Specific and label-free sensing of enzyme-dna interactions using biological transistors

Abstract

The present disclosure provides bio-transistors for sensing target molecules. More specifically, the present disclosure provides systems and methods for determining the presence and / or quantity of target molecules, specifically, protein-based target molecules, such as proteineous factors, specifically T7 primase, that bind nucleic acid molecules in a biological sample. The present disclosure uses nucleic acid molecules as the affinity molecule, thereby providing screening methods for therapeutic compounds and diagnosis and monitoring of conditions associated with the target molecules.

Description

[0001] SPECIFIC AND LABEL-FREE SENSING OF ENZYME-DNA INTERACTIONS

[0002] USING BIOLOGICAL TRANSISTORS

[0003] TECHNOLOGICAL FIELD

[0004] The present disclosure relates to bio-transistors for sensing target molecules. More specifically, the present disclosure provides systems and methods for determining the presence and / or quantity of target molecules based on calibration data, specifically by using nucleic acid-based affinity moieties.

[0005] BACKGROUND ART

[0006] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0007] [1] US20230022648.

[0008] [2] Bhattacharyya, I. M. et al. A New Approach toward the Realization of Specific and Label-Free Biological Sensing Based on Field-Effect Devices. Adv Electron Mater (2022) doi: 10.1002 / aelm.202200399.

[0009] [3] Bhattacharyya, I. M. et al. A new approach towards the Debye length challenge for specific and label-free biological sensing based on field-effect transistors. Nanoscale 14, 2837-2847 (2022).

[0010] [4] Ron, I. et al. Label-free and specific detection of active Botulinum neurotoxin in 0.5 μL drops with the meta-nano-channel field-effect biosensor. Sens Actuators B Chem 393, 134171 (2023).

[0011] [5] Samanta, S. et al. Specific and Label-Free Sensing of Prostate-Specific Antigen (PSA) from an Ultrasmall Drop of Diluted Human Serum with the Meta-Nano-Channel Silicon Field-Effect Biosensor. Adv Mater Technol (2023) doi: 10.1002 / admt.202202200.

[0012] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter. BACKGROUND

[0013] Field-effect transistors for biological sensing (bioFET) have been the scope of continuous research for the last 40 years. The motivation for bioFET sensing is the inherent amplification capabilities to transduce small surface potential variations into significant variations in drain-source current IDS)- Moreover, bioFET suggests the bio-convergence of the microelectronic industry with biological sensing, which entails robustness, mature- technology, low noise-levels, low cost, and ultimate capabilities for multiplexed sensing in ultra-small samples.

[0014] The leading bioFET sensing mechanism involves the electrostatic perturbation at the sensing area upon the introduction of specific molecules, which is then transduced by the bioFET into a variation in source-drain current IDS- This electrostatic perturbation is due to charges and / or dipoles associated with the chemical or physical adsorption of biomolecules at the sensing area, the generation of new bonds between the biomolecules and the surface, or any redistribution of electrostatic charges at the sensing area upon.

[0015] The inventors previously developed a biosensor to shorten and optimize the Debye length at an interface between the sensing region and the fluid [1]. This biosensor includes a semiconductor active region; a sensing region configured to contact a fluid; and multiple electrodes that comprise decoupling electrodes and additional electrodes. The decoupling electrodes may be configured, wherein operating in a first mode, to prevent a formation of a top conductive channel within the semiconductor active region; and the additional electrodes are configured, wherein operating in the first mode, to independently control (i) one or more properties of one or more other conductive channels formed within the semiconductor active region, and (ii) a Debye length at an interface between the sensing region and the fluid [1].

[0016] The bioFET was employed for sensing different molecules [2, 3, 4, 5].

[0017] Primase is an enzyme that recognizes a specific DNA sequence and serves as a model for other proteins and enzymes that bind to specific DNA sequences. Primase recognizes single-stranded DNA and provides readout upon specific binding, which makes an ideal model for specific DNA-protein interactions (such as transcription factors that bind through the major and minor grooves of double-stranded DNA). GENERAL DESCRIPTION

[0018] The inventor's model system of the present disclosure consists of a DNA strand composed of a primase recognition sequence (5’-GTC-3’), and the primase domain of gene product 4 of bacteriophage T7 binds the GCT. The specific binding of primase to the DNA recognition sequence is monitored. The bioFET employed for the label-free detection of primase-DNA is composed of lateral side gates for the determination of the channel conductivity and a solution electrode for the determination of the solution potential. The BioFET is realized with silicon-on-insulator (SOI) wafer technology, and the active sensing area is biofunctionalized with the DNA strands described above.

[0019] Thus, the first aspect of the present disclosure relates to a bio-transistor system comprising at least one transistor unit and a control unit. The transistor unit comprises (i) at least one channel, (ii) source and drain electrodes, (iii) at least one gate electrode and (iv) at least one active region located in proximity to the channel region and carrying affinity moieties. The at least one active region is configured for accepting at least one sample. The transistor unit also comprises (v) at least one additional electrode positioned to be in electrical contact with the sample. The control system comprises at least one processor and memory circuitry. The control system is configured and operable for performing one or more measurements of the sample. Each measurement comprises maintaining a selected electric potential on the at least one additional electrode and determining current transmission profile through the at least one channel with respect to potential variation of the at least one gate electrode. The control unit thereby configured to determine data on the presence and / or quantity of one or more target molecules in the sample. In some embodiments, the affinity moiety comprises at last one nucleic acid-based molecule. In yet some further embodiments, the target may be a proteineous target.

[0020] Another aspect of the present disclosure relates to a battery comprising two or more of the bio-transistor system as defined above.

[0021] Another aspect of the present disclosure relates to a method for determining presence and / or quantity of at least one target molecule in at least one sample. The method comprising: (a) contacting the at least one sample with a bio-transistor having an active region (e.g., a modified active region) carrying affinity moieties, or a battery comprising at least two of the bio-transistors; (b), performing one or more measurements, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; and (c), processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample and determining presence and / or quantity of the one or more target molecules in accordance with pre-stored calibration data.

[0022] Another aspect of the present disclosure relates to a screening method for identifying a compound that modulates the interaction of an affinity moiety with a target molecule in at least one sample. The method comprising: (a) contacting the at least one sample with a bio- transistor system, in the presence and the absence of at least one candidate compound. The bio-transistor having an active region (e.g., a modified active region) carrying selected at least one type of affinity moieties; (b) performing one or more measurements for each sample, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; (c) processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample; (d) determining presence and / or quantity of the one or more target molecules in accordance with pre-stored calibration data, thereby determining a target molecule value for the sample in the presence of the candidate compound, and a target molecule value in the absence of the candidate compound; and (e) determining that the candidate compound is a modulator of the interaction between the affinity moiety and the target molecule, if the target molecule value obtained for the sample in the presence of the candidate compound is different from the target molecule value obtained in the absence of the candidate compound.

[0023] Another aspect of the present disclosure relates to a diagnostic method for determining a physiological and / or environmental condition or state of a subject and / or a media and / or a habitat. The method comprising: (a) contacting the at least one sample with a bio-transistor having an active region (e.g., a modified active region) carrying affinity moieties, or a battery comprising at least two of said bio-transistors; (b) performing one or more measurements, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; (c) processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample; (d) determining presence and / or quantity of the one or more target molecules in accordance with pre-stored calibration data, thereby obtaining a target molecule value for the sample; and (e) determining that the subject and / or media and / or habitat display said physiological and / or environmental condition or state, if the at least one target molecule value obtained for the sample in step (d), is positive or negative with respect to a reference target molecule value pre-determined for the physiological and / or condition or state, or with respect to a target molecule value determined for at least one control sample.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non- limiting example only, with reference to the accompanying drawings, in which:

[0026] Figure 1. schematically exemplifies a bio-transistor system according to some embodiments of the present disclosure.

[0027] Figure 2. schematically exemplifies a bio-transistor system formed from a plurality of transistor units according to some embodiments of the present disclosure.

[0028] Figure 3. exemplifies method actions for determining presence and / or quantity of one or more target molecules according to some embodiments of the present disclosure.

[0029] Figure 4. Illustrations of the MNC biosensor.

[0030] Figure 5. IDS-VGLcurves for selected VGFvalues and for 0.5 μL drops of 3% neutral solution spiked with primase concentrations ranging from 1 fM to 10 μM. The corresponding Inormcurves are presented along-side the IDS-VGLcurves.

[0031] Figure 6. Extracted calibration curves for the specific and label-free sensing of primase- DNA interaction in neutral solution. Figure 7. IDS-VGLcurves for selected VGFvalues measured for MNC biosensor biofunctionalized with anti-AFP antibodies (background measurement)

[0032] IDS-VGLcurves for selected VGFvalues measured for MNC biosensor biofunctionalized with anti-AFP antibodies. Each data point is an average of 64 measurements (16 drops, each drop is measured 4 times), and the error bars are the corresponding standard deviations (see inset).

[0033] Figure 8A-8D. Process of Si / SiO2surface biofunctionalization with anti-AFP antibodies

[0034] Fig. 8A. An illustration showing the process of Si / SiO2surface biofunctionalization. The corresponding contact angle measurements are also shown.

[0035] Fig. 8B. Ellipsometry measurements and contact angle measurements of Si / SiO2samples post various modification steps.

[0036] Fig. 8C. EIS measurements showing the real and imaginary capacitances for the various modification steps.

[0037] Fig. 8D. IDS-VGLfor various VGFvalues for unmodified MNC device and biofunctionalized MNC biosensor. The data points and the error bars (see inset) reflect the averages and standard deviations of 12 measurements (3 drops each measured 4 times).

[0038] Figure 9A- 9C. IDS-VGLfor selected VGFvalues for MNC biosensor modified with anti- AFP molecules

[0039] Fig. 9A. IDS-VGLfor selected VGFvalues for MNC biosensor modified with anti-AFP molecules. The measurements are performed in 1 : 100 diluted serum. The data points reflect an average of 42 measurements (Total of 14 drops each measured 3 times), and the error bars are the respective standard deviations (see inset).

[0040] Fig. 9B. The second derivatives of the curves presented in Figure 11 A where a peak reflects the excitation of a conducting channel. The dependence of channel excitation on gates’ voltage configuration is shown.

[0041] Fig.9C(i)-9C(iv). Non-specific measurements for: AFP introduced to an unmodified MNC biosensor (Fig. 9C(i)), AFP introduced to an MNC biosensor modified with APTMS (Fig. 9C(ii)), hCG molecules introduced to an MNC biosensor modified with anti-AFP antibodies (Fig. 9C(iii)), and PSA molecules introduced to an MNC biosensor modified with anti- AFP antibodies (Fig. 9C(iv)). All measurements are performed in 1: 100 diluted serum. The insets present magnifications showing the error bars reflecting the standard deviations of the measured populations.

[0042] Figure 10A-10C. IDS-VGLfor selected VGFvalues for different concentrations of AFP molecules

[0043] Fig. 10A. IDS-VGLfor selected VGFvalues for 10 concentrations of AFP molecules. The measurements are performed in 1: 100 diluted serum. Each concentration (=drop) is measured 4 times. The insets show the error bars reflecting the standard deviations. The non-specific signals are accounted for and removed.

[0044] Fig. 10B. The extracted Readout corresponding to the curves presented in Figure 10A. The insets show the corresponding error bars. The labels at the top of the graphs indicate the corresponding conducting channels.

[0045] Fig. 10C. An illustration showing one possible mechanism for the Readout polarity switch induced by the double layer electric field.

[0046] Figure 11A-11B. Readout calibration curves for AFP molecules

[0047] Fig. 11A. Readout calibration curves. The illustrations on the right reflect the channel configurations. The vertical dashed grey lines indicate the calibration threshold.

[0048] Fig. HB(i)-llB(ii). The dependency of the calibration threshold on VGF(Fig. llB(i)). The non-dependency of the calibration threshold on VGL(Fig. llB(ii)). The shift between the lines for VGL= 0, -0.5, -1 and -1.5 V are only for the purpose of visualization.

[0049] Figure 12. AFP sensing performance

[0050] The figure presents Table 2, that shows a summary of the MNC biosensor sensing performance for specific and label-free detection of AFP.

[0051] The AFP amino acid sequence is denoted by SEQ ID NO: 4.

[0052] These and other aspect of the present disclosure will become apparent by the hand of the following description. DETAILED DESCRIPTION OF EMBODIMENTS

[0053] Biosensing based on biological field-effect transistors (bioFET) is a promising technology toward specific, label-free, and multiplexed sensing in ultra-small samples. The leading bioFET sensing mechanism is the electrostatic perturbation induced at the sensing area upon the introduction of specific molecules. In the current disclosure, the inventors employ a unique biological field-effect transistor (BioFET) for the sensing of DNA-based molecules, using DNA-primase interaction. The BioFET is composed of lateral side gates to control the conducting channel, and a solution electrode to control the interaction. High- end specific and label-free sensing of DNA-primase binding is demonstrated with a limit- of-detection of 1 fM, a dynamic range of 10 orders of magnitude and with excellent linearity and sensitivity.

[0054] One of the major infections in dairy cattle is bovine mastitis (BM), a prominent inflammatory disease associated with inflammation in the udder tissues [Djabri, B., et al. Vet Res 33, 335-357 (2002)]. Several methods are currently available or suggested for detection of dairy cattle infected with BM [Ball H J & Greer D. Vet Rec. 129, 507-509 (1991); Chagunda, M. G., et al. Journal of Dairy Research 73, 431-440 (2006); Nirala, N. R. & Shtenberg, G. Appl SurfSci 566, 150700 (2021); Kitchen, B. J., et al. Journal of Dairy Research 45, 15-20 (1978); Nirala, N. R. & Shtenberg, G. Spectrochim Acta A Mol Biomol Spectrosc 257, 119769 (2021); Nirala, N. R., et al. Taianta 239, 123087 (2022); Pemberton, R. M., et al. Analyst 126, 1866-1871 (2001); Kumar, D. N., et al. Taianta 220, 121439 (2020); Kumar, D. N., et al. ACS Sens 5, 1969-1976 (2020)].

[0055] As indicated above, the present disclosure provides a bio-transistor system configured and operable for detection of presence and / or quantity of one or more selected molecules. An exemplary configuration of bio-transistor system 100 is illustrated in Fig. 1. The system includes at least one transistor unit 110 including source 120 and drain 130 regions associated with respective source 124 and drain 134 electrodes, and a channel region 140 between them. The transistor unit 110 also includes one or more gate electrodes 144, located and configured to apply selected electric field on the channel 140 region to thereby enable switching of the transistor unit 110 by affecting charge carriers within the channel 140. Typically, as exemplified herein the source 120, drain 130 and channel 140 regions are placed on a back insulator layer 160 and covered by a top insulator 170. Further, in some embodiments, the at least one gate electrode may be a side (e.g., lateral) gate electrode with respect to the channel region 140, alternatively or additionally, the at least one gate electrode may be positioned between one of the source and drain electrodes 124 and 134 and the active region 150. The active region 150 is located on top of the channel region 140, electrically insulated from the channel region 1 40 by the top insulator 170. The at least one gate electrode 144 is generally separated from the channel region 140 by a gate insulator, which may be a portion of top insulator 170. In some configurations, the transistor unit 110 may be formed of one or more semiconductor materials such as silicon, having selected doped regions with selected p-type or n-type doping. For example, the active layer defining the source 120 and drain 130 regions and the channel region 140 between them may be formed of silicon semiconductor having varying p- and n-type doping regions.

[0056] The transistor unit 110 further includes an active region 150 located in vicinity (e.g., above) a region of the channel 140 and separated from the channel, e.g., by top insulator 170. The active region 150 is formed of a region of top insulator 170, which is modified to carry selected one or more types of binding molecules, or affinity moieties, selected to bind one or more target molecules. This configuration provides the bio-transistor system 100 with capabilities for detection of the selected target molecules. The transistor unit 110 is configured to accept a liquid sample 50, e.g., a liquid drop, positioned on, or in contact with, the active region 150. In some configurations, the active region 150 may be surrounded by edge 152 configured to limit flow of liquid sample and prevent contact of the sample with electrodes of the unit 110 such as gate electrodes 144. The edge 152 may be formed of any electrically insulating material and may be a part of top insulator 170.Transistor unit 110 may also include at least one additional electrode 154 positioned to be in electric contact with the liquid sample 50, the additional electrode 154 is also referred to as quasi-reference electrode or sample electrode.

[0057] As indicated above, transistor unit 110 may be formed of silicone including n- and p-doped silicon regions forming the channel region 140 as well as source 120 and drain 130 regions. Top 170 and bottom 160 insulators may be formed of silicon oxide layers. It should be noted that in some other embodiments, the transistor may be formed of selected one or more other semiconductor materials as the case may be.

[0058] Bio-transistor system 100 may also include a control system 500. Control system 500 may include one or more processor and memory circuitries (PMC) 510 and may also include an electric circuit 52 configured to selectively apply electric potential and / or transmit electric current through the electrodes of transistor unit 110 to enable bio-transistor system 100 to determine data on presence and / or quantity of target molecules in the sample 50.

[0059] In this connection PMC 510 may include one or more processors and memory, and may include, or be associated with, an I / O interface for receiving input data and for transmitting output instructions and / or data for operation of the bio-transistor system 100. PMC 510 is operatively connected to the I / O interface and is configured to provide processing necessary for operation of the bio-transistor as described herein. PMC 510 is configured to execute one or more functional operations in accordance with computer readable instructions implemented on a computer readable medium (e.g., non-transitory memory) being pre-stored at the memory of PMC 510 or at a separate computer readable medium.

[0060] In this connection, PMC 510 may operate the electric circuit 520 for selectively providing electric potential to the electrodes of transistor unit 110, and for determining data on current flow between the source electrode 124 and drain electrode 134. In this connection the control unit 500 is configured and operable for performing one or more measurements of the sample 50 to determine data on presence and / or quantity of target molecules in the sample 50.

[0061] In some embodiments, the one or more measurements may include applying selected electric potential to one or the additional electrode 154 and / or the at least one gate electrode 144 and determining current flow between the source 124 and drain 134 electrodes in response to variation of the selected electric potential. More specifically, in some embodiments, each of the one or more measurements may include operation of the control system to maintain a selected electric potential VGFon the at least one additional electrode 154 to maintain a selected potential on the sample 50. Additionally, while maintaining a selected potential VGFon the sample 50, the control system 500 may operate for determining current transmission profile between the source 124 and drain 134 electrodes with respect to varying potential VGon at least one gate electrode 144.

[0062] Interaction of the target molecules with the one or more affinity moieties (typically being nucleic acids, amino acid, small molecules, carbohydrate-based molecules, lipid-based molecules, or any combination thereof) selected to specifically bind (directly or indirectly) the at least one target molecule in the sample (or vice versa), generate certain electric potential affecting current flow through the channel region 140 over gate and / or sample potential. Accordingly, presence and / or quantity of the target molecules interacting with the affinity molecules affects current transmission through the channel region 140. Accordingly, the PMC 510 may determine variation in current flow between the source 124 and drain 134 electrodes and utilize pre-stored calibration data to determine data on presence and / or quantity of target molecules in the sample.

[0063] Further, in some embodiments, each of the one or more measurements may include operation of the control system to maintain a selected electric potential VGFon electrode 154 to maintain a selected potential on the sample 50. Additionally, while maintaining a selected potential VGFon the sample, the control system 500 may operate for determining current transmission profile between the source 124 and drain 134 electrodes with respect to varying potential VGon at least one gate electrode 144. For example, this may be done by applying a selected potential between the source 124 and drain 134 electrodes and determining level of current transmission between the source 120 and drain 130 regions through the channel 140 with respect to potential VGapplied to the at least one gate electrode 144. Typically, each of the one or more measurements may be performed using a different selected potential VGFapplied on the sample 50.

[0064] As described below, the present disclosure is based on the inventors’ understanding that interactions between target molecules and selected affinity moieties located on the active region 150 of the transistor unit 110 affect the transistor source-gate transmission in certain conditions. The interactions between target molecules and affinity moieties causes variation in distribution of electric charges resulting in variation of electric fields around the active region 150, which also affects the channel region 140. Accordingly, each measurement may include collecting data on current to gate potential (I-VG) characteristics of the transistor unit 110 for given sample potential VGF.

[0065] Typically, the control system 500 may be configured to perform a selected number of one or more, or two or more measurements, using different sample potentials VGF, and to store collected data on current for different gate potential values for each measurement. This measurement data, indicative of transistor unit 110 I-VGcharacteristics for one or more different sample potential values is indicative of presence and / or quantity of the target molecules in the sample 50.

[0066] To this end, the control system 500 may include pre-stored data, e.g., pre-stored at the memory of PMC 510, or stored in a remote location accessible view network communication, for calibrating transistor unit 110 characteristics to data on presence and / or quantity of target molecules in the sample 50. The pre stored data may be indicative of I- VGcharacteristics of the transistor unit 110 as a function of presence and / or quantity of the target molecules for one or more given sample potentials VGF. Additionally, or alternatively, the pre stored data may be indicative of a relation between current transmission through the channel as a function of sample potential VGF, for one or more given gate voltage VGvalues. After determining data on presence and / or quantity of the target molecules, the control system 500 may operate to generate output signal indicative of the data and provide the output signal via I / O interface to be presented to an operator using user interface and / or for use in further processing.

[0067] As indicated above, the bio-transistor system 100 utilizes selected affinity moieties placed (adsorbed, attached) on an active region 150 located in vicinity to the channel region 140. The active region 150 may be separated from semiconductor region of the channel region 140 by an electric insulating layer 170, such that electrostatic changes at the active region 150 may apply electric fields affecting the channel region 140.

[0068] Selection of the affinity moieties determine one or more target molecules to be identified using the bio-transistor system 100. To enable detection of a plurality of different target molecules, the present disclosure may also provide a bio-transistor system 100 including a plurality of transistor units, each carrying a selected different type of affinity moieties, selected to interact with one or more target molecules, including e.g., one or more different target molecules. It should be understood that in some embodiments, each of the affinity moieties is specific for one target molecule. Thus, in some embodiments the disclosed bio transistor system may comprise at least one affinity moiety, and in some embodiments, a plurality of affinity moieties that are specific for one or more target molecule. Each of the affinity moieties is specific for one target molecule, but for each target molecule it is possible to have more than one either identical or different affinity moiety. In yet some further embodiments, the disclosed bio transistor system may comprise different affinity moieties specific for more than one target molecule, wherein each of the affinity moiety is specific for one target molecule. In this connection reference is made to Fig. 2 exemplifying a bio-transistor array configuration of system 100. The system includes a selected number of transistor units 110a to 110n, each having source and drain regions 120 and 130 and a channel region between them, and active region 150 carrying the selected affinity moieties, gate electrode 144, and an additional electrode (not shown) positioned to be in contact with a sample positioned on the active region 150. The plurality of transistor units 110a- 11 On are connected to a control system 500 and are independently operated for determining data on presence and / or quantity of the respective target molecules as described herein.

[0069] In some embodiments, the bio-transistor system 100 may include a sample channel configured for receiving a liquid sample and channeling portions of the liquid samples to different active regions of a plurality of transistor units as illustrated in Fig. 2. To avoid variation in electric potential between the sample portions located on the different active regions, the channel may include a selected number of electrical insulating valves configured to close prior to performing measurements on the samples.

[0070] Fig. 3 exemplifies a method for determining presence and / or quantity of at least one type of target molecules according to some embodiments of the present disclosure in a way of a block diagram. As shown, the method includes providing selected affinity moieties in contact (e.g., adsorbed, attached) with an active region 3010 of one or more transistor units. The bio-transistor unit may be treated with the selected affinity moieties during production and provided for use in determining presence and / or quantity of the selected target molecules. To determine presence and / or quantity of at least one type of target molecules, the method includes contacting a sample with the active region of the one or more transistor units 3020 and operating the transistor unit to perform one or more measurements 3030. Generally, each measurement includes maintaining a sample potential 3032, using one or more additional electrodes being in electric contact with the sample, and determining current-voltage characteristics of the transistor unit 3034. As indicated, the method may include a selected number of measurements, typically using different one or more potentials VGFvalues applied to the sample. Following one or more (or two or more) measurements, the method includes processing the measurement data and determining data on presence and / or quantity of the target molecules 3040. The processing may generally include using pre-provided calibration data indicative of variation in transistor current- voltage characteristics for given amounts of the target molecules in the sample.

[0071] Accordingly, the system and method of the present disclosure may utilize predetermined calibration data, determined for given activity region 150 carrying given amount of affinity moieties of selected type. In some examples the calibration data may be in the form of a multi-dimensional calibration data including data on variation of the source-drain current with respect to different gate potentials VGand to different sample potentials VGF. Accordingly, ss indicated above, variation of the sample potential VGFmay be used as a parameter for determining presence and / or quantity of the target molecules. In some other embodiments, variation of the sample potential VGFmay provide for varying detection range for difference ranges of quantity of the target molecule, where for each value of sample potential, variation of gate potential VGand the relation between gate potential and source-drain current provides the calibration for quantity of target molecules in the sample.

[0072] Referring back to Fig. 3, the method of the present disclosure utilizes performing one or more measurements of the sample. As indicated above, each measurement includes maintaining sample potential VGFat a selected value and determining source-drain current for selected range of gate potentials VG. Typically, the different measurements utilize different values of the sample potential VGF. It should be understood that the measurement may also utilize a selected, generally constant, source-drain voltage to eliminate, or at least significantly reduce effects of the potential variation on the interaction between the affinity moieties and target molecules. Additionally, variation of sample potential VGFmay affect concentration of the target molecules within the sample, e.g., forming layers of different concentrations of the target molecules, thereby limiting detection accuracy. To solve this issue, the present technique may utilize a selected number of different sample potential VGFvalues, and respective calibration data.

[0073] Accordingly, the systems and methods of the present disclosure provide for determining presence and / or quantity of selected target molecules within a sample. As described above, the present technique utilizes calibration data, determined in accordance with known concentrations of the target molecules. Typically, the calibration data may be determined in accordance with amount and / or density of affinity moieties associated with the active region 150.

[0074] In accordance with structure of the transistor unit 110, variation in source-drain current, associated with gate potential VG, and sample potential VGF, may be associated with concentration of charge carrier in the channel region 140. In some configurations of the transistor unit 110, variation of gate potential VG may affect size and / or shape of the channel region 140 and accordingly affect current transmission through the channel. An exemplary structure of transistor unit suitable for affecting channel shape and dimensions by variation of gate potential VG is described in US 2023 / 0022648, that is a previous patent application by the inventors, incorporated herein for reference with respect to configurations of the transistor unit.

[0075] Thus, the first aspect of the present disclosure relates to a bio-transistor system comprising at least one transistor unit and a control unit. The transistor unit comprises (i) at least one channel, (ii) source and drain electrodes, (iii) at least one gate electrode and (iv) at least one active region located in proximity to the channel region and carrying at least one affinity moiety. It should be noted that each affinity moiety is specific for a target molecule. Specifically, each of the affinity moieties is specific for one target molecule. In yet some further embodiments, the active region may carry two or more different affinity moieties, each specific for one target molecule. In some embodiments, different affinity moieties may be specific for the same target molecule. Alternatively, different affinity moieties may be specific for different target molecules, wherein each of the affinity moieties is specific for one target molecule. The at least one active region is configured for accepting at least one sample (e.g., a liquid sample). The transistor unit also comprises (v) at least one additional electrode positioned to be in electrical contact with the sample. The control system comprises at least one processor and memory circuitry. The control system is configured and operable for performing one or more measurements of the sample. Each measurement comprises maintaining a selected electric potential on the at least one additional electrode and determining current transmission profile through the at least one channel with respect to potential variation of the at least one gate electrode. The control unit thereby configured to determine data on the presence and / or quantity of one or more target molecules in the sample.

[0076] In some embodiments of the present disclosure, the control system is configured to performed two or more measurements utilizing two or more different selected electric potentials applied to the at least one additional electrode.

[0077] In some other embodiments, the control system comprises pre-stored calibration data comprising data on electric transmission through the channel for given gate electrode potential with respect to one or more selected electric potentials applied to the at least one additional electrode.

[0078] In some further embodiments, the control system comprises pre-stored calibration data comprising data on electric transmission trough the channel with respect to variation of the gate electrode potential.

[0079] It should be noted that the pre-stored calibration data may be generated from different concentrations of the target molecule in a sample. More specifically, the pre-stored calibration data may be generated from at least one concentration of target molecule. Still further, the pre-stored calibration data may be generated from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or more different concentrations of the target molecule in the sample.

[0080] In some embodiments of the present disclosure, the bio-transistor system comprising a plurality of two or more transistor units comprising respective plurality of two or more active regions carrying two or more different or identical types of affinity moieties.

[0081] In some other embodiments, the active region is separated from the channel region by an electrical insulator layer.

[0082] In some further embodiments, the at least one gate electrode is electrically insulated from the active region. Still further, in some embodiments, the control system comprises at least one electrical circuit coupled vie electrical connection to the transistor unit and configured to provide selected electric potentials to electrodes of the transistor unit.

[0083] In some embodiments of the present disclosure, the at least one affinity moiety comprises at least one of: a nucleic acid-based molecule, an amino acid-based molecule, a small molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof and the affinity moiety specifically binds, either directly or indirectly, the at least one target molecule in the sample.

[0084] In some embodiments, the affinity moiety comprises, or is derived from a component of an affinity pair. The term "affinity moiety" or "affinity molecule" as used herein, or "affinity pair" (as used herein after), refers to a corresponding binding couple biomolecule partners. The term "Affinity" or "binding affinity" is the strength of the binding interaction between a binding biomolecule to its binding partner or target (e.g., a protein, DNA or small molecule). Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions. The smaller the KD value, the greater the binding affinity of the binding molecule for its target. The larger the KD value, the weaker the target molecule and affinity molecule are attracted to and bind to one another. Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, and hydrophobic and van der Waals forces between the two molecules. In addition, binding affinity between a binding molecule and its target molecule may be affected by the presence of other molecules. The affinity pair is at least one of: proteineous factor-specific nucleic acid binding site, aptamer-aptamer target, enzyme-substrate, receptor-ligand, or any combination thereof. It should be noted that each affinity moiety is specific for one target molecule. In some embodiments, the affinity moiety and the target molecule recognized by the affinity moiety form together an affinity pair. The term "specific" and "specifically bind" is as described herein after. In some further embodiments, the target molecule comprises at least one of: an amino acid- based molecule, a small molecule, a nucleic acid-based molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof.

[0085] As indicated above, the affinity molecule, and / or the target molecule and / or the candidate compound disclosed herein after in connection with the screening methods, may be a nucleic acid-based, amino acid-based, a small molecule-based, a carbohydrate-based and / or a lipid-based. The term "-based" as used herein refers to the at ;least one affinity moiety or molecule, and / or to the target molecule, which is composed of the building block specified prior to "-based". For example, amino acid-based affinity molecule / s and / or target molecule / s, are affinity molecule / s and / or target molecule / s, which are composed of amino acids, such as proteins, peptides, glycoproteins, lipoproteins, etc. Still further, nucleic acid- based affinity molecules and / or target molecule / s, are affinity molecule / s and / or target molecule / s, which are composed of nucleic acids such as polynucleotides, aptamers, etc. Small molecule-based affinity molecule / s and / or target molecule / s are affinity molecule / s and / or target molecule / s, which are composed of small molecules, which are low molecular weight organic compound, having a molecular weight lower than 900 Daltons. Carbohydrate-based affinity molecules are affinity molecules which are composed of carbohydrates such as monosaccharides, disaccharides, oligosaccharides, and polysaccharide as well as proteoglycans, glycoproteins, etc. lipid-based molecules are affinity molecule / s and / or target molecule / s, which are composed of lipids such as fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, proteolipids, and others.

[0086] As indicated above, in some embodiments the at least one affinity moiety and / or the at least one target molecule of the disclosed bio transistor systems and methods, may be, or may comprise a nucleic acid molecule. The term “nucleic acid”, “nucleic acid sequence”, or "polynucleotide" and “nucleic acid molecule” refers to polymers of nucleotides, and includes but is not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA / RNA hybrids including polynucleotide chains of regularly and / or irregularly alternating deoxyribosyl moieties and ribosyl moieties (i.e., wherein alternate nucleotide units have an —OH, then and — H, then an —OH, then an — H, and so on at the 2' position of a sugar moiety), and modifications of these kinds of polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included. The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. Preparation of nucleic acids is well known in the art. It should be appreciated that the invention may further refer to polyribonucleotide. The term "polyribonucleotide" refers to a polynucleotide comprising two or more modified or unmodified ribonucleotides and / or their analogs. The term "polyribonucleotide" is used interchangeably with the term "oligoribonucleotide”.

[0087] As indicated above, in some embodiments the at least one affinity moiety and / or the at least one target molecule of the disclosed bio transistor systems and methods, may be, or may comprise a small molecule. A small molecule in the context of the present disclosure refers to a low molecular weight organic compound, having a molecular weight lower than 900 Daltons, and in some embodiments less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, the small molecule is less than about 900 daltons (Da), 800 Da 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a saccharide. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and / or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups. Small molecules often comprise one or more cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups.

[0088] In accordance with the present disclosure, when referring to a small molecule it includes also crystalline and amorphous forms of those compounds, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as mixtures thereof. "Crystalline form" or "polymorph," as used herein include all crystalline and amorphous forms of a small molecule, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms, as well as mixtures thereof, unless a particular crystalline or amorphous form is referred to.

[0089] In accordance with the present disclosure, the term "small molecule" may include pharmaceutically acceptable forms of the recited compounds, including chelates, non- covalent complexes, prodrugs, and mixtures thereof. Further and in accordance with the preset disclosure, the term "small molecule" includes also pharmaceutically acceptable forms of a particular molecule and as such the term small molecule also encompasses pharmaceutically acceptable salts.

[0090] As indicated above, in some embodiments the at least one affinity moiety and / or the at least one target molecule of the disclosed bio transistor systems and methods, may be, or may comprise an amino acid-based molecule. An "amino acid-based molecule", as used herein refers to a molecule that is primarily composed of amino acids or derivatives of amino acids. Amino acids are organic compounds that contain both an amino group (-NH2) and a carboxyl group (-COOH), along with a unique side chain specific to each amino acid. Amino-acid based molecules in accordance with the present disclosure encompass various structures including peptides, that are short chains of amino acids linked by peptide bonds (typically consist of 2 to 50 amino acids), polypeptides, that are intermediate-length chains of amino acids that can fold into specific structures and may function independently or as part of a larger protein complex and proteins, that are composed of long chains of amino acids, typically consisting of 50 or more amino acids, and fold into specific three- dimensional structures that determine their function in biological processes. An example for an affinity moiety-target pair composed of a protein include, but is not limited to the antibody-antigen, receptor-ligand and peptide aptamers and targets thereof. Still further, it should be understood that amino-acid-based molecule as used in the present disclosure further encompasses amino acid derivatives, specifically, molecules derived from amino acids through chemical modifications.

[0091] In yet some further embodiments, the at least one affinity moiety and / or the at least one target molecule of the disclosed bio transistor systems and methods, may be, or may comprise a carbohydrate-based molecule. A carbohydrate-based molecule, as used herein, is a chemical compound that primarily consists of carbohydrate components, which are organic molecules made up of carbon (C), hydrogen (H), and oxygen (O) atoms, usually with the hydrogen and oxygen atoms in a ratio of 2: 1, as in water. Carbohydrates are one of the four main classes of biomolecules and can be simple sugars (monosaccharides like glucose and fructose), double sugars (disaccharides like sucrose and lactose), or complex carbohydrates (polysaccharides like starch, cellulose, and glycogen).

[0092] Still further, in some embodiments, the at least one affinity moiety and / or the at least one target molecule of the disclosed bio transistor systems and methods, may be, or may comprise a lipid-based molecule.

[0093] A lipid-based molecule, as used herein, is a chemical compound primarily composed of lipids, which are a diverse group of hydrophobic or amphipathic organic molecules. Lipids are insoluble in water but soluble in nonpolar solvents. It should be understood that the present disclosure encompasses any affinity moiety and / or target molecule composed of or comprising a lipid-based molecule, of any type, for example, triglycerides, that consist of three fatty acids attached to a glycerol backbone, phospholipids, that have two fatty acids and a phosphate group attached to glycerol, steroids, that have a structure based on a carbon skeleton with four fused rings (e.g., Cholesterol), glycolipids, that contain a carbohydrate group attached to a lipid, and fatty Acids, that are carboxylic acids with a long hydrocarbon chain, which can be saturated (no double bonds) or unsaturated (one or more double bonds).

[0094] In some further embodiments, the target molecule comprises, or is derived from a component of an affinity pair. The term "target molecule" as used herein refers to at least one specific molecule in a sample for which determining its presence and / or quantity is desired. This target molecule will be the corresponding partner of the affinity moiety. A target molecule as used herein may be either a single molecule or a combination or a complex of two or more molecules or subunits forming a specific recognition partner recognized and bound by the specific affinity moiety of the disclosed bio-transistor systems.

[0095] The affinity pair comprises at least one of: proteineous factor-specific nucleic acid binding site, aptamer-aptamer target, antigen-antibody, enzyme-substrate, receptor-ligand, or any combination thereof.

[0096] In some embodiments, the affinity moiety comprises at least one nucleic acid molecule. Such nucleic acid molecule forms a specific recognition and binding site, for at least one target molecule.

[0097] In some embodiments, various molecules may interact and bind nucleic acids and thus may be used in the present disclosure as the target molecule.

[0098] In some embodiments, the target molecule may be at least one nucleic acid-binding protein. In yet some further embodiments, the target molecules may be a small molecule, that can be natural or synthetic molecules that can bind to DNA and alter its function. Some examples of small molecules that bind to DNA may include antibiotics, that bind DNA and prevent microorganisms (e.g., bacteria) from replicating, chemotherapeutic drugs that may damage DNA or interfering with DNA replication.

[0099] In yet some further embodiments, the target molecule may comprise an amino acid-based molecule, specifically, a protein that may bind nucleic acid molecule, or specific binding site or sequence within a nucleic acid molecule such as DNA. To name but few, such protein-based target molecules may be any proteineous molecule that display a catalytic activity or a regulative activity upon binding thereof to the specific binding site. For example, transcription factors, that are proteins that regulate gene expression by binding to specific DNA sequences and either activating or repressing the transcription of genes. Histones, that package DNA into chromosomes in eukaryotic cells. Polymerases, Nucleases, DNA methyl transferases, that modify the structure and / or function of the nucleic acid molecule, for example, by extending (polymerase), tethering (ligase), cleaving (nuclease), adding methyl groups to DNA, and / or DNA repair enzymes.

[0100] Thus, in some embodiments, an affinity pair, for the affinity moiety and target molecule, may be a proteineous factor-specific nucleic acid binding site binding-pair. More specifically, a proteineous factor-specific nucleic acid binding site refers to a specific sequence or structure within a nucleic acid molecule (DNA or RNA) that is recognized and bound by a particular protein factor, that may be a protein, often a regulatory protein such as a transcription factor, RNA-binding protein, or enzyme, that interacts with nucleic acids. Non-limiting examples for such proteins, are proteins involved in various cellular processes, including gene expression, replication, and RNA processing. The nucleic acid binding site, that may be used in the present disclosure as the affinity moiety, is a specific region on a nucleic acid (DNA or RNA) where the protein binds. The binding site is typically a specific sequence of nucleotides or a particular structural motif that the protein recognizes and interacts with.

[0101] As indicated above, the affinity pair of the present disclosure is based on DNA binding sites. More specifically, these are characterized by specific sequences of nucleotides recognized by DNA-binding proteins through complementary interactions. The binding sites may in some embodiments comprise palindromic sequences (common in binding sites for proteins that function as dimers). DNA-binding proteins often recognize specific motifs within the binding site, that may include consensus sequences that are critical for the binding specificity and affinity of the protein. Common recognition motifs in proteins encompassed by the present disclosure include helix-turn-helix, zinc fingers, and leucine zippers, which fit into the DNA's major groove. Transcription factor binding site for example, are typically composed of a short sequence of 6-10 nucleotides, such as the TATA box (TATAAA) found in many eukaryotic promoters. The TATA-binding protein (TBP) recognizes and binds to this sequence. Additional embodiments for protein binding sites within a nucleic acid molecule include operator site, for example, the lac repressor that binds the lac operator sequence (01 ) in the presence of lactose.

[0102] In yet some further alternative embodiments, an affinity pair, for the affinity moiety and target molecule, may be an aptamer-aptamer target binding pair. More specifically, an aptamer- aptamer target binding pair, as used herein, refers to the specific and high-affinity interaction between an aptamer and its corresponding target molecule. Aptamers according to some embodiments, are short, single-stranded DNA or RNA molecules that can fold into unique three-dimensional structures, allowing them to bind selectively to a wide range of targets, including proteins, small molecules, ions, and even whole cells. Thus, in some embodiments, the affinity moiety of the disclosed bio transistor systems is, composed of, or comprising, at least one aptamer, for example, a nucleic acid-based aptamer.

[0103] "Aptamers" are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities (KD in the pM to μM range), and are sometimes classified as "chemical antibodies" or "antibody mimics". The nucleic acid-based structure of aptamers, which are mostly oligonucleotides, is very different from the amino acid-based structure of antibodies, which are proteins. Aptamers are usually obtained by selection from a large random sequence library, using methods well known in the art, such as SELEX and / or Molinex. In SELEX, the best aptamers from a starting DNA library made of about a quadrillion different randomly generated pieces of DNA or RNA are repeatedly selects. After SELEX, the chemistry of the aptamers might be mutated or changed and another selection can be done, or a rational design processes might be used to engineer improvements.

[0104] Aptamers are optimized to achieve a variety of beneficial features. The most important feature is specific and sensitive binding to the chosen target. Some aptamers are engineered to fit into a biosensor or in a test of a biological sample. In various embodiments, aptamers may include single-stranded, partially single-stranded, partially double-stranded or double- stranded nucleic acid sequences; sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides and nucleotides comprising backbone modifications, branch points and non-nucleotide residues, groups or bridges; synthetic RNA, DNA and chimeric nucleotides, hybrids, duplexes, heteroduplexes; and any ribonucleotide, deoxyribonucleotide or chimeric counterpart thereof and / or corresponding complementary sequence. In certain specific embodiments, aptamers used by the invention are composed of deoxyribonucleotides.

[0105] In some embodiments, the aptamer that may be applicable herein may optionally comprise a spacer between the nucleic acid sequence and the reactive group. The spacer may be an alkyl chain such as (CH2)6 / 12, namely comprising six to twelve carbon atoms.

[0106] Aptamer targets can include small molecules and heavy metal ions, larger ligands such as proteins, and even whole cells. Accordingly, the target molecule recognized by such affinity moiety may be a protein, small molecule, ion, and even a whole cell. It should be further noted that aptamer-target binding is mediated by non-covalent interactions, including hydrogen bonds, electrostatic interactions, Van der Waals forces, and sometimes hydrophobic interactions.

[0107] It should be understood that although specifically directed at affinity moiety that comprises at least one nucleic acid molecule (e.g., binding site, aptamer and the like), the present disclosure further encompasses the use of any of the disclosed affinity pairs, for example, an affinity pair may be an antibody-antigen pair. An "antibody Ab)'' , also known as an "immunoglobulin (7g)", is a large, Y-shaped glycoprotein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule called an "antigen". Each tip of the "Y" of an antibody contains a paratope (analogous to a lock) that is specific for one particular epitope (analogous to a key) on an antigen, allowing these two structures to bind together with precision. The term "antibody" as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Exemplary categories of antigen-binding domains that can be used in the context of the present invention include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds a particular antigen or antigen-binding scaffolds. The antigen binding domains in accordance with the invention may recognize and bind a specific antigen or epitope. It should be therefore noted that the term “binding specificity”, ’’specifically binds to an antigen”, “specifically immuno-reactive with”, “specifically directed against” or “specifically recognizes”, when referring to an antigen or particular epitope, refers to a binding reaction which is determinative of the presence of the epitope in a heterogeneous population of proteins and other biologies.

[0108] The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. Still further, as indicated above, an "antigen-binding domain" can comprise or consist of an antibody or antigen-binding fragment of an antibody.

[0109] Still further, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein. An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0110] In some embodiments, the affinity moiety and the specific target molecule may form an affinity pair composed of an enzyme- substrate. More specifically, "affinity pair" highlights the complementary nature of the enzyme and substrate, where the enzyme's active site is specifically shaped to recognize and bind its substrate, resulting in a high-affinity interaction and high specificity. This interaction is central to the enzyme's catalytic function and involves specificity, that is meant herein that each enzyme binds to a particular substrate or a group of closely related substrates, affinity, that relates to the strength of the binding between an enzyme and its substrate is referred to as affinity. High-affinity binding ensures that the substrate is efficiently captured by the enzyme. This binding is often mediated by non-co valent interactions such as hydrogen bonds, ionic interactions, and Van der Waals forces.

[0111] Enzymes are usually much larger than their substrates. Sizes range from just 62 amino acid residues, for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal fatty acid synthase. Only a small portion of their structure (around 2-4 amino acids) is directly involved in catalysis: the catalytic site. This catalytic site is located next to one or more binding sites where residues orient the substrates. The catalytic site and binding site together compose the enzyme's active site. The remaining majority of the enzyme structure serves to maintain the precise orientation and dynamics of the active site. Enzyme structures may also contain allosteric sites where the binding of a small molecule causes a conformational change that increases or decreases activity. Enzymes must bind their substrates before they can catalyze any chemical reaction. Specificity is achieved by binding pockets with complementary shape, charge and hydrophilic / hydrophobic characteristics to the substrates. Enzymes can therefore distinguish between very similar substrate molecules to be chemoselective, regioselective and stereospecific. The enzyme component of the enzyme-substrate affinity pair can be a whole enzyme, and / or any fragments thereof, such as the enzyme's active site or the enzyme's binding site as long as it's binding capability is maintained.

[0112] Thus, in some embodiments, the formation of the enzyme-substrate complex, is referred to herein as binding of the target molecule to the affinity moiety that is detected and / or quantified by the bio transistor systems and methods of the present disclosure. Formation of the enzyme-substrate complex is followed by catalysis and release of the products from the enzyme's active site.

[0113] In yet some further alternative embodiments, an affinity pair, for the affinity moiety and target molecule, may be a receptor-ligand binding pair, specifically, where the affinity moiety is or comprises a receptor where the target molecule is the ligand or vice versa. More specifically, a receptor-ligand affinity pair refers to the specific and often high- affinity interaction between a receptor (a protein molecule usually located on the cell surface or within a cell) and a ligand (a molecule that binds to the receptor). Specificity of this binding as used herein is meant that each receptor typically binds to a particular ligand or a group of structurally similar ligands, thereby forming the receptor-ligand complex, that is detected and quantified by the disclosed bio transistor systems and methods, and often triggers a conformational change in the receptor, initiating a cascade of intracellular events. Example that may be useful in the present disclosure may include hormone receptors, neurotransmitter receptors, and the like. "Receptors" are chemical structures, composed of protein, that receive and transduce signals that may be integrated into biological systems. These signals are typically chemical messengers which bind to a receptor and produce physiological responses such as change in the electrical activity of a cell. Receptor proteins can be classified by their location. Cell surface receptors also known as transmembrane receptors, include ligand-gated ion channels, G protein-coupled receptors, and enzyme-linked hormone receptors. Intracellular receptors are those found inside the cell and include cytoplasmic receptors and nuclear receptors. A molecule that binds to a receptor is called a "ligand" and can be a protein, peptide , or another small molecule.

[0114] In some embodiments, the affinity moiety comprises at least one nucleic acid sequence comprising at least one binding site and the target molecule comprises at least one proteineous factor that specifically recognizes and binds the nucleic acid binding site. As indicated above, the target recognition moiety, that is also referred to herein as an affinity moiety, comprise in some embodiments binding site that is specifically recognized by, and bound by the target proteineous factor. "Specifically binds" as used herein refers to an affinity moiety which interacts with a specific target molecule, while avoiding interactions with other molecules. Examples of moieties with specific binding capabilities include for example, nucleic acid binding sites, aptamers, as well as antibodies, enzymes and receptors. The target recognition component, specifically, the nucleic acid sequence (or molecule) specifically binds the target molecule in any stoichiometric ratio. In some embodiments, one target recognition component binds at least one, at least two, at least three, at least four, at least five, at least ten, at least hundred, at least thousand and even more, target molecules. In some embodiments, the target-recognition component, specifically, binding site, comprises more than one moiety, wherein the plurality of moieties may be the same and / or may be different (e.g. different binding sites, etc).

[0115] In some further embodiments, the proteineous factor that is the target molecule that is a proteineous involved in and / or catalyzes at least one of: nucleic acid synthesis, replication, transcription, translation, correction, repair and / or editing. In some embodiments, the target molecule is a factor that is involved in nucleic acid replication.

[0116] Nucleic acid replication is a fundamental biological process essential for the duplication of genetic material in living organisms. This process ensures that each daughter cell receives an identical copy of the parent cell's DNA during cell division. Nucleic acid replication is the biological process by which a cell duplicates its DNA or RNA, producing two identical copies from one original molecule. This process is essential for cellular division, growth, and reproduction, ensuring that each daughter cell inherits an accurate copy of the genetic material. Replication relies on a pre-existing nucleic acid strand (the template) to direct the synthesis of a new complementary strand. The sequence of the template strand dictates the sequence of the new strand via base pairing: adenine (A) pairs with thymine (T) in DNA or uracil (U) in RNA, and cytosine (C) pairs with guanine (G). Key enzymes and proteins that participate DNA replication include DNA Polymerases, that add nucleotides to the growing DNA strand in a 5' to 3' direction, Helicase, that unwinds the DNA double helix at the replication fork, Primase, that synthesizes RNA primers to initiate DNA synthesis, Ligase, that seals nicks in the DNA backbone, joining Okazaki fragments on the lagging strand and Topoisomerase, that relieves the torsional strain generated ahead of the replication fork.

[0117] Still further, as noted above, the disclosed target molecule is a protein involved in DNA replication. DNA replication is a multi-step process that includes initiation, that begins at origins of replication and is governed by initiator proteins that recognize the origins, leading to the formation of the replication complex and unwinding of the DNA. The next step involves DNA elongation, where the DNA polymerases add nucleotides to the RNA primers, extending the new DNA strand. Synthesis occurs continuously on the leading strand and discontinuously on the lagging strand, forming Okazaki fragments. The final step, termination when the entire template has been copied, and the replication machinery disassembles

[0118] It should be understood that replication of nucleic acids in accordance with the present disclosure further encompasses RNA replication, and the associated proteins. Thus, in some embodiments, the affinity moiety used by the present disclosure may be an RNA molecule, and the target molecule may be any protein that participates in RNA replication, for example, RNA-dependent RNA polymerase, that synthesizes RNA from an RNA template (e.g., in some viruses and RNA-based systems).

[0119] In some specific embodiments, the proteineous factor is at least one polymerase.

[0120] More specifically, as used herein, a polymerase is an enzyme that catalyzes the polymerization of nucleotides into nucleic acids, forming long chains of DNA or RNA. These enzymes are crucial for the processes of DNA replication, RNA transcription, and various DNA repair mechanisms, ensuring the accurate synthesis and maintenance of genetic material. Polymerases include DNA Polymerases, that use deoxyribonucleotides for synthesizing DNA, RNA Polymerases that synthesize RNA from ribonucleotides, and reverse Transcriptases that synthesize DNA from an RNA template. In some embodiments, DNA polymerases applicable as the at least one target molecule in accordance with the present disclosure, may include the prokaryotic DNA polymerase I, II, and III and the Eukaryotic Polymerases a, δ, and ε. Still further, RNA polymerase that are encompassed by the present disclosure as the target molecule may include Polymerase I (that Synthesizes ribosomal RNA (rRNA), RNA Polymerase II, that synthesizes messenger RNA (mRNA) and RNA Polymerase III, that synthesizes transfer RNA (tRNA) and other small RNAs. In some further specific embodiments, the polymerase is RNA polymerase. In yet some further embodiments, the RNA polymerase is a primase. More specifically, Primase, as used herein, is an RNA polymerase enzyme that synthesizes short primer RNA sequences that serve as starting points for DNA synthesis, providing a free 3'-OH group for DNA polymerases to extend. Primase is essential for initiating the replication of both the leading and lagging strands during DNA replication.

[0121] In some embodiments of the present disclosure, the proteineous factor is at least one primase and the affinity moiety comprises nucleic acid sequence comprising at least one primase biding site. Primase is an RNA polymerase enzyme that synthesizes short RNA sequences called primers. These primers serve as starting points for DNA synthesis, providing a free 3'-OH group for DNA polymerases to extend. Primase is essential for initiating the replication of both the leading and lagging strands during DNA replication. Primase synthesizes short RNA primers, typically 10-15 nucleotides long, which are complementary to the DNA template strand. In prokaryotes, primase is part of the primosome complex. In eukaryotes, primase is associated with DNA polymerase a in the DNA polymerase a-primase complex. Multiple RNA primers are synthesized by primase along the lagging strand template to initiate the synthesis of Okazaki fragments. Each fragment requires a new primer for DNA polymerase to extend. Primase specifically synthesizes RNA primers during DNA replication, distinct from other RNA polymerases that synthesize longer RNA molecules. Primase is crucial for the initiation of DNA synthesis, as DNA polymerases cannot begin synthesis de novo and require a primer with a free 3'-OH group.

[0122] Proper functioning of primase is essential for the accurate and efficient replication of the genome, ensuring genetic information is faithfully transmitted during cell division.

[0123] DNA polymerases cannot initiate synthesis of DNA de novo, requiring a 3 '-hydroxyl terminated primer positioned at the catalytic site. During DNA replication, primers are synthesized by a class of enzymes designated DNA primases. DN A primases catalyze the synthesis of short oligoribonucleotides that can then be used as primers by DNA polymerase. DNA primases play roles in the loading of DNA helicase, temporal regulation of replication, and handoff of the primer to the DNA polymerase.

[0124] Based on their sequences and structures, DNA primases can be grouped into two families, the prokaryotic and the eukaryotic / archaeal primases. All are encompassed by the present disclosure. Prokaryotic primases contain six conserved sequence motifs. An N-terminal zinc-binding domain (ZBD) formed by motif I is a determinant for recognition of a specific sequence in DNA. Motifs II— VI are located in the C-terminal RNA polymerase domain (RPD) that consists of two subdomains. The C-terminal topoi somerase-primase (TOPRIM) fold contains the active site where the metal-mediated condensation of nucleotides occurs. The N-terminal subdomain of the RPD is less conserved, and its role remains elusive. In some embodiments, the primase used herein is a T7 primase. Bacteriophage T7 DNA helicase / primase (also known as Gp4), an ATP-dependent DNA helicase and primase that is essential for viral DNA replication and recombination. Primase activity synthesizes short RNA primers at the sequence 5'-GTC-3' on the lagging strand that the polymerase elongates using dNTPs and providing the primase is still present. The characteristics of the protein distribution suggest prophage matches in addition to the phage matches. A zinc binding domain found in the N-terminal region of the bacteriophage T7 Gp4 and P4 alpha protein. P4 is a multifunctional protein with origin recognition, helicase and primase activities. DNA primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. In some embodiments, T7 primase as used herein comprises the amino acid sequence as denoted by SEQ ID NO: 2, or any homologs or derivatives thereof.

[0125] In some embodiments the affinity moiety that specifically binds the T7 primase used herein as the target molecule is a specific T7 primase recognition sequence comprising 5’-GTC- 3’ at the sensing area. In yet some further embodiments, the recognition sequence, specifically the affinity moiety used herein may comprise two or more repeats of the 5’- GTC-3’ sequnce. In yet some specific and non-limiting embodiments, the affinity moiety may comprise the nucleic acid sequence as denoted by SEQ ID NO: 1.

[0126] In other embodiments, the proteineous factor is at least one transcription factor.

[0127] The term transcription factor as used herein, refers to a protein that binds to specific DNA sequences to regulate the transcription of genetic information from DNA to messenger RNA (mRNA). These proteins play a crucial role in gene expression by either promoting or inhibiting the transcription process. Transcription factors function by recognizing and binding to promoter or enhancer regions of target genes, recruiting or blocking the RNA polymerase enzyme, and interacting with other proteins to modulate the transcriptional activity.

[0128] In some embodiments of the present disclosure, the at least one sample is a biological sample, an environmental sample and / or a screening test sample.

[0129] A "sample" as used herein may be any biological and / or environmental sample. A "biological sample" is a biological material collected from living and / or deceased organisms and / or living and / or dried out plants or their environment. There are many different types of biological samples, including biofluids, tissue, cells and other. Biological samples can be obtained from the body via several different methods such as excretion (e.g. urine), secretion (e.g. breast milk) or extraction (e.g. blood). Non limiting examples of biological samples include blood, bile, bone marrow aspirate, breast milk / mammary gland milk, Cerebral Spinal Fluid (CSF), feces, plasma, saliva, semen, serum, sputum, sweat, as well as oral, nasal and vaginal fluids (typically collected using a swab), and synovial fluid, tears and urine. Biological samples include also cells such as epithelial cells, fibroblasts, immune cells (e.g. T cell, B cells, NK cells etc.), peripheral blood mononuclear cells (PBMCs), red blood cells (RBCs), buffy coat, bone marrow mononuclear cells, dissociated tumor cells, mesenchymal stem cells, myoblasts, hepatocytes, etc as well as tissues. Cell samples are collected and isolated from either tissue samples, biofluid samples, or biopsy samples. Depending on the cell type, specific cell isolation protocols are required in order to obtain the purified cell sample.

[0130] Biological material collected from plants may be for example any sample derived from the leaf, roots, stems, nectar, seeds and / or fruits of any plant.

[0131] "Environmental sample" refers to any sample derived from any media or material in the environment. The most common environmental samples are air, water, soil, biological materials, and wastes (liquids, solids or sludges) such as sewage. Environmental sampling is typically performed to determine the presence of Hazardous Materials in any media or material, including indoor or outdoor air, soil, groundwater, surface water or building materials. Still further, in some embodiments, a sample may comprise any food product or any byproduct derived from any industrial activity or facility.

[0132] Samples also includes material that is extracted and / or prepared from any of the above biological and / or environmental samples using standard methods such as RNA, DNA, protein lysates, cell-free DNA (cfDNA), etc.

[0133] As indicated herein, in some embodiments, the disclosed bio transistor system may be useful in screening for compounds that may modulate the interaction between the two components of the binding pair, specifically, any molecule that decreases or alternatively decreases the interaction between the affinity moiety and the target molecule. This aspect will be elaborated in the screening method described herein after. Accordingly, the sample may further comprise a candidate compound that is evaluated for its ability to change the interaction (e.g., binding) of the affinity moiety (e.g., a nucleic acid molecule), and its specific target (e.g., protein factor such as the primase). Such candidate molecule may be any molecule. Thus, in some further embodiments, the at least one sample further comprise at least one candidate compound that modulates the interaction between the affinity moiety and the target molecule. The candidate compound comprises at least one of: an amino acid- based molecule, a nucleic acid-based molecule, a small molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof. It should be noted that these molecules are as described herein in the present disclosure.

[0134] In some embodiments, the affinity moiety comprises a nucleic acid sequence comprising at least one primase binding site, the target molecule is a primase, and the sample is a screening test sample comprising the primase, optionally, a predetermined amount of primase, and a candidate compound that potentially inhibits binding of primase to the primase binding site.

[0135] In some further embodiments, the target molecule is associated with at least one pathologic disorder in a subject.

[0136] In some additional embodiments, the pathologic disorder is at least one of: a disorder caused by a pathogenic agent, a proliferative disorder, an inflammatory disorder, a metabolic disorder, a neurodegenerative disorder and an autoimmune disorder.

[0137] In some further embodiments, the pathogenic agent is at least one of bacteria, archaea, virus, fungi, algae, parasite, protists, and worms.

[0138] In some specific embodiments, the pathogenic agent is bacteria, and the target molecule is a bacteriophage T7 primase.

[0139] In some embodiments, the affinity moiety is an aptamer specific for a target molecule associated with at least one pathologic disorder in a subject and / or an environmental contamination.

[0140] Another aspect of the present disclosure relates to a battery comprising two or more of the bio-transistor system as defined above.

[0141] Another aspect of the present disclosure relates to a method for determining presence and / or quantity of at least one target molecule in at least one sample. The method comprising: (a) contacting the at least one sample with a bio-transistor having an active region, e.g., a modified active region, carrying at least one affinity moiety, or a battery comprising at least two of the bio-transistors. It should be noted that each of the affinity moiety is specific for one target molecule. Next in step (b), performing one or more measurements, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; and (c) processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample and determining presence and / or quantity of the one or more target molecules in accordance with pre-stored calibration data.

[0142] In some embodiments of the present disclosure, the bio-transistor used by the method comprising: (i) at least one channel; (ii) source and drain electrodes; (iii) at least one gate electrode; and (iv) at least one additional electrode positioned to be in electrical contact with the sample. The at least one active region is located in proximity to the channel region, separated from the channel region by an electrically insulating layer. The control unit thereby configured to determine data on presence and / or quantity of one or more target molecules in the sample.

[0143] In some further embodiments, the method comprising performing two or more measurements, wherein each measurement comprises applying respective selected different potential on the sample.

[0144] In yet some further embodiments, the biosensor used in the disclosed methods is the biosensor as defined in the present disclosure. Thus, in some embodiments, the present disclosure provides a diagnostic method for detecting any proteineous target molecule, using a nucleic acid-based affinity moiety, for example, a nucleic acid binding site, or alternatively, a nucleic acid aptamer that is specific for any proteineous target molecule, or any small molecule or any of the target molecules disclosed by the present disclosure.

[0145] Another aspect of the present disclosure relates to a screening method for identifying a compound that modulates the interaction of an affinity moiety with a target molecule in at least one sample. The method comprising: (a) contacting the at least one sample with a bio- transistor system, in the presence and / or in the absence of at least one candidate compound. The bio-transistor having an active region (e.g., a modified) carrying at least one affinity moiety. It should be noted that in some embodiments, the one or more affinity moieties are selected moieties. In yet some further embodiments, the bio transistor or battery used by the disclosed methods may comprise one or more different or similar or identical affinity moieties. It should be further noted that each one of the at least one affinity moiety is specific for one target molecule. In step (b), performing one or more measurements for each sample, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; and (c) processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample; (d) determining presence and / or quantity of the one or more target molecules in accordance with pre-stored calibration data, thereby determining a target molecule value for the sample in the presence of the candidate compound, and a target molecule value in the absence of the candidate compound; and (e) determining that the candidate compound is a modulator of the interaction between the affinity moiety and the target molecule, if the target molecule value obtained for the sample in the presence of the candidate compound is different from the target molecule value obtained in the absence of the candidate compound.

[0146] In some embodiments of the present disclosure, the bio-transistor used by the method comprising: (i) at least one channel; (ii) source and drain electrodes; (iii) at least one gate electrode; and (iv) at least one additional electrode positioned to be in electrical contact with the sample. The at least one active region is located in proximity to the channel region, separated from the channel region by an electrically insulating layer. The control unit thereby configured to determine data on presence and / or quantity of one or more target molecules in the sample. In some embodiments, the biosensor used in the disclosed methods is the biosensor as defined in the present disclosure.

[0147] In some further embodiments, performing one or more measurements comprises performing two or more measurements, wherein each measurement comprises applying respective selected different potential on the sample.

[0148] In some further embodiments, the affinity moiety of the bio sensor system used by the screening method of the present disclosure comprises at least one of: a nucleic acid-based molecule, an amino acid-based molecule, a small molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof, and the affinity moiety specifically binds (directly or indirectly) the at least one target molecule in the sample.

[0149] In some embodiments, the affinity moiety of the bio sensor system used by the screening method of the present disclosure comprises or is derived from a component of an affinity pair. The affinity pair is at least one of: proteineous factor-specific nucleic acid binding site, aptamer-aptamer target, antibody-antigen, enzyme-substrate, receptor-ligand, or any combination thereof. In some embodiments of the present disclosure, the disclosed screening method is particularly applicable for screening of a compound that inhibits the interaction of the affinity moiety with the target molecule. More specifically, a compound that "inhibits the interaction" as used herein, refers to a compound that reduces, prevents, blocks, impedes, suppresses, prevents, hinders and / or restricts interaction between the at least one affinity moiety and the target molecule either partially, or completely. For example, any inhibition of between about 10-50%, 51-80%, 81-99% or even, of 100% of the interaction or binding between the affinity moiety and the target molecule.

[0150] In some embodiments of the present application, the target molecule of the affinity moiety of the bio sensor system used by the screening method of the present disclosure comprises at least one of: an amino acid-based molecule, a small molecule, a nucleic acid-based molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof.

[0151] In some further embodiments, the target molecule comprises, or is derived from a component of an affinity pair, said affinity pair comprises at least one of: proteineous factor-specific nucleic acid binding site, aptamer-aptamer target, antibody-antigen, enzyme-substrate, receptor-ligand, or any combination thereof.

[0152] In some embodiments of the present application, the candidate compound comprises at least one of: a small molecule, an amino acid-based molecule, a nucleic acid-based molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof.

[0153] In some further embodiments, the affinity moiety comprises at least one nucleic acid sequence comprising at least one binding site and the target molecule comprises at least one proteineous factor that specifically recognizes and binds the nucleic acid binding site. In some embodiments, the proteineous factor is involved in and / or catalyzes at least one of: nucleic acid synthesis, replication, transcription, translation, correction and / or editing. In some further embodiments, the proteineous factor is involved in nucleic acid replication. In other specific embodiments, the proteineous factor is at least one polymerase.

[0154] It should be understood that the polymerase may be RNA polymerase.

[0155] In some further specific embodiments, the proteineous factor is at least one primase and the affinity moiety comprises at least one primase biding site.

[0156] Still further, in some embodiments, the affinity moiety of the bio sensor system used by the screening method of the present disclosure is a specific T7 primase recognition sequence comprising 5’-GTC-3’ at the sensing area. In yet some further embodiments, the T7 primase recognition sequence used as the at least one affinity moiety in the bio transistor systems and methods of the present disclosure may comprise two or more repeats of the 5’-GTC-3’ motif. In yet some further embodiments, such affinity moiety may comprise the nucleic acid sequence as denoted by SEQ ID NO: 1.

[0157] In some other embodiments, the proteineous factor is at least one transcription factor.

[0158] In some embodiments of the present application, the at least one sample is a biological sample, an environmental sample and / or a screening test sample.

[0159] In some further embodiments, the biological sample is derived from a eukaryotic organism. In some embodiments, the methods and systems of the present disclosure may be applicable for any organism of the biological kingdom Animalia. In more specific embodiments, such organism may be any unicellular or multicellular invertebrate or vertebrate organism. More specifically, invertebrates, may be organisms of the Phylum Porifera - Sponges, the Phylum Cnidaria - Jellyfish, hydras, sea anemones, corals, the Phylum Ctenophora - Comb jellies, the Phylum Platyhelminthes - Flatworms, the Phylum Mollusca - Molluscs, the Phylum Arthropoda - Arthropods, the Phylum Annelida - Segmented worms like earthworm and the Phylum Echinodermata - Echinoderms. Still further, in some embodiments, the methods of the present disclosure may be applicable for any vertebrate organism, specifically, any organism derived from any of the vertebrates groups that include Fish, Amphibians, Reptiles, Birds and Mammals (e.g., Marsupials, Primates, Rodents and Cetaceans). In some particular embodiments, the methods of the present disclosure may be applicable for a mammal (specifically, at least one of a human, Cattle, rodent, domestic pig (swine, hog), sheep, horse, goat, alpaca, lama and Camels).

[0160] More specifically, in some embodiments, as indicated herein, the methods of the present disclosure may be applicable for a vertebrate organism. Vertebrates comprise all species of animals within the subphylum Vertebrata (chordates with backbones). The animals of the vertebrates group include Fish, Amphibians, Reptiles, Birds and Mammals (e.g., Marsupials, Primates, Rodents and Cetaceans).

[0161] Vertebrates represent the overwhelming majority of the phylum Chordata, with currently about 66,000 species described. Vertebrates include the jawless fish and the jawed vertebrates, which include the cartilaginous fish (sharks, rays, and ratfish) and the bony fish.

[0162] Still further, in some embodiments, the subject of the of the preset disclosure may be any one of a human or non-human mammal, an avian, an insect, a fish, an amphibian, a reptile, a crustacean, a crab, a lobster, a snail, a clam, an octopus, a starfish, a sea-urchin, jellyfish, and worms.

[0163] In more specific embodiments, the subject of the present disclosure may be a mammal. In yet some further embodiments, such mammalian organisms may include any member of the mammalian nineteen orders, specifically, Order Artiodactyla (even-toed hoofed animals), Order Carnivora (meat-eaters), Order Cetacea (whales and purpoises), Order Chiroptera (bats), Order Dermoptera (colugos or flying lemurs), Order Edentata (toothless mammals), Order Hyracoidae (hyraxes, dassies), Order Insectivora (insect- eaters), Order Lagomorpha (pikas, hares, and rabbits), Order Marsupialia (pouched animals), Order Monotremata (egg-laying mammals), Order Perissodactyla (odd-toed hoofed animals), Order Pholidata, Order Pinnipedia (seals and walruses), Order Primates (primates), Order Proboscidea (elephants), Order Rodentia (gnawing mammals), Order Sirenia (dugongs and manatees), Order Tubulidentata (aardvarks).

[0164] In yet some further embodiments, the present disclosure may be applicable for any organism of the order primates. More specifically, primates are divided into two distinct suborders, the first is the strepsirrhines that includes lemurs, galagos, and lorisids. The second is haplorhines - that includes tarsier, monkey, and ape clades, the last of these including humans. In yet some further embodiments, the present disclosure may be applicable for any organism of the subfamily Homininae, that includes the hylobatidae (gibbons) and the hominidae that includes ponqunae (orangutans) and homininae [gorillini (gorilla) and hominini ((panina(chimpanzees) and hominina (humans))]. Thus, in some embodiments, a subject as disclosed herein relates to a human subject. In some embodiments, the human subject may be of any sex, ethnic group, age or physical or mental condition.

[0165] In some specific embodiment, the methods of the present disclosure may be applicable for a mammal that may be at least one of a Cattle, domestic pig (swine, hog), sheep, horse, goat, alpaca, lama and Camels.

[0166] More specifically, the subject the present disclosure as well as the methods disclosed herein above offer great economic advantage for any industrial or agricultural use of animals, specifically, livestock. Thus, in some specific embodiments, the present disclosure may be applicable for mammalian livestock, specifically those used for meat, milk and leather industries. Livestock are domesticated animals raised in an agricultural setting to produce labor and commodities such as meat, eggs, milk, fur, leather, and wool. The term includes but is not limited to Cattle, sheep, domestic pig (swine, hog), horse, goat, alpaca, lama and Camels. Of particular interest are cattle applicable in the meat and milk industry, as well as in the leather industry. More specifically, in certain embodiments, the subject of the present disclosure may be Cattle, colloquially cows, that are the most common type of large, domesticated ungulates, that belong to the Bovidae family.

[0167] In yet some further embodiments, the organism applicable in the methods of the present disclosure, may be avian organisms. In yet some further specific embodiments, the present disclosure may be suitable for birds. More specifically, domesticated and undomesticated birds are also suitable organisms for the present disclosure.

[0168] Still further, in some embodiments the organism of the biological kingdom Plantae may be a dioecious plant, specifically, a plant presenting biparental reproduction. In some specific embodiments, the plant may be of the family Cannabaceae, specifically, any one of Cannabis (hemp, marijuana) and Humulus (hops). In more specific embodiments, the plant of the family Cannabaceae may be Cannabis (hemp, marijuana). In yet some further embodiments, the plant of the family Cannabaceae may be Humulus (hops). In some embodiments, any plants are applicable in the present disclosure, for example, any model plants such as, Arabidopsis, Tobacco, Solanum licopersicum, Solanum tuberosum. In yet some further embodiments, Canola, Cereals (Corn wheat, Barley), rice, sugarcane, Beet, Cotton, Banana, Cassava, sweet potato, lentils, chickpea, peas, Soy, nuts, peanuts, Lemna, Apple, may be applicable in the present disclosure.

[0169] A non-comprehensive list of useful annual and perennial, domesticated or wild, monocotyledonous or dicotyledonous land plant or Algae - (i.e unicellular or multicellular algae including diatoms, microalgae, ulva, nori, gracilaria), applicable in accordance with the present disclosure may include but are not limited to crops, ornamentals, herbs (i.e., labiacea such as sage, basil and mint, or lemon grass, chives), grasses (i.e., lawn and biofuel grasses and animal feed grasses), cereals (i.e., rice, wheat, rye, oats, corn), legumes (i.e. soy, beans, lentils, chick peas, peas, peanuts), leafy vegetables (i.e. kale, bok-choi, cress, lettuce, spinach, cabbage), Amaranthacea (i.e. sugar beet, beet, quinoa, spinach), Compositea (i.e. sunflower, lettuce, aster), Malvaceae (i.e. cotton, cacao, okra, hibiscus), cucurbits (i.e., cucumber, squash, melon, watermelon), Solanaceous species (i.e tobacco, potato, tomato, petunia and pepper), Umbellifera (i.e. carrot, celery, dill, parsley, cumin), Crucifera (i.e., oilseed rape, mustard, brassicas, cauliflower, radish), Sesame, the monocot Aspargales (i.e. onion, garlic, leek, asparagus, vanilla, lilies, tulips, narcissus), Myrtacea (i.e., Eucalyptus, pomegranate, guava), Subtropical fruit trees (i.e. Avocado, Mango, Litchi, papaya), Citrus (i.e. orange, lemon, grapefruit), Rosacea (i.e. apple, cherry, plum, almond, roses), berry-plants (i.e. grapes, mulberries, blueberries, raspberry, strawberry), nut trees (i.e. macademia, hazelnut, pecan, walnut, chestnuts, brazil nut, cashew), banana and plantain, palms (i.e., oil-palm, coconut and dates), evergreen, coniferous or deciduous trees, woody species.

[0170] For example, at least one organism of the biological kingdom Animalia or of the biological kingdom Plantae. In some specific embodiments, the eukaryotic organism of the biological kingdom Animalia is a mammalian subject.

[0171] In some embodiments of the present disclosure, the disclosed screening method is particularly applicable for screening of a compound that inhibits the interaction of the affinity moiety with the target molecule. As indicated above, a compound that "inhibits the interaction" as used herein, refers to a compound that reduces, prevents, blocks, impedes, suppresses, prevents, hinders and / or restricts interaction between the at least one affinity moiety and the target molecule either partially, or completely. For example, any inhibition of between about 10-50%, 51-80%, 81-99% or even, of 100% of the interaction or binding between the affinity moiety and the target molecule.

[0172] In some embodiments of the present disclosure, the target molecule is associated with at least one pathologic disorder in a subject.

[0173] In some further embodiments, the pathologic disorder is at least one of: a disorder caused by a pathogenic agent, a proliferative disorder, an inflammatory disorder, a metabolic disorder, a neurodegenerative disorder and an autoimmune disorder.

[0174] In some embodiments, the pathogenic agent is at least one of bacteria, archaea, virus, fungi, algae, parasite, protists, and worms.

[0175] In yet some specific embodiments, the methods and bio transistor systems of the present disclosure may be applicable for diagnosing, monitoring and treating an infectious disease caused by bacterial pathogens. More specifically, a prokaryotic microorganism includes bacteria such as Gram positive, Gram negative and Gram variable bacteria and intracellular bacteria. Examples of bacteria contemplated herein include the species of the genera Treponema sp., Borrelia sp., Neisseria sp., Legionella sp., Bordetella sp., Escherichia sp., Salmonella sp., Shigella sp., Klebsiella sp., Yersinia sp., Vibrio sp., Hemophilus sp., Rickettsia sp., Chlamydia sp., Mycoplasma sp., Staphylococcus sp., Streptococcus sp., Bacillus sp., Clostridium sp., Corynebacterium sp., Proprionibacterium sp., Mycobacterium sp., Ureaplasma sp. and Listeria sp.

[0176] Still further, examples of bacteria contemplated herein include the phylum Bacteroidota, more specifically the genus Bacteroidaceae and Phocaeicola.

[0177] Particular species include Bacteroides fragilis, Bacteroides thetaiotaomicron, Phocaeicola dorei, Akkermansia muciniphila , Bacteroides uniformis, Bacteroides ovatus, Bacteroides stercoris, Bacteroides cellulosilyticus, Bacteroides caccae, Bacteroides eggerthii, , Bacteroides intestinalis, Bacteroides clarus, Bacteroides fragilis_A, Bacteroides finegoldii, Bacteroides faecis, Bacteroides massiliensis, Bacteroides togonis, Bacteroides nordii, Bacteroides salyersiae, Bacteroides intestinalis_A, Bacteroides ndongoniae, Bacteroides sp003545565, Bacteroides sp905207245, Bacteroides bouchesdurhonensis, Bacteroides fluxus, Bacteroides gallinarum, Bacteroides stercorirosoris, Bacteroides graminisolvens, Bacteroides pyogenes, Bacteroides oleiciplenus, Bacteroides sp002491635, Bacteroides cutis, Bacteroides sp900547205, Bacteroides acidifaciens, Bacteroides sp905197435, Bacteroides neonati, Bacteroides sp014385165 and / or Parabacteroides distasonis.

[0178] Particular species include Mycoplasma pulmonis, Salmonella typhimurium, Treponema pallidum, Borrelia burgdorferi, Neisseria gonorrhea, Neisseria meningitidis, Legionella pneumophila, Bordetella pertussis, Escherichia coli, Salmonella typhi, Shigella dysenteriae, Klebsiella pneumoniae, Yersinia pestis, Vibrio cholerae, Hemophilus influenzae, Rickettsia rickettsii, Chlamydia trachomatis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus anthracis, Clostridium botulinum, Clostridium tetani, Clostridium perfringens, Corynebacterium diphtheriae, Proprionibacterium acnes, Mycobacterium tuberculosis, Mycobacterium leprae and Listeria monocytogenes. A lower eukaryotic organism includes a yeast or fungus such as but not limited to Candida albicans, Pneumocystis carinii, Aspergillus, Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Trichophyton and Microsporum, are also encompassed by the invention. A complex eukaryotic organism includes worms, insects, arachnids, nematodes, aemobe, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Trypanosoma brucei gambiense, Trypanosoma cruzi, Balantidium coli, Toxoplasma gondii, Cryptosporidium or Leishmania.

[0179] In yet some further embodiments, the bio transistor systems and methods of the present disclosure may be applicable for any infectious disorders caused by a viral pathogen or a virus. The term "virus" as used herein, refers to obligate intracellular parasites of living but non-cellular nature, consisting of DNA or RNA and a protein coat. Viruses range in diameter from about 20 to about 300 nm. Class I viruses (Baltimore classification) have a double-stranded DNA as their genome; Class II viruses have a single-stranded DNA as their genome; Class III viruses have a double-stranded RNA as their genome; Class IV viruses have a positive single-stranded RNA as their genome, the genome itself acting as mRNA; Class V viruses have a negative single- stranded RNA as their genome used as a template for mRNA synthesis; and Class VI viruses have a positive single-stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. It should be noted that the term “viruses” is used in its broadest sense to include viruses of the families Flaviviruses, Alphaviruses, Togaviruses, Coronaviruses, Hepatitis D, Orthomyxoviruses, Paramyxoviruses, Rhabdovirus. Still further, more specific embodiments relate to Influenza viruses A and B, coronaviruses (e.g. SARS-COV2), Ebola viruses, adenoviruses, papovaviruses, herpesviruses: simplex, varicella-zoster, Epstein-Barr (EBV), Cowpox viruses, Cytomegalo virus (CMV), pox viruses: smallpox, vaccinia, hepatitis B (HBV), rhinoviruses, hepatitis A (HBA), poliovirus, respiratory syncytial virus (RSV), Middle East Respiratory Syndrome (MERS), rubella virus, hepatitis C (HBC), arboviruses, rabies virus, measles virus, mumps virus, human deficiency virus (HIV), HTLV I and II, flaviviruses such as Dengue virus, west nile virus, yellow fever virus, and Zika virus.

[0180] More specifically, in certain embodiments the methods and bio transistor systems of the present disclosure may be suitable for disorders caused by fungal pathogens. The term "fungi" (or a “fungus”), as used herein, refers to a division of eukaryotic organisms that grow in irregular masses, without roots, stems, or leaves, and are devoid of chlorophyll or other pigments capable of photosynthesis. Each organism (thallus) is unicellular to filamentous and possess branched somatic structures (hyphae) surrounded by cell walls containing glucan or chitin or both and containing true nuclei. It should be noted that "fungi" includes for example, fungi that cause diseases such as ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, paracoccidio-idoinycosis, and candidiasis.

[0181] As noted above, the present invention also provides for methods and bio transistor systems for the diagnosis and monitoring of a pathological disorder caused by “parasitic protozoan”, which refers to organisms formerly classified in the Kingdom “protozoa”. They include organisms classified in Amoebozoa, Excavata and Chromalveolata. Examples include Entamoeba histolytica, Plasmodium (some of which cause malaria), and Giardia lamblia. The term parasite includes, but not limited to, infections caused by somatic tapeworms, blood flukes, tissue roundworms, ameba, and Plasmodium, Trypanosoma, Leishmania, and Toxoplasma species. As used herein, the term “nematode” refers to roundworms. Roundworms have tubular digestive systems with openings at both ends. Some examples of nematodes include, but are not limited to, basal order Monhysterida, the classes Dorylaimea, Enoplea and Secernentea and the “Chromadorea” assemblage.

[0182] In some further embodiments, the pathogenic agent is bacteria, and the target molecule is primase. Specifically, bacteriophage T7 primase.

[0183] In some embodiments of the present disclosure, the affinity moiety comprises a nucleic acid sequence comprising at least one primase binding site, the target molecule is a primase, and the method is for screening a compound that inhibits the interaction of primase to its nucleic acid binding site. According to some embodiments, such compounds may be useful as antimicrobial agent, specifically, antibiotic agents.

[0184] Antibiotics represent a fundamental class of antimicrobial agents, indispensable for the treatment of bacterial infections. These agents function by targeting specific bacterial processes and structures, either inhibiting bacterial growth or directly killing the bacteria. The diverse mechanisms of action, which include inhibition of cell wall synthesis, protein synthesis, DNA replication, and membrane integrity. Antibiotic agents include beta- lactams, aminoglycosides, tetracyclines, macrolides, fluoroquinolones, sulfonamides, glycopeptides, oxazolidinones, lipopeptides, and polymyxins.

[0185] In some embodiments, the candidate compound is a small molecule compound.

[0186] In some embodiments of the present disclosure, the compound that inhibits the interaction of primase to its nucleic acid binding site may be applicable as an anti-bacterial agent.

[0187] In some embodiments, the screening method is for identifying anti-bacterial compounds.

[0188] In some embodiments, where the target molecule is a transcription factor, the candidate compound may be a compound that inhibits the binding of the transcription factor to its binding site. According to such embodiments, the inhibitor may be used for blocking specific transcription of genes associated with a pathologic disorder transcribed by the transcription factor.

[0189] In some embodiments, the affinity moiety is an aptamer specific for a target molecule associated with a with at least one pathologic disorder in a subject and / or an environmental contamination. In some embodiments, the biological sample is derived from a eukaryotic organism. For example, at least one organism of the biological kingdom Animalia or of the biological kingdom Plantae. In some specific embodiments, the eukaryotic organism of the biological kingdom Animalia is a mammalian subject.

[0190] Another aspect of the present disclosure relates to a diagnostic method for determining a physiological and / or environmental condition or state of a subject and / or a media and / or a habitat. The method comprising: (a) contacting the at least one sample with a bio-transistor having an active region (e.g., modified) carrying affinity moieties, or a battery comprising at least two of said bio-transistors; (b) performing one or more measurements, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; (c) processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample; and (d) determining presence and / or quantity of the one or more target molecules in accordance with pre-stored calibration data, thereby obtaining a target molecule value for the sample; and (e) determining that the subject and / or media and / or habitat display said physiological and / or environmental condition or state, if the at least one target molecule value obtained for the sample in step (d), is positive or negative with respect to a reference target molecule value pre-determined for the physiological and / or environmental condition or state, or with respect to a target molecule value determined for at least one control sample.

[0191] In some embodiments of the present disclosure, the bio-transistor system of the diagnostic method is as defined above. Thus, the disclosed methods involve in the first step determination of the level of specific target molecule to obtain the value for each target in the sample, as will be elaborated herein after. The next step involves determination if the expression value is positive or negative. It should be understood that determination of a "positive" or alternatively "negative" value of the target molecule levels and / or amount with respect to a standard value or a control value may involve in some embodiments comparison of the value determined for the quantity and / or mount) of the target molecule of the examined sample as obtained in step (d), with the amount / level value of the target obtained for a control sample, or from any established or predetermined value of the target molecule level and / or amount and / or quantity (e.g., a standard value) obtained from a known control (either healthy controls or of subjects suffering from a pathological disorder). Thus, in some embodiments, "positive" is meant a value that is higher, increased, elevated, overexpressed in about 5% to 100% or more, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, when compared to the amount / level / quantity value of the reference and / or the standard value of a healthy control, any other suitable control or any other predetermined standard. Still further, a "negative" value in some embodiments may be a reduced, low, non-existing or lack of expression of a target molecule in about 5% to 100% or more, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, when compared to the value of the amount of the target molecule in a healthy control, any other suitable control or any other predetermined standard.

[0192] As used herein, “healthy controls” or “healthy population” may refer to a population of subjects that does not suffer from a disease of interest or refer to a population before appearance of a disease of interest. In some embodiments, the value of the target molecule in a control population refers to a baseline level of the target molecule of a healthy population or to a baseline level of the target molecule before appearance of a disease in a studied population. In some other embodiment, a “healthy control” or “control” may refer to the to a baseline level of the target molecule before appearance of a disease in a specific subject.

[0193] In yet some further embodiments, "positive", specifically, higher, elevated levels and / or amount and / or quantity when compared to a control, the subject is classified as a subject that display a specific disorder as indicated herein for each of the target molecules.

[0194] It should be appreciated that a "reference value" or a "Standard” , or a "predetermined standard” or a "predetermined reference value" as used herein, denotes either a single standard value or a plurality of standards with which the level of at least one of the target molecule / s from the tested sample is compared. The standards may be provided, for example, in the form of discrete numeric values or in the form of a comparative curve prepared on the basis of such standards (standard curve). Thus, in yet more specific embodiments, the method of the invention involves comparing the values of the amount and / or quantity of the target molecule / s determined for the tested sample with predetermined standard values or cutoff values, or alternatively, with the values of at least one control sample. As used herein the term “comparing” denotes any examination of the level and / or amount and / or quantity values obtained in the samples disclosed herein as detailed throughout in order to discover similarities or differences between at least two different samples. It should be noted that in some embodiments, comparing according to the present disclosure encompasses the possibility to use a computer-based approach.

[0195] Step (a) of the disclosed methods involves the action of contacting the bio transistors of the present disclosure with the examined sample. The term “contacting” means to bring, put or incubates together. As such, a first component, e.g., affinity moiety is contacted with a second component, specifically, the target molecule when the two components are brought or put together, e.g., by touching them to each other or combining them. In the context of the present disclosure, the term "contacting" includes all measures or steps which allow interaction between the at least one of the affinity moieties of at least one of the target molecules. The contacting is performed in a manner so that the at least one of affinity moieties of at least one of the target molecules, can interact with or bind to the target molecule in the tested sample. The binding will preferably be non-covalent, reversible binding, e.g., binding via salt bridges, hydrogen bonds, hydrophobic interactions or a combination thereof.

[0196] In some other embodiments, the physiological state and / or condition of a subject comprises pathological condition / s and / or health condi tion / s in the subject.

[0197] In some further embodiments, the pathological condition is at least one immune-related disorder. The immune-related disorder is an infectious disease caused by a pathogenic agent.

[0198] In some further embodiments, the immune-related disorder includes an infectious disease, an inflammatory disease, an autoimmune disease, a neurodegenerative disease, a proliferative disease.

[0199] In some embodiments of the present disclosure, the target molecule is associated with at least one pathologic disorder in a subject. In some embodiments, the immune-related disorder is at least one of an infectious disease, a proliferative disorder, an inflammatory disease, an autoimmune disorder, a metabolic disorder and a neurodegenerative disease.

[0200] An "Immune-related disorder" or "Immune- mediated disorder", as used herein encompasses any condition that is associated with the immune system of a subject, more specifically through inhibition of the immune system, or that can be treated, prevented, or ameliorated by reducing degradation of a certain component of the immune response in a subject, such as the adaptive or innate immune response. An immune-related disorder may include infectious condition (e.g., by a pathogen, specifically, viral, bacterial, or fungal infections), inflammatory disease, autoimmune disorders, immunodeficiency (e.g., primary or a secondary) metabolic disorders and proliferative disorders, specifically, cancer. In some embodiments, the immune-related disorder is an infectious disease caused by a pathogenic agent.

[0201] In some embodiments, the immune-related disorder applicable in the methods of the present disclosure may be at least one infectious disease. An infectious disease as used herein encompasses any infectious disease caused by a pathogenic agent, specifically, a pathogen. More specifically, such infectious disease may be any pathological disorder caused by a pathogen. As used herein, the term “pathogen” refers to an infectious agent that causes a disease in a subject host. Pathogenic agents include prokaryotic microorganisms, lower eukaryotic microorganisms, complex eukaryotic organisms, viruses, fungi, mycoplasma, prions, parasites, for example, a parasitic protozoan, yeasts, or a nematode, as well as toxins and venoms, as indicated herein before.

[0202] In some further embodiments, the affinity moiety comprises a nucleic acid aptamer specific for the target molecule.

[0203] In some aspects thereof, the present disclosure further provides personalized therapeutic methods that comprise a diagnostic step using the bio-transistor systems and methods of the present disclosure, followed by administration of a therapeutic compound, for example, any compound that inhibits the interaction of primase to its binding site (or any target effector that participate in DNA replication), thereby may interfere with bacterial DNA replication. It is to be understood that the terms "treat”, “treating”, “treatment" or forms thereof, as used herein in accordance with the personalized therapeutic methods, mean preventing, ameliorating or delaying the onset of one or more clinical indications of disease activity in a subject having a pathologic disorder. Treatment refers to therapeutic treatment. Those in need of treatment are subjects suffering from a pathologic disorder. Specifically, providing a "preventive treatment" (to prevent) or a "prophylactic treatment" is acting in a protective manner, to defend against or prevent something, especially a condition or disease.

[0204] The term “treatment or prevention” as used herein, refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, an immune-related condition and illness, immune-related symptoms or undesired side effects or immune-related disorders. More specifically, treatment or prevention of relapse or recurrence of the disease, includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and / or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms. It should be appreciated that the terms "inhibition", "moderation", “reduction”, "decrease" or "attenuation" as referred to herein, relate to the retardation, restraining or reduction of a process by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more.

[0205] With regards to the above, it is to be understood that, where provided, percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.

[0206] The term "amelioration" as referred to herein, relates to a decrease in the symptoms, and improvement in a subject's condition brought about by the methods according to the present disclosure, wherein said improvement may be manifested in the forms of inhibition of pathologic processes associated with the immune-related disorders described herein, a significant reduction in their magnitude, or an improvement in a diseased subject physiological state.

[0207] The term "inhibit" and all variations of this term is intended to encompass the restriction or prohibition of the progress and exacerbation of pathologic symptoms or a pathologic process progress, said pathologic process symptoms or process are associated with.

[0208] The term "eliminate" relates to the substantial eradication or removal of the pathologic symptoms and possibly pathologic etiology, optionally, according to the methods of the present disclosure described herein.

[0209] The terms "delay", "delaying the onset", "retard" and all variations thereof are intended to encompass the slowing of the progress and / or exacerbation of a disorder associated with the immune-related disorders and their symptoms slowing their progress, further exacerbation or development, so as to appear later than in the absence of the treatment according to the present disclosure.

[0210] Another aspect of the present disclosure relates to a diagnostic kit. The diagnostic kit comprising: (a) at least one bio-transistor system, and optionally, at least on of: (b) at least one control sample; and (c) at least one therapeutic agent. The bio-transistor system comprises at least one transistor unit and a control system. The transistor unit comprising: (i) at least one channel; (ii) source and drain electrodes; (iii) at least one gate electrode; (iv) at least one active region located in proximity to the channel region and carrying at least one affinity moiety. In some embodiments, each affinity moiety is specific for a target molecule. The at least one active region is configured for accepting at least one sample; and (v) at least one additional electrode positioned to be in electrical contact with the sample. The control system comprising at least one processor and memory circuitry. The control system is configured and operable for performing one or more measurements of the sample, wherein each measurement comprises maintaining a selected electric potential on the at least one additional electrode and determining current transmission profile through the at least one channel with respect to potential variation of said the least one gate electrode. The control unit thereby configured to determine on the presence and / or quantity of one or more target molecules in the sample.

[0211] In some embodiments, the kit is adapted for performing the methods described above. All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0212] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. Thus, as used herein the term "about" refers to ± 10 %.

[0213] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of" and "consisting essentially of". The phrase "consisting essentially of" means that the composition or method may include additional ingredients and / or steps, and / or parts, but only if the additional ingredients and / or steps do not materially alter the basic and novel characteristics of the claimed composition or method. Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0214] It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging / ranges between" a first indicate number and a second indicate number and "ranging / ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0215] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical, and medical arts.

[0216] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments unless the embodiment is inoperative without those elements.

[0217] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0218] Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0219] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. EXAMPLES

[0220] Experimental procedures

[0221] Materials

[0222] All chemicals and reagents used in this study were obtained from Sigma Aldrich Chemical Company (St. Louis, MO, USA) and Acros Organics (Geel, Belgium) without further purification or drying. Nucleoside triphosphates (NTPs) were sourced from New England Biolabs (NEB). Radiolabeled ATP, [a-32P]ATP (3000 Ci / mmol), was purchased from PerkinElmer. Protein purification was performed using Fast Protein Liquid Chromatography (FPLC) with Ni-NTA (affinity chromatography) and S200 (size exclusion chromatography) columns.

[0223] Device fabrication. The silicon part of the MNC biochip was fabricated using silicon-on- insulator (SOI) wafers in a CMOS-flavor process. Each MNC biochip contains 5 MNC biosensors. The thicknesses of the SOI device layer and the buried oxide (BOX) are 145 nm and 400 nm, respectively. The device layer is n-type doped to 1017cm'3. The p-type regions (associated with the lateral gates) are degenerated, and the source and drains are degenerated n-type regions. The distance between p-type regions is 700 nm (which defines the maximum width of the channel), and the length of the channel is 10 pm. Standard CMOS silicidation is performed in all regions in which the aluminum metal lines interface the silicon to ensure low resistance ohmic contacts. The gate dielectric (sensing area) is 5.5 nm of thermal oxide. The MNC biochip is passivated with 1 pm of SiO2except the electrical pads and the sensing area.

[0224] Formation of the biorecognition layer composed of a primase recognition sequence. The biorecognition layer (used as active region) was firstly developed and physically characterized on Si / SiO2 (10 nm SiO2) substrates. The substrates were sonicated for 2 minutes and rotated for 1 minute with ethyl acetate, acetone, 2-propanol, and deionized (DI) water, successively. The substrates were immersed in piranha solution (4: 1 H2SO4: H2O2) for 4 minutes for the purpose of surface activation. The substrates were rinsed with DI water, N2 dried, and immersed for 3 hours in a 0.1% v:v solution of 3- aminopropyltrimethoxysilane (APTMS) in methanol. Leftover APTMS was discarded with multiple cycles of methanol sonication and rinsing. The samples were hydrolysed for 24 hours and baked for 1 hour at 120 °C for durability and stabilization. The substrates were placed in a 0.5% glutaraldehyde (GA) crosslinker solution, and afterwards immersed for 12 hours at 4°C in a 10 rnM pH 7.4 Tris-Buffered Saline (TBS) containing Ipg / ml of T7 primase recognition sequence. Electrochemical Impedance Spectroscopy (EIS, Palmsens 4, Palmsens Inc.) was used to characterize the various modification steps. The measurements were performed in a pH 7.4 0.1 mM PBS. The sample was measured in a three-electrode 0.8 mL electrochemical cell. The back side of the silicon substrate (the working electrode) was manually scratched and contacted with a conductive carbon paint. SEC-C Pt Gauze working electrode (ALS Japan) was the selected counter electrode, and RE- IBP (Ag / AgCl) by ALS Japan was the selected reference electrode. Further surface physical characterization was performed with spectroscopic ellipsometry (Alpha-SE Ellipsometer, J.A. Woollam), and contact angle tensiometer (Model OCA 20, dataphysics). The above biofunctionalization steps were performed on the MNC bioFETs.

[0225] Electrical measurements. A fully equipped probe station was employed for the electrical measurements. Probe needles were used to contact the MNC biochip aluminum pads to the source measuring units (SMUs) (B 1500 semiconductor parameter analyzer, Keysight Ltd). Measurements are carried out with 0.5 μL drops of various buffers, including TE buffer, binding buffer (comprising 30mM tris HC1 pH 7.5, 6.5mM MgCl2, 30mM K-glutamate, 6mM DTT, 65 μM rNTPs, and 100μM ATP), and the primase storage buffer (containing 50mM tris pH 7.5, 0.1mM DTT, ImM EDTA, 100mM NaCl, and 10% glycerol), which are drop-cast onto the bioFET chip. The gating of the sample was performed by an Ag / Ag+ quasi-reference electrode realized from a commercial reference electrode (012171 RE-7, ALS Co., Ltd); the glass holder of the reference electrode was detached, and the exposed Ag wire was decorated with an Ag / AgCl ink (011464, ALS Co., Ltd). The quasi-reference electrode was mounted on a probe holder and connected to the B 1500 SMU for potential determination (VGF) and leakage current measurement.

[0226] Sensing measurements are conducted with 0.5 μL starting with a 1 fM concentration of primase, which is applied and measured. After each measurement, the drop is collected with a clean room towel before applying and measuring the next higher concentration. Protein Expression and Purification

[0227] Escherichia coli BL21 (DE3) cells were transformed with genes encoding the T7 phase primase fragment. The bacteria were cultured in LB medium supplemented with 50 ug / L kanamycin until they reached an OD600 of 0.6-0.7. Protein expression was induced with 0.5 mM IPTG, and the cultures were incubated with shaking at 16°C for 18 hours. Cells were harvested by centrifugation at 4700 rpm for 30 minutes, and the resulting pellet was resuspended in buffer A (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM imidazole, 10% v / v glycerol, and 0.5 mM dithiothreitol). Lysis was performed using a pressure homogenizer and sonication. The lysate was clarified by centrifugation at 14000 rpm for 60 minutes, filtered through a 0.45-μM filter, and loaded onto a Ni-NTA column (HisTrap FF, GE Healthcare). The column was equilibrated with buffer A, and the protein was eluted using a linear gradient of imidazole (10-400 mM) in buffer A. Fractions containing the T7 primase fragments were pooled, treated with 1 mM EDTA and precipitated with ammonium sulfate (0.5 g / mL) for at least six hours. The precipitate was collected by centrifugation at 14000 rpm for one hour, resuspended in buffer B (50 mM Tris-HCl pH 8, 100 mM NaCl, 10% v / v glycerol, and 0.5 mM dithiothreitol), and loaded onto a Superdex S200 column (GE Healthcare). The protein was eluted with buffer B. Fractions containing purified T7 primase fragments were combined, dialyzed against buffer B containing 50% glycerol, and stored at -20 °C.

[0228] T7 phase primase activity assay

[0229] A 10 μL reaction mixture containing 4 μM 5’ GGGTCAAAAAAAAAA 3’, as denoted by SEQ ID NO: 3, 250 μM NTPs (CTP, GTP, and UTP), radiolabeled a-32P ATP, and 500 nM T7 primase fragment in buffer (40 mM Tris-HCl, pH 7.5, 10 mM MgC12, 10 mM DTT, and 50 mM KGlu) was incubated at 30°C for 30 minutes. The reaction was stopped by adding an equal volume of sequencing buffer (98% formamide, 0.1% bromophenol blue, 20 mM EDTA). Samples were then analyzed on a 25% polyacrylamide gel containing 7 M urea and visualized via autoradiography to detect the short RNA primers synthesized by the primase. Formation of the biorecognition layer composed of anti-AFP antibodies. The MNC decorated with devices were biofunctionalized and transformed into MNC biosensors. The biofunctionalization was first developed on plain silicon samples (2 X 2 cm) 5 nm of SiO2. The various involved biofunctionalization steps were characterized by electrochemical impedance spectroscopy (EIS) (Palmsens 4, Palmsens Inc.), contact angle tensiometer (Model OCA 20, dataphysics), and spectroscopic ellipsometry (J.A. Woollam Alpha-SE Ellipsometer). The samples were successively cleaned with ethyl acetate, acetone, and 2- propanol for 2 min in a bath sonicator for each cleaning step. The cleaning was followed by surface activation with piranha for 4 minutes (4: 1 ratio of H2SO4:H2O2), DI water wash and N2 drying. The next step was surface chemical modification with 3- aminopropyltrimethoxysilane (APTMS) linker molecules. The samples were incubated in APTMS solution (0.1% v:v in methanol) for 3 hours, followed by three cycles of 3 minutes each of sonication in methanol. The samples were hydrolyzed by 24 hours incubation in DI water, dried and placed in an oven for 1 hour at 120°C [M. Zhu, et al. , Langmuir 2012, 28, 416-423]. Prior to the binding of the anti-AFP antibodies, the APTMS was modified with glutaraldehyde (GA) (SAB4501531-100UG, Sigma Aldrich). Finally, the samples were incubated for 12 hours in 1 pg / ml solution (10 mM PBS, pH 7.4) of anti-AFP (Abeam, AB-ab3980). The MNC chips were biofunctionalized in a similar manner. The various involved biofunctionalization steps were characterized by electrochemical impedance spectroscopy (EIS) (Palmsens 4, Palmsens Inc.), contact angle tensiometry (Model OCA 20, dataphysics), and spectroscopic ellipsometry (J.A. Woollam Alpha-SE ellipsometer). The contact angle of anti-AFP modified chip is 52°± 0.5° and 2.2 ± 0.8 nm is the measured thickness of the anti-AFP layer.

[0230] AFP electrical and sensing measurements. The MNC devices were electrically measured on a probe station with probe needles contacting the chip metal pads to source measuring units (SMUs) of the B1500 semiconductor parameter analyzer by Keysight Ltd. A neutral solution of 0.1 mM pH 7.4 phosphate-buffered saline (PBS) with a volume of 0.5 μL was manually applied with a pipette to the MNC sensing area. The drop was electrically contacted with a quasi-reference electrode made of an Ag / Ag+reference electrode (012171 RE-7, ALS Co., Ltd) where the glass holder was removed by exposing the Ag wire which was coated with an Ag / AgCl ink for reference electrode (011464, ALS Co., Ltd). The quasi-reference electrode (VGF) was connected to an additional B1500 SMU for determining potential of the solution. The quasi-reference electrode was mounted on a probe manipulator. The drop lifetime before evaporation is 2-3 minutes, which provides a time window for the various current-voltage (I-V) measurements. The I-V measurements were repeated during the drop lifetime, and the measurements were performed for successive applied drops in order to confirm and establish repeatability. The measurements in neutral solution were performed solely during the process of MNC device biofunctionalization in order to validate device functionality post modification. The excellent repeatability of unmodified MNC devices and biofunctionalized MNC biosensors ensure the buffer capacity of the small solution drops [R. E. G. Van Hal, et al. Sensors Actuators B 1995, 24-25, 201], as well as the stability of the quasi-reference electrode for possible drop-to-drop variations [L. R. F. Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley And Sons Inc., 2008].

[0231] The AFP sensing measurements were performed with 0.5 pF drops of 1: 100 diluted serum obtained from Kaplan Medical Center blood bank. The dilution of the serum was performed with Tris-Buffered saline (TBS) 0.1 mM pH 7.4. Mixtures of diluted serum spiked with different concentrations of AFP (Abeam, ab 114216) were prepared. The serum drop was biased in the same manner described for the neutral solution drop. The stability of the quasi- reference electrode with respect to drop-to-drop variations of the diluted serum [L. R. F. Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley And Sons Inc., 2008] , and non-specific adsorption of biomolecules (on the quasi-reference electrode) was addressed by the non-specific measurements (Figure 9C). Successive I- V measurements were performed for a diluted serum drop in order to validate and establish repeatability. Afterwards, the diluted serum drop was collected with a clean- room wipe, and a new drop spiked with a higher AFP concentration is introduced on the MNC sensing area, and the procedure was repeated. The non-specific measurements were performed in the following manner: AFP was introduced to an unmodified MNC device, and AFP was introduced to an MNC modified with APTMS. Also, human chorionic gonadotropin (hCG) hormone (Sigma- Aldrich, hCG-C1063), and prostate specific antigen (PSA) (Sigma Aldrich, P3338-25UG) physiological biomarkers were introduced to MNC biosensor modified with anti- AFP antibodies. hCG was selected as a different glycoprotein type of biomarker with a dimer consisting of a 145 amino acid beta- subunit that is unique to hCG and a 92 amino acid alpha-subunit, having a molecular weight -37 kDa. Similarly, PSA is a 237 amino-acids long single chain glycoprotein with a molecular weight of ~28 kDa. AFP is a glycoprotein which consists of a polypeptide chain with 591 amino acids and a carbohydrate chain with a molecular mass of -68.8 kDa. For all the non-specific measurements, the concentrations of the biomarkers (AFP, PSA and hCG) were 10 ng / ml, 100 ng / ml, and 1 μg / ml corresponding with the highest AFP concentrations (equivalent to 105pM, 1.05nM and 10.5nM, respectively) considered in this work for the specific measurements. The excellent repeatability of the non-specific measurements also removes the concern of pH fluctuations due to the presence of biomolecules in the drops [R. E. G. Van Hal, et al. Sensors Actuators B 1995, 24-25, 201].

[0232] EXAMPLE 1

[0233] MNC biosensor biofunctionalized with a primase recognition sequence

[0234] Figure 4 presents an illustration of the employed bioFET. The conduction channel of the bioFET channel is formed at the channel volume and is composed of electron majority carriers. The channel volume is laterally bound on both sides with p-type regions forming pn junctions. The p-type regions are electrically connected to aluminum lines and form the lateral gates (VGL). The backgate (VGB) is grounded throughout the current study. The solution potential is determined by a commercial quasi-reference electrode (VGF).

[0235] The bioFET is functionalized on active regions thereof with a biorecognition layer composed of surface-bound DNA with a specific T7 primase recognition sequence containing 5’-GTC-3’ at the sensing area. The primase recognition sequence bound to the active surface comprises the nucleic acid sequence: 5’ / 5AmMC6 / TG TCT TGA TTC GCT TGA CGC TGG TGG TGG TGG GTG TGT GTG GGTCTT TTG TTT GTG GTG G 3', (5' Amino Modifier C6), the oligo sequence is as denoted by SEQ ID NO: 1. The biofunctionalization follows conventional amine-based protocols. EXAMPLE 2

[0236] IDS-VGLcurves for selected VGFvalues performed for MNC biosensor biofunctionalized with a primase recognition sequence

[0237] The sensor surface of MNC bioFETs was first cleaned with ethyl acetate, acetone and isopropanol. Then the sensing area activated with piranha (4:1 H2SO4:H2O2) solution for 4 minutes, and then ringed with DI water and N2 dried. Activated sensor then immersed in 0.1% v:v solution of 3 -aminopropyl trimethoxysilane (APTMS) and methanol. The sensor hydrolysed for overnight and cured at 12°C for Ih. Afterward, the sensor immersed in 10 mM PBS (Phosphate-buffered saline) containing 10 pg / ml of DNA1 for 12h at 4°C. Finally, the sensor was washed with 10 mM PBS and dried with N2.

[0238] Figure 5 shows IDS-VGLcurves for selected VGFvalues and for 0.5 μL drops of 3% neutral solution (PBS 7.4 0.1 mM) spiked with primase concentrations ranging from 1 fM to 10 μM. The drops are applied in increasing order from lowest primase concentration to highest. Figure 5 also shows the corresponding Inormcurves defined as Inorm= (Iprimase - Ibaseiine) / Ibaseiinewhere Iprimaseand Ibaseiineare IDSvalues measured for a sample spiked with primase and neutral solution without primase, respectively. As shown in the figure, variation in primase concentration affects the IDS-VGLcurves where the sensitivity is affected by sample potential VGF.

[0239] Figure 6 presents the extracted calibration curves for specific and label-free sensing of primase-DNA binding.

[0240] As shown, a number of calibration curves are determined for different gate potential VGL and sample potential VGFare determined. Accordingly, to provide selected dynamic range for detection of primase concentration, the bio-transistor system may operate using one or more (or two or more) different values of sample potential VGF, while determining source- drain current to a selected number of gate potential values VGL(or VGin accordance with specific one or more gates being used). EXAMPLE 3

[0241] MNC biosensor biofunctionalized with anti-AFP antibodies (background measurements)

[0242] Figure 7 shows the dependency of IDSon VGLfor different VGFvalues measured with a 0.5 μL drop of 1 : 100 diluted serum for an unmodified MNC device. Each curve was an average of 16 drops and each drop was measured 4 times with a total of 64 measurements. The inset shows a magnification of the data points showing the errors bar reflecting the standard deviations of the 64 measurements. The data presents an excellent repeatability of an unmodified MNC device in 1: 100 diluted serum. The repeatability removes the concern of drop-to-drop pH fluctuations [R. E. G. Van Hal, J. C. et al. Sensors and Actuators B, 1995, 24-25, 201-205] and establishes the potential stability of the quasi-reference electrode under possible drop-to-drop variations [L. R. F. Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley and Sons Inc., 2008]. The dependency of IDSon VGLand VGFis detailed below in Figure 9 for MNC biofunctionalized with AFP-antibodies.

[0243] EXAMPLE 4

[0244] Process of Si / SiO2surface biofunctionalization with anti-AFP antibodies

[0245] Figure 8A illustrates the main steps towards sensing area biofunctionalization with anti- AFP antibodies [I. M. Bhattacharyya, et al. Nanoscale, 2022, 14, 2837-2847; I. M. Bhattacharyya, et al. Adv. Electron. Mater. 2022, n / a, 2200399]. In short, the Si / SiCb sensing area was activated with piranha followed by surface chemical modification with APTMS linker molecules, and final surface-tethering of the anti-AFP antibodies (see Experimental procedure). Figure 8A also presents the corresponding contact angle measurements. The surface modifications were developed and characterized using 1 cm x 1 cm samples of silicon substrates decorated with 5 nm SiO2(Figure 8B-8C). Figure 8B presents contact angle measurements for Si / SiO2samples post piranha activation, post APTMS modification and surface biofunctionalization with anti-AFP antibodies. The measurements are performed at three locations on the samples with two measurements performed at each location, such that the data points and the error bars reflect the averages and standard deviations, respectively, of six measurements. The contact angle dependency on the various surface modifications follows behavior well documented previously [R. G. Frieser, J. Electrochem. Soc. 1974, 121, 669; Z.-Z. Liu, et al. Thin Solid Films 2008, 517, 635]. Figure 8B also shows ellipsometry measurements performed on the same Si / SiCL samples. First, the SiCF sample is measured and the 5 nm SiCF thickness is validated. Afterwards, the sample is measured post APTMS modification where the SiCF is fixed and the APTMS thickness is fitted. Finally, the sample is measured post surface biofunctionalization with anti-AFP antibodies where both the SiCF and the APTMS thicknesses are fixed to the measured values and the antibody layer is fitted. The mean square error (MSE) for all measurements is smaller than 2. The thicknesses presented in Figure 8C reflect the expected values of APTMS and the antibody layer [I. M. Bhattacharyya, et al. Nanoscale 2022, 14, 2837; K. Bierbaum, et al. Langmuir 1995, 11, 512; I. M. Bhattacharyya, et al. Adv. Electron. Mater. 2022, 2200399]. Figure 8C presents the EIS measurements of the imaginary capacitance (C”) vs. the real capacitance (C’) for unmodified Si / SiO2, post APTMS modification and post biofunctionalization with anti- AFP antibodies. Three distinct curves are shown, which reflect the effect of the modifications on the sensing area. The capacitance post APTMS modification is not significantly different from the unmodified sample, but the presence of an antibody layer is well reflected in the formation of an additional semi-circle characteristic of the generation of an additional layer. Finally, the biofunctionalization is applied to an MNC biosensor and Figure 8D shows IDSVS. VGLfor different VGFvalues performed for unmodified, and MNC modified with anti-AFP antibodies. All the measurements are performed for 0.5 μL drops of 0.1 mM 7.4 pH PBS solution. Three drops are measured for the unmodified MNC biosensor, and three drops are measured for the modified MNC biosensor, and each drop is measured four times. Figure 8D presents an excellent robustness and repeatability of a modified MNC device in 0.1 mM 7.4 pH PBS solution, as well as asserts the stability of the quasi-reference electrode under possible drop-to-drop variations [L. R. F. Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley And Sons Inc., 2008]. Finally, the repeatability addresses the concern of possible drop-to-drop pH fluctuations [R. E. G. Van Hal, J. C. T. Eijkel, P. Bergveld, Sensors Actuators B 1995, 24-25, 201]. EXAMPLE 5

[0246] IDS-VGLfor selected VGFvalues for MNC biosensor modified with anti-AFP molecules Figure 9A presents IDS-VGLfor the selected 6 VGFvalues of MNC biosensor modified with anti-AFP measured in 1: 100 diluted serum. The contact angle of anti-AFP modified chip was 52°± 0.5° and the measured thickness of the anti-AFP layer was 2.2 ± 0.8 nm.

[0247] The data points and the error bars (see inset) reflect the averages and standard deviations, respectively, of 14 measured drops where each drop is measured 3 times. Note the excellent repeatability and robustness of the biofunctionalized MNC biosensor in 1:100 diluted serum. Furthermore, the repeatability reflects the buffering capability of the 0.5 μL drops of 1: 100 diluted serum [R. E. G. Van Hal, J. C. T. Eijkel, P. Bergveld, Sensors Actuators B 1995, 24-25, 201], as well as the stability of the quasi-reference electrode under possible drop-to-drop variations [L. R. F. Allen J. Bard, Electrochemical Methods: F ndamentals and Applications, 2nd Edition, John Wiley And Sons Inc., 2008]. Figure 9B presents the corresponding second derivatives of the curves presented in Figure 9A where each peak represents the excitation of a conducting channel [I. M. Bhattacharyya, et al. Nanoscale 2022, 14, 2837]. For VGF=0 V and 0.5 V, top and middle channels were normally-on, and the back channel was excited for higher VGLvalues. Note that lower VGFvalues force a higher voltage for the excitation of the back channel. IDSis zero for VGF=-0.5 V and low VGL, the middle channel is excited upon VGLincrease (at VGL— 0.6 V), and the top channel is excited afterwards (at VGL~0.4 V). Obviously, lower VGFvalues shift the excitation of the middle and top channels to higher VGLvalues. Also, Figure 9B illustrates the excitation sequence of the 3 channels for the various applied voltages. Importantly, as described above, although 3 channels are discussed, in practice each combination of VGLand VGFinduces the formation of IDSof a different shape and size.

[0248] The measurements were performed in 1:100 diluted serum, which implies protein background concentrations of 600-800 μg ml-1 (primarily albumin and globulins). More control measurements are provided in Figure 9C. Figure 9C(i)-9C(iv) show the non- specific response of the MNC biosensor for the introduction of AFP to an unmodified MNC biosensor, the introduction of AFP to an APTMS-modified MNC biosensor, and for the introduction of PSA and hCG to anti-AFP-modified MNC biosensors. The measurements are performed for 0.5 μL drops of 1: 100 diluted serum spiked with the respective target molecule concentration. In each case, the target molecule concentrations for hCG are 0.27 nM, 2.70 nM and 27.0 nM and for PSA those are 0.35 nM, 3.50 nM and 35.0 nM in accordance with the 3 highest concentrations selected for AFP-specific sensing (0.11 nM, 1.05 nM and 10.5 nM). The IDS-VGLcurves are very dense and the distinction between the different concentrations can be ascertained from the insets, where every data point is the average of 4 measurements performed for each drop (=concentration) and the error bars represent the corresponding standard deviations. In all cases, the negligible non-specific signals and the excellent repeatability remove the concerns of: 1) a sensing signal due to serum pH fluctuations induced by the presence of biomolecules [R. E. G. Van Hal, J. C. T. Eijkel, P. Bergveld, Sensors Actuators B 1995, 24-25, 201], 2) a sensing signal originating from physical adsorption of target molecules on the quasi-reference electrode, 3) a sensing signal due to effect of drop-to-drop variations on the quasi-reference electrode potential [L. R. F. Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley And Sons Inc., 2008], and 4) a sensing signal due to non-specific adsorption of target molecules on the MNC biosensor sensing area.

[0249] EXAMPLE 6

[0250] IDS-VGLfor selected VGFvalues for different concentrations of AFP molecules

[0251] Next, real-time, specific, and label-free sensing of AFP molecules, from 0.5 μL drops of 1: 100 diluted serum spiked with various AFP concentrations, was demonstrated with the MNC biosensor. Figure 10A shows IDS-VGLcurves for the 6 selected VGFvalues, on both linear and logarithmic scales, and for all 10 AFP concentrations. The data points and the error bars (see insets) reflect the averages and standard deviations, respectively, of 4 measurements performed for each drop (=concentration). In order to confirm that the measured IDS-VGLcurves do reflect specific signals, the following procedure was followed. First, it is ensured that the variation between one drop to the second (variation between successive AFP concentrations) is not due to the natural variation between two successive drops. Hence, the IDSdifference between two adjacent concentrations, for each VGFand VGLvalues, is required to be higher than the average difference between the 14 drops of Figure 9A. Second, it is ensured that the variation between one drop to the next is not due to non- specific signals. Hence, the IDSdifference between a given AFP concentration and the baseline (diluted serum without AFP), for each VGFand VGLvalues, is required to be higher than the corresponding non-specific measurements presented in Figure 9C. In this manner the following concerns are removed: sensing signals originating from pH fluctuations induced by the target molecules or drop-to-drop variation in pH [R. E. G. Van Hal, J. C. T. Eijkel, P. Bergveld, Sensors Actuators B 1995, 24-25, 201], non-specific adsorption on the quasi-reference electrode and the MNC biosensor sensing area, and the effect of drop-to- drop variation on the quasi-reference electrode potential [L. R. F. Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley And Sons Inc., 2008].

[0252] Figure 10B shows the Readout curves corresponding to the IDS-VGLcurves of Figure 10A. The Readout was calculated as (lDSAFP-lDSbaselme)lDSbaselme, where lDSAFPis the measured IDSfor a given AFP concentration and for set values of VGFand VGL, and lDSbaselmeis the IDSmeasured for 1 : 100 diluted serum without AFP spiking. Importantly, the x-axis scales were adjusted to ensure that values reflecting a null IDS(noise level) are not considered. The relevant excited channels are also marked in Figure 10B (T for top channel, M for middle channel, and B for back channel). First, the dependency of the 10.5 aM concentration, for VGF=-0.5 V and VGL=-0.7 V, is 75% which corresponds with an excited middle channel. Note how the Readout drops with increasing VGLwhich corresponds to the widening of the middle channel. Also, the Readout for the same 10.5 aM concentration is just about nulled for VGF=-2V and VGL=0.2 V which corresponds also to an excited middle channel. However, the former middle channel is very different in shape from the latter. The latter middle channel is further removed from the front interface (VGF=-2 V vs. VGF=-0.5 V), and it is also considerably wider compared with the former channel (VGL=0.2 V vs. VGL=-0.7 V). This is the important merit of the MNC biosensor: the variety of available channels provides multitude of ways to couple the random electrostatic distribution of the biological interactions at the sensing area, and the consequential surface potential distribution, with the electrodynamics of the underlying conducing channels to provide a meaningful IDS- Next, the effect of active sensing was demonstrated and discussed. All measured channels exhibit positive Readout values which imply an overall total positive charge at the sensing area, except the middle channel of VGF=-2 V. For this middle channel of VGF=-2 V the Readout switches polarity: for AFP concentration range of 10.5 fM-10.5 nM the Readout increases with AFP concentration increase, but it is negative for 10.5 fM-10.5 pM and positive for 105 pM-10.5 nM. The switch in Readout polarity suggests that the four gates do not only affect the transduction of the sensing area potential into IDS, but also affects the interactions themselves, refereed to as active sensing. In other words, if the total charge distribution of the complexes at the sensing area is positive, then the Readout must be positive and cannot take negative values. As a corollary, a switch in Readout polarity suggests that the biological interactions at the sensing area are affected by the gating configuration (active sensing). The effect of VGLon biological interactions does not seem probable as VGLproduces horizontal electric fields in the silicon device layer. On the other hand, VGFaffects the double layer (which is also affected by the biological entities and interactions) adjacent to the sensing area. Hence, VGFdetermines the double layer electric field, the pH level and the ionic strength, all can potentially affect biological interactions at the sensing area [L. R. F. Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley And Sons Inc., 2008; I. M. Bhattacharyya, G. Shalev, ACS Sensors 2020, 5, 154]. Therefore, it is suggested that VGF=-2 V produces a double layer environment which results in total negative charge and a Readout decrease. Note that VGFalso affects the Debye screening length at the sensing area (by the determination of surface ionic strength), but in this case the Readout is not expected to switch polarity. Figure 10C illustrates one possible mechanism to induce a switch in Readout polarity which relates to the direction of the double layer electric field determined by VGF- Such a switch in the polarity of the electric field can affect the orientation of the biological complexes and to generate either positive or negative Readout. Of course, the effect of the double layer electric field on the orientation of the target molecules also depends on the density of the surface-bound complexes, as presented in Figure 10C. Active sensing with the MNC biosensor is provided by the solution potential which affects the biological interactions indirectly by determining the conditions at the double layer. Still, more research is needed to underpin the mechanism by which VGFaffects the biological interactions at the sensing area. EXAMPLE 7

[0253] Readout calibration curves for AFP molecules

[0254] Figure 11A shows the Readout calibration curves for the considered channel configurations. The illustrations on the right of the calibration curves show the cross- sections, midway between the source and drain, of the various channels visualizing the size, shape, and location of the corresponding channels. Note, that each horizontal line represents a calibration curve obtained for a different channel configuration. Also, dashed vertical grey lines indicate the ‘calibration threshold’ of the calibration curves which marks the onset of the linear region of the dynamic range. Two interesting observations are in place. First, VGLdetermines the sensitivity (the slope of the linear region) and lower VGLgenerates higher sensitivity. This directly reflects the effect of channel configuration on sensing performance. Second, active sensing is allowed as VGFaffects the calibration threshold value. The dependency of the calibration threshold on VGFis presented in Figure llB(i). Evidently, VGFaffects the calibration threshold significantly in the range of three orders of magnitude in AFP concentration from 105 fM to 10.5 pM. The importance of the calibration threshold is with respect to its association with Kd. Kdis defined in accordance with the law of mass action in equilibrium: [ligand]- [receptor] -kon= [1 igand: receptor] -koff, where [ligand] and [receptor] are the concentrations or activities of the reactants, [ligand:receptor] is the concentration or activity of the complexes, and konand koffare rate constants for the forward and reverse reactions, respectively. Kdis defined as Kd= ([ligand] - [receptor]) / [ligand:receptor], such that Kd= [ligand] reflect a state in which half of the receptors are unbound and half are bound to ligands [D. B. Finlay, S. B. Duffull, M. Glass, Br J Pharmacol. 2020, 177, 1472]. Therefore, experimentally, Kdis extracted from calibration curves by intersecting the ligand concentration with the middle of the calibration curve linear region, halfway between calibration threshold and saturation. However, need to keep in mind that this description does not apply accurately to the current work as the surface-bound anti-AFP antibodies, at the MNC biosensor sensing area, do not interact with all the AFP molecules present in the 0.5 pF drop, as the surface occupied by the drop is significantly greater than the sensing area. Still, following the conventional definition of Kd, as provided above, a shift in the calibration thresholds presented in Figure 11A directly implies a shift in Kd, and the calibration threshold shift from 105 fM to 10.5 pM suggests a shift from a higher binding affinity to a lower binding affinity, respectively. Figure llB(ii) shows the lack of dependency of the calibration threshold value on VGL- This lack of dependency is expected as VGLaffects solely the electron charge carriers in the silicon, and its effect on the potential of the solid-biological interface is negligible. On the other hand, the dependency of the calibration threshold on VGFis expected, as VGFdetermines ѱ0 and the double layer conditions in terms of pH, ionic strength and electric field, each of these can potentially affect the ligand-receptor interaction.

[0255] A summary of the sensing performance is provided in Figure 12 that discloses Table 2, which presents the dependency of the sensitivity, linearity (R2), dynamic range, and LOD on channel configuration. The sensitivity, which is the slope of the linear fit in units of Readout per decade of concentration (%dec-1), and the LOD, defined as the lowest concentration with an IDSgreater than the average IDSplus three standard deviations of the background diluted serum, are extracted in accordance with the IUPAC conventions [M. Nic, J. Jirat, et al. IUPAC Compendium of Chemical Terminology: Gold Book, IUPAC, Research Triagle Park, NC, 2.1.0., 2009]. The requirements for the presented performances are a dynamic range of at least four orders of magnitude, and R2higher or equal to 0.96. Highest sensitivity of 25.98 Readout / dec, for a dynamic range of 1.05 pM-10.5 nM, was measured for a narrow middle channel (VGF=-0.5 V, VGL=-0.6 V). Note that the sensitivity exhibits a maxima behavior in terms of VGF(the sensitivity decreases for VGFsmaller or greater than -0.5 V), and it is always higher for a narrow middle channel (small VGLvalues). Therefore, VGF=-0.5 V provides the optimal double layer in terms of sensitivity, and VGL=- 0.6 V provides the most efficient transduction of the surface potential, induced by the biological interactions, to an electronic signal. On the other hand, VGF=0.5 V provides the highest dynamic range from 105 fM to 10.5 nM, and, similar to the other VGFvalues, lower VGLvalues conclude an enhanced sensitivity.

[0256] The dependency of the sensing performance on channel configuration is shown. The criteria for channel configuration selection are R2≥0.96 and a dynamic range of at least 4 orders of magnitude. The LOD and the dynamic range are extracted in accordance with the IUPAC definition [M. Nic, et al. IUPAC Compendium of Chemical Terminology: Gold Book, IUPAC, Research Triagle Park, NC, 2.1.0., 2009].

[0257] It should be noted that 105fM, 1.05pM, 10.5pM, 105pM, 1.05nM, and 10.5nM AFP corresponds to lOpg / ml, lOOpg / ml, Ing / ml, lOng / ml, lOOng / ml and Ipg / ml AFP, respectively.

Claims

CLAIMS:

1. A bio-transistor system comprising: a. at least one transistor unit comprising: i. at least one channel; ii. source and drain electrodes; iii. at least one gate electrode; and iv. at least one active region located in proximity to the channel region and carrying at least one affinity moiety, each of said affinity moiety is specific for a target molecule; said at least one active region is configured for accepting at least one sample; and v. at least one additional electrode positioned to be in electrical contact with the sample; and b. a control system comprising at least one processor and memory circuitry, wherein said control system is configured and operable for performing one or more measurements of the sample, wherein each measurement comprises maintaining a selected electric potential on the at least one additional electrode, and determining current transmission profile through the at least one channel with respect to potential variation of said at least one gate electrode; said control unit thereby configured to determine data on the presence and / or quantity of one or more said target molecule / s in the sample.

2. The bio-transistor system of claim 1 , wherein the control system is configured to performed two or more measurements utilizing two or more different selected electric potentials applied to the at least one additional electrode.

3. The bio-transistor system of claim 1 or 2, wherein said control system comprises pre-stored calibration data comprising data on electric transmission through the channel for given gate electrode potential with respect to one or more selected electric potentials applied to the at least one additional electrode.

4. The bio-transistor system of any one of claims 1 to 3, wherein said control system comprises pre-stored calibration data comprising data on electric transmission trough the channel with respect to variation of the gate electrode potential.

5. The bio-transistor system of any one of claims 1 to 4, comprising a plurality of two or more transistor units comprising respective plurality of two or more active regions carrying two or more different or identical types of affinity moieties.

6. The bio-transistor of any one of claims 1 to 5, wherein the active region is separated from the channel region by an electrical insulator layer.

7. The bio-transistor system of any one of claims 1 to 6, wherein the at least one gate electrode is electrically insulated from the active region.

8. The bio-transistor system of any one of claims 1 to 7, wherein the control system comprises at least one electrical circuit coupled vie electrical connection to the transistor unit and configured to provide selected electric potentials to electrodes of the transistor unit.

9. The bio-transistor system of any one of claims 1 to 8, wherein said affinity moiety comprises at least one of: a nucleic acid-based molecule, an amino acid-based molecule, a small molecule, a carbohydrate-based molecule, a lipid-based molecule or any combination thereof, and wherein each of said at least one affinity moiety specifically binds said target molecule in said sample.

10. The bio-transistor system of any one of claims 1 to 9, wherein said at least one affinity moiety comprising, or is derived from a component of an affinity pair, said affinity pair is at least one of: proteineous factor-specific nucleic acid binding site, aptamer- aptamer target, antibody-antigen, enzyme-substrate, receptor-ligand, or any combination thereof.

11. The bio-transistor system of claim 1 to 10, wherein said target molecule comprises at least one of: an amino acid-based molecule, a small molecule, a nucleic acid-based molecule, a carbohydrate-based molecule, a lipid-based molecule or any combination thereof.

12. The bio-transistor system of any one of claims 1 to 11 , wherein said target molecule comprises, or is derived from a component of an affinity pair, said affinity pair comprises at least one of: proteineous factor-specific nucleic acid binding site, aptamer- aptamer target, antibody-antigen, enzyme-substrate, receptor-ligand, or any combination thereof.

13. The bio-transistor system of any one of claims 1 to 12, wherein said affinity moiety comprises at least one nucleic acid sequence comprising at least one binding site andwherein said target molecule comprises at least one proteineous factor that specifically recognizes and binds said nucleic acid binding site.

14. The bio-transistor system of claim 13, wherein said proteineous factor is involved in and / or catalyzes at least one of: nucleic acid synthesis, replication, transcription, translation, correction and / or editing.

15. The bio-transistor system of claim 14, wherein said proteineous factor is involved in nucleic acid replication.

16. The bio-transistor system of claim 15, wherein said proteineous factor is at least one polymerase.

17. The bio-transistor system of claim 16, wherein said proteineous factor is at least one primase and wherein said affinity moiety comprises nucleic acid sequence comprising at least one primase biding site.

18. The bio-transistor system of claim 14, wherein said proteineous factor is at least one transcription factor.

19. The bio-transistor system of any one of claims 1 to 18, wherein said at least one sample is a biological sample, an environmental sample and / or a screening test sample.

20. The bio-transistor system of claim 19, wherein said at least one sample further comprises at least one candidate compound that modulates the interaction between said affinity moiety and said target molecule, wherein said candidate compound comprises at least one of: an amino acid-based molecule, a nucleic acid-based molecule, a small molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof.

21. The bio-transistor system of claim 19, wherein said affinity moiety comprises a nucleic acid sequence comprising at least one primase binding site, said target molecule is a primase, and wherein the sample is a screening test sample comprising said primase and a candidate compound.

22. The bio-transistor system of any one of claims 1 to 21, wherein said target molecule is associated with at least one pathologic disorder in a subject.

23. The bio-transistor system of claim 22, wherein said pathologic disorder is at least one of: a disorder caused by a pathogenic agent, a proliferative disorder, an inflammatory disorder, a metabolic disorder, a neurodegenerative disorder, and an autoimmune-disorder.

24. The bio-transistor system of claim 23, wherein said pathogenic agent is at least one of bacteria, archaea, virus, fungi, algae, parasite, protists, and worms.

25. The bio-transistor system of claim 24, wherein said pathogenic agent is bacteria and wherein said target molecule is bacterial primase.

26. A battery comprising two or more of the bio-transistor system as defined in any one of claims 1 to 25.

27. A method for determining presence and / or quantity of at least one target molecule in at least one sample, comprising: a. contacting the at least one sample with a bio-transistor having an active region carrying at least one affinity moiety, or a battery comprising at least two of said bio- transistors, wherein each affinity moiety is specific for a target molecule; b. performing one or more measurements, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; and c. processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample and determining presence and / or quantity of said target molecule / s in accordance with pre-stored calibration data.

28. The method of claim 27, wherein the bio-transistor comprising: i.at least one channel; ii. source and drain electrodes; iii.at least one gate electrode; and iv.at least one additional electrode positioned to be in electrical contact with the sample; wherein the at least one active region is located in proximity to the channel region, separated from the channel region by an electrically insulating layer, and wherein the control unit thereby configured to determine data on presence and / or quantity of said target molecule / s in the sample.

29. The method of claim 27 or 28, wherein said performing one or more measurements comprises performing two or more measurements, wherein each measurement comprises applying respective selected different potential on the sample.

30. The method of any one of claims 27 to 29, wherein said bio-transistor is as defined by any one of claims 1 to 25.

31. A screening method for identifying a compound that modulates the interaction of at least one affinity moiety with a target molecule in at least one sample, comprising: a. contacting said at least one sample with a bio-transistor system, in the presence and the absence of at least one candidate compound, said bio-transistor having an active region carrying at least one affinity moiety, wherein each affinity moiety is specific for a target molecule; b. performing one or more measurements for each sample, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; and c. processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample; d. determining presence and / or quantity of the one or more target molecules in accordance with pre-stored calibration data, thereby determining a target molecule value for said sample in the presence of said candidate compound, and a target molecule value in the absence of said candidate compound; and e. determining that said candidate compound is a modulator of the interaction between the affinity moiety and the target molecule, if the target molecule value obtained for said sample in the presence of said candidate compound is different from the target molecule value obtained in the absence of said candidate compound.

32. The screening method of claim 31, wherein the bio-transistor comprising: i. at least one channel; ii. source and drain electrodes; iii. at least one gate electrode; andiv. at least one additional electrode positioned to be in electrical contact with the sample; wherein the at least one active region is located in proximity to the channel region, separated from the channel region by an electrically insulating layer, and wherein the control unit thereby configured to determine data on presence and / or quantity of one or more target molecules in the sample.

33. The screening method of claim 31 or 32, wherein said performing one or more measurements comprises performing two or more measurements, wherein each measurement comprises applying respective selected different potential on the sample.

34. The screening method of any one of claims 31 to 33, wherein said bio-transistor is as defined by any one of claims 1 to 25.

35. The screening method of any one of claims 31 to 34, wherein said at least one affinity moiety comprises at least one of: a nucleic acid-based molecule, an amino acid- based molecule, a small molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof, and wherein each of said at least one affinity moiety specifically binds a target molecule of said at least one target molecules in said sample.

36. The screening method of any one of claims 31 to 35, wherein said affinity moiety comprises, or is derived from a component of an affinity pair, said affinity pair is at least one of: proteineous factor-specific nucleic acid binding site, aptamer-aptamer target, enzyme-substrate, receptor-ligand, or any combination thereof.

37. The screening method of claim 31 to 36, wherein said target molecule comprises at least one of: an amino acid-based molecule, a small molecule, a nucleic acid-based molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof.

38. The screening method of any one of claims 31 to 37, wherein said target molecule comprises, or is derived from a component of an affinity pair, said affinity pair comprises at least one of: proteineous factor-specific nucleic acid binding site, aptamer- aptamer target, antibody-antigen, enzyme-substrate, receptor-ligand, or any combination thereof.

39. The screening method of any one of claims 31 to 37, wherein said candidate compound comprises at least one of: a small molecule, an amino acid-based molecule, anucleic acid-based molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof.

40. The screening method of any one of claims 31 to 39, wherein said affinity moiety comprises at least one nucleic acid sequence comprising at least one binding site and wherein said target comprises at least one proteineous factor that specifically recognizes and binds said nucleic acid binding site.

41. The screening method of claim 40, wherein said proteineous factor is involved in and / or catalyzes at least one of: nucleic acid synthesis, replication, transcription, translation, correction and / or editing.

42. The screening method of claim 41, wherein said proteineous factor is involved in nucleic acid replication.

43. The screening method of claim 42, wherein said proteineous factor is at least one polymerase.

44. The screening method of claim 43, wherein said proteineous factor is at least one primase and wherein said affinity moiety comprises at least one primase biding site.

45. The screening method of claim 41 , wherein said proteineous factor is at least one transcription factor.

46. The screening method of any one of claims 31 to 45, wherein said at least one sample is a biological sample, an environmental sample and / or a screening test sample.

47. The screening method of any one of claims 31 to 46, for screening of a compound that inhibits the interaction of said affinity moiety with said target molecule.

48. The screening method of any one of claims 31 to 47, wherein said target molecule is associated with at least one pathologic disorder in a subject.

49. The screening method of claim 48, wherein said pathologic disorder is at least one of: a disorder caused by a pathogenic agent, a proliferative disorder, an inflammatory disorder, a metabolic disorder, a neurodegenerative disorder and an autoimmune-disorder.

50. The screening method of claim 49, wherein said pathogenic agent is at least one of bacteria, archaea, virus, fungi, algae, parasite, protists, and worms.

51. The screening method of claim 50, wherein said pathogenic agent is bacteria and wherein said target molecule is bacterial primase.

52. The screening method of claim 51 , wherein said affinity moiety comprises a nucleic acid sequence comprising at least one primase binding site, said target molecule is a primase, and wherein the method is for screening a compound that inhibits the interaction of primase to its nucleic acid binding site.

53. The screening method of claim 52, wherein said compound that inhibits the interaction of primase to its nucleic acid binding site is an anti-bacterial agent.

54. The screening method of any one of claims 31 to 53, for identifying anti-bacterial compounds.

55. A diagnostic method for determining a physiological and / or environmental condition or state of a subject and / or a media and / or a habitat, comprising: a. contacting the at least one sample with a bio-transistor having an active region carrying at least one affinity moiety, or a battery comprising at least two of said bio- transistors, wherein each affinity moiety is specific for a target molecule; b. performing one or more measurements, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; c. processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample; d. determining presence and / or quantity of the one or more target molecules in accordance with pre-stored calibration data, thereby obtaining a target molecule value for said sample; and e. determining that the subject and / or media and / or habitat display said physiological and / or environmental condition or state, if the at least one target molecule value obtained for said sample in step (d), is positive or negative with respect to a reference target molecule value pre-determined for said physiological and / or environmental condition or state, or with respect to a target molecule value determined for at least one control sample.

56. The diagnostic method of claim 55, wherein said bio-transistor system is as defined in any one of claims 1 to 25.

57. The diagnostic method of claim 55 or 56, wherein said physiological state and / or condition of a subject comprises pathological condi tion / s and / or health condi tion / s in said subject.

58. The diagnostic method of claim 57, wherein said pathological condition is at least one immune-related disorder, said immune-related disorder is an infectious disease caused by a pathogenic agent.

59. The diagnostic method of claim 55 to 58, wherein said target molecule is associated with at least one pathologic disorder in a subject.

60. The diagnostic method of claim 59, wherein said affinity moiety comprises a nucleic acid aptamer specific for said target molecule.

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