Patent Application
20230236147
The present invention generally relates to the field of sensing devices and methods for quantifying analytes. Specifically, the present invention relates to sensors based on field-effect transistors comprising semiconducting two-dimensional nanosheets, methods of preparation thereof and methods for determining the concentrations of target analytes in a liquid sample, such as a biological sample.
Electrochemical sensors have become an important topic in the field of applied research as well as in medical and biological applications, due to their advantages over classical analytical methods including ease of operation, low cost and compact size. Biosensors are a subgroup of chemical sensors comprising biological host molecules as recognition elements coupled to a chemical or physical transducer. Highly selective biosensors present a powerful tool for real-time measurement of a variety of analytes for a wide range of applications such as food safety, environmental monitoring, drug screening and diagnosis.
These sensors may comprise recognition elements, capable of specifically and selectively detecting or measuring the amount of a given analyte. Among these recognition elements are molecules with a biological function, such as enzymes, antibodies, aptamers and the like, which provide an advantageous alternative to the detection of specific analytes, owing to their availability and biocompatibility. In particular, the variety of enzymes and enzymatic cascade reactions makes these recognition elements applicable to a wide range of analytes.
Two-dimensional (2D) semiconducting materials such as graphene, hexagonal boron nitride (h-BN) and black phosphorus have been attracting increasing interest during the last years due to their physicochemical, optical and electronic properties. The use of these materials for diagnostics applications is promoting overcome the current boundaries of sensitivity, response time and sample processing. In particular, graphene, a sheet of hexagonally arranged carbon atoms, is one of the most attractive 2D materials for biosensing devices due to its high conductivity, large specific area and great chemical stability.
Biosensors comprising graphene transistors are described in the patent literature, such as in US 2013/306934 A1, US2016/025675 A1, US 2016/334399 A1 and WO 2019/231224 A1. While graphene has distinct conductive properties that provide an advantageous detection sensitivity, the materials often present high heterogeneity, resulting in variable physical properties such as transconductance, minimal current, maximal resistance and threshold voltage. This results in decreased repeatability of sensor fabrication methods and lower sensor stability, features that must be improved for the use of graphene transistors as reliable sensors in medical diagnostics. Further disadvantages of the devices of the prior art are: i) the lack of a biosensor based on liquid-gated reduced graphene oxide field-effect transistors (rGO-FETs) having an arrangement suitable for detection using small volume samples; ii) the lack of a biosensor based on rGO-FETs with a gate electrode adapted for stable and reproducible measurements; iii) the lack of a biosensor based on rGO-FETs comprising a nanoarchitecture suitable for the immobilization of a broad number of recognition elements without loss of their biological activity and having anti-fouling properties; iv) the lack of a biosensor based on rGO-FETs comprising a highly stable nanoarchitecture suitable for the non-covalent immobilization of recognition elements to the semiconducting transistor channel; v) the lack of a method of preparation for multiple and coplanar rGO-FETs, for multiple analyte diagnostics; vi) the lack of a rGO-FET suitable for being post-modified with microfluidic layers for Lab-on-a-Chip (LoC) measurements.
Lastly, direct covalent attachment of recognition elements to 2D semiconducting materials induces lattice defects that hinder charge transport and, thus, impair the signal transduction. For example, graphene covalent functionalization is accomplished using “oxidative” defects, such as carboxylates or hydroxyl groups (see, e.g., S. Niyogi, E. Bekyarova, M. E. Itkis, H. Zhang, K. Shepperd, J. Hicks, M. Sprinkle, C. Berger, C. N. Lau, W. A. Deheer, E. H. Conrad, R. C. Haddon, Spectroscopy of covalently functionalized graphene, Nano Lett. 10 (2010) 4061-4066. https://doi.org/10.1021/n11021128). The higher the degree of defects present, the less charge transport and sensor sensitivity will be observed in a biosensing device. Therefore, it would be advantageous that the attachment of recognition elements to 2D semiconducting materials for biosensing devices is done by non-covalent, i.e. supramolecular approaches.
There is therefore a need to provide a sensor based on transistors of 2D semiconducting materials having improved reproducibility, increased stability and sensitivity that requires only a small volume of biological sample and that is suitable for multiple analyte (multiplex) diagnostics and post-modification with microfluidic layers for LoC measurements. In addition, there is a need to provide sensors comprising a nanoarchitecture suitable for the immobilization of a large number of recognition elements without loss of their biological activity and having antifouling properties, wherein the nanoarchitecture further amplifies the specific sensor signal. Furthermore, there is a need to provide sensors comprising a highly stable nanoarchitecture suitable for the non-covalent immobilization of recognition elements to the semiconducting transistor channel. Lastly, there is a need to provide a portable system for measuring the concentration of a specific analyte in a biological sample using the provided sensor.
The present invention offers a solution to the shortcomings of the prior art by providing a sensor of increased stability, obtained by a pre-treatment of graphene oxide sheets in solution. In addition, the gate, source and drain electrodes of the sensor are coplanar, with the source and drain electrodes being interdigitated. The sensor is provided with an interfacial nanoarchitecture suitable for the immobilization of a broad number of recognition elements without loss of their biological activity. This nanoarchitecture also amplifies the specific sensor signal. Further, the sensor is provided with a polymeric coating that allows for the elimination of signals that are non-specific to a target analyte, such as those due to ionic strength, temperature, interferents, etc.
With this configuration, measurements for the detection and quantification of molecules of clinical interest can be achieved in sample volumes as low as 1 μL, in a small and simpler-to-use sensor.
Therefore, in a first aspect, the invention provides a sensor comprising a field-effect transistor of a semiconducting material, the material being in the form of two-dimensional nanosheets, comprising a source electrode, a drain electrode and a gate electrode, and an interfacial nanoarchitecture comprising a recognition element, a structural element and a polymeric coating, wherein the gate electrode of the transistor is coplanar with the drain electrode and the source electrode of the transistor.
In preferred embodiments of the invention, the material is selected from graphene, reduced graphene oxide, few-layer graphene, twisted bilayer graphene, conducting polymers, transition metal dichalcogenides, black phosphorous, and hexagonal boron nitride. In a further preferred embodiment, the material is reduced graphene oxide (rGO).
Optionally, the sensor comprises a second field-effect transistor comprising a polymeric coating. Further, the sensor may optionally comprise more than two field-effect transistors on the same substrate. Preferably, the field-effect transistors are reduced graphene oxide field-effect transistors.
In an embodiment, the recognition element is located at a distance up to 100 nm from a semiconducting material surface of the transistor. The recognition element is held immobilized by the structural element of the interfacial nanoarchitecture. The structural element is attached to a semiconducting two-dimensional nanosheet by one or more supramolecular binding-points. In a preferred embodiment, the semiconducting material is reduced graphene oxide.
Preferably, the source and drain electrodes of the transistor are interdigitated electrodes. In preferred embodiments, the electrodes are made of a conductive material selected from gold, platinum, graphite, silver, conducting polymers and combinations thereof.
In a preferred embodiment, the gate electrode is made of a conductive material selected from gold, platinum, graphite, silver, conducting polymers and combinations thereof and comprises a coating of Ag/AgCl.
In preferred embodiments, the recognition element is a substance selected from an enzyme, an antibody, an aptamer, clustered regularly interspaced short palindromic repeats (CRISPR) with a CRISPR associated protein (Cas), an ion-selective molecule, a high-affinity binding-protein and combinations thereof. Preferably, the substance is selected from urease, acetylcholinesterase, creatinine deiminase, streptavidin, avidin, valinomycin, tridodecylamine, an antibody or aptamer capable of binding an analyte selected from the group consisting of ferritin, Interleukin 6 (IL-6), SARS-CoV-2 spike protein, SARS-CoV-2 nucleocapsid (N) protein, follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), estradiol, Luteinizing hormone (LH), fragments thereof, and modified fragments thereof.
In a preferred embodiment, the structural element comprises a substance selected from a polyelectrolyte, a polymer, a cross-linker, a heterofunctional nanoscaffold and combinations thereof. Preferably, the heterofunctional nanoscaffold comprises a substance selected from vinylsulfonated-polyamine (VS-PA), streptavidin, avidin and combinations thereof.
Preferably, the polymeric coating comprises a substance selected from polyethylene-glycol (PEG), a polyethylene-glycol derivatized polymer, a substance comprising polyethylene-glycol, a zwitterionic polymer, a fluoropolymer, a hydrogel and combinations thereof.
In a second aspect, the invention provides a system comprising:
Preferably, the calculating means for calculating the concentration comprise an algorithm for processing a sensor response and/or eliminating means for eliminating interfering signals.
The system preferably comprises an interface for displaying the value of the concentration of the target analyte in the biological sample.
In a third aspect, the invention provides a method for preparing a sensor comprising field-effect transistors, the method comprising the steps of:
In preferred embodiments of the third aspect, the invention provides a method for preparing a sensor comprising reduced graphene oxide field-effect transistors, the method comprising the steps of:
In a fourth aspect, the invention provides a method for detecting an analyte in a biological sample using the system according to the second aspect, comprising the steps of:
The invention further provides a method for indirectly detecting an analyte in a liquid sample using the system according to the second aspect, comprising the steps of:
In preferred embodiments, the enzyme-labeled secondary recognition element is dissolved in the liquid sample.
In preferred embodiments of all the aspects below, the nanoarchitechture is such that a recognition element is located at a distance up to 100 nm from a semiconducting material surface of the transistor. Preferably, a structural element is attached to the semiconducting material by one or more supramolecular binding-points.
In a fifth aspect, the invention provides a method for characterizing a recognition element by studying its interactions with ligands using the system according to the second aspect, the method comprising the steps of:
In preferred embodiments of this fifth aspect, the method comprises the steps of:
In other preferred embodiments of this fifth aspect, the method comprises the steps of:
In a sixth aspect, the invention provides a method for preparing heterofunctional nanoscaffolds attached to a semiconducting two-dimensional nanosheet surface by one or more supramolecular binding-points, the method comprising the steps of:
In preferred embodiments, the polymer containing primary amines is selected from polyallylamine, polyethyleneimine, polybutenylamine, polylysine and polyarginine, and copolymers thereof.
The invention will be described in further detail below, with reference to the accompanying figures and by means of non-limiting examples.
The term “sensor” as used herein relates to a device that can be used to detect or measure a physical property, e.g. to qualitatively and quantitatively determine the amount of a specific compound in a solution of interest. The sensors provided herein comprise transistors, which amplify an electronic signal related to the presence and quantity of the specific compound.
The term “semiconducting two-dimensional nanosheets” as used herein relates to a nanostructured material having semiconducting properties and a thickness in a scale ranging from 0.3 to 100 nm from which a FET may be manufactured. Non-limiting examples of these materials include substances as graphene, reduced graphene oxide (rGO), few-layer graphene, twisted bilayer graphene, conducting polymers, transition metal dichalcogenides, black phosphorous, and hexagonal boron nitride (h-BN). The terms “semiconducting material” and “semiconductor” may be used interchangeably in the present disclosure.
As used herein, the term graphene oxide (GO) relates to a material produced by oxidation of graphite by methods well known by one of ordinary skill in the art. Reduced graphene oxide (rGO) is the form of GO that is processed by chemical, thermal and other methods in order to reduce its oxygen content, as described in further detail below.
A field-effect transistor (FET) is a type of transistor which uses an electric field to control the flow of current. FETs comprise at least three electrodes or terminals: source, gate, and drain. FETs control the flow of current by applying a voltage to the gate electrode, which in turn alters the conductivity between the drain and source electrodes.
Consequently, the terms “field-effect transistor comprising semiconducting two-dimensional nanosheets” and “reduced graphene oxide field-effect transistor (rGO-FET)” as used herein relate respectively to a FET that comprises a semiconducting material channel, the semiconducting material being in the form of two-dimensional nanosheets and to a FET that comprises a reduced graphene oxide channel as described in further detail below.
The term “interfacial nanoarchitecture” refers to arrangements of at least a recognition element, a structural element and a polymeric coating, forming the interface between the sensor and the medium to be sensed and having a nanometric characteristic length, which can be obtained by one of the nanoconstruction techniques described in further detail below. The term “multivalent heterobifuctional nanoscaffold” relates to a specific interfacial nanoarchitecture obtained as described in further detail below.
As used herein, the term “supramolecular binding-point” refers to a non-covalent bond mediated by one or more of the following forces: hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions and electrostatic interactions.
The invention provides sensors comprising rGO-FETs that use a very low sample volume, in the range of about 1 μL to about 5000 μL, preferably 1 μL to about 1000 μL. To this end, aside from the transistors, the sensors further comprise a substrate as well as a receptacle for the solution to be analyzed that comprises the target molecule or analyte, e.g. a liquid sample, such as biological sample obtained from a patient.
The substrate may be made of a material suitable for supporting the other sensor components, with non-limiting examples including glass, silicon (Si) and silicon dioxide (SiO2)
The sample may be generally a liquid sample or a biological sample, such as blood, blood serum, saliva, urine and the like. These biological substances contain certain analytes that might be of interest in the diagnosis and treatment of a physiological or psychological condition in a patient.
The invention further provides a system and a method for the instantaneous measurement of a target analyte concentration in a solution, e.g. a liquid or biological sample. The system can be considered an online front-back-end laboratory (OLFBE-LAB).
The system provided by the invention can comprise the modules that can be observed in
As previously mentioned, in an embodiment of the present invention, the bioFET sensor comprises at least three contact terminals or electrodes and a receptacle for the solution to be analyzed. The concentration of a target analyte modifies the transfer curve or FET response of the bioFET sensor, from which the concentration can be quantified. The bioFET and its receptacle are configured to achieve measurements using between 1 and 1000 μL of the solution comprising the target analyte.
The FETmeter provides energy to the bioFET sensor by providing a combination of drain and source tensions and gate-source tensions that are controlled by software. By measuring the output current between the drain and the source electrodes, a “transfer curve” or a “FET response” can be obtained, which is dependent on the target analyte concentration. The FETmeter comprises means for transferring data to an operator terminal, e.g. a wireless network to transfer the data to a portable computer, a tablet, a smartphone or the like. The FETmeter may use any type of wireless, or personal area network to directly transfer any data, including but not limited to the raw data and/or the processed data, to an operator terminal which may include a personal computer, a laptop computer, a PDA, smartphone, tablet or the like. The FETmeter may also transfer any data, including but not limited to the raw data or the processed data, to any cloud-based storage service via wireless connection, either using an integrated WiFi chip, an integrated Internet of things (IoT) chip, or a mobile carrier SIM card integrated into the design.
In a still preferred embodiment of the invention, the FETmeter itself may also act as an operator terminal by displaying the data and the controls on a display, such as a LED, LCD, AMOLED display or the like, optionally comprising tactile capabilities to allow the user to interact with the device.
The FETmeter casing is purposefully designed to accommodate a bioFET at a specific location within the casing. In an embodiment of the invention, the bioFET rests at an indentation specifically designed for this purpose, allowing the bioFET to be electrically and chemically separated from any interfering stimuli.
An algorithm based on signal acquisition, processing, analysis, and curve fitting, yields the value of the target analyte concentration starting from the obtained transfer curves or from other curves of the FET response. The main “landmarks” that can be obtained from the FET response comprise the transconductance, the Dirac point (also called the charge neutrality point, CNP), the minimum current of the transfer curve (or the maximum resistance), the current between the drain and the source electrodes (Ids), among others.
The operator terminal, e.g. a smartphone, tablet, or the like, contains the clinical history of the patient and can eventually transfer relevant data to the patient's physician.
The clinical data can be stored either within the operator terminal or within a HCI such as in a cloud-based storage service and can be transferred by the patient to the patient's physician either in its raw format, as a spreadsheet, or as a PDF file containing either the latest measurements or the entire history of measurements.
The bioFET sensor provided by the invention comprises a FET, as illustrated in
With regards to
The FET is completed by bathing, i.e. being immersed in an electrolytic solution, since the electrolyte acts as an electrical connection between the gate terminal and the graphene.
In an embodiment of the present invention, in order to render the graphene sensitive to the precise detection of a specific target analyte, the graphene is modified by means of a recognition element, e.g. substance complementary to the target analyte. The electric current between the drain and source terminals will be a function of the number of analyte molecules coupled to the complementary substance.
In order to render the bioFET less sensitive to variables exogenous to the analyte determination objective variables, a dual-bioFET can be provided, as seen in
With reference to
A schematic representation of the analog module of the FETmeter can be seen in
In order to obtain the FET transfer curve or the FET response, used to extract the data to enter in the algorithm that calculates the target analyte concentration, the FETmeter excites the gate terminal with a controlled voltage by the microprocessor, in accordance with the software.
The FET transfer curve is obtained by measuring the current between the source and drain electrodes (i.e., the current flowing through the rGO) while the voltage between the gate and the source electrodes is swept using a potential function and the voltage between the drain and the source electrodes is maintained constant. FET responses can be obtained with the FETmeter by applying potential functions to the sensor. The FETmeter records the generated current in the drain-source circuit and in the source-gate circuit, which by means of an analog-to-digital converter (ADC) enters the microprocessor that uses the calculation algorithm to calculate the target analyte concentration.
In the dual-FET configuration, according to the present invention, a controlled voltage is applied between the drain terminals and the source terminals and the currents generated in the main circuits are measured in each of the elements. These signals are recorded by means of ADCs that enter the microprocessor, which applies the exogenous variable cancellation algorithm, and in turn applies the target analyte concentration calculation algorithm.
In the multi-FET configuration comprising n FETs (n=1, 2, 3, . . . ), analog multiplexers controlled by the microprocessor may be provided to commutate the gate and source terminals of each transistors, in order to sequentially measure the currents of each FET, from FET1 to FETn. Such an arrangement is illustrated in
The sweeping voltages in order to obtain the bioFET transfer curve is a continuous voltage in the range of 0.1 to 1000 mV, preferably 100 mV between the drain and source terminals, and a ramp between the gate and the source terminals, as can be seen in
As can be seen in
In order to achieve increased symmetry in the measurement process, the polarity of the drain and source voltage and of the gate voltage are reversed as shown in
Another available tool is the sequential disconnection of each of the dual-FET elements, in order to cancel out the crossing currents, as illustrated in
With the disconnection of each terminal, the electric potential of the contact interphases can be measured, which are considered by the algorithm in order to cancel out said electric potentials during calculation of the target analyte concentration.
The invention further provides a method to prepare a sensor comprising rGO-FETs.
As a first step, a graphene oxide solution is homogenized by means of a two-stage centrifugation process. In this step, a graphene solution with a concentration in the range of 1 to 4000 μg/mL, preferably 80 μg/mL in distilled water is brought to a pH above 2 by the addition of an alkali compound. The solution is sonicated for about 10 min and then centrifuged at 100 to 2000 rpm, preferably 600 rpm for about 90 min. Once separated, the supernatant is then centrifuged at 4000 to 14000 rpm, preferably 8000 rpm for about 15 min. The supernatant for the latter centrifugation process is discarded and the precipitate or sediment is brought to the initial solution volume using ultrapure water. The resulting solution is sonicated for about 15 min.
Subsequently, the gate terminal, i.e. an Ag/AgCl electrode is prepared in the same plane as the drain and source terminal, i.e. all three terminals or electrodes are coplanar. Since the FETs are “liquid-gated”, the all-coplanar FET design advantageously allows the miniaturization of the sensor, as well as the use of a reduced sample volume.
The gate electrode may be prepared by electroplating or by inkjet printing using inks comprising Ag/AgCl nanoparticles.
In the electroplating process, an electro-reduction potential of −10 to −4000 mV, preferably −130 mV is applied between the gate terminal and an Ag0 wire, while simultaneously an electro-oxidation potential of 0 to 4000 mV, preferably 400 mV is applied between the interdigitated electrodes and the Ag0 wire for about 12 minutes in an electrolytic solution comprising Ag2SO4.
According to the present invention, the FETs are prepared from reduced graphene oxide and substrates comprising glass, Si or SiO2 and having coplanar gate, drain and source terminals or electrodes made of a conductive material selected from gold, platinum, graphite, silver and combinations thereof, wherein the drain and the source electrodes are interdigitated, as described above.
Preparation of the FETs comprises incubating the substrates comprising the gate, drain and source electrodes, i.e. the chips, in a solution of (3-aminopropyl)triethoxysilane (APTES) in an organic solvent at a concentration of 0.1% to 10%, preferably 2% during about 1 hour. The APTES-modified chips are then further incubated in the GO solution, prepared as indicated above, for about 1 hour. The obtained chips are washed with deionized water in order to remove GO in excess.
The GO layers thus deposited onto the chip surface are subsequently reduced by exposure to hydrazine vapors at a temperature between 50 and 120° C., preferably 80° C., for about 12 hours in a closed recipient.
The chip is then placed in a stove at a temperature of 50° C. to 500° C., preferably 200° C., for about 2 hours.
Subsequently, a partial oxidation of the Ag0 gate electrode to form AgCl is carried out by electroplating in a chloride containing solution, e.g., a NaCl solution at 3 M or a KCl solution at 3 M, or the like, and by applying an oxidation potential of about 150 mV (versus an Ag/AgCl reference) to the Ag0 gate electrode, while a reduction voltage of about −100 mV (versus an Ag/AgCl reference) is applied to the interdigitated electrodes for about 60 seconds.
The steps above are carried out for single-FET, dual-FET and multi-FET sensor configurations as described above, prepared from substrates comprising coplanar gate, drain and source electrodes, wherein the drain and source electrodes are interdigitated electrodes.
Each of the electroplating and the partial-oxidation processes can be carried out using the FETmeter device to obtain single-FET, dual-FET and multi-FET sensors with the Ag/AgCl coating.
An interfacial nanoarchitecture is prepared over the rGO-FET, as illustrated for example in
As illustrated by Examples 2 and 3 below, advantageous results of immobilizing the recognition element through a structural element which is attached to a semiconducting nanosheet by one or more supramolecular binding-points include: i) supramolecular bonds do not disrupt the band structure or the chemical structure of the semiconducting two-dimensional nanosheet. Thus, the semiconducting properties of the FET sensor are not deteriorated, and the sensor retains its maximum charge carrier mobility, transconductance and sensitivity. ii) Multivalent supramolecular interactions between the structural element and the semiconducting nanosheet ensure a nanoarchitecture with high stability. This feature enables a better reproducibility and the regeneration of the biosensor surface for its reuse.
According to the present invention, the step of providing this interfacial nanoarchitecture comprises the surface modification of the FET with the recognition element, the structural element, and the antifouling polymer layer, by employing nanoconstruction techniques. Layer-by-layer assembly, self-assembly, polymer salt-complex, spin-coating, drop-casting are examples of the nanoconstruction techniques based non-covalent binding that can be used for the preparation of the interfacial nanoarchitecture of the invention.
Unexpectedly, surprisingly improved sensing features can be obtained by a synergistic effect between the interfacial nanoarchitecture and the FET. This effect is illustrated in Example 3 below, which describes a polyelectrolyte/enzyme nanoarchitecture prepared onto the rGO-FET for the sensing of urea, resulting in an improved sensitivity of the biosensor due to the synergy between the functionality of the polyelectrolyte used in the nanoarchitecture and the field-effect of the sensor. This synergistic effect is also illustrated in Examples 4 and 5, which describe a multivalent heterofunctional nanoscaffold to immobilize antibodies onto rGO-FET for the sensing of antigens, resulting in improved sensitivity, stability and with diminished non-specific adsorption.
For interfacial nanoarchitectures onto rGO-FETs prepared using the layer-by-layer nanoconstruction technique, in an embodiment, this step comprises submerging rGO-FETs obtained in the previous steps into a solution of a charged pyrene molecule, preferably 1-pyrenesulfonate, in dimethylformamide (DMF) at a concentration between 0.1 and 10 mM, preferably 3 mM. A polyelectrolyte is then assembled by submerging the rGO-FET into a solution of the polyelectrolyte, preferably selected from polyethyleneimine (PEI), poly(allylamine hydrochloride) (PAH) or poly(diallyldimethylammonium chloride) (PDADMAC) at a concentration between 0.05 and 50 mg/mL, preferably 2 mg/mL for about 10 minutes, with subsequent washing using distilled water.
The rGO-FET modified with the polyelectrolyte is then submerged into a solution comprising the recognition element for about 30 min and washed with distilled water. In the case of an enzyme solution, the concentration may be between 0.05 and 50 mg/mL, preferably 1 mg/mL. This step promotes the adsorption of the recognition element onto the rGO surface.
The recognition element may be chosen according to its complementarity with the target analyte to be detected or measured. For example, the enzyme urease may be used as the recognition element for the analyte urea, an antibody can be used as the recognition element for a corresponding antigen, etc. In different embodiments of the present invention, other recognition elements suitable for the preparation of the interfacial nanoarchitecture on the rGO-FET comprise aptamers, clustered regularly interspaced short palindromic repeats (CRISPR) with a CRISPR associated protein (Cas) and ionophores.
These last steps using the polyelectrolyte and recognition elements can be repeated in alternation to increase the surface concentration of the recognition element. Preferably, the finished assembly comprises a polyelectrolyte outermost layer.
As an alternative embodiment, the interfacial nanoarchitecture is obtained by modifying the graphene channel or the gate electrode of the rGO-FET with a coating comprising an ion-selective membrane. The ion selective membrane comprises a combination of an ion-selective molecule (ionophore) as the recognition element, a hydrophobic electrolyte, a plasticizer and a high molecular weight hydrophobic polymer as the structural element. This modification can provide a sensor for detection of ions such as K+, Cl−, Ca2+, PO43− and the like. For the preparation of a potassium-selective membrane, an ion-selective cocktail comprising o-nitrophenyl octyl ether (o-NPOE) (65.88%), polyvinyl chloride (PVC) (33%), potassium ionophore I, also named valinomycin, (1%), and potassium tetrakis (p-chlorophenyl) borate (KTPCIB) (0.22%) in 2.5 mL of Tetrahydrofuran (THF) can be employed. The ion-selective cocktail is used for the modification of rGO-FET by spin-coating, drop-casting, or dip-coating.
In the interfacial nanoarchitecture, the distance of the recognition element to the surface of the rGO can be modified using nanoconstruction techniques such as the layer-by-layer assembly of polyelectrolytes and recognition elements. By employing these techniques, it is possible to locate the recognition elements onto the rGO surface with nanometric precision.
The recognition element can be at a distance of up to 100 nm from the rGO surface. Advantageously, the rGO-FET sensors with recognition elements comprised in an interfacial nanoarchitecture as provided by the present invention have an amplified sensibility compared to those rGO-FET sensors wherein the recognition element is directly linked to the rGO or graphene surface, i.e. without an interfacial nanoarchitecture. Without being bound by any theory, this sensitivity amplification occurs since rGO-FETs sensors transduce any field-effect change onto the rGO interface within a distance in the range of 0 to 100 nm. Therefore, when a target analyte is recognized using the recognition elements comprised in the nanoarchitecture, prepared as described in the present invention, the response and sensitivity are maximized. Moreover, the structural element of the nanoarchitecture may be functional polymers that change its charge when the recognition event occurs, triggering a second effect of signal amplification. On the other hand, when the recognition element is directly linked to the rGO surface, the potential response is not exploited.
As a finishing step, in an embodiment of the invention, the obtained nanoarchitecture comprising the recognition element and the structural element is post-modified by deposition of an antifouling polymer coating. To this end, a solution comprising an antifouling polymer, such as a polyethylene-glycol (PEG), a polyethylene-glycol derivatized polymer, a zwitterionic polymer, a fluoropolymer, e.g., a tetrafluoroethylene-perfluoro-3,6-dioxa-4-methyl-7-octenesulfonic acid copolymer, at a concentration of about 0.5% by weight is dropped over the nanoarchitecture which contains the recognition element and is on the rGO-FET, and subsequently dried at a temperature of about 4° C. An anti-fouling film that covers the nanoarchitecture is thus obtained, thereby enhancing the stability of the deposited layers, as well as preventing the non-specific adsorption of molecules other than the target analyte, that may result in interfering electronic signals.
By constructing the interfacial nanoarchitecture onto the rGO-FET as provided by the present invention, a broad variety of target analytes can be determined. A few non-limiting examples of target analytes that can be quantified are:
The detection process using a FET sensor modified with a nanoachitecture comprising the recognition element is illustrated in
In specific embodiments, the sensor provided by the invention may be obtained by the synthesis of vinylsulfonated-polyamines (VS-PA) scaffolds linked, from one side, to graphene through multivalent π-π interactions with pyrene groups, and, from the other side, to lectins or antibodies (as recognition elements) and PEG (as the antifouling element) through covalent bonds.
These heterobifunctional scaffolds are constructed by three simple surface modifications: i) pyrenebuthanoic acid succinimidyl ester (PBSE) adsorbs onto graphene; ii) polyamines in aqueous solutions react quickly with PBSE, forming a multipoint attached film through pyrene groups; iii) remaining primary amine groups are modified with divinylsulfone (a well-known crosslinker of —SH, —NH2 and OH— groups via Michael type addition) to obtain VS-PA grafted onto graphene.
Non-limiting examples of the polyamines that can be used for the heterobifunctional scaffolds are homopolymers or copolymers of polyallylamine, polyethyleneimine, polybutenylamine, polylysine and polyarginine.As detailed in the examples set forth below, the heterobifunctional interface construction was monitored step-by-step by spectroscopic ellipsometry and contact angle. Vinylsulfonation of polyamines was proved by Raman spectroscopy and, then, its reactivity to hydrophilic molecules containing —SH, —NH2 or OH— groups was demonstrated by contact angle. Furthermore, it is shown by FETs measurements that the VS-PA preparation does not affect the semiconducting propiertes of graphene. Attachment of recognition elements to VS-PA was optimized firstly for concanavalin A (ConA), a model lectin with high affinity to glycans. For the present disclosure, the stability of the interfacial nanoarchitecture in surfactant content media, its antifouling properties and the recognition of a glycoprotein was studied by ellipsometry and surface Plasmon resonance spectroscopy (SPR). Finally, scaffolds containing monoclonal antibodies (mAbs) against SARS-CoV-2 spike protein (mAb-SARS-CoV-2 spike) and human ferritin (mAb-FTH1) were studied by SPR for the specific recognition of their target molecules. KD equilibrium constants and a simple method to reuse the interface are also provided herein.
The method disclosed herein are also used to obtain sensors based on FETs obtained from other semiconducting materials in two-dimensional nanosheets, such as graphene, few-layer graphene, twisted bilayer graphene, conducting polymers, transition metal dichalcogenides, black phosphorous, and hexagonal boron nitride. Due to the similarity of dimensional aspect ratio, semiconducting properties, and molecular conformation properties between these semiconducting materials, the interfacial nanoarchitecture comprising a structural element attached to the semiconducting nanosheets by one or more supramolecular binding-points can be used with any of the previously mentioned semiconducting materials in two-dimensional nanosheets in order to obtain sensors based on FETs. Moreover, the methods described herein for the construction of FET-based sensors with interfacial nanoarchitectures can be applied with to this group of materials, which will be readily apparent to the skilled person. It is important to note that the disadvantages of the devices of the prior art described herein as compared to rGO-FET sensors are also present in the devices of the prior art comprising FET sensors of other semiconducting materials in two-dimensional nanosheets. In concordance, the improvements obtained by the use of the methods described herein for rGO-FET sensors can be also observed for other for other semiconducting two-dimensional nanosheets FET sensors. Thus, the invention provided by the present disclosure solves not only needs for rGO-FET sensors, but also for other semiconducting two-dimensional nanosheets FET sensors.
A rGO-FET sensor provided by the invention, as described above, was prepared using the following compounds and equipment.
The FETs were prepared from graphene oxide and glass substrates having coplanar gate, drain and source terminals or electrodes made of gold.
As a first step, a graphene oxide solution was homogenized by means of a two-stage centrifugation process. In this step, a graphene solution with a concentration of 80 μg/mL in distilled water was brought to a pH above 8 by the addition of NaOH. The solution was sonicated for about 10 min and then centrifuged at 600 rpm for 90 min. Once separated, the supernatant was then centrifuged at 8000 rpm for 15 min. The supernatant for the latter centrifugation process was discarded and the precipitate or sediment was brought to the initial solution volume using ultrapure water. The resulting solution was sonicated for 15 min.
Subsequently, the gate terminal, i.e. an Ag/AgCl electrode was prepared in the same plane as the drain and source terminal, by the electroplating process. An electro-reduction potential of −130 mV was applied between the gate terminal and an Ag0 wire, while simultaneously an electro-oxidation potential of 400 mV was applied between the interdigitated electrodes and the Ag0 wire for 12 minutes in an electrolytic solution comprising Ag2SO4.
Preparation of the FETs comprised incubating the substrates comprising the gate, drain and source electrodes, i.e. the chips, in a solution of (3-aminopropyl)triethoxysilane (APTES) in absolute ethanol at a concentration of 2% during 1 hour. The APTES-modified chips were then further incubated in the GO solution prepared as indicated above for 1 hour. The obtained chips were washed with deionized water in order to remove GO in excess.
The GO layers thus deposited onto the chip surface were subsequently reduced by exposure to hydrazine vapors at a temperature of 80° C., for 12 hours in a closed recipient. The chip was then placed in a stove at a temperature of 200° C., for 2 hours.
Subsequently, a partial oxidation of the Ag0 gate electrode to form AgCl was carried out by electroplating in a 3 M NaCl solution and by applying an oxidation potential of 150 mV (versus an Ag/AgCl reference) to the Ag0 gate electrode, while a reduction voltage of −100 mV (versus an Ag/AgCl reference) was applied to the interdigitated electrodes for 60 seconds.
The scanning electron microscopy (SEM) characterization of the rGO-FET is shown in
The X-ray photoelectron spectroscopy spectrum of the chemically reduced rGO is shown in
The drain-source current (Ids) as a function of the gate-source potential (Vg) for this rGO-FET was measured with the FETmeter in buffer solution (1 mM KH2PO4, 4 mM KCl, 27 mM Na2CO3 and 88 mM NaCl and pH=7.4) using as gate electrode both an external Ag/AgCl wire (dashed line,
As shown in
Moreover,
Supramolecular scaffold of vinylsulfonated-polyamines (VS-PA) onto graphene covered surfaces were prepared by sequential surface modification steps: i) graphene modified substrates/sensors (rGO) were incubated in a dimethylformamide (DMF) solution containing 5 mM 1-pyrenebuthanoic acid succinimidyl ester (PBSE) for 2 h. Then, washed with DMF and dried; ii) PBSE-modified graphene substrates/sensors (hereafter PBSE/rGO) were then incubated in 2 mg/mL Polyallilamine (PAH) or polyethilenimine (PEI) at pH=10 for 1 h, washed with deionized water and dried; iii) the VS-PA scaffolds (hereafter VS-PA/PBSE/rGO), were obtained by incubating the polyamine-modified PBSE/rGO substrates/sensors in 5% divinylsulfone (DVS) solution in carbonate buffer (0.5 M Na2CO3, pH=11) for 1 h, washed with deionized water and dried.
Binding of small motifs to VS-PA/PBSE/rGO substrates/sensors was carried out as follows: a) mannose binding (through —OH groups) was done by incubating the substrates/sensors in a 10% wt mannose in carbonate buffer solution (0.5 M Na2CO3, pH=11) for 18 h. b) Cysteine binding (through —SH groups) was done by incubation of the substrates/sensors in 10 mM cysteine in PBS pH=7.4 for 12 hs. c) Lysine binding (through —NH2 groups) was done by incubation of the substrates/sensors in 10 mM L-Lysine in borate buffered saline solution (BBS, 10 mM sodium borate, 140 mM NaCl pH=9.0) for 12 h.
Binding of proteins and antibodies to VS-PA/PBSE/rGO was carried out as follows: i) the substrates/sensors were incubated in 100 μg/mL protein or antibody solution in BBS pH=9.0 buffer for 5 h (at this pH, the Cys and Lys groups of the protein will react covalently with VS-PA), and then washed with BBS. ii) The antifouling of the substrates/sensors was done by incubating them in 0.2 mM PEG-NH2 (10 kDa) solution in BBS pH=9.0 for 2 h and washing with BBS. iii) Substrates/sensors were finally incubated in 100 mM ethanolamine (ETA) solution in BBS pH=9.0 for 30 minutes, to block vinyl sulfone remaining groups, wash with BBS and store in buffer.
Spectroscopic Ellipsometry (SE) was employed to study the scaffold construction in a step-by-step manner, since it is a highly sensitive, non-destructive, and reproducible technique. After PBSE modification, polyamines were attached to the surface by reaction of their ammino groups with the succinimide group from PBSE. Particularly, low- and high-molecular-weight (Mw) poly(allylamine) (PAH, 17.5 and 140 kDa) and poly(ethylenimine) (PEI, 25 and 750 kDa) were used. For the construction of the optical model, a Cauchy layer was introduced on top of the rGO layer, and its thickness was adjusted until optimal fit.
As it can be seen through the thickness evolution of the PA layer in
Firstly, CA measurements reveal PBSE being anchored to rGO as a decrease in the contact angle by 7.4% (from 78.8°±0.7° to 73.0°±0.5°). Furthermore, PA inclusion is also manifested as a decrease in the CA, but also suggesting that PEI coverage is more efficient than when using PAH; while PAH decreases the CA just by ˜5%, indicating a poor coverage of the PBSE/rGO surface, PEI anchoring seems to be more homogeneous, with a marked decrease of the CA by almost 30%.
PA films were then exposed to divinyl sulfone (DVS), allowing their —NHx groups to react with the vinylic groups from DVS. The thickness of this PA-VS layer was estimated through SE, by adjusting the previous thickness of the Cauchy layer. As it can be seen in
Mannosylation was used for proving the degree of modification of the vinyl sulfonated-polyamines scaffolds. As displayed in
Raman spectra were collected for DVS and PEI solutions, as well for VS-PEI sample (solid), with an excitation wavelength of 785 nm and they are shown in
These reactions were employed for by covalently docking different motifs, such as mannose (through —OH groups), cysteine (through —SH groups) and lysine (through —NH2 groups) to VS-PEI scaffolds. To this end, VS-PEI scaffolds were treated in buffer solutions adjusted at different pH values, pH=11 for mannose, pH=7.4 for cysteine and pH=9 for lysine. The incubation of the VS-PEI modified substrates in mannose, lysine or cysteine solutions leads to a marked decrease in the contact angle (45±2° for mannose, 40±2° for cysteine and 33.0±0.3° for lysine) as shown in
The modification of VS-PEI scaffolds with PEG was studied by in-situ surface plasmon resonance (SPR) spectroscopy. With a flow of 20 μL/min, three consecutive injections of 0.2 mM PEG-NH2 solution for 12 minutes followed by buffer for 3 minutes were performed. As it can be seen in
As a model platform, Concanavalin A (Con A, a well-known lectin protein with high lysine content and without cysteine residues) was employed, and PEGylation (PEG-NH2) was performed afterwards thus providing antifouling capability to the architecture, as will be described in further detail below. Finally, for neutralizing the remaining vinylic groups, substrates were exposed to ethanolamine (ETA) solution. The covalent modification of VS-PA scaffolds with ConA, PEG-NH2, and ETA, was evaluated by SE and CA measurements. As it can be seen at
To avoid point-to-point variability for masking any effect during the covalent modification of VS-PA scaffolds with ConA, followed by PEG-NH2 and ethanolamine (ETA) modifications, was monitored by in-situ SPR. From the SPR study, it was observed more than 95% of the protein attached to VS-PA scaffolds during the first 40 minutes. Nevertheless, since the covalent reaction to lysine groups is relatively slow, the incubation of VS-PA scaffolds in the protein solution was maintained during 5 h. PEGylation was performed elapsed that time, showing no desorption but a slight increase of the θSPR, confirming SE results above discussed: that both ConA anchorage to VS-PEI and later PEGylation steps are taking place. In the monopyrene PBSE approach of the prior art, a decrease of θSPR is observed during the PEGylation and ETA steps, suggesting some protein desorption. Therefore, ConA bound to VS-PA scaffolds was more stable than those to PBSE.
For testing the stability of the architecture, the PEGylated ConA/VS-PEI scaffolds were incubated in a solution of the non-ionic surfactant Triton X-100 0.2% and monitoring the changes in the thickness of the film by SE measurements during 24 h, analyzing the global thickness at different time intervals. For each measurement, the substrates were rinsed with the appropriated buffer and dried. As shown in
To analyze the antifouling performance of the PEGylated structures, a reference point was stablished by exposing rGO surfaces to 1 μM bovine serum albumin (BSA) solution in BBS pH=9 for 30 min and rinsed with BBS and deionized water, and dried. The non-specific interaction of BSA with rGO, was manifested in SE measurements as the apparition of a 1.88 nm Cauchy layer. PEGylated PBSE/rGO substrates were first exposed to ETA for blocking the remaining reactive groups, and then exposed to BSA solution as previously stated. The thickness difference of the Cauchy layer after BSA exposure (1.6 nm) was normalized towards the value found for rGO, as shown in
Having shown the structural stability and antifouling capability of the PEGylated VS-PEI scaffold, it is necessary to explore whether the recognition capacity of the docked biomacromolecules is retained or not. To this end, the biological affinity between ConA (a carbohydrate-binding protein) and glucose oxidase (GOx) (a carbohydrate-residues high content protein) was evaluated in-situ by SPR. GOx solutions of increasing concentrations were injected at 20 μL/min and monitored by graphene SPR sensors modified with ConA by the VS-PA approach. The response at the steady states (i.e. at the equilibrium) of the surface plasmon resonance angle shifts (ΔθSPR) were obtained as a function of the protein concentration (see J. Katrlik, R. S̆krabana, D. Mislovic̆ová, P. Gemeiner, Binding of d-mannose-containing glycoproteins to d-mannose-specific lectins studied by surface plasmon resonance, Colloids Surfaces A Physicochem. Eng. Asp. 382 (2011) 198-202. https://doi.org/10.1016/j.colsurfa.2011.01.020), which is shown in
To demonstrate that the construction of supramolecular VS-PA scaffolds does not disrupt the semiconducting properties of graphene, GFETs characteristic transfer curves before and after each modification step were carried out. In
The VS-PEI platform was also assessed for antibody-antigen interfacial recognition onto graphene. To this end, monoclonal antibodies (mAbs) specific against SARS-CoV-2 spike protein (mAb-SARS-CoV-2 spike) and human ferritin (mAb-FTH1) were anchored to VS-PEI.
The covalent modification of VS-PEI scaffolds with mAb-SARS-CoV-2 spike protein or mAb-FTH1, followed by PEG-NH2 and ETA modifications, was monitored by SPR. Surface mass densities of 436±30 ng/cm2 were estimated for both mAbs, as illustrated in
For the case of the mAb-FTH1 biointerface (
Finally, the regeneration of the mAb recognition sites was studied by incubation in glycine 10 mM pH=2.
The rGO-FET sensor obtained in Example 1, together with the FETmeter and the algorithm that calculates the target analyte concentration, was used to set up a system as provided by the invention to determine the urea concentration in a solution.
In this example, interfacial nanoarchitectures were made from the polycation PEI and the enzyme urease prepared onto rGO-FETs by the layer-by-layer nanoconstruction technique. First, the rGO-FETs were submerged into a solution of 1-pyrenesulfonate, in dimethylformamide (DMF) at a concentration of 3 mM. A polyelectrolyte was then assembled by submerging the rGO-FET into a solution of polyethyleneimine (PEI) at a concentration of 2 mg/mL at pH 8.5 for about 10 minutes, with subsequent washing using distilled water. The rGO-FET modified with the polyelectrolyte was then submerged into a solution comprising urease as a recognition element for 30 min and washed with distilled water. The urease concentration was 1 mg/mL. This step promotes the adsorption of the recognition element onto the rGO surface.
These last steps, using the polyelectrolyte and enzyme, were repeated in alternation three times to increase the surface concentration of the recognition element. The nanoarchitecture was finished with a PEI outermost layer. By means if this nanoarchitecture, the urea sensors had an improved sensitivity obtained by an unexpected synergy between the polymer-enzyme nanoarchitecture and the rGO-FET sensor. In this case, the PEI polyelectrolyte contains weak-bases as functional groups that decrease their positive charge as the urea catalysis proceeds, resulting in an amplification of the field-effect response.
Surface plasmon resonance (SPR) experiments were performed to demonstrate the nanoconstruction of the PEI/Urease multilayer nanoarchitecture, as illustrated in
The urea response of the sensor prepared with the nanoarchitecture onto the rGO-FET was monitored for different urea concentrations. Results are shown in
Other nanoarchitectures prepared onto the rGO-FET were studied by Surface plasmon resonance (SPR), for example the construction of a PDADMAC/Urease multilayer nanoarchitecture as illustrated in
The rGO-FET sensor obtained in Example 1, together with the FETmeter and the algorithm that calculates the target analyte concentration, was used to set up a system as provided by the invention to determine the potassium concentration in solution.
In this example, the nanoarchitecture comprising the recognition element was obtained by modifying the graphene channel or the gate electrode of the rGO-FET with a coating comprising an ion-selective membrane. The ion selective membrane comprised a combination of an ionophore as the recognition element, a hydrophobic electrolyte, a plasticizer and a high molecular weight hydrophobic polymer as the structural element. We prepared an ion-selective cocktail by dissolving 660 mg of o-NPOE (65.88%), 330 mg of PVC (33%), 10 mg of potassium ionophore I, also named valinomycin, (1%), and 2.23 mg of KTPCIB (0.22%) in 2.5 mL of Tetrahydrofuran (THF). Then, 100 μL of the ion-selective cocktail was drop-casted onto the graphene channel.
The biorecognition platforms were prepared as follows:
The measurements were performed employing an electrolyte-gated setup in HEPES 1 mM, NaCl 10 mM, CaCl2 0.05 mM, pH 7.4 buffer solution. The IDS current was registered while both VDS and VGS were fixed at 100 mV and −250 mV respectively. The cell was filled with 200 μL buffer solution and the measurements were started. Volumes of different Ferritin concentration solutions prepared in the same buffer were added to the cell to reach the final Ferritin concentration. The accumulative relative drain-source current change (|%ΔIDS|) was calculated by computing the difference between the current values immediately before and after the injection of the analyte solution. The curves obtained are illustrated in
In
The biorecognition platforms were prepared as follows:
The drain-source current was registered while applying both fixed VDS=50 mV and VGS=−250 mV and the enzyme urease was used to reveal the presence of ferritin. If the sample contained human ferritin (solid line in
The biorecognition platforms were prepared as follows:
The measurements were performed employing an electrolyte-gated setup in PBS pH 7.4×0.1 (×0.1 implies a 1/10 dilution) buffer solution. The IDS current was registered while both VDS and VGS were fixed at 100 mV and −250 mV respectively. The cell was filled with 200 μL buffer solution and the measurements were started. Volumes of different Spike concentration solutions prepared in the same buffer were added to the cell to reach the final Spike concentration. The accumulative relative drain-source current change (%ΔIDS) was calculated by computing the difference between the current values immediately before and after the injection of the analyte solution.
In
Moreover, the dashed line in
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2021/054656 | 5/27/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63030558 | May 2020 | US |
