WO2025019760A1

WO2025019760A1 - Polymer substrate conductive flow cell

Info

Publication number: WO2025019760A1
Application number: PCT/US2024/038712
Authority: WO
Inventors: Trevor J. MORIN; Namal NAWANA; Robert Stevens; Jeremy BURBIDGE
Original assignee: Sapphiros Llc
Priority date: 2023-07-20
Filing date: 2024-07-19
Publication date: 2025-01-23

Abstract

In one aspect, the present disclosure relates to a system for characterizing a biological polymer, which includes a porous polymeric substrate having a plurality of pores each of which extends from an inlet at top surface of the substrate to an outlet at a bottom surface of the substrate, where each of the pores is configured to provide a channel for passage of the biological polymer from a respective inlet to a respective outlet thereof. A plurality of capture linkers is coupled to the top surface of the substrate such that each of the capture linkers is associated with one of the pores and is configured to capture the biological polymer. Further, a plurality of electrodes is coupled to the top surface such that each of the pores is associated with at least one of said plurality of electrodes such that application of a voltage to said at least one electrode causes the captured biological polymer to pass through the channel of the respective pore. The system further includes a plurality of detecting elements each of which is in communication with the outlet of a respective one of said pores and configured to generate a signal indicative of at least one characteristic of the biological polymer as the polymer passes through the respective one of said pores.

Description

POLYMER SUBSTRATE CONDUCTIVE FLOW CELL

Prior Filed Application

This application claims the benefit of priority to U.S. Provisional Application No. 63/528,044, filed on July 20, 2023, the content of which is incorporated by reference herein in its entirety.

Background

The present disclosure is generally related to systems and methods for characterizing biological polymers, and more particularly to such systems and methods that can be employed to sequence such polymers.

Long-range DNA and RNA sequencing technologies have revolutionized genomics research and have significant implications for the medical community. These sequencing techniques offer several advantages over traditional short-read sequencing methods.

There are also numerous clinical applications for long-range sequencing technologies, including clinical diagnostics. By way of example, such technologies can aid in the identification of disease-causing genetic valiants, including those present in non-coding regions of the genome. Long-read sequencing can also be employed to detect pathogens, monitor viral evolution, and investigate microbial communities in complex infections. These applications have the potential to enhance disease diagnosis, inform treatment decisions, and improve patient outcomes.

Some impediments, however, remain with respect to further adoption of such long-range sequencing technologies, such as price and ease of manufacture.

Summary

In one aspect, the present disclosure relates to a system for characterizing a biological polymer, which includes a porous polymeric substrate having a plurality of pores each of which extends from an inlet at a top surface of the substrate to an outlet at a bottom surface of the substrate, where each of the pores is configured to provide a channel for passage of the biological polymer from a respective inlet to a respective outlet thereof. A plurality of capture linkers is coupled to the top surface of the substrate such that each of the capture linkers is associated with one of the pores and is configured to capture the biological polymer. Further, a plurality of electrodes is coupled to the top surface such that each of the pores is associated with at least one of said plurality of electrodes such that application of a voltage to said at least one electrode causes the captured biological polymer to pass through the channel of the respective pore. The system further includes a plurality of detecting elements each of which is in communication with the outlet of a respective one of said pores and configured to generate a signal indicative of at least one characteristic of the biological polymer as the polymer passes through the respective one of said pores.

In various embodiments, the characteristic of the biological polymer comprises identity of one or more polymeric units within a respective one of said pores as the polymer passes through that pore. In some such embodiments, the signals generated by any one of the detecting elements can be analyzed to gather information regarding the sequence of the polymeric units of the biological polymer. In some embodiments in which the same biological polymer is introduced into the pores, the signals associated with the detecting elements of those pores can be combined so as to obtain a sequence of the polymeric units with enhanced statistical accuracy.

In various embodiments, each of the detecting elements includes a graphene portion, which is electrically insulated from the graphene layers of the other detecting elements. By way of example, such electrical insulation of the graphene layers can be achieved by depositing a patterned electrically insulating layer (e.g., a patterned SiCF layer) on a graphene layer so as to divide the graphene layer into a plurality of electrically-insulated portions each corresponding to one of the detecting elements.

In various embodiments, at least one of the detecting elements can include any of semiconducting and a metallic detection layer that is coupled to the bottom surface of the polymeric substrate, e.g., deposited on the bottom surface of the polymeric substrate or positioned in proximity of the bottom surface, e.g., at a distance in a range of about 1 to about 10 mm. A source electrode and a drain electrode can be electrically coupled to the detection layer for applying a voltage or a current to that layer. A variation of the target characteristic of the biological polymer as the polymer passes through a respective pore associated with that detecting element modulates an electrical property of the detection layer, e.g., its electrical conductivity, which can be detected, e.g., via a variation in the current flowing through the detection layer. In various embodiments, the system can further include a measurement circuitry that is in communication with at least one of the source and the drain electrode for detecting the change in the electrical property, which can be, for example, an electrical impedance of the detection layer.

By way of example, and without limitation, the detection layer can include any of graphene, graphene oxide, an allotrope of graphene and any derivatives or combinations thereof. In various embodiments, the detection layer includes any of silver, boron, molybdenum and derivatives or combinations thereof.

In various embodiments, the detection layer includes a mono-atomic layer or a multi- atomic layer.

In various embodiments, the biological polymer includes an oligonucleotide and the characteristic of the oligonucleotide includes the identity of one or more nucleotides thereof. For example, as the oligonucleotide passes through a pore, a plurality of detection signals can be generated, each indicative of several nucleotides passing through the pore. The detection signals can be compiled to generate the nucleotide sequence of the oligonucleotide. By way of example, the oligonucleotide includes any of DNA and RNA.

In various embodiments, the biological polymer includes a protein and the characteristic of the protein includes a sequence of amino acids thereof.

In various embodiments, the electrodes used for driving the biological polymer into the respective pores can be individually addressable, i.e., each electrode (or electrode pair) can be activated independent of the other electrodes (or electrode pairs).

In various embodiments, the system can further include at least one voltage source, e.g., a DC voltage source, for applying voltages (e.g., DC voltages) to the electrodes. A plurality of conductive paths can electrically connect the DC voltage source to the electrodes.

A controller in communication with the DC voltage source can send control signals to the DC voltage source for adjusting voltages applied to the electrodes.

In various embodiments, at least one compound (herein also referred to as a stabilizing compound) can be housed within each of the pores (or at least some of the pores) for controlling passage of the biological polymer through the pores. By way of example, the stabilizing compound can be a biological compound, a synthetic compound, or combinations thereof. In various embodiments, the biological compound can be any of a protein and a protein complex. In some such embodiments, the protein or the protein complex can provide a channel through which the biological polymer can be translocated across the pore. By way of example, and without limitation, the protein can be any of MspA protein and DNA helicase, among others. In some embodiments, the protein complex can be a ribosome.

In various embodiments, the biological compound can include two units, where a first unit can be positioned within the pores and a second unit can be attached to an end of the biological polymer, where the application of a voltage to the electrodes associated with the pores can cause the biological polymer and the attached second unit to enter the pores such that the second unit docks with the first unit to form the biological compound within the pore.

The pores can be distributed randomly or according to a regular array in the polymeric substrate.

In some embodiments, the stabilizing compound can be distributed randomly among the pores. For example, the porous polymeric substrate can be exposed to a medium containing the stabilizing compound such that the compound finds its way into some of the pores but not others. In such embodiments, a sequential activation of the electrodes (or electrode pairs) associated with the pores and the analysis of the respective detection signals can allow distinguishing the pores containing the compound from those that do not contain the compound.

In a related aspect, a method of characterizing a biological polymer is disclosed, which includes capturing the biological polymer on a top surface of a polymeric substrate, the polymeric substrate comprising a plurality of pores, passing the biological polymer (or at least a portion thereof) through at least one of the plurality of pores formed in the polymeric substrate, and generating an electrical signal indicative of at least one characteristic of the biological polymer as the polymer passes through the pore.

In various embodiments, a plurality of capture linkers, each associated with a respective one of the plurality of pores, formed on the top surface of the polymeric substrate is used to capture the biological polymer on the top surface of the polymeric substrate. In various embodiments, the passing of the biological polymer through a pore is achieved via application of an electric field to the polymer. By way of example, the electric field can be generated by application of a voltage across two electrodes coupled to the top surface of the polymeric substrate in proximity of an inlet of the pore.

In various embodiments of the above method, the rate of passage (translocation) of the biological polymer through the plurality of the pores can be controlled, e.g., via a compound positioned within the pore. Such a compound can provide a channel through which the biological compound can pass to traverse the pore. For example, for a given voltage applied across a pair of electrodes associated with a pore, such a compound can reduce the rate of the polymer passage through the pore.

In various embodiments, the biological compound can be any of a protein and a protein complex. In some such embodiments, the protein or the protein complex can provide a channel through which the biological polymer can be translocated across the pore. By way of example, and without limitation, the protein can be any of MspA protein and DNA helicase, among others. In some embodiments, the protein complex can be a ribosome.

In some embodiments, the electrodes (or electrode pairs) associated with the pores can be activated in a temporal sequence, i.e., they can be activated one at a time, such that the signals generated by the detecting elements can be acquired in different time intervals, each associated with a pore corresponding to the activated electrode(s).

In various embodiments, the pores can be distributed randomly throughout the polymeric substrate. As noted above, in addition or alternatively, the exposure of the porous substrate to a medium containing a stabilizing compound can result in introduction of that compound into a random subset of the pores.

In various embodiments, the signals (e.g., electrical signals) associated with the detecting elements can be combined to achieve a statistical measure of the characteristic of the polymer being determined. Further understanding of various aspects of the present teachings can be obtained by reference to the following detailed description in conjunction with the associated drawings, which are described briefly below.

Brief Description of the Drawings

FIG. 1A is a side schematic view of a cartridge used in a system according to an embodiment of the present teachings,

FIG. IB is a block diagram of an embodiment of a system according to the present teachings,

FIG. 1C schematically depicts a patterned insulating layer deposited on a graphene layer (graphene sheet) for generating a plurality of graphene regions that are electrically insulated from one another,

FIG. 2A is a schematic view of a GFET that can be employed in various embodiments of the present teachings,

FIG. 2B is an example of an I-V curve associated with the GFET depicted in FIG. 2A,

FIG. 3 is a perspective view of a cartridge according to an embodiment having a porous polymeric substrate, wherein a biological compound is disposed in the pores for controlling the passage of a biological polymer through the pores,

FIG. 4A is a schematic perspective view of a pore of a porous polymeric substrate in which a protein is disposed at a time at which a biological polymer tethered to a helicase protein has been captured by a capture linker coupled to the top surface of the polymeric substrate,

FIG. 4B is a schematic perspective view of the pore depicted in FIG. 4A after the application of a voltage to an electrode pair associated with the pore causes the biological polymer and the respective tethered helicase protein to be pushed into the pore such that the helicase docks with the protein to form a protein complex through which the biological polymer can pass, FIG. 4C is a schematic perspective view of the pore depicted in FIGS. 4A and 4B at a time when a portion of the biological polymer has exited the pore and another portion of the polymer is within the pore,

FIGS. 5A, 5B, and 5C depict examples of hypothetical I-V curves associated with the stages depicted in FIGS. 4A, 4B, and 4C, respectively,

FIG. 6 is a perspective view of a pore of a porous substrate according to an embodiment, illustrating exemplary sizes of the pore, the electrodes and a biological compound positioned in the pore,

FIG. 7A is a top view of a porous polymeric substrate according to an embodiment depicting a plurality of electrodes positioned on the top surface of the polymeric substrate,

FIG. 7B is a bottom view of the porous polymeric substrate shown in FIG. 7A showing a a grid of electrically isolated regions of graphene and randomly inserted protein complexes in the pores of the polymeric substrate (illustrated with X’s),

FIG. 8 is a schematic view of a bottom surface of a polymeric substrate according to an embodiment of the present teachings depicting a graphene layer deposited on the bottom surface of the polymeric substrate, and

FIGS. 9A and 9B arc schematic views of embodiments of a cartridge according to the present teachings having a plurality of graphene-based detecting elements, where the detecting elements share a common gate electrode.

Detailed Description

The present disclosure is generally related to methods and systems for determining one or more characteristics of a biological polymer and more particularly to methods and systems for rapid long-range sequencing of such polymers, e.g., DNA and RNA. As discussed in more detail below, in some embodiments, graphene field-effect transistors (GFETs) operationally coupled to porous polymeric substrates can be employed for achieving sequencing of biological polymers. In various embodiments, the adoption of GFETS for such applications can provide sensitive devices for rapid and low-cost sequencing of a variety of biological polymers, including DNA, RNA, protein, among others. Such sequencing can find a variety of applications, including unraveling of structural variations, improving genome assembly, enabling phasing of genetic variants, and the study of RNA transcriptomcs, among others.

Various terms are used herein in accordance with their ordinary meanings in the art. The term “about,” as used herein, denotes a variation of at most 10% around a numerical value. The term “substantially,” as used herein, denotes a deviation, if any, from a complete state and/or condition of at most 10%. The terms “top” and “bottom” are used herein to distinguish two opposed surfaces of a polymeric substrate and are not intended to indicate necessarily a particular orientation.

Without any loss of generality and for ease of description, various embodiments will be discussed below assuming that the biological polymer is a double stranded DNA (dsDNA). It should, however, be understood that the present teachings can be employed for examining and determining characteristics of a variety of biological polymers, including sequencing such polymers. Again, by way of example and without limitation, the characteristic of the polymer may be the identity of the polymeric units forming the polymer.

With reference to FIGS. 1A and IB, a system 100 according to an embodiment of the present teachings includes a cartridge 102 having a porous substrate 104 (a polymeric substrate, in this embodiment) that extends between a top surface 104a and a bottom surface 104b. A plurality of pores 106 is distributed throughout the polymeric substrate 104. Each of the pores 106 extends across the width of the polymeric substrate 104 from an inlet 106a positioned at the top surface 104a to an outlet 106b positioned at the bottom surface 104b. The polymeric substrate 104 can be formed using a variety of different materials. Some examples of such materials include, without limitation, silica and/or alumina.

A plurality of capture linkers 108 is coupled to the top surface of the polymeric substrate 104 such that each of the capture linkers is associated with one of the pores. By way of example, and without limitation, the capture linkers may be covalently coupled to various moieties on the top surface of the polymeric substrate. In various embodiments, the capture linker can specifically bind to the target biological polymer 101 and capture that polymer. For example, for capturing a target oligonucleotide, the capture linker can be an oligonucleotide having a nucleotide sequence that is complementary to the nucleotide sequence of a portion of the target oligonucleotide. Tn another embodiment, the capture linker can be an aptamer that can specifically bind to a target protein.

While in some embodiments the same capture linker is associated with the pores, in other embodiments, the capture linkers associated with some pores may be different from the capture linkers associated with the other pores. In other words, while in some embodiments the cartridge can be used to characterize one target polymer, in other embodiments the cartridge may be employed to characterize multiple target polymers.

A plurality of electrically conductive electrodes 200 is coupled to the top surface of the polymeric substrate such that the electrodes are pair-wise associated with the pores. In various embodiments, each of the electrically conductive electrodes can be in the form of a metallic electrode deposited on the top surface of the polymeric substrate. As discussed in more detail below, the application of a voltage across each pair of the electrodes can drive the target biological polymer captured by the capture linker into the respective pore. In this example, a DC voltage source 202 operating under the control of a controller 204 can apply the requisite DC voltages to the electrodes to generate an electric field, which can in turn cause the translocation of the biological polymer across the lumen of the pores. By way of example, and without limitation, the DC voltage can be in a range of about 100 mV (millivolts) to about 300 mV.

In various embodiments, each electrode pair associated with one of the pores is individually addressable such that each electrode pair can be activated independent of the other electrode pairs so as to drive the biological polymer into the respective pore.

With continued reference to FIG. 1A, a plurality of detecting elements 206 are in communication with the outlets of the pore 106 such that each pore is associated with one of the detecting elements. The detecting elements are electrically isolated from one another so as to ensure that a detection signal generated by each of the detecting elements can be associated with a particular pore, and more particularly with a biological polymer passing through that pore.

By way of example, in this embodiment, each detecting element 206 includes a graphene layer 208 that extends between a source electrode 210 and a drain electrode 212. In this embodiment, the detecting elements are coupled via a plurality of posts 213 to the polymeric substrate and are offset relative to the bottom surface of the polymeric substrate to allow the biological polymer to exit the pores, e.g., to be collected by a collection well (not shown).

With reference to FIGS. IB and 1C, in this embodiment, the graphene portions (regions) associated with the different pores are electrically insulated from one another via a patterned insulating layer 214. By way of example, in this embodiment, a graphene layer can be formed on an underlying electrically insulating substrate 215, e.g., a SIMOX substrate, and a patterned layer of an electrically insulating material, such as SiCh, can be formed over the graphene layer, e.g., via photolithography, such that each exposed graphene portion forms a part of the GFET corresponding to one of the pores.

With reference to FIGS. 2A and 2B and as noted above, in this embodiment, each graphene detector functions as a graphene field effect transistor (GFET) having a graphene layer, a source electrode, a drain electrode and a gate electrode that can be used to control the electrical conductivity of the graphene layer. In various embodiments, a DC potential (e.g., 100 mV) can be applied between the source and the drain electrode to establish a current through the graphene layer. The application of a voltage to the gate electrode generates an electric field that extends into the graphene layer, thereby modulating its conductivity. The variation of the current flowing through the graphene layer as a function of the gate voltage can be measured to provide an I-V curve exhibiting a minimum at the charge neutrality point (Dirac point), as shown in FIG. 2B. The proximity of an analyte, such as a biological polymer, to the graphene layer, e.g., the portion of a biological polymer within pore, can change the local charge density around the graphene layer, resulting in a shift in at least one electrical property, e.g., the electrical conductance, of the graphene layer. In some embodiments, such a shift can be detected, e.g., as a change in the current flowing through the graphene layer. By way of example, in such embodiments, by monitoring the change in the current, e.g., via the V_out feedback transistor, a target characteristic, e.g., the sequence of the polymeric units, passing through the respective pore can be determined.

By way of example, the detected change may be compared with previously-measured calibration data to determine the polymeric units causing that change. By way of example, when sequencing an oligonucleotide, the calibration data can provide the expected change in the conductivity of the graphene layer for different nucleotide sequences. For example, if the pore length is such that a sequence of three nucleotides can fit within the pore, calibration data can provide expected changes to the conductivity of the graphene layer for different 3-nuclcotidc sequences, e.g., AAT or TTG, etc.

In various embodiments, a compound (herein also referred to as stabilizing compound) can be disposed in the pores of the polymeric substrate for controlling the movement of the biological polymer through the pores. For example, the compound can be a biological molecule or molecular complex, a synthetic modified molecule, an altered biological molecule or combinations thereof. For example, the biological compound can provide a channel through which the biological polymer can be translocated through the pore.

By way of illustration, FIG. 3 shows a cartridge 300 having a plurality of pores 302 in which a biological compound 304 is disposed. The compound 304 can control (regulate) the translocation of the biological polymer through the pores. For example, the compound 304 can provide a channel through which the biological polymer can pass. By way of example, and without limitation, the compound 304 can be a biological molecule, such as a protein, a peptide, an antibody, an antigen, a nucleic acid, a peptide nucleic acid, locked nucleic acids, morpholinos, sugars, lipids, glycophosphoinositols, lipopolysaccharides, and synthetic variants thereof, among others. By way of further examples, the compound 304 can be an ion channel, a nucleoside channel, a transmembrane receptor, and variations thereof. In some embodiments, the compound, e.g., a protein, can be stabilized within the pore due to the hydrophobic (aliphatic) nature of the polymeric material. As noted above and further discussed below, a biological polymer of interest can be captured by a linker attached to the top surface of the polymeric substrate and can be fed into a pore and ratcheted through the pore by applying a voltage to the electrodes coupled to the top surface of the polymeric substrate. For example, the application of a voltage to the electrodes can generate an electric field that repels the biological polymer and hence drives the biological polymer in the pores.

In various embodiments, the biological polymer can exhibit selective binding to the compound 304, e.g., under certain environmental conditions, such as pH and/or temperature. In this embodiment, the compound 304 includes two units that can come together as the biological polymer is driven into a pore, as discussed in more detail below. In various embodiments in which the two units are proteins, the unit coupled to the biological polymer is referred to as the motor protein and the unit positioned in a pore and with which the motor protein docks is referred to as the biological protein.

For example, the biological compound 304 can include a helicase that is coupled to a protein, such as MspA. In various embodiments, the protein can be disposed within the pores and the biological polymer can be prepared so as to be tethered to the helicase protein. The introduction of the biological polymer, via application of a voltage to the electrodes, into a pore can result in the helicase docking with the protein, thereby providing the biological compound, which in turn provides a channel through which the biological polymer is translocated.

FIGS. 4A, 4B, and 4C show a pore 400 at three temporal points during the use of the cartridge for sequencing a DNA segment 402, which is tethered to a helicase protein 404. The capture linker 108 associated with the pore 400 can capture the DNA segment. The application of a voltage across two electrodes 400a/400b deposited on the top of the polymeric substrate in proximity of the pore’s inlet can generate an electric field that can drive the DNA segment and its associated helicase protein into the pore. The helicase protein can dock with the protein positioned within the pore, thereby forming a protein complex that provides a channel through which the DNA can traverse, as shown in FIG. 4B.

As the DNA segment traverses through the pore (FIG. 4B and 4C), the graphene detector associated with the pore can generate an electrical signal indicative of the nucleotides within the pore. For example, the I-V (current-voltage) response of the graphene detector can be sensitive to different stages of the transversal of the DNA segment through the pore.

For example, FIG. 5A shows a hypothetical example of the drain current versus the gate voltage of the graphene detector prior to the introduction of the DNA segment into the pore (FIG. 4A). FIG. 5B in turn shows the drain- v.s. -gate IV curve associated with the introduction of a few nucleotides of the DNA segment, e.g., an AAT nucleotide sequence in this example, into the pore while FIG. 5C shows the respective drain-v.s.-gate IV curve at a time when the AAT nucleotide sequence has passed through the pore and has been replaced with another nucleotide sequence. The helicase unwinds the DNA as to allow ssDNA to pass through . The drain-v.s.-gate IV curve can provide a unique signature associated with the nucleotide sequence within the pore (e.g., a sequence of 3 or 4 nucleotides). As the DNA traverses through the pore, the electric signatures generated by the graphene detector can be collected and analyzed to derive a nucleotide sequence of the DNA segment.

In general, the sizes of the polymeric substrate and the pores distributed therein can be selected based on the requirements of a particular application. For example, as shown in FIG. 6, the height of the polymeric substrate, i.c., the distance between the top and the bottom surface of the polymeric substrate, which corresponds to the length of the pores can be in a range of about 2000 to about 10,000 nm (2 - 10 microns). Further, the diameter of the pores can be in a range of about 30 nm to about 1000 nm. In some embodiments, the roughness of the polymeric top surface, e.g., characterized as root-mean -square variation of the surface height, can be in a range of about 10 nm to about 500 nm. In some embodiments, the porosity of the polymeric substrate can be 30% or more, e.g., 40% or more, 50% or more or 60% or more. By way of example, the porosity of the porous polymeric substrate can be determined through the use of nitrogen adsorption porosimetry and accurate measurement of the nanoporous layer by scanning electron microscopy measurement of ion milled step edges or other accurate methods. For example, the porosity of the porous polymeric substrate can be measured according to ISO 15901-2:2022 Pore size distribution.

Further, the height of the electrodes used to drive the biological polymer into the pores can be in a range of about 10 nm to about 500 nm. In the example schematically depicted in FIG. 6, the biological compound has a height of about 12 nm, with a bottom width of about 4 nm and a top width of about 10 nm, though the biological compound, or a synthetic compound, can have other sizes as well.

In some embodiments, the electrodes can be formed on the surface of the porous polymeric substrate via deposition of an electrically conductive printable composition on that surface. By way of example, and without limitation, such a conductive printable composition can include electrically conductive flakes distributed within a liquid. By way of example, and without limitation, the flakes can include any of silver, graphene, boron nitride, or a mixture thereof and can have a length in a range of about 0.1 microns to about 3 microns, a thickness in a range of about 0.8 nm to about 100 nm, and an aspect ratio in a range of about 5:1 to about 3750:1. As noted above, in various embodiments, the electrodes for driving the biological molecules into the pores arc individually addressable. This allows the application of a localized voltage to the electrodes such that the resulting field would push the biological polymer into a respective pore. Further, in various embodiments, these electrodes are placed sufficiently far from one another to eliminate (or reduce below an acceptable threshold) cross-talk between them. Thus, in embodiments in which a compound is used to regulate the passage of the biological polymer through the pores, only those pores containing the compound, e.g., a protein complex, can generate the detection signals providing sequence information.

Referring again to FIG. 1A, in various embodiments, the controller 204 can activate the electrodes 200 one-by-one in a temporal sequence. The electrical signals generated by the respective detecting elements can be transmitted to an analysis module 201, e.g., an ASIC in this embodiment, that operates under the control of the controller 204 and analyzes the data to generate information regarding the characteristic of interest of the polymer, e.g., the sequence of its polymeric units.

In some such embodiments in which GFETs are employed, rather than utilizing electrically separated graphene portions each corresponding to a particular pore, a common graphene layer can be used across the pores with a common source and drain electrode. Each pore will be, however, associated with its own gate electrode. Alternatively, in some embodiments, the electrodes for driving the biological polymer into the pores can be activated in a temporal sequence. Although the graphene layer is shared among the pores, the one-by-one activation of the electrodes driving the biological polymer into the pores allows associating an electronic signal generated via a change in the conductivity of the graphene layer with a particular pore. In other words, the interrogation of the wells in a temporal sequence allows the graphene layer to be shared among multiple pores.

As noted above, in some embodiments, a stabilizing compound, e.g., a protein, may be randomly inserted into the plurality of the pores. Thus, only a subset of the pores that contain the stabilizing compound can provide sequence information regarding the biological polymer. FIG. 7A identifies such pores containing the stabilizing compound with X’s.

FIG. 7B in turn schematically depicts a plurality of metal electrodes (represented by dots) that are coupled to the top surface of the polymeric substrate. As discussed above, in various embodiments, the electrodes are placed far enough from one another so as to minimize (and preferably eliminate) cross-talks between the electrodes. In some such embodiments, the electrodes coupled to the top surface of the polymeric substrate can be activated, e.g., in a temporal sequence, to drive the biological polymer into the pores. Only the electrodes associated with the pore containing the stabilizing compound generate signals that can be utilized to infer information regarding the desired characteristic of the biological polymer, e.g., the sequence of its polymeric units.

In various embodiments, a graphene layer can be directly deposited on a bottom surface of a porous polymeric substrate according to the present teachings. By way of example, FIG. 8 schematically depicts such a graphene layer deposited onto the bottom surface of the polymeric substrate. Similar to the embodiment depicted in FIG. 1A, a patterned insulator layer (not shown in this figure) can be deposited on the graphene layer so as to generate a plurality of electrically isolated graphene regions each of which is associated with one of the pores. In this embodiment, a plurality of openings are generated in the graphene layer such that each opening is in a substantial register with the outlet of a respective pore to allow a biological polymer to exit the pore. By way of example, and without limitation, the openings in the graphene layer can be formed via exposure of the graphene layer to laser radiation. See, e.g., Nano Let. 2023, 23, 11, 4893-4900, Publication Date: May 16, 2023

“Nanoprocessing of Self-Suspended Monolayer Graphene and Defect Formation by Femtosecond-Laser Irradiation,” which is incorporated herein by reference in its entirety.

In various embodiments, a common gate electrode can be used for all of the graphene detecting elements. By way of example, FIG. 9A shows a common gate electrode 900 patterned on the graphene that is on the polymeric substrate. FIG. 9B shows a common gate electrode 900’ fabricated on the bottom side of the polymeric substrate and covered by a graphene layer.

By way of example, in some embodiments, the porous layer can include alumina and/or silica. In some embodiments, the porous layer can have a surface roughness of about 500 nm or less, e.g., about 200 nm or less, or about 100 nm or less. As used herein, surface roughness refers to an arithmetic surface roughness (Ra) and is typically measured by the method described in ISO 21920-2:2021 (Part 1: Surface and its parameters). By way of example, the porous substrate can include alumina and/or silica nanoparticles. In another embodiment, the porous layer may be formed of polypropylene. An example of a porous layer can be, for example, OLMEC Paper available from InnovaArt.

In some embodiments, the graphene layers of the detecting elements can be formed on the porous substrate using a printable medium comprising graphene flakes and a liquid. By way of example, the flakes can have a length in a range of about 0.1 pm to about 3 pm, a thickness of 0.8 nm to 100 nm, and an aspect ratio of from 5:1 to 3750:1. Further, the conductive layers functioning as electrodes for applying electrical signals to the graphene layers can also be in the form of a printable medium that can be applied to the graphene layer. For example, the printable medium can include metal flakes, e.g., silver flakes, and a liquid that can be printed onto the graphene layers of the detecting elements.

The following Example is provided for illustrative purposes of deposition of metal on a substrate using a printable medium.

Example

Ames Goldsmith Nano Platelet Silver flake S0010-NM2 (630g) can be mixed with Zeller & Gmelin Hydrotek Transparent White (118.5g) and water (63g). The mixing process can remove all particulate agglomerations to create a free flowing silver flexographically printable ink. The ink can be loaded onto a standard flexographic printing press fitted with a 6 VOL anilox metering roller. The nanoalumina or nanosilica coated substrate can be fed into the press from a roll. A flexographic printing plate with the desired pattern can be mounted onto a 22-inch repeat plate cylinder. Ink can be loaded into the ink reservoir. Upon operation, ink is wetted onto the anilox roller and the silver ink is transferred from the surface of the wetted anilox to the moving substrate by the flexoplate on the plate cylinder. The ink passes through a forced hot air dryer and infrared lamp to dry the ink in order to prevent loss of definition and coating the rollers in the press with silver ink. The printed substrate is collected in a roll format ready for transfer to die attach or further print processes. Serpentine track test devices can be used to measure the sheet resistance of the printed silver layer using a two probe test with an Ohm meter.

Those having ordinary skill in the art will appreciate that various changes can be made to the above embodiments without departing from the scope of the invention. 1

Claims

What is claimed is:

1. A system for examining a biological polymer, comprising: a porous polymeric substrate having a plurality of pores each of which extends from an inlet at a top surface of the substrate to an outlet at a bottom surface of the substrate, each of said pores configured to provide a channel for passage of the biological polymer from a respective inlet to a respective outlet thereof, a plurality of capture linkers coupled to said top surface of the substrate such that each of the capture linkers is associated with one of said pores and is configured to capture the biological polymer, a plurality of electrodes coupled to said top surface such that each of the pores is associated with at least one of said plurality of electrodes such that application of a voltage to said at least one electrode causes the captured biological polymer to pass through the channel of the respective pore, and a plurality of detecting elements each of which is in communication with the outlet of a respective one of said pores and configured to generate a signal indicative of at least one characteristic of said biological polymer as the polymer passes through the respective one of said pores.

2. The system of Claim 1, wherein said characteristic of the polymer comprises identity of one or more polymeric units within said channel of a respective one of said pores as the polymer passes through that pore.

3. The system of Claim 1, further comprising an insulator layer deposited on at least a portion of the upper surface of the polymeric substrate for electrically isolating the pores from one another.

4. The system of any one of Claims 1 - 3, wherein at least one of said detecting elements comprises: any of a semiconducting and a metallic detection layer disposed on at least a portion of said bottom surface of the polymeric substrate, a source electrode electrically coupled to said detection layer, a drain electrode electrically coupled to said detection layer, wherein a variation of said characteristic of the biological polymer as the polymer passes through a respective pore associated with the at least one of said detecting elements modulates an electrical property of said detection layer.

5. The system of Claim 4, further comprising a measurement circuitry in communication with at least one of said source and said drain electrode for detecting the change in said electrical property.

6. The system of Claim 5, wherein said electrical property comprises an electrical impedance of said detection layer.

7. The system of Claim 4, wherein said detection layer comprises any of graphene, graphene oxide, an allotrope of graphene and any derivatives or combinations thereof.

8. The system of Claim 4, wherein said detection layer comprises any of silver, boron, molybdenum and any derivatives or combinations thereof.

9. The system of Claim 4, wherein said detection layer comprises a mono-atomic layer.

10. The system of Claim 4, wherein said detection layer comprises a multi-atomic layer.

11. The system of Claim 1, wherein said biological polymer comprises an oligonucleotide.

12. The system of Claim 11, wherein said characteristic of the oligonucleotide comprises one or more nucleotides present within the channel of a respective one of said pores.

13. The system of Claim 1, wherein said biological polymer comprises a protein.

14. The system of Claim 13, wherein said characteristic of the protein comprises one or more amino acids of said protein present within the channel of a respective one of said pores.

15. The system of Claim 11, wherein said oligonucleotide comprises any of DNA and RNA.

16. The system of Claim 1, wherein said electrodes are individually addressable.

17. The system of Claim 16, further comprising at least one DC voltage source for applying DC voltages to said plurality of electrodes.

18. The system of Claim 17, further comprising a controller in communication with said at least one DC voltage source for adjusting DC voltages applied to said plurality of electrodes.

19. The system of Claim 18, further comprising a plurality of electrically conductive paths extending from said DC voltage source to said plurality of electrodes.

20. The system of Claim 1, further comprising at least one stabilizing compound housed within each of said pores for controlling passage of said biological polymer through said pores.

21. The system of Claim 20, wherein said stabilizing compound comprises a biological compound.

22. The system of Claim 21, wherein said biological compound comprises any of a protein and a protein complex.

23. The system of Claim 22, wherein any of said protein and said protein complex provides a channel through which the biological polymer can pass.

24. The system of Claim 22, wherein said protein comprises any of MspA and DNA helicase.

25. The system of Claim 22, wherein said protein complex comprises a ribosome.

26. The system of Claim 21, wherein said biological compound comprises a first unit positioned within each of said pores and a second unit attached to an end of said biological polymer, wherein application of a voltage to an electrode of each of said pores causes the captured polymer including the second unit of the biological compound to enter that pore such that the second unit docks to said first unit to form said biological compound within the pore.

27. The system of Claim 1, wherein said pores are randomly distributed within said polymeric substrate.

28. The system of Claim 1, wherein said pores are distributed according to a regular array in said polymeric substrate.

29. A method of characterizing a biological polymer, comprising: capturing the biological polymer on a top surface of a polymeric substrate, the polymeric substrate comprising a plurality of pores; passing the biological polymer through at least one of said plurality of pores formed in the polymeric substrate; and generating an electrical signal indicative of at least one characteristic of the biological polymer as the biological polymer passes through a channel of said pore.

30. The method of Claim 29, wherein a plurality of capture linkers, each associated with a respective one of the plurality of pores, formed on the top surface of said substrate is used to capture the biological polymer on the top surface of polymeric substrate.

31. The method of Claim 30, further comprising controlling the rate of passage of the biological polymer through the plurality of pores.

32. The method of Claim 31, wherein controlling the rate of passage of the biological polymer through the plurality of pores comprises reducing the rate of passage of the biological polymer through the plurality of pores.

33. The method of claim 31, wherein controlling the rate of passage of the biological polymer through the pore is done using a stabilizing compound positioned in the pore.

34. The method of claim 33, wherein said stabilizing compound comprises any of a biological and a synthetic compound.

35. The method of claim 30, wherein the generated electrical signal is a change in an electrical property of a detecting element.