Patent Application
20200300842
The present application relates to the field of microelectronic sensors based on open-gate pseudo-conductive high-electron-mobility transistors (PC-HEMT) and their use in sorbent, immunosorbent and sorbent cell assays and for measuring biological cell dynamics.
The polarization doped high-electron-mobility transistor (HEMT) is a field effect transistor (FET) in which two layers of different bandgap and polarisation field are grown upon each other forming a hetero-junction structure. As a consequence of the discontinuity in the polarisation field, surface charges are created at the interface between the layers of the hetero junction structure. If the induced surface charge is positive, electrons will tend to compensate the induced charge resulting in the formation of the channel. Since in the HEMT, the channel electrons are confined in a quantum well in an infinitely narrow spatial region at the interface between the layers, these electrons are referred to as a two-dimensional electron gas (2DEG). This special confinement of the channel electrons in the quantum well actually grants them two-dimensional features, which strongly enhance their mobility surpassing the bulk mobility of the material in which the electrons are flowing.
The HEMTs based on the layers of III-V semiconductor materials, such as gallium nitride (GaN) and aluminium gallium nitride (AlGaN), have recently been developed with a view to high-voltage and high-power switching applications. The high voltages and high switching speeds allow smaller, more efficient devices, such as home appliances, communications and automobiles to be manufactured. To control the density of electrons in the 2DEG channel and to switch the HEMT on and off, the voltage at the gate of the transistor should be regulated.
Many commercially available AlGaN/GaN-based HEMT structures have a negative VT, resulting in a “normally-on” operation mode at 0V gate potential. They are called “depletion-mode transistors” and used in various power switching applications when the negative voltage must be applied on the gate in order to block the current. However, for safe operation at high voltage or high-power density, in order to reduce the circuit complexity and eliminate standby power consumption, HEMTs with “normally-off” characteristics are preferred.
Several techniques to manufacture the normally-off HEMTs have been reported. Burnham et al in “Gate-recessed normally-off GaN-on-Si HEMT using a new O2-BCl3 digital etching technique”, Phys. Status Solidi C, Vol. 7, 2010, No. 7-8, pp. 2010-2012, proposed normally-off structures of the recessed gate type. In this structure, the AlGaN barrier layer is etched and the gate is brought closer to the interface between the AlGaN barrier layer and the GaN buffer layer. As the gate approaches the interface between the layers, the VT increases. The normally-off operation of the transistor is achieved once the depletion region reaches the interface and depletes the 2DEG channel at zero gate voltage. The major advantages of these HEMTs are lower power consumption, lower noise and simpler drive circuits. These HEMTs are currently used, for example, in microwave and millimetre wave communications, imaging and radars.
Chang et al in “Development of enhancement mode AlN/GaN high electron mobility transistors”, Appl. Phys. Lett., Vol. 94, 2009, No. 26, p. 263505, proposed using a very thin AlGaN barrier instead of etching the relatively thick barrier layer to approach the AlGaN/GaN interface. This structure also achieves the normally-off operation by approaching the transistor gate towards the AlGaN/GaN interface. Chen et al (2010) in “Self-aligned enhancement-mode AlGaN/GaN HEMTs using 25 keV fluorine ion implantation”, in Device Research Conference (DRC), 2010, pp. 137-138, proposed to use the fluorine-based plasma treatment method. Although many publications have adopted various methods to achieve normally-off devices with minimum impact on the drain current, they unfortunately sacrificed device turn-on performance.
Sorbent assays, including enzyme-linked immunosorbent assay (ELISA) and molecularly-imprinted sorbent assays, are plate-based assays designed for detecting and quantifying chemical and biochemical substances such as peptides, proteins, antibodies hormones and small organic molecules. In a traditional ELISA, an antigen is immobilised to a solid surface and then conjugated to an antibody that is linked to an enzyme. There are three types of ELISA, where each type of the assay can be used qualitatively to detect the presence of an antibody or antigen. Alternatively, a standard curve based on known concentration of an antibody or antigen can be prepared, from which the unknown concentration of a sample can be determined.
Detection is optical and accomplished by assessing the linked enzyme activity via incubation with a substrate to produce an optically measurable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction. A detection enzyme or other tag can be linked directly to the primary antibody or introduced through a secondary antibody that recognises the primary antibody. It may be linked to a protein such as streptavidin if the primary antibody is biotin labelled. The most commonly used detection enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options. The choice of substrate strongly depends upon the required assay sensitivity and the instrumentation available for signal-detection, such as spectrophotometer, fluorometer or luminometer.
Traditional ELISAs can be performed with a number of modifications to the basic procedure. The key step, immobilisation of an antigen of interest is accomplished by direct adsorption to the assay plate or indirectly via a capture antibody that has been immobilised on the plate. The antigen is then detected either directly (primary antibody is labelled) or indirectly (secondary antibody is labelled).
Among various standard assay formats, where differences in both capture and detection were the concern, it is important to differentiate between particular strategies that exist specifically for the detection step. Irrespective of the specific method by which an antigen is captured on the plate (either by direct adsorption to the surface, or through a pre-coated “capture” antibody, as in a sandwich format), it is the detection step (direct or indirect detection) that largely determines the sensitivity of the assay.
In-Cell ELISA is performed with cells that are plated and cultured overnight in standard microplates. After the cultured cells are fixed, permeabilised and blocked, target proteins are detected with antibodies. This is an indirect assay format, where secondary antibodies are either fluorescent (for direct measurement by a fluorescent plate reader or microscope) or enzyme-conjugated (for detection with a soluble substrate using a plate reader).
In most cases (in most biosensors or bioassays), molecular receptors bound to the transistor surface or to the plate surface are spatially separated from this surface by molecular cross-linkers or proteins of approximately 5-15 nm length. The molecule charges are therefore screened from the sensing surface by dissolved counter ions. As a result of the screening, the electro-static potential that arises from charges on the analyte molecule exponentially decreases to zero with increasing the distance from the sensing surface. This screening distance is defined as a “Debye length”, and it must be carefully selected when designing the receptor layer of any biosensor or sorbent assay in order to ensure the optimal sensing. For example, when detecting molecules in blood serum, the typical Debye screening length is 0.78 nm at temperature 36° C. with an electrical permittivity of 74.5 for water. This means that after this length, the Debye screening length is given by:
where n0 is the bulk concentration of the electrolyte, ϵr is the relative dielectric permittivity of the solvent (in case of water at 36° C. a value of 74.5), ϵ0 is the permittivity of the vacuum, kb is the Boltzmann constant, T is the temperature, z is the ion charge, and e is the elementary charge.
The screening length means that an electrical field originating from a point charge is dropped to its 1/e value (29%) in this length. Because of this limitation, charges from larger biomolecules (5-15 nm) cannot be detected in a serum sample. To overcome this problem, the charges should be attracted closer to the sensor or plate surface by using very short length receptors or by operating the sensor in completely desalted buffers for electronic molecular detection.
Currently, the Debye length limitation can be overcome by modification of the receptors and controlling the immobilisation density over the sensor sensing surface. Elnathan et al (in “Biorecognition Layer Engineering: Overcoming Screening Limitations of Nanowire-Based FET Devices”, Nanoletters 12, 2012, pp. 5245-5254) described this approach in detail and demonstrated the increased sensitivity of their sensor to troponin detection directly from serum for the diagnosis of acute myocardial infarction. However, the method proposed by them still requires receptor modification, which is a cumbersome biochemical procedure.
The present inventors have now unexpectedly found that performing a sorbent assay, such as In-Cell ELISA, on the sensor of the present invention, makes it possible to sense beyond the Debye screening length without modification of the receptors.
The present application describes embodiments of a microelectronic device for sorbent, immunosorbent or cell sorbent assay, or flow cytometry, comprising:
In a particular embodiment, the microelectronic device of the present invention further comprises a Vivaldi antenna electrode or metamaterial electrode, such as Aharonov-Bohm antenna electrode, said Vivaldi antenna electrode or said metamaterial electrode being placed on the top layer between said source and drain contact in the open gate area of the transistor and capable of detecting electrical signals in the frequency range of 30 GHz to 300 THz.
In some embodiments, the PC-HEMT of each microelectronic sensor, in the open gate area, is not coated with a molecular or biomolecular layer and is capable of remotely detecting target (analyte) gases, chemical compounds or biomolecules from the environment. In other embodiments, the PC-HEMT further comprises at least one molecular or biomolecular layer immobilised within the open gate area of the transistor and capable of binding or adsorbing target (analyte) gases, chemical compounds or biomolecules from the environment. The molecular or biomolecular layer is selected, for example, from a cyclodextrin, 2,2,3,3-tetrafluoropropyloxy-substituted phthalocyanine or their derivatives. In certain embodiments, the molecular or biomolecular layer comprises capturing biological molecules, such as primary, secondary antibodies or fragments thereof against certain proteins to be detected, or their corresponding antigens, enzymes or their substrates, short peptides, specific DNA sequences, which are complimentary to the sequences of DNA to be detected, aptamers, receptor proteins or molecularly imprinted polymers.
In a particular embodiment, the PC-HEMT multilayer heterojunction structure comprises one of the following A-D structure:
The PC-HEMT source and drain contacts may be ohmic or non-ohmic. When the source and drain contacts are non-ohmic, the electrical metallisations of the transistor are capacitively-coupled to the 2DEG or 2DHG conducting channel for inducing displacement currents, thereby creating said non-ohmic source and drain contacts. In a particular embodiment, the transistor further comprises a dielectric layer deposited on top of said multilayer hetero-junction structure. In a specific embodiment, the thickness of the PC-HEMT top (barrier or buffer) layer in the open gate area is 6 to 7 nm, or 6.2 nm to 6.4 nm; and the surface of said top layer has a roughness of 0.2 nm or less, or 0.1 nm or less, or 0.05 nm or less. In another embodiment, the multilayer heterojunction structure further comprises a piezoelectric electro-optical crystal (EOC) transducer adapted to be brought into a contact with a medium to be sensed and adapted to be illuminated with a polarised light.
In a particular embodiment, the microelectronic device of the present invention is a microelectronic microwell plate (multiwell plate or microplate), and each of said microelectronic sensors is integrated at the bottom of its corresponding well of said microwell plate. In one embodiment, the microelectronic device of the present invention is a microelectronic microwell plate for sorbent, immunosorbent or cell sorbent assay, or flow cytometry, comprising:
In a further embodiment, a method for sorbent, immunosorbent or cell sorbent assay of a sample containing a chemical compound or a biological compound to be tested in a gas phase or in a liquid phase comprises the following steps:
Non-limiting examples of the chemical compound and biological compounds analysed by the method of the present invention are:
In yet further embodiment, a method for in-vitro measurement of cell dynamics comprises the following steps:
Various embodiments may allow various benefits and may be used in conjunction with various applications. The details of one or more embodiments are set forth in the accompanying figures and the description below. Other features, objects and advantages of the described techniques will be apparent from the description and drawings and from the claims.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Disclosed embodiments will be understood and appreciated more fully from the following detailed description taken in conjunction with the appended figures. The drawings included and described herein are schematic and are not limiting the scope of the disclosure. It is also noted that in the drawings, the size of some elements may be exaggerated and, therefore, not drawn to scale for illustrative purposes. The dimensions and the relative dimensions do not necessarily correspond to actual reductions to practice of the disclosure.
b: 0V gate potential, and
In the following description, various aspects of the present application will be described. For purposes of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the present application. However, it will also be apparent to one skilled in the art that the present application may be practiced without the specific details presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the present application.
The term “comprising”, used in the claims, is “open ended” and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. It should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It needs to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a device comprising x and z” should not be limited to devices consisting only of components x and z. Also, the scope of the expression “a method comprising the steps x and z” should not be limited to methods consisting only of these steps.
Unless specifically stated, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within two standard deviations of the mean. In one embodiment, the term “about” means within 10% of the reported numerical value of the number with which it is being used, preferably within 5% of the reported numerical value. For example, the term “about” can be immediately understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. In other embodiments, the term “about” can mean a higher tolerance of variation depending on for instance the experimental technique used. Said variations of a specified value are understood by the skilled person and are within the context of the present invention. As an illustration, a numerical range of “about 1 to about 5” should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges, for example from 1-3, from 2-4, and from 3-5, as well as 1, 2, 3, 4, 5, or 6, individually. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Unless otherwise clear from context, all numerical values provided herein are modified by the term “about”. Other similar terms, such as “substantially”, “generally”, “up to” and the like are to be construed as modifying a term or value such that it is not an absolute. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those of skilled in the art. This includes, at very least, the degree of expected experimental error, technical error and instrumental error for a given experiment, technique or an instrument used to measure a value.
As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and/or clarity.
It will be understood that when an element is referred to as being “on”, “attached to”, “connected to”, “coupled with”, “contacting”, etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, “directly on”, “directly attached to”, “directly connected to”, “directly coupled” with or “directly contacting” another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.
As used herein, the term “cell dynamics” includes any spatially and temporally regulated molecular events and processes occurring in biological cells. This includes but not limited to cellular behaviours such as cytokinesis, chemotaxis, cell division, cell differentiation, cell signalling, cell movement and cell contractility, transcriptional changes and changes in synaptic strength dependent upon spatially localised, temporally dynamic biochemical reactions. Tens of thousands of these events, such as changes in protein or lipid phosphorylation, localisation, and binding occur every second. The sensors of the present invention are capable of revealing how the cell and molecular dynamics lead to normal development or to diseases and help in understanding and preventing these diseases at the earliest stages and in the frame of the point-of-care diagnostics.
Reference is now made to
The term “2DEG” mentioned in the present description and claims should not be understood or interpreted as being restricted to the two-dimensional electron gas. As stated above and will be explained later in this application, the two-dimensional hole gas may also be a possible current carrier in a specific heterojunction structure. Therefore, the term “2DEG” may be equally replaced with the term “2DHG” without reference to any particular PC-HEMT configuration.
The source and drain contacts connecting the PC-HEMT to the electric circuit may be ohmic or non-ohmic (capacitively-coupled, as will be described below). In one embodiment,
Further,
“Capacitive coupling” is defined as an energy transfer within the same electric circuit or between different electric circuits by means of displacement currents induced by existing electric fields between circuit/s nodes. In general, ohmic contacts are the contacts that follow Ohm's law, meaning that the current flowing through them is directly proportional to the voltage. Non-ohmic contacts however do not follow the same linear relationship of the Ohm's law. In other words, electric current passing through non-ohmic contacts is not linearly proportional to voltage. Instead, it gives a steep curve with an increasing gradient, since the resistance in that case increases as the electric current increases, resulting in increase of the voltage across non-ohmic contacts. This is because electrons carry more energy, and when they collide with atoms in the conducting channel, they transfer more energy creating new high-energy vibrational states, thereby increasing resistance and temperature.
When electrical metallisations are placed over single-crystalline or polycrystalline semiconductor material, the “Schottky contact” or “Schottky barrier contact” between the metal and the semiconductor occurs. Energy of this contact is covered by the Schottky-Mott rule, which predicts the energy barrier between a metal and a semiconductor to be proportional to the difference of the metal-vacuum work function and the semiconductor-vacuum electron affinity. However, this is an ideal theoretical behaviour, while in reality most interfaces between a metal and a semiconductor follow this rule only to some degree. The boundary of a semiconductor crystal abrupt by a metal creates new electron states within its band gap. These new electron states induced by a metal and their occupation push the centre of the band gap to the Fermi level. This phenomenon of shifting the centre of the band gap to the Fermi level as a result of a metal-semiconductor contact is defined as “Fermi level pinning”, which differs from one semiconductor to another. If the Fermi level is energetically far from the band edge, the Schottky contact would preferably be formed. However, if the Fermi level is close to the band edge, an ohmic contact would preferably be formed. The Schottky barrier contact is a rectifying non-ohmic contact, which in reality is almost independent of the semi-conductor or metal work functions.
Thus, a non-ohmic contact allows electric current to flow only in one direction with a non-linear current-voltage curve that looks like that of a diode. On the contrary, an ohmic contact allows electric current to flow in both directions roughly equally within normal device operation range, with an almost linear current-voltage relationship that comes close to that of a resistor (hence, “ohmic”).
Reference is now made to
An electrical connection to the 2DEG channel shown in
The third option would be the use of the photo effect that may also induce an electric current in the 2DEG channel. In order to couple the light excitation with the electronic effects in the conductive 2DEG channel, a photo effect in a silicon layer should be created. Regarding the direct photo effect, it is well known that light can only be absorbed when the energy of the absorbed photon (E=hv) is large enough for an electron to be excited into the valence band. In that case, E is the photon energy, h is Planck's constant and v is the frequency of the photon. The frequency is coupled to the wavelength λ of light by the constant speed of light c=λv. Typically, the bandgap of silicon at room temperature is 1.12 eV, which means that silicon becomes transparent for wavelength larger than 1240 nm, which is the near infrared range.
For smaller wavelength (i.e. larger energy of the photons), electron/hole pairs are generated leading to a photocurrent. In the fully-depleted, intrinsically doped silicon structures, this results in a higher charge carrier density and consequently, higher sensitivity. For these structures, light is adsorbed in the whole visible range making such devices ideal photodetectors. The mechanism that allows the silicon semiconductor to become photosensitive to irradiation with light has already been described in literature. In the direct photo effect, it can be tuned by the size, crystalline direction and surface termination. These effects actually originate from two-dimensional quantum confinement of electrons in the nano-sized 2DEG structure.
Although irradiation of the silicon structure with light of larger wavelengths with photon energies below the bandgap does not have enough energy to excite carriers from the valence to the conduction band in bulk silicon, the electron/hole pairs can also be generated between the valence band and surface states, and the donor-like surface trap states can still be formed (see the definition and explanation of the surface trap states below). The electrons actually deplete these holes trapped at the surface and hence, modulate the gate field. The photogenerated holes are confined to the centre of the silicon structure by the gate field, where they increase the conduction of the 2DEG channel, because of the band bending. The holes increase the channel conductivity for a certain lifetime until they are trapped (recaptured) at the surface. The gain of the transistor can be extremely huge if this re-trapping lifetime is much longer than the holes transit time.
If the source and drain contacts are non-ohmic (capacitively-coupled), in order to electrically contact the 2DEG channel underneath, which is about 7-20 nm bellow metallisations (14), the AC frequency regime is used. The capacitive coupling of the non-ohmic metal contacts with the 2DEG channel is normally induced at the frequency higher than 30 kHz. In the case of the non-ohmic contacts, the DC readout cannot be performed. Instead, the AC readout or impedance measurements of the electric current flowing through the 2DEG channel are carried out.
Thus, the significant features of the PC-HEMT structure are that:
The same transistors of the embodiments shown in
The PC-HEMT shown in
In general, mechanical sensors, much like pressure sensors, are based on the measurement of the externally induced strain in the heterostructures. The pyroelectric properties of group-III-nitrides, such as gallium nitride (GaN), allow two mechanisms for strain transduction: piezoelectric and piezoresistive. The direct piezoelectric effect is used for dynamical pressure sensing. For measurements of static pressure, such sensors are not suitable due to some leakage of electric charges under the constant conditions. For static operation, the piezoresistive transduction is more preferable.
Piezoresistive sensors using wide band gap materials have been previously employed using hexagonal silicon carbide bulk materials for high temperature operation. The piezoresistivity of GaN and AlGaN structures was found to be comparable to silicon carbide. However, piezoresistivity can be further amplified by HEMT structure, as taught by Martin Eickhoff et al in “Piezoresistivity of AlxGa1-xN layers and AlxGa1-xN/GaN heterostructures”, Journal of Applied Physics 90, 2001, 3383.
For piezoresistive strain sensing at relatively lower pressures (or pressure differences), diaphragm or membranes should be used, where the external pressure is transferred into a changed internal strain caused by bending, as shown in
Eickhoff et al (2001) conducted the first experiments on AlGaN/GaN hetero-structures where the 2DEG channel confined between the upper GaN and AlGaN barrier layer and demonstrated the linear dependence of the 2DEG channel resistivity on the applied strain. Moreover, a direct comparison to cubic SiC and a single AlGaN layer clearly demonstrated the superior piezoresistive properties of the latter. From these results, it is clear that the interaction of piezoelectric and piezoresistive properties improves the sensitivity of pressure sensors by using GaN/AlGaN heterostructures confined with the 2DEG channel.
The sensor configuration shown in
The electrical metallisations (14) connect the PC-HEMT to an electric circuit and allow electric current to flow between the source and drain contacts. The electrical metallisations (14) are made of metal stacks, such as Cr/Au, Ti/Au, Ti/W, Cr/Al and Ti/Al. The Cr or Ti layers of the metal stack is, for example, of 5-10 nm thickness, while the second metal layer, such as Au, W and Al, is of 100-400 nm thickness. The actual metallisations (14) are chosen according to the established technology and assembly line at a particular clean room fabrication facility. The source and drain ohmic contacts are usually made of metal stacks, such as Ti/Al/Mo/Au, Ti/Al/Ni/Au, Ti/Au and Ti/W having the thickness of 15-50 nm. The non-ohmic contacts on the other hand are capacitively coupled to the conducting 2DEG channel (13) via displacement currents (19).
In yet further embodiment, substrate layer (10) comprises a suitable material for forming the barrier layer and is composed, for example, of sapphire, silicon, silicon carbide, gallium nitride or aluminium nitride. The hetero-junction structure (11, 12) is deposited on the substrate layer (10), for example, by a method of metalorganic chemical vapour deposition (MOCVD), and it forms a two-dimensional electron gas (2DEG) channel (13) in the close proximity to the interface between the buffer layer (11) and the top barrier layer (12). The top barrier layer (12) then may be either recessed or grown as a thin layer between the source and drain contacts, thereby forming an open gate area.
The 2DEG/2DHG channel (13) formed near the interface between the buffer layer (11) and the barrier layer (12) serves as a main sensitive element of the transistor reacting to a surface charge and potential. The 2DEG/2DHG channel (13) is configured to interact with very small variations in surface or proximal charge or changes of electrical field on the barrier layer/liquid-air or barrier layer/metal/liquid-air interfaces interacting with the donor-like surface trap states of the barrier layer. This will be defined and discussed below in detail.
“Open gate area” of the PC-HEMT is defined as an area between the source and drain contacts of the transistor which is directly exposed to a conductive medium, such as liquid or gas capable of conducting current. An example of the conductive liquid is an electrolyte saline solution. In this case, instead of the fixed gate voltage, which is normally applied to a gate electrode, a reference potential is applied to the electrolyte-semiconductor system, via an optional reference electrode that is dipped into the electrolyte. As a result, in the absence of the physical gate, the electrolyte itself becomes an open gate of the transistor. This will be explained in more detail below.
The specific thickness of the top barrier layer (12) in the open gate area is achieved by either dry etching the semiconductor material of the barrier layer (12), i.e. recessing the layer in the open gate area with the etching rate of 1 nm per 1-2 min in a controllable process, or coating the buffer layer (11) in the open gate area with an ultrathin layer of the III-V semiconductor material. In order to increase the charge sensitivity of the transistor, the surface of the recessed ultrathin barrier layer is post-treated with plasma (chloride) epi-etch process. Consequently, the natively passivated surface is activated by the plasma etch to create an uncompensated (ionised) surface energy bonds or states, which are neutralized after MOCVD growing.
The top barrier layer recessed or grown in the open gate area to 5-9 nm is optimised by minimising the roughness of the top semiconductor layer to 0.2 nm and less. The resulted structure was surprisingly found to significantly enhance sensitivity of the sensor. This specific thickness of 5-9 nm of the top barrier layer in the open gate area with the roughness of 0.2 nm or less corresponds to the “pseudo-conducting” current range between normally-on and normally-off operation modes of the transistor and requires further explanation.
“Pseudo-conducting” current range of the HEMT is defined as an operation range of the HEMT between its normally-on and normally-off operation modes. “Trap states” are states in the band-gap of a semiconductor which trap a carrier until it recombines. “Surface states” are states caused by surface reconstruction of the local crystal due to surface tension caused by some crystal defects, dislocations, or the presence of impurities. Such surface reconstruction often creates “surface trap states” corresponding to a surface recombination velocity. Classification of the surface trap states depends on the relative position of their energy level inside the band gap. The surface trap states with energy above the Fermi level are acceptor-like, attaining negative charge when occupied. However, the surface trap states with energy below the Fermi level are donor-like, positively charged when empty and neutral when occupied. These donor-like surface trap states are considered to be the source of electrons in the formation of the 2DEG channel. They may possess a wide distribution of ionization energies within the band gap and are caused by redox reactions, dangling bonds and vacancies in the surface layer. A balance always exists between the 2DEG channel density and the number of ionised surface donors which is governed by charge neutrality and continuity of the electric field at the interfaces.
Thus, the donor-like surface traps formed at the surface of the barrier layer of the HEMT are one of the most important sources of the 2DEG in the channel. However, this only applies for a specific barrier layer thickness. In a relatively thin top barrier layer, the surface trap state is below the Fermi level. However, as the top barrier layer thickness increases, the energy of the surface trap state approaches the Fermi energy until it coincides with it. The thickness of the top barrier layer corresponding to such situation is defined as “critical”. At this point, electrons filling the surface trap state become pulled to the channel by the strong polarisation-induced electric field found in the barrier to form the 2DEG instantly.
If the surface trap states are completely depleted, further increase in the barrier layer thickness will not increase the 2DEG density. Actually, if the 2DEG channel layer fails to stretch the barrier layer, the later will simply relax. Upon relaxation of the barrier layer, crystal defects are created at the interface between the buffer layer and the barrier layer, and the piezoelectric polarisation instantly disappears causing deterioration in the 2DEG density.
In order to illustrate the above phenomenon of pseudo-conducting current, reference is now made to the following figures. As mentioned above,
In view of the above, it is clear that the mechanism of the 2DEG depletion based on recessing the top barrier layer is strongly dependent on the donor-like surface trap states (or total surface charge). As the thickness of the barrier layer decreases, less additional external charge is needed to apply to the barrier layer surface in order to deplete the 2DEG channel. There is a critical (smallest) barrier thickness, when the 2DEG channel is mostly depleted but still highly conductive due to a combination of the energy barrier and the donor surface trap states energy. At this critical thickness, even the smallest energy shift at the surface via any external influence, such as surface reaction, charging etc., leads immediately to very strong 2DEG depletion. As a result, the surface of the top barrier layer at this critical thickness is extremely sensitive to any smallest change in the electrical field of the surroundings.
Thus, it has been found that the recess of the top layer in the open gate area from 9 nm down to 5 nm drastically reduces the 2DEG density, brings the transistor to the “near threshold” operation and results in highly increased surface charge sensitivity. The specific 5-9 nm thickness of the transistor's top layer responsible for its surprising pseudo-conducting behaviour gives the transistor the incredible sensitivity. So, when it comes into a contact with an ionic fluid or body skin, it opens up the gate to be able to do the ultrasensitive sensing. This thickness must be optimised for significantly enhancing sensitivity of the sensor. This specific thickness of the top layer was surprisingly found to correspond to the “pseudo-conducting” current range between normally-on and normally-off operation modes of the 2DEG channel and requires further explanation.
The top layer is recessed to this specific thickness after subjecting to short plasma activation by an ultra-low damage reactive-ion etching technique using inductively-coupled plasma (ICP) with a narrow plasma-ion energy distribution. Such short plasma treatment allows much lower roughness of the surface, which is a function of the semiconductor vertical damage depth during the plasma etching process. Such low surface roughness (about 0.2 nm and less) can be achieved only via this ICP-RIE ultra low damage etching process with a narrow plasma-ion energy distribution, and this inherently results in a very low vertical damage depth to the top layer, which allows the minimal surface scattering and minimal surface states-2DEG channel interaction with the maximum signal-to-noise ratio of the sensor. Thus, the depth effect of the vertical sub-nanometre damage to the top recessed layer, due to an ultra-low damage ICP-RIE etching process with a very narrow plasma-ion energy distribution, is the only way to optimally achieve the required sub-nanometre roughness of the semiconductor surface. This inherently results in an adjustable pseudo-conductive working point with the highest charge sensitivity ever possible. This depth effect is always inherent to the sub-nanometre roughness of the semiconductor surface, which was measured using AFM (atomic force microscope).
Thus, in addition to the recessed top layer thickness, roughness of the top layer surface is another very important parameter that has not been previously disclosed. It has been surprisingly found that the roughness of the top layer surface (in the open gate sensitive area) bellow 0.2 nm prevents scattering of the donor-like surface trap states. Thus, combination of these two features: 5-9 nm thickness of the top layer in the open gate area and strongly reduced roughness of its surface (bellow 0.2 nm) make the sensor incredibly sensitive.
In a certain aspect, the method for manufacturing of the PC-HEMTs of the present invention comprises the following steps:
Reference is now made to
The atomic layer etching (ALE) performed in Step 8 of the manufacturing process is the most important stage in the process. As mentioned above, it allows the controlled recess of a top layer, removing a single atomic layer-by-layer, where the etch thickness is in the order of magnitude of a single atomic monolayer. As explained above, such ultra-low damage to the top layer of the heterogeneous structure, when the actual surface roughness is controlled by a single atomic monolayer, allows to achieve the sub-nanometre roughness (about 0.2 nm and less) of the top layer when its thickness is only few nanometres (5-9 nm). There are no known ways in the semiconductor technology which would allow to achieve such low roughness at this particular thickness of the semiconductor layer. Therefore, the manufacturing method developed by the present inventors is unique and made it possible to unexpectedly arrive to the pseudo-conducting structures of the present invention.
The ALE process sequence consists of repeated cycling of process conditions. The total amount of material removed is determined by the number of repeated cycles. Each cycle is typically comprised of four steps: adsorption, first purge, desorption and second purge. During the adsorption step of the cycle, reactive species are generated in the reactor (for example, upon plasma excitation), adsorbed by, and react with material on the wafer. Due to the self-limiting process, and with the proper choice of reactants and process conditions, reaction takes place with only a thin layer of material, and the reaction by-products are formed. This step is followed by purging of the reactor to remove all traces of the reactant. Then the by-product desorption takes place due to bombardment of the wafer surface by noble gas ions with a tightly controlled energy. Again, by-products are purged from the reactor, and the wafer is ready for the last two (optional) steps of the manufacturing process.
Reference is now made to
In a further aspect, the hetero-junction structure may be a three-layer structure consisting of two GaN layers and one AlGaN layer squeezed between said buffer layers like in a sandwich, wherein the top layer is a buffer layer. This may lead to formation of the two-dimensional hole gas (2DHG) in the top GaN layer above the AlGaN layer which results in reversing polarity of the transistor compared to the two-layer structure discussed above.
In general, polarity of III-V nitride semiconductor materials strongly affects performance of the transistors based on these semiconductors. The quality of the wurtzite GaN materials can be varied by their polarity, because both the incorporation of impurities and the formation of defects are related to the growth mechanism, which in turn depends on surface polarity. The occurrence of the 2DEG/2DHG and the optical properties of the hetero junction structures of nitride-based materials are influenced by the internal field effects caused by spontaneous and piezo-electric polarizations. Devices in all of the III-V nitride materials are fabricated on polar {0001} surfaces. Consequently, their characteristics depend on whether the GaN layers exhibit Ga-face positive polarity or N-face negative polarity. In other words, as a result of the wurtzite GaN materials polarity, any GaN layer has two surfaces with different polarities, a Ga-polar surface and an N-polar surface. A Ga-polar surface is defined herein as a surface terminating on a layer of Ga atoms, each of which has one unoccupied bond normal to the surface. Each surface Ga atom is bonded to three N atoms in the direction away from the surface. In contrast, an N-polar surface is defined as a surface terminating on a layer of N atoms, each of which has one unoccupied bond normal to the surface. Each surface N atom is also bonded to three Ga atoms in the direction away from the surface. Thus, the N-face polarity structures have the reverse polarity to the Ga-face polarity structures.
As described above for the two-layer heterojunction structure, the barrier layer is always placed on top of the buffer layer. The layer which is therefore recessed in the two-layer heterojunction structure is the barrier layer, specifically the AlGaN layer. As a result, since the 2DEG is used as the conducting channel and this conducting channel is located slightly below the barrier layer (in a thicker region of the GaN buffer layer), the hetero-junction structure is grown along the {0001}-direction or, in other words, with the Ga-face polarity. However, as explained above, the physical mechanism that leads to the formation of the 2DEG is a polarisation discontinuity at the AlGaN/GaN interface, reflected by the formation of the polarisation-induced fixed interface charges that attract free carriers to form a two-dimensional carrier gas. It is a positive polarisation charge at the AlGaN/GaN interface that attracts electrons to form 2DEG in the GaN layer slightly below this interface.
As noted above, polarity of the interface charges depends on the crystal lattice orientation of the hetero-junction structure, i.e. Ga-face versus N-face polarity, and the position of the respective AlGaN/GaN interface in the hetero-junction structure (above or below the interface). Therefore, different types of the accumulated carriers can be present in the hetero-junction structure of the embodiments.
In case of the three-layer hetero-junction structure, there are four possible configurations:
Thus, there are four hetero-junction three-layer structures implemented in the transistor of the embodiments, based on the above configurations:
In all the above structures, the deposition of a dielectric layer on top might be beneficial or even necessary to obtain a better confinement (as in case of the N-face structures). As shown in
The preferable structures of the embodiments are structures “B” and “C”. In the structure “B”, the 2DHG conducting channel formed in the top GaN layer, which has a higher chemical stability (particularly towards surface oxidation) than the AlGaN layer. Concerning the structure “C”, the 2DEG conducting channel might be closer to the surface. Therefore, the electron mobility might be lower than in the 2DEG structure with the Ga-face polarity. In general, the polarity of the heterostructure can be adjusted by the choice of the substrate (e.g. C-face SiC) or by the growth conditions.
Based on the above, one of the aspects of the present application is an open-gate pseudo-conductive high-electron mobility transistor (PC-HEMT) for performing a sorbent assay, in particular In-Cell ELISA, and for measuring cell dynamics, comprising either:
The PC-HEMT of the present invention may further comprise a Vivaldi antenna electrode placed on the top layer between said source and drain contact in the open gate area of the transistor in order to enable detection of electrical signals in the frequency range of 30 GHz to 300 THz. Alternatively, the PC-HEMT of the present invention may additionally comprise (instead of the Vivaldi antenna) a metamaterial electrode, such as an Aharonov-Bohm antenna electrode placed on the top layer between said source and drain contact in the open gate area of the transistor in order to enable detection of electrical signals in the frequency range of 30 GHz to 300 THz. These types of the PC-HEMTs (with a Vivaldi antenna electrode or metamaterial electrode) are described in co-pending patent application U.S. Ser. No. 16/793,839 and PCT/IB2020/051836, respectively, incorporated herein by reference.
As mentioned above, an electrical connection of the multilayer heterojunction structure to the 2DEG or 2DHG channel can be realised via capacitive coupling to the electrical metallisations through a Schottky barrier contact. In that case, since the source and drain contacts are non-ohmic (i.e. capacitively-coupled), the DC readout cannot be carried out. Therefore, in order to electrically contact the 2DEG/2DHG channel underneath, about 5-20 nm bellow the electrical metallisations, the AC-frequency regime must be used. In other words, the AC readout or impedance measurements of the electric current flowing through the 2DEG/2DHG-channel should be performed in this particular case. The capacitive coupling of the non-ohmic metal contacts with the 2DEG/2DHG channel becomes possible only if sufficiently high AC frequency, higher than 30 kHz, is applied to the metallisations. To sum up, the electrical metallisations, which are capacitively coupled to the 2DEG/2DHG channel utilise the known phenomenon of energy transfer by displacement currents. These displacement currents are induced by existing electrical fields between the electrical metallisations and the 2DEG/2DHG conducting channel operated in the AC frequency mode through the Schottky contact as explained above.
Reference is now made to
In
Ion flow is illustrated in
The above phenomenon occurring at the PC-HEMT surface, discovered by the present inventors, is defined as an “intra-fluid ionic” interaction or formation of an “ionic cloud”. Thus, if the PC-HEMT connected to a circuit is immersed into an ion conductive fluid (being liquid or gas), then ions of the fluid start electro-dynamically react to any external charge by their movement and form the ionic cloud. Being in direct contact or close proximity to the barrier layer surface, the charge sensitivity is thus tremendously enhanced by this ionic cloud. The fluid actually acts in this case as an antenna matching the 2DEG transducer perfectly. Electric charges generated in any environment, as well as their super position dipoles forming the ionic cloud, are projected to this fluid antenna, in which the transistor is immersed. Sensing of the electric charges with the PC-HEMT of the present invention is therefore possible in a contactless manner, when the molecules are at some distance from the surface of the transistor. This clearly allows to overcome a usual “sensing noise” of any traditional biosensor having reporter molecules attached to the surface of the sensor, as will be discussed below.
To sum-up, in a direct current (DC) mode, the top layer-to-fluid-interface of the PC-HEMT is in charge equilibrium, where the 2DEG is directly incorporated as a balancing polarisation element. Once an external electric charge (originated from dipole molecules forming a layer or an ionic cloud, or two neutral molecules creating a dipole pair under London forces and also forming an ionic cloud) is introduced into the electrolyte fluid environment, the net charge equilibrium is shifted resulting in a change in the electron density and mobility. In case of the pseudo-conducting 2DEG channel, it becomes easily modulated and the strongest amplification phenomenon is observed. Sensitivity of the PC-HEMT in this case is so ultra-high that it allows detecting neutral molecules diffusing to the surface and coupling to the PC-HEMT top layer surface via a getter effect changing the surface trap states. The getter effect actually allows the sensor based on the PC-HEMT of the present invention to collect free gases by adsorption, absorption or occlusion.
In a radiofrequency (RF) mode, when the electric current in the 2DEG/2DHG channel is alternating (AC), the near-field and displacement current coupling effects at electrochemical double layers take place. In that case, the super-Debye interactions allow detection of any ion types selectively at MHz frequency range and ion solvation shells and resonance frequencies of intra-fluid ion-ion interaction at GHz range.
As mentioned above, in most cases (in most biosensors), molecular receptors bound to the transistor surface are spatially separated from this surface by molecular cross-linkers or proteins of approximately 5-15 nm length. Therefore, the aforementioned charges are screened from the sensing surface by dissolved counter ions. As a result of the screening, the electro-static potential that arises from charges on the analyte molecule exponentially decreases to zero with increasing the distance from the sensing surface. This screening distance is defined as a “Debye length”, and it must be carefully selected when designing the receptor layer of any ISFET in order to ensure the optimal sensing. For example, when detecting molecules in blood serum, the typical Debye screening length is 0.78 nm at temperature 36° C. with an electrical permittivity of 74.5 for water. The screening length means that an electrical field originating from a point charge is dropped to its 1/e value (29%) in this length. Because of this limitation, charges from larger biomolecules (5-15 nm) cannot be detected in a serum sample. To overcome this problem, the charges should be attracted closer to the sensor surface by using very short length receptors or by operating the sensor in completely desalted buffers for electronic molecular detection.
The present inventors surprisingly found that it is actually possible to sense beyond the Debye screening length, without any modification of the receptors and even without any functionalisation of the top layer surface of the PC-HEMT with (bio)molecular receptors, by operating the PC-HEMT of the present invention at high frequencies and using a combined transducer principle. The present inventors have recently discovered that the precise identification of biomolecules and cells can be obtained by combination of a precise monitoring and control of the main parameters, temperature, pH and ionic strength with an array of electronically identical PC-HEMTs of the present invention.
Since the main limitation in the DC readout mode is the Debye screening of charges, the DC alone is not really suitable for sensing of biomolecules and cells and mainly depends on the charge carried by the target biomolecules. The present inventors proposed to overcome these limitations by adding the AC readout with a frequency sweep up to 1 MHz or higher. Opposite to the DC readout, the charges of the target molecules have a negligible influence on the sensing in the AC mode.
In addition, the AC readout can also detect the presence of the bound molecules or cells. The AC sensing mode has the same basis as the impedance spectroscopy. It shows the change of the sensor's surface capacitance and resistance which contains information about the binding of the target molecule, as well as the ‘number’ (concentration) of the bound molecules or cells.
Thus, the AC electronic readout combined with the DC readout is useful for enzymatic, electrochemical and affinity sensing when both charged and uncharged molecules are involved. When operated at higher frequencies (more than 1 MHz), the problematic Debye screening can be overcome, and also larger molecules can be sensed.
In a further aspect of the present invention, the combined transducer principle defined herein as a “multiparametric readout” includes: DC electronic readout of the sensor, AC electronic readout of the sensor and temperature sensing. The PC-HEMT-based sensor of the present invention therefore further comprises a reference or counter electrode and characterised with respect to its electronic properties and the measurement configuration for molecular sensing applications. The main features of the sensors of the present invention are determined by the transfer characteristics and the output characteristics at room temperature. The transfer characteristics shows the drain current of the transistors as a function of their source voltage at constant drain-source voltages.
In general, the term “transfer function” (TF) is a mathematical representation to describe inputs and outputs of black box models. In order to describe the frequency response of the sensor, a counter electrode and the first amplifier stage are considered as a black box element with a certain frequency response. Since the analogue transistor amplification is exploited in the present invention, the instant model is described with a term “transistor transfer function” (TTF). The TTF is defined as a mathematical ratio between the input (Vstim) and the output signal (Vout) of an electrical, frequency-dependent system. Its frequency response H(jω) is defined as follows:
wherein ω is the angular frequency and j is the imaginary unit.
Thus, the TTF can be used to investigate impedance (defined as the ratio between voltage and applied current) or capacitance (defined as the capability of a capacitor to store charges) changes, caused by binding of molecules or cells onto the surface of the transistor.
The DC electronic readout is based on the transfer characteristics and is carried out in a liquid medium. The sensor in a DC readout mode is biased by a certain drain-source voltage while a voltage sweep is done through a reference electrode, and it senses the charges at the sensor surface. The resulting transfer characteristics reflect the characteristic behaviour of the sensor, as well as its surface condition, and are used to detect target molecules or cells on the sensor surface. In addition to the DC mode, the AC electronic readout is used to enable the multiparametric readout. When operated at higher frequencies (more than 1 MHz), the Debye screening can then be overcome, and also larger molecules and cells can be sensed.
To sum up, at any solid state/electrolyte interface, the capacitive and resistive elements of the sensor form an electrochemical surface potential originated from an interaction between the surface trap states and a double layer capacity, while the interaction between the 2DEG and the surface trap states originates from tunneling and electrostatics. It has now been surprisingly found that operation of the PC-HEMT sensor as an open gate field-effect transistor is not required in order to modulate the surface electrochemical potential within the barrier layer/electrolyte system.
In a specific embodiment, the microelectronic sensor of the present invention for performing sorbent, immunosorbent, sorbent or cell assays and for measuring cell dynamics comprises the following components:
The ADC card may be any suitable analogue-to-digital converter data logger card that can be purchased, for example, from National Instruments® or LabJack®. Optionally, the current amplifier can be operated directly with current flowing via the conducting 2DEG channel into the amplifier with small input resistance of 1MΩ at gain higher than 104 and only 1Ω at gains lower than 200. This setup may directly amplify the electric current modulation in the 2DEG channel originated from external body charges.
In a specific embodiment, the wireless connection module may be a short-range Bluetooth® or NFC providing wireless communication between the sensor and the readout module for up to 20 m. If the connection module is Wi-Fi, the connection can be established with a network for up to 200 nm, while GSM allows the worldwide communication to a cloud. The external memory can be a mobile device (such as a smartphone), desktop computer, server, remote storage, internet storage or cloud.
Alternatively, the PC-HEMT of the invention may be based on a piezoelectric electro-optical crystal (EOC) transducer. The PC-HEMT based on the EOC piezoelectric substrate exhibits the highest coupling between electrical and mechanical energy compared to all other varieties of substrates. Additionally, such a substrate also has the advantages of having a high velocity-shift coefficient and a very high electromechanical coupling coefficient, K2, which yields a greater mass sensitivity in comparison with the same regular SAW device on any other piezoelectric substrates. The EOC may be any suitable electro-optical crystalline material such as LiNbO3, which is brought into a contact with a medium to be sensed. The EOC is then illuminated with a polarised light.
In case of the LiNbO3 crystalline material, the wavelength of the polarised light is about 400-600 nm. Modulated light from the light source illuminates the substrate with the EOC, and then falls on the 2DEG structure. The 2DEG structure is ultrasensitive to an incident light creating the p-n-pairs in the top recessed layer and as a result, strongly affecting the 2DEG conductivity. In general, irradiation of the 2DEG structure with light switches the 2DEG channel from normally-off to a pseudo-conducting or normally-on state. Therefore, being in a close proximity to the ionic cloud, the EOC is capable of changing its light absorbance strongly affecting electrical current in the 2DEG channel, thereby resolving any smallest light intensity changes coming from the EOC transducer. Depending on the excitation light wavelength, the position of the sensor relative to the incident light beam can be changed. For instance, in case of IR light (700-1500 nm), the sensor should be placed perpendicularly to the light beam for achieving the highest sensitivity. The parasitic charging of the EOC is compensated via the electrodes attached to the crystal. Additionally, a variety of light filters in front of the sensor can be utilised. Thus, the use of the EOC configuration of the PC-HEMT of the invention makes it possible to drastically increase sensitivity of the sensor to an electrical charge.
In a further specific embodiment, the microelectronic sensor of the present invention for performing sorbent, immunosorbent, sorbent or cell assays and for measuring cell dynamics comprises the following components:
In some embodiments, the sensors of the present application can be used for portable long-time-operation solution within remote cloud-based service. The portable sensor of an embodiment should have a very small power consumption saving the battery life for a prolong usage. In this case, the non-ohmic high-resistive contacts capacitively connecting the sensor to an electric circuit are preferable. The non-ohmic contacts actually limit an electric current flowing through the 2DEG/2DHG channel by having an electrical resistance 3-4 times higher than the resistance of the 2DEG/2DHG-channel, thereby reducing electrical power consumption without sacrificing sensitivity and functionality of the sensor. Thus, the use of non-ohmic contacts in some embodiments of the sensor of the present application is a hardware solution allowing minimising the power consumption of the device. In another embodiment, the power consumption of the device can be minimised using a software algorithm managing the necessary recording time of the sensor and a battery saver mode, which limits the background data and switches the wireless connection only when it is needed.
In a further embodiment, a method for sorbent, immunosorbent or cell sorbent assay of a sample containing a chemical compound or a biological compound to be tested in a gas phase or in a liquid phase, comprises the following steps:
The sensors of the present invention have been proved to be an effective tool for rapid drug screening with accurate results and in point-of-care diagnostics. The present invention also provides methods for identifying targets of a drug in a cell by comparing the effects of the drug on a cell, the effects on a cell of modifications to a target of the drug, and the effects of the drug on a cell which has had the target modified.
Among other applications, the sensors of the present invention are useful for studying the effect of pharmaceuticals on a human heart. Neuronal and cardiac muscle cells (cardiomyocytes) are commonly used for initial medical trials in pharmacological examinations. To obtain results similar to the response of cells in the human body, human-induced pluripotent stems (iPS) are most often used. To get FDA approval, the new drug should not have a harmful effect on the heart. In addition, the benefits achieved must be greater than the damage done.
The flow of ions in or out of the cell as well as the action potential can be measured by performing electrophysiological techniques as patch-clamping. Calcium flow is an important factor for the physiological function of the human body, as it affects the flow of ions and, therefore, signal transmission. As a result, multiple diseases are caused by impaired calcium metabolism. As already mentioned, drugs can have a negative effect on the human body, for example, by changing the action potential of the cell, which is activated by the flow of ions and which leads to a contraction of the heart. Therefore, it is very important to measure cell contraction, since any pathological contraction indicates impaired cell function. A huge drawback of methods such as patch-clamping is that only the ion flux can be detected, but not the contractility of the cells.
In contrast, the sensors of the present invention can be used to detect the cell dynamics, including contractility of cells. This is done by growing cells directly on the surface of the sensor. Cell movement is measured electronically in real time. In addition, the sensor collects all the information received from the cell signal because the signal is not filtered. This is a great advantage, for example, against optical methods that use specific algorithms to analyse the obtained optical result. Thanks to an electronic readout, immunostaining and expensive measuring equipment are not required to obtain results. In the following experimental examples supporting the present invention, cardiomyocytes are used primarily because they give a strong and clearly defined electrical signal as well as a visible contraction, which can be correlated to that signal.
Cardiomyocytes were digested and isolated from chopped neonatal mouse heart (having age of 1-3 days). They were seeded on the sensor coated with fibronectin. After 3-5 days in culture, cardiomyocytes started beating on the sensor. Depending on the number of cells used (1 k to 10 k per sensor), cells beat in independent clusters (low density) or synchronise completely (high density). The cells stayed active for several weeks. The protocol for cardiomyocytes isolation using enzyme digestion of neonatal heart tissue with the Pierce Primary Cardiomyocytes Isolation Kit is available online at ThermoFischer Scientific®. The active cardiomyocytes were measured under controlled conditions in incubator at 37° C. and 5% CO2 with the sensor shown in
Several electronic configurations of the sensor were used in the present experiments. In the configuration shown in
In the configuration shown in
In another configuration of the present embodiments, the differential voltage is kept zero so that the bridge is always balanced. The resistance changes on the variable resistor to keep the differential voltage zero are proportional to the resistance change of the sensor. In this configuration the voltage amplifier is optional. Microcontroller constantly monitors and adjusts the closed loop feedback to the programmable resistors to keep the bridge balanced.
Reference is now made to
Reference is now made to
Thus, the present invention provides a method for in-vitro measurement of a cell dynamics, said method comprising:
In one of the aspects of the present invention, a microelectronic microwell plate for sorbent, immunosorbent or cell sorbent assay comprises:
In some embodiments, a method for sorbent, immunosorbent or sorbent cell assays comprises the following steps:
Electronic ELISA experiments using the sensor of the present invention and supporting the present invention are presented below. In all the electronic ELISA experiments, the assay kits were purchased from EUROIMMUN®. The assay kits contained washing buffer, capture analyte, blocking solution and target analyte. The buffer preparation was not necessary since all necessary buffers were provided by EUROIMMUN®. The composition of these buffers is confidential and cannot be disclosed. All the samples were prepared according to the proprietary protocols provided by EUROIMMUN®. These protocols are also confidential and cannot be disclosed. Each experiment was performed according to the proprietary protocol provided by EUROIMMUN® and cannot be disclosed either.
In general, the following experimental procedure in each experiment was carried out:
In each experiment, two sensors were used either modified or incubated with the positive sample, and two sensors were used either modified or incubated with the negative sample. Volume of measuring cavity in each sensor was 80 μL. Three or four electronic channels were used for each negative or positive sample, and the readout was done simultaneously. Raw IDS data was generated for positive and negative samples. The corrected data was obtained by a baseline correction in each channel, which was done by creating exponential model. The baseline was subtracted from the raw data.
Reference is now made to
Charges formed in a liquid medium sensed by ISFET-type sensors come from the dissolved molecules. Depending on the pH value of the liquid and the molecules' isoelectric point, the dissolved molecules exhibit a global charge. However, this charge may be non-uniformly distributed over the molecule. In addition, the molecules have different sizes and a different 3D structure. Therefore, it is very important that:
The sensor of the present invention is different in all the aforementioned aspects. As noted above, it has recently and surprisingly been found by the present inventors that the PC-HEMT of the invention is capable of overcoming the Debye length limitation. The overall design of the PC-HEMT enables its additional operation in the frequency domain and helps to stabilise the electronic readout when recording even very small DC changes. Therefore, the sensor based on the PC-HEMT of the present invention can be used in impedance spectroscopy. Combination of potentiometric and impedimetric readout enables a more reliable sensing of molecules with the potential to sense beyond the Debye screening of electrical charges in an electrolyte solution, which is usually the limiting factor in most of the sensors having only potentiometric or conductometric readout.
As mentioned above, the PC-HEMT of the present invention can be optionally functionalised with different molecules (receptors), which are capable of binding to a target (analyte) molecule, for sensing. As a result, the PC-HEMT-based sensor of the invention can be used for label-free detection of target (analyte) molecules by monitoring changes in the electric current of the transistor caused by variations in the charge density or the impedance at the open gate-electrolyte interface. However, a more interesting approach is when the PC-HEMT is not functionalised, but still capable of sensing target molecules or biomolecules. As discussed above, sensing of the electric charges with the transistor of the present invention is possible in a contactless manner, when the molecules are at some distance from the surface of the transistor. This clearly allows to overcome a usual “sensing noise” of any traditional biosensor having reporter molecules attached to the surface of the sensor. Thus, the PC-HEMT-based sensor of the present invention can overcome the Debye-length limitation and tremendously increase the signal-to-noise ratio of the sensor and consequently, enhance sensitivity.
Moreover, the PC-HEMT-based sensor of the present invention does not require any surface modification with reporter molecules or any plate treatment or washing procedures or protocols. The sensing can be performed directly and immediately after immersing the sensor into any media being tested. This is in huge contrast to modern bioassays, such as ELISA or qPCR, which require hours and multistep laboratory procedures to perform. In addition, while most of the modern bioassays are single-use and require utilisation of the used materials and plates, the present sensor can be easily and immediately re-used after washing.
The above examples of the electronic ELISA of the present invention clearly indicate that many different analytes can be successfully detected with the sensor of the present invention. The sensor can distinguish between positive and negative patient serum with ultra-high sensitivity without having it modified. The sensor indicates biological activity at the sensor surface and can measure different concentrations of an analyte.
All the known HEMT devices are classified today in two general types: normally-on, and normally-off. Applicant has now discovered a third type (PC-HEMT), which is both normally-on and normally-of at the same time. It is a resonance semiconductor device operating based on a quantum superposition principle, hence its incredible sensitivity (switching between on and off takes no time).
The PC-HEMT-based sensors of the present invention are characterized in that the thickness of the top layer of the heterojunction structure in the open gate area is 5-9 nanometres (nm) and the surface of said top layer has a roughness of 0.2 nm or less, wherein the combination of said thickness and said roughness of the top layer is suitable for creating a quantum electronic effect of operating said 2DEG or 2DHG channel simultaneously in both normally-on and normally-off operation modes of the channel, thereby making said transistor suitable for conducting electric current through said channel in a quantum well between normally-on and normally-off operation modes of the transistor.
The amplification of the electrical current in the PC-HEMTs observed under any small external effect is tremendous. Every tiny electrical potential or piezoelectric impact, or a single molecule charge, even remotely (from the distance up to half a meter) immediately destroys this quantum equilibrium and either closes the channel or opens it. This is the quantum resonance effect that was observed unexpectedly when Applicant was working on improving the surface flatness of the top layer. When 0.2 nm roughness was reached by recessing the top layer down to 5 nm, removing atom layer by atom layer with short plasma pulses (etching) using Applicant's proprietary technique (described herein), an unusually drastic increase of the signal was suddenly registered. When approximately 4 nm was reached, this effect disappeared and the transistor returned its normal characteristics as a normally-off HEMT.
Following this surprising experimentally observed phenomenon, Applicant tested various ranges of thickness and roughness of the top layer and arrived to the conclusion that this incredibly high amplification of the HEMT sensitivity occurs only in the range of 5-9 nm thickness of the top layer. However, in order to obtain and observe this hypersensitivity of the HEMT, the top layer surface should be flat having roughness 0.2 nm and less. If such low roughness is not reached, this quantum effect cannot be created and observed.
This application claims priority to U.S. 62/821,882 entitled ELECTRONIC SORBENT ASSAY filed on Mar. 21, 2019. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62821882 | Mar 2019 | US |
