an additional control mechanism is required and which is provided in this disclosure.

In accordance with the present disclosure, it is also used probes measuring tunneling current in the resistive layer and the second probe measures another tunneling current in the solution or in the active layer. There can be more than one probe in the active layer. Hence, at least two parameters of the tunneling current are also recorded. These parameters can be used to adjust the input voltage. This disclosure allows minimizing the impact of the electro-magnetic field and electrical currents on the chemical- and bio-composition of the active layer. This is of high importance in medical diagnostics because more tests can be performed on the same sample without destroying it. In addition, the present method allows obtaining more information about bio-chemical materials and performing a more quantitative test.

The adaptive system can be described mathematically. Suppose, we have two variables, f and g, and consider their product h=fg. For simplicity, we omit coefficients responsible for correct dimension units and supposing the variables f and g are normalised functions. The change of h is described by its derivative dh=gdf+fdg. In the case of present disclosure, h is a photonic light intensity; f is related with the electronic properties and electrical charge, and g is related with concentration of excited molecules that emit light. In order to obtain a maximum value h, the derivative dh/dt should be positive. There can be variations of dh/dt, but it is needed to obtain a maximum h within the shortest time. The algorithm can be described as follows: based on the observed value dh, we set a new value fi+i=fi+d‘ f where coefficient d1 is an adaptive coefficient. A new value ^+ι=&+ιίί+ι=&+ι(ίί+^ίί)=&+ιίί(1+^). After N iterations we get hN+i=gi+ifi+i=gN+ifi(l+d1)(l+d1+1)...(l+dN+1). So, we have a set of values hi, hi+i, .. ,hN+i and the last value has to have its maximum. This means that at maximum dh/dt=0. Or, in our adaptive process

Hence, the condition for obtaining the maximum value h is

Taking into account that

where mN is the adaptive photonic coefficient, we obtain

Because gN 0 then one can rewrite

One can see that the electronic function can be replaced with the adaptive electronic coefficients or adaptive electronic function d1, the optical function is described by the adaptive optical coefficients m1. The real systems are core complex because photon light emission is correlated with the number of excited molecules, and the emitted photons reduce the number of excited molecules:

In the case of one pulse generation, the function g is continuously decreasing with time. Its decay is described by the lifetime τι. The lifetime depends on the number of excited molecules and light intensity. It also depends on the energy of the excited molecule.

In the case of a multi-pulsed generation, the system can simultaneously generate new excited molecules. In a state of maximum photon emission with maximum lifetime, the number of new excited molecules shall be equal to the number of emitted photons. In this case

The additional term gei is correlated with the number of new excited molecules. At maximum photoemission we get

or

This is the additional condition that is used to find the maximum photonic efficiency. The maximum photonic efficiency is characterised not only by the

number of excited states, but also the maximum lifetime. Therefore, a more accurate function that characterises the process is hr. If any of its components, h or τ, is very small, then the integrated photonic process is not effective. Then, the most effective excited photon emission process is obtained at hr = maximum.

Sample chip

Various technical solutions for the medical ECL sampling devices are known. They can be special cassettes including all necessary reagents, lab-on-chip devices, a chip made on semi-conductive basement, or a simple sampling stick with proper electrical connections. In the context of the electrical excitation and measuring unit according to the present invention use of a micro ECL sample device that requires very small amount of chemicals, reagents and tiny electrical contacts is possible because this invention enables probe technique that allows measuring electrical currents without influence on functionality of the device. Particularly, this invention enables measuring the tunneling current of hot electrons by using at least one probe. This probe is placed on top of the resistive layer (see Figure 3). The tunneling current measured by this probe indicates the dynamics of the tunneling of hot electrons. A small number of hot electrons should be sufficient to create suitable amount of excited molecules producing satisfactory ECL signal. More precisely, the number of hot electrons shall be equal to the number of molecules in the ground state in the vicinity of the oxide layer. Because the number of molecules varies with time, the number of hot electrons shall be adjusted accordingly. The tunneling current measured by a probe indicates the number of hot electrons capable to react with the molecules.

Injection of excess of hot electrons onto the ECL sample system can cause misbalance in the electrical charge distribution reducing efficiency of the sample device. Therefore, a second probe (2) is optionally placed at narrow distance from the surface of the resistive layer (see figure 3), preferably at a distance of 100 to 200 nm from the resistive layer. The majority of ECL processes occurs in distance having thickness of below 50 nm. The effective distance can be increased to 200 nm or even to 500 nm with proper adjustments of injection parameters. Compared to the whole distance between cathode and anode (solution phase) the ECL reaction layer is extremely small. Whenever the test system is based on bioaffinity measurement principle, the analytes will concentrate into very narrow space near cathode surface, and so-called proximity effect is achieved which enables the exploitation of the homogeneous assay principle without washing out of the unbound label. One of the main advantages of the excitation and measuring device according to the present disclosure is to be able to focus the hot electrons in a narrow distance specific to the test system in concern.

In the case when all injected hot electrons are reacting with the label molecules in distance below 100-200 nm, there will be no electrical current detected by the probe (2) which locates at this distance. In case when the number of hot electrons exceeds the number of the molecules in the ground state, the rest of the hot electrons create electrical current in the probe (2). Probe (1) detecting incoming electron flow and probe (2) are thus effectively used to find out the proper parameters of the ECL process.

The ECL sample device can be produced as in the prior art or by using semiconductor fabrication techniques including material deposition, patterning, etching and thermal treatment. The substrate can be a silicon wafer or another suitable material that has desired mechanical and electrical properties. Silicon is favorable because it is standard material in semiconductor industry for fabrication of integrated circuits, sensors and other micro devices. The process starts from deposition of bottom layers, for example, by chemical vapor deposition, evaporation or magnetron sputtering in vacuum. After the first layer is prepared, it is treated by lithography applying to a photoresist. The photoresist is a temporal layer on top of the deposited one. After the lithography treatment, the photoresist is partially removed revealing the patterns of the deposited material that are etched away. The etching removes the reminder of the photoresist as well. After this step, the first layer is patterned. Then the procedure is repeated for making the second layer and so on. In general any of the prior art methods in semiconductor industry can be exploited.

Light detection unit

The light detection unit is a photodetector device with supplied measurement electronic circuitry. In one embodiment, the light detection unit is a photomultiplier tube (PMT). It placed in the vicinity of the ECL region of the sample device. While electrical pulses induce photon emissions, the generated photons are transformed and amplified back to electrical pulses which are measured. The output signal of PMT is directly correlated with the light intensity of the ECL process. The PMT output signals, according to the present disclosure, adjust the feedback of the excitation parameters.

In another embodiment, the light detection unit is a semiconductor avalanche photodiode (APD) which is even more suitable than PMT for integration of the electronics. ADP's measurement electronics circuitry can be integrated with other electronic circuitry on the same chip as ASIC microchip.

In a yet another embodiment, the light detection unit is a single photon detection device based on Si p-n structure called Silicon Photodiode (SiPD). When photons move across silicon, they can be absorbed and transfer their energies to electrons (bound electrons). The absorbed energy allows the electrons to transit from the valence band into the conduction band, creating charge carries, electron-hole pairs. The absorption depth of a photon in silicon correlates with its energy and this correlation is used to measure the photon wavelength. SiPD can detect, not only intensity of the ECL light, but also measure the light spectrum. Silicon absorbs photons in a wide range of wavelengths within a depth of a few tens of microns and thus suits well as a photodetector device in the adaptive instrument according to the present disclosure.

Another regime of operating SiPD is realized at high electrostatic field when the charge carriers have gained significant energies and can generate secondary carriers in the depletion layer through impact ionization. This operating principle is used in a single photon avalanched photodiode (SPAD). It operates at reverse bias at breakdown region. Hence, it needs some quenching time to return to the effective gaining.

There can be still other types of photodetectors, for example, those based on the nanotube technique wherein the energy transition from photon type to electron type is realized in a special geometrical complex of nano wires. However, all these described photodetectors can be applied as a part of the instrument of the present disclosure.

Excitation unit

The present disclosure deviates the most distinctly from the known art in the construction of the electrical excitation unit. However, the signal/pulse generator part can be chosen from practically any signal generator operating or achieved to operate at voltage area of pulses from 1 V to 100 V and 0.01-10 mA range with frequency of pulses from nano- to milliseconds. Whereas the signal generator is of standard, it must include, according to the present disclosure, inputs which allow dynamic regulation of the each of the parameters of voltage, current, and frequency and duration of the excitation pulses.

The electronics circuitry of the pulsed generator can be realized in different arrangements. It can be an external generator connected to the ECL device using wires or probes, or it can be a separate ASIC micro-chip integrated together with the ECL device.

Programmable control unit A programmable control unit is the core of the measuring and excitation system for generating hot electron flow to various purposes. If it is used for measurement of light produced by hot electrons into solutions, the control unit comprises, electrical excitation unit, an optical detection unit, time-resolved spectroscopy unit, electronics measurement unit and an interface system. Figure 3 shows schematically the main parts of the instruments and their interconnections. The essential part is the control unit which provides voltage pulses and measurement of the tunneling currents. An example of the programmable unit for measuring extremely small electrical current, for example, can be the Keithly 236 instrument which can be programmed so that it will provide pulsed voltages and accurate measurements of small electrical currents of nA and less. A compact system made on a chip (SOC) electronics can be designed to carry out similar functions.

If external measurement devices are used, their drivers are integrated into one control software program. The software program, for example, can be developed using Lab VIEW programmable language.

If the devices are integrated in one electronics block with integrated circuit components mounted on PCB boards, then the software program can be similarly designed using any standard programming languages. In this case, the electronics components are supplied with communication commands allowing control using the interface.

The communication bus can be a GPIB, USB or other. The GPIB bus is mainly used with external instruments that are supplied with the GPIB control drives.

Role of adaptive system in the present disclosure

Adaptive technique is known in microelectronics, robotics, statistics, etc. to find a proper function of the system. In many cases, parameters of the system are not known a priory, for example, a telescope for scanning the sky. It receives a signal, optical or radio, from a particular direction. Based on the output signal, it continues searching the object with more accurate angular resolution (in case of reasonable signal) or changes the direction with bigger change of direction. A feedback technique is a method allowing a system to obtain a result during shortest possible time. Theoretically, a telescope can scan the sky with the best angle resolution. However, it will take too long time to scan the whole sky. The sky can change, some objects can disappear and new objects can arise.

Therefore, it is important to get a scanning algorithm that allows a fast scanning with crude angle resolution and change to fine scanning when an object is detected.

In microelectronics, the devices are characterized with certain electrical and physical parameters. Having known behavior of an individual device, one can develop more complex systems comprising the individual devices. Here the devices have functions that are predicted, or tested. The feedback in electronics systems is used to find out proper conditions for operation of the system.

Consider a system wherein certain components have uncertainty parameters of the main functions. The functions include optical, electronic, and chemical function. Due to the uncertainty, traditional feedback methods do not function effectively. The present disclosure does not present exact parameters characterizing the system. Moreover, the present system comprises electrical, chemical, and optical functions which depend on each other. Thus, there is a system with variable parameters and limited lifetime. The limited lifetime means that the ECL decays are of the same order as the measurement time and the chemical decay. However, it is possible to assume that there exists a narrow region comprising exact parameters with small variations, where the system is functioning. Therefore, a special feedback which will help to transfer the system into optimally functioning condition is required. In the present disclosure, we are describing a method of fast analysis of the system and feedback mechanism allowing transferring the system into functioning condition during shortest possible period. The system comprises a source of hot electrons, electrochemical material, chemical-optical material, a power supplier and measuring devices. Each component of the system operates during limited period of time with different starting times. Moreover, the source of hot electrons, electrochemical material and chemical-optical material are characterized with a limited lifetime. This means that the whole system is functioning during a limited period of time. The present invention enables turning the system into functioning stage during the shortest times. The system includes the following parameters: voltage pulse for power generation, concentration of hot electrons depends of voltage pulse, time and location and material, electro-chemically active material depends on concentration of molecules and hot electrons, optically active sensing material depends on concentration of excited molecules and optical luminescence, resistive oxide film and interface layers, as well as electrical contacts and probes.

In order to turn the system into the functioning stage, pulsed voltage is applied to generate hot electrons and transfer them into the electro-chemically active layer. The transfer mechanism occurs through the electron-tunneling through a band-gap resistive (e.g. oxide) layer. At the same time when a suitable portion of the hot electrons are transferred into the electro-chemically active layer, a portion of molecules are chemically transferred into pre-excited state capable to interact with hot electrons. When these two events happen within correct time frame, the pre-excited molecules react with the hot electrons with formation of excited molecules. The excited molecules emit photons which is the luminescence process. The luminescence decay varies depending on energy of the excited molecules and their concentration. Thus, the system is functioning when the concentration of the excited molecules is sufficient to produce luminescence with long lifetime. During the luminescence process, the excited molecules transfer into ground state through number of energy states. Some of the light emitting label compounds react irreversibly with nearby molecules or decompose and cannot be re-excited. The redox potential of the local chemical milieu becomes too destructive to the label molecules in the case of too high concentration of hot electrons or their too high energy. Some of the nearground energy states are not effective for excitation. Therefore, not all of these molecules participate in the next round of the excitation process. The more excitation rounds, the less concentration of the molecules participating in the ECL process. Thus, applying high-power voltage pulses is not optimal solution and adaptive means must be applied. There exists a combination of parameters which make the system functioning. An algorithm of this invention allows us to find out these parameters through the opto-electrical feedback. The feedback is based on electrical and optical measurements. The electrical feedback is based on measurements of the tunneling current in the band-gap material indicating the flow of hot electrons. The optical feedback is based on optical time-resolved spectroscopy measurements indicating the concentration of excited molecules and life-times.

The adaptive system deals with variable parameters in which the initial values are zero except concentration of molecules at state 0 and thermal fluctuations. The molecules at state 0 do not emit light. In order to emit light, they have to change their energy state from 0 to 1, or excited state. The number of excited energy states has to be considered as well. Therefore, it was assumed here that the function Ντ is proportional to the number of the molecules at state 0 which is a constant for any particular case.

The adaptive mechanism is required to quickly find the working point at which the integrated value \Nrdt has a maximum value. Because of a dynamically fast system in which the number of hot electrons is variable and the number of excited molecules is variable too, it needs to have a fast estimation of the ECL process. Therefore, a number of fast feedback steps are performed to obtain excitation voltage parameters for the threshold pulse voltage at which the ECL process corresponds to Nthiithi =C1= constant. After this stage, during the second stage of the feedback when ECL intensity is the main feedback parameter, and the feedback from the photonic measurements is used to obtain the maximum of the integrated value \Nrdt corresponding to Cl. Then a new pair of parameters is found NthiTthi =C2 and a new maximum of the integral \Nrdt corresponding to C2 is found and so on. The system is functioning when after few feedback steps the integral \Nrdt is found for Ck which is near the theoretical maximum.

In practice, good results are obtained within 10% of variation from the maximum value. The diagrams Fig. 5 - Fig. 7 allow us to obtain a number of sub-integrals:

Each sub-integral provides information about dynamics of the ECL process at a specific time allowing quick response of the power unit. Supposing 0\t0 Ni A dt is very low, no waiting is needed until the total measurement time. Instead, the system can already react after the time t0 and the voltage pulse can be adjusted at once (increased). The same is applied to other specific times: tb 2ti.. .ifr. Formally such complicated procedure is required because the working point is only very narrow area incorporating thickness of the active layer, concentration of excited molecules, hot-electron flow. The last two, concentration of hot electrons and the number of excited molecules shall be correlated at the working point. In the non-correlated situation, for example, when the number of hot electrons is too small, only

fraction of the whole number of the molecules will be excited. As a result, intensity of the ECL will be much lower than at possible maximum. On the other hand, if the number of hot electrons is much larger than the number of molecules in the active layer, then excess of the hot electrons will transfer their kinetic energy to heat and unproductive side chemical reactions. The latter will result in big noise oscillations in the ECL curve and degradation of the active layer and even the resistive layer. The problem is that there is no exact number of excited molecules providing the optimal ECL and after the degradation mentioned above, the system will not work at all. Thus, the techniques according to the present disclosure allow to obtain the working point at the shortest times with minimal influence to the active layer. ECL output is dependent on the number of excited molecules in a definite space. This dependency is revealed in ECL decay curves after electrical pulses. ECL decays very fast just after the excitation, see Fig.4, typically during several microseconds, until the time ti in the figure, and then slow down until the time t2, typically in a few milliseconds. Systems which are characterized with longer life-times and larger number of excited molecules, produce higher ECL with slower decays. It is more convenient to represent ECL decays as a rotating vector of the ECL intensity. The rotation speed is correlated with time. This representation creates a spiral-like curve. A slowly decaying ECL process produces more circles and decay parameters can be obtained easily for different paths. Figs 5-8 show dynamics of the process for different times in detail. Particularly, one can automatically obtain the lifetimes of the system at different stages when the ECL vector intersects, for example, the horizontal axis.

The dynamics of the adaptive system is revealed in Fig. 8. This figure shows how the function Ντ develops with time at different parameters.

Fig. 10 shows typical behavior of ECL in terbium-based systems. The figure shows correlation between ECL and concentration of Tb-chelates for different voltages. ECL is not much dependent on voltage at low concentration of Tb-chelates, around IE-12 M. On the other hand, at higher concentration of Tb-chelates this dependency is more prominent, especially for IE-7 M. It can be noticed, that ECL intensity is extremely sensitive to voltage for last case. The correlation between ECL intensity and voltage for different concentration of Tb shows that the ECL mainly changes between 5 and 10 Volts and saturates at larger voltages in the experimental system in concern.

The present invention is further illustrated by the following non-limiting Examples.

EXAMPLE

General construction and function of the device

Figure 1 illustrates the prior art and Fig. 2 the present invention. The principle of the process control is shown in Fig. 2a and 2b. There are three stages of the control: 1) before observation of ECL (tunneling spectroscopy in Figure 2a), 2) during observation of ECL (time resolved spectroscopy in Figure 2a) and 3) during relaxation process after time-resolved spectroscopy is finished (relaxation spectroscopy in Figure 2a). During the first stage 1), the electrical and tunneling currents are measured at anode and at few probe locations. The probes, the number of which may be one to several, are tiny metal contacts, size of which are significantly smaller than the dimensions of the device (for example of hundreds of square micrometers), Thus, the probes do not change any electrical or optical processes taking place in the device. In addition, voltages applied to the probes are in the range of 1-25V and do not impact on the charge flow in the device. There can be one or more probes. The probe does not need to have a mechanical contact with other conductive surfaces of the device. It can be suspended in the space or have a mechanical contact with resistive surfaces. However, the probes are placed in locations where currents are meaningful to be measured including the tunneling currents. One of the locations is in the vicinity of the light emission region. Another location is the one where hot electron energies are measured before their reaction with molecules. The probes thereby provide information about electro-dynamical processes. Particularly, during stage 1 (see above), the values of the tunneling currents indicate the presence of hot electrons. If the currents are very small, the system is programmed to increase the amplitude of the pulsed voltage to a next value in accordance to a calibration curve obtained from measurements made for basic molecules, chelates without a sample using the same device. There can be few similar steps until ECL is observed. This means that the system has moved to the stage 2. During this stage, time-resolved spectroscopy provides data on photonic processes. If the light intensity is too low, the feedback mechanism provides new values of the pulsed voltage, amplitude and duration. The feedback steps are repeated few times until optimal time-resolved characteristics are obtained. During this stage, the measurements of electric currents including tunneling currents produce additional information about electro-dynamic processes. When the time-resolved spectroscopy measurements are made, the optical detection system is switched off and the system is returning to the initial stage. This is the relaxation process, stage 3). The electrical probing measurements still continues collecting information about electro-dynamical processes. For example, they can indicate leakage currents, destroyed materials, or a new charge distribution in the system and relaxation time.

Control software

It is described here the principle of function of the feedback system between separate units.

The control software architecture is shown in Fig. 9a, b and c. The initial parameters are voltage pulse, duration, frequency, amplitude and DC offset of the voltage supply as well as chemical composition of the ECL layer. The software program tracks all the parameters and compares them with the measured data. A first block of comparison is the tunneling feedback. The measured data are tunneling current 1, duration, frequency, light intensity, and tunneling current 2. The two components of the tunneling currents come from two different probes. The measured data are compared then with the initial measured data, or the data measured at previous stage. The comparison is carried out in the feedback analysis block which calculates the adaptive coefficients and corrections to the new values of the voltage supply. The output data comprise the data array of the new parameters, Ντ and measured data for the corresponding time step. All the output data enter the loop again for the new measurement. In the tunneling feedback block analysis, it is assumed that the ECL intensity is low, so that the adaptive coefficients are calculated based on the tunneling currents. This loop will increase number of hot electrons injected into the ECL solution layer. After the ECL is sufficient, and the adaptive coefficient Ντ reaches a threshold value, the photonic feedback is used too. This represents a typical adaptive control to a novel application. The measured data for comparison remain the same, but the new values are adjusted so to obtain a maximum of the adaptive coefficients including Ντ. The logic of the adaptive process is explained in a block diagram of Fig. 9c.

Claims

What is claimed is:

1. A device capable of generating highly energetic or hot electrons into solutions, wherein the device comprises: i) an electrical pulse generator, ii) a reaction cavity having a solution into which hot electrons can be injected as a result of electrical pulses via anode and cathode electrodes by (i), iii) the cavity (ii) including at least one of the following sensors: photon emission sensor, sensor measuring electron flow to the cavity (ii) over tunneling and solution and sensor measuring electron flow at the distance of 50-500 nm from the electrode surface; iv) adaptive system making decisions for adjusting and controlling the pulse generator attaining the adjustment of the pulse generator (i) to amplitude, duration and frequency values of the pulses which are the most favorable producing hot electron flow into solution in the cavity (ii).
2. The device of claim 1, wherein the adaptive system makes a decision based on previous data obtained before the decision, predictive model, an algorithm and instant data.
3. The device of claim 2, wherein the adaptive system makes a decision based on an external function.
4. The device of claim 3, wherein the external function is independent of any chemical, physical or optical process.
5. The device of any one of claims 1 to 4 wherein the system makes a number of decisions until the parameters of the system provide a maximum efficiency of the device.
6. The device of any one of claims 1 to 5 , wherein the decisions are made in a shorter time than any chemical, electrical or optical process is completed.
7. A method to provide and control a flux of highly energetic or hot electrons into solutions in a device involving more than two different processes, wherein the method comprises an adaptive system making decisions for adjusting and controlling electric pulses generated by a pulse generator to attain the most favorable amplitude, duration and frequency of the pulses to produce hot electron flow into the solution in the cavity.
8. The method of claim 7, wherein the different processes are photonic, electronic and chemical processes.
9. A computer program product embodied on a computer readable storage medium, the computer program product comprising instructions executable by one or more processors to control production of a flux of highly energetic hot electrons into solutions by an adaptive system in a device involving at least photonic, electronic and chemical processes, wherein the adaptive system makes decisions based on pervious data obtained before the decision, a predictive model, instant data and optionally an external function.