122 is positioned along the channel 15 in an area to which at the opposite side of the channel there are adjacent the first gate electrodes 11 and 21 and only one of the source or drain of the transistors 10, 20, but not both the source and drain of both transistors 10, 20. In other words, the common second gate electrode 122 is located at the bottom side of the channel 15 between the source electrode 13 of the detection transistor 10 and the drain electrode 24 of the reference transistor 20, overlaps with the area of the first gate electrodes 11, 21 and has a width that is smaller than the distance between the source electrode 13 of the detection transistor 10 and the drain electrode 24 of the reference transistor 20.

The transistors 10 and 20 may be arranged on the common substrate 126 and covered with a common encapsulation 127, exposing the first gate electrodes 11, 21 of both transistors, the detection transistor 10, and the reference transistor 20. Furthermore, the biosensing unit B may include multiple pairs of detection transistor-reference transistor, which can further increase the assay's sensitivity.

The first gate electrode 11 of the detection transistor 10 can be connected with a sensitive substrate 31a, 31b with functionalization species—suitable for interaction with the target substance. The functionalization species of the sensitive substrate 31a, 31b may be chemical molecules e.g. enzymes, antigens, nucleotide sequences e.g. DNA, or RNA, amino-acids sequences, or chemical groups, e.g. —COOH, —NH₂, —OH, which are capable of interaction with the target substance. For example, for the target substance being glucose, the sensitive substrate 31a, 31b may comprise glucose oxidase molecules serving as the functionalization species. The sensitive substrate 31a, 31b may also comprise a base material (inert to the target substance)—for suitable adherence of the functionalization species within the sensitive substrate 31a, 31b.

The first gate electrode 21 of the reference transistor 20 can be connected with an insensitive substrate 32a, 32b (FIGS. 3A, 4) without functionalization species, thus, the insensitive substrate 32a, 32b is unsuitable for interaction with the target substance. However, the insensitive substrate 32a, 32b is configured to detect at least one of the external factors which may impact the detection—distorting the results. For example, the insensitive substrate 32a, 32b may be configured to measure pH of the sample, thereby providing a reference that enables the minimization of interferents' influence on the measurement results. Preferably, the insensitive substrate 32a, 32b is made of the same base material connected to the electrode 21 as the base material of the sensitive substrate 31a, 31b-connected to the electrode 11 (however, the base material of the insensitive substrate 32a, 32b does not include the functionalization species).

The base material for both the sensitive and insensitive substrates, denoted as 31a, 31b, 32a, 32b, may be composed of a polymer, resin, or oligomer. For instance, the base material could be a conductive membrane polymer, such as Naftion®, which is directly attached to the first gate electrode, 11, 21. For the sensitive substrate, 31a, 31b, the membrane polymer may include enzymes, such as glucose oxidase for interaction with glucose.

The first gate electrode, 11, of the detection transistor, 10, can be connected with the sensitive substrate, 31a, and the first gate electrode, 21, of the reference transistor, 20, can be connected with the insensitive substrate, 32a, 32b. This connection can be achieved either by physical or chemical binding, ensuring that the substrate, 31a, 32a, once placed on the first gate electrode, 11, 21, is undetachable. Consequently, the biosensing unit B of the biosensor becomes disposable. This is depicted schematically in FIG. 1A for the detection transistor, 10, and in FIG. 3A for the biosensing unit B. Here, the sensitive substrate, 31a, and the insensitive substrate, 32a, are arranged as layers directly on the first gate electrode, 11, 21, respectively. The sensitive substrate, 31a, as well as the insensitive substrate, 32a, may be bonded to the first gate electrode, 11, 21, through chemical bonding, such as covalent bonds, ionic bonds, etc.

In another embodiment, the substrates, 31b, 32b, can be detachably connected to the respective first gate electrodes, 11, 21, via a conductive path (FIG. 6A). This allows for repeated connection and disconnection of the first gate electrode, 11, and the respective substrate (sensitive and insensitive, 31b, 32b), making the sensitive and insensitive substrate, 31b, 32b, disposable. The advantage of disposable substrates, 31b, 32b, is that the biosensing unit B of the biosensor, and thus, all the transistors can be used multiple times, allowing for multiple assays during the lifetime of the biosensing device. This can significantly reduce the cost of single assays and limit waste generation.

The conductive path, which makes both sensitive and insensitive substrates, 31b, 32b, disposable, can take various forms, including but not limited to conductive films, wiring, junction connectors, an electrolytic connection, etc. Furthermore, the detachable disposable substrates, 31b, 32b, may collectively form a single disposable strip (FIG. 5—Unit A). This strip comprises a sheet of material, for example, made of the base material, or comprising the base material with a sensitive zone arranged thereon with the functionalization species, and an insensitive zone without functionalization species. This allows both the sensitive substrate, 31b, and the insensitive substrate, 32b, to be simultaneously subjected to interaction with a single sample suspected to contain the target substance. This provides unified conditions of the assay for the entire biosensing unit B of the biosensor, as shown schematically in FIG. 4.

The assay for detecting the target substance with a biosensing kit, comprising the biosensor with the biosensing unit B, and the disposable unit A, comprising the sensitive substrate, 31b, and optionally further comprising the insensitive substrate, 32b, is shown schematically in FIG. 5. In phase one (I), the strip (unit A) with the sensitive substrate 31b and the biosensor are provided (for the example where the biosensing unit comprises only the detection transistor 10—the detachable strip comprises only the sensitive substrate 31b, and for the example where the biosensing unit further comprises the reference transistor 20—the detachable strip further comprises the insensitive substrate 32b, which are provided in two zones of the strip). In phase two (II), the user attaches the disposable strip to the biosensing unit using the conductive path. This ensures that the disposable strip is connected with the first gate electrode, 11, of the detection transistor, 10, and optionally with the first gate electrode, 21, of the reference transistor, 20, if present, as shown schematically in FIG. 6A. In phase three (III), the user brings the sample, suspected to contain the target substance, into contact with the detachable strip, both the sensitive and insensitive zones at once. For example, a saliva sample can be drawn directly from the mouth by holding the strip in the user's mouth for a specified period, typically from 1 to several seconds (as shown in FIG. 5—phase III). The units C-E of the biosensor conduct signal gathering and calculation, with the result being directly displayed on a screen of the biosensor, as shown in FIG. 5—phase four (IV). Additionally, the obtained data can be wirelessly transferred to an external device (such as a mobile phone, tablet, etc.) and processed externally by dedicated applications. The corresponding assays can be conducted for samples in various forms, e.g., blood, sweat, urine. The sample can be brought into contact with the sensitive substrate, 31b, (and optionally with the insensitive substrate, 32b, if provided in the disposable strip—unit A), prior to, during, or after connecting unit A with unit B of the biosensing kit, using the conductive path.

Detection Method

The assay is based on measuring the output characteristics: I_DS(drain-source current) as a function of V_DS(drain-source voltage) of the detection transistor, 10. The drain current (I_DS) changes as a function of the concentration of the target substance, which is attributed to changes in the electrical potential (modulation of the depletion region at the gate-channel Schottky region) on the first gate electrode, 11, 31a, 31b. This corresponds with the electrochemistry of interactions of the functionalization species of the sensitive substrate, 31a, 31b, and the target substance.

For instance, for glucose detection, the enzyme (glucose oxidase) can be contained in the sensitive substrate for selectively oxidizing glucose. Due to the oxidation reaction, gluconolactone and H2O2 particles are created as the reaction products, as described in Equation 1:

$\begin{matrix} Glucose + O_{2} \frac{(glucose oxidase)}{\to} gluconolactone + H_{2} O_{2} & (1) \end{matrix}$

Next, the H2O2 dissociates into protons (H+), electrons (e−), and O2, as described in Equation 2:

$\begin{matrix} H_{2} O_{2} \to O_{2} + 2 H^{+} + 2 e^{-} & (2) \end{matrix}$

The net result of the above two reactions, according to Equations 1 and 2, is that a stoichiometric quantity of protons is liberated, which is directly proportional to the number of glucose molecules oxidized by the enzyme. The protons, being charged, affect a depletion region of the detection transistor, and therefore affect the drain-source current (I_DS) (FIG. 6B). As presented in FIG. 6B, at 0% of the target substance (e.g., glucose) in the sample subjected to assay, the channel is nearly pinched off and the current flowing through the channel is low. As the target substance (e.g., glucose) concentration increases, the depletion region thickness (Wd) decreases, subsequently increasing the width of the conduction channel (Wc). This leads to a decrease in the barrier potential and the resistivity between the source electrode and the drain electrode in the channel, which is followed by an increase in the drain-source current (I_DS).

FIGS. 2A-2C graphically illustrate the electrical measurements that can be taken during the assay with a biosensor. This biosensor includes a biosensing unit B, which features a detection transistor 10. As previously mentioned, the detection transistor measures the target substance by assessing output characteristics: I_DS=f (V_DS) of the biosensor. The detection transistor (MESFET) has two gate electrodes: the first gate electrode and the second gate electrode. It also includes a channel between the electrodes that operates under a constant bias of the second gate electrode.

In the absence of polarization of the second gate electrode, the initial gate-source voltage (VGS) is 0, and the drain-source voltage (V_DS) is also 0. Under these conditions, the detection transistor is in thermodynamic equilibrium and no current flows. A depletion layer exists beneath the first-gate Schottky junction. By increasing V_DS>0 V, the source-drain current (I_DS) will flow due to the potential difference between the source electrode and the drain electrode, as shown in FIG. 2A. Initially, for low V_DS, the channel resistance remains constant, and I_DSis linearly dependent on V_DSaccording to Ohm's law. As V_DSincreases, the Schottky barrier becomes increasingly reverse biased, causing the voltage to drop across the channel. This potential difference results in an expansion of the depletion layer at the drain electrode side of the channel Consequently, the channel narrows at the drain electrode side, leading to the rounding off of the source-drain current (I_DS). At a specific voltage, known as the saturation drain-source voltage (V_DSsat), the width of the depletion layer equals the channel thickness, resulting in the channel being pinched off, as depicted in FIG. 2B. At this drain-source voltage (V_DS), the source electrode and the drain electrode are completely separated by a reverse-biased depletion region. Despite the pinch-off and the resulting lack of charges, the source-drain current (I