Multi-modal nanopore system for biosensing and sequencing

Abstract

A multi-modal nanopore system for biosensing and sequencing. In some embodiments, the system includes a first nanopore device, the first nanopore device including: a first chamber and a second chamber in a planar substrate; a nano-fluidic channel connecting the first chamber and the second chamber; a first electrode and a second electrode forming a nanopore between the first electrode and the second electrode; an optical coupling element configured to couple an electromagnetic beam with the nanopore; a first bias circuit for applying a bias to the first electrode and the second electrode, to form a transverse electric field in the nanopore; and a current-measuring circuit configured to measure a current between the first electrode and the second electrode.

Description

MULTI-MODAL NANOPORE SYSTEM FOR BIOSENSING AND SEQUENCINGCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] The present application claims priority to and the benefit of U.S. Provisional Application No. 63 / 596,218, filed November 3, 2023, entitled "MULTI-MODAL NANOPORE SYSTEM FOR BIOSENSING AND SEQUENCING", the entire content of which is incorporated herein by reference.FIELD

[0002] One or more aspects of embodiments according to the present disclosure relate to sensing and sequencing, and more particularly to a system and method for sensing and sequencing of biopolymers.BACKGROUND

[0003] Biopolymers, such as protein molecules, deoxyribonucleic acid (DNA) molecules, and ribonucleic acid (RNA) molecules may play multiple roles in biological systems. Such a molecule may have various characteristics of interest, including, for example, the sequence of building blocks (e.g., moieties) in the molecule, and modifications, such as epigenetic modifications on deoxyribonucleic acid (DNA).

[0004] It is with respect to this general technical environment that aspects of the present disclosure are related.SUMMARY

[0005] According to an embodiment of the present disclosure, there is provided a system, including: a first nanopore device, the first nanopore device including: a first chamber and a second chamber in a planar substrate; a nano-fluidic channel connecting the first chamber and the second chamber; a first electrode and a second electrode forming a nanopore between the first electrode and the second electrode; an optical coupling element configured to couple an electromagnetic beam with the nanopore; a first bias circuit for applying a bias to the first electrode and the second electrode, to forma transverse electric field in the nanopore; and a current-measuring circuit configured to measure a current between the first electrode and the second electrode.

[0006] In some embodiments, the system further includes: a third electrode and a fourth electrode in the first chamber and the second chamber, respectively; and a second bias circuit, for generating an electroosmotic flow, a longitudinal electric field, or the combination of both, for causing a molecule to translocate from the first chamber to the second chamber.

[0007] In some embodiments: the system includes one or more surfaces functionalized to reduce a translocation speed of the molecule, or the system is configured to apply an electromagnetic field to slow the translocation speed of the molecule, or the system includes one or more molecular motors to control the translocation speed of the molecule.

[0008] In some embodiments, the system includes one or more surfaces functionalized to reduce a translocation speed of the molecule.

[0009] In some embodiments, a surface of the one or more surfaces is a surface of the first electrode.

[0010] In some embodiments, the surface is functionalized with a thiol containing molecule further including a functional group selected from a polyethylene glycol, a 4(5)- (2- mercaptoethyl)-1 H-imidazole-2-carboxamide (ICA), and an alkyl group.

[0011] In some embodiments, the system is configured to apply an electromagnetic field to slow the translocation speed of the molecule.

[0012] In some embodiments, the electromagnetic field is an optical electromagnetic field.

[0013] In some embodiments, the optical electromagnetic field is an optical electromagnetic field of a laser directed at the nanopore.

[0014] In some embodiments, the electromagnetic field is an electric field.

[0015] In some embodiments, the electric field is a field resulting from a bias applied by the first bias circuit.

[0016] In some embodiments, the optical coupling element includes a total internal reflection objective having a focus at the nanopore.

[0017] In some embodiments, the system includes: a nanopore device array including the first nanopore device, and a group of lenses for relaying and projecting an image of the nanopore device array onto the ends of a plurality of optical fibers.

[0018] In some embodiments, the group of lenses images each of the nanopores of the nanopore device array onto a respective one of the ends of the fibers.

[0019] In some embodiments, the system further includes a spectrometer connected to the optical fibers.

[0020] According to an embodiment of the present disclosure, there is provided a method, including: operating a system including a first nanopore device, the first nanopore device including: a first chamber and a second chamber in a planar substrate; a nano-fluidic channel connecting the first chamber and the second chamber; a first electrode and a second electrode forming a nanopore between the first electrode and the second electrode; an optical coupling element configured to couple an electromagnetic beam with the nanopore, the operating including: applying a first bias to the first electrode and the second electrode, to form a transverse electric field in the nanopore; and measuring a current between the first electrode and the second electrode.

[0021] In some embodiments, the method further includes applying a second bias to a third electrode in the first chamber and a fourth electrode and the second chamber, to cause a molecule to translocate from the first chamber to the second chamber.

[0022] In some embodiments, the method further includes measuring a Raman scattering signal from the molecule.

[0023] In some embodiments, the method further includes etching a sacrificial layer from within a precursor chip, to form the nano-fluidic channel and the cavity between the first electrode and the second electrode.

[0024] In some embodiments, the method further includes adjusting a gap between the first electrode and the second electrode by a reversible electrodeposition process.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] These and other features and advantages of the present disclosure will be appreciated and understood with reference to the specification, claims, and appended drawings wherein:

[0026] FIG. 1 is a schematic drawing of a system for sensing and sequencing of biopolymers, according to an embodiment of the present disclosure;

[0027] FIG. 2 is a schematic drawing of a system and method for reversible electrodeposition, according to an embodiment of the present disclosure;

[0028] FIG. 3 is a schematic drawing of a system for sensing and sequencing of biopolymers, according to an embodiment of the present disclosure;

[0029] FIG. 4 is a schematic drawing of a system for sensing and sequencing of biopolymers, according to an embodiment of the present disclosure;

[0030] FIG. 5A is a schematic drawing of a system for sensing and sequencing of biopolymers including nano-fluidic guides, according to an embodiment of the present disclosure;

[0031] FIG. 5B is a graph of potential profiles, according to an embodiment of the present disclosure;

[0032] FIG. 6A is a schematic drawing of a portion of a system for sensing and sequencing of biopolymers, according to an embodiment of the present disclosure, where a local transverse electric field is configured to slow down or stall a translocating molecule between a first and a second electrodes;

[0033] FIG. 6B is a schematic drawing of a portion of a system for sensing and sequencing of biopolymers, according to an embodiment of the present disclosure, where a local transverse electric field is configured to allow a translocating molecule to pass between a first and a second electrodes; and

[0034] FIG. 7 is a schematic cross-sectional view of a portion of a system for sensing and sequencing of biopolymers, according to an embodiment of the present disclosure.DETAILED DESCRIPTION

[0035] The detailed description set forth below in connection with the appended drawings is intended as a description of exemplary embodiments of a nanopore systemprovided in accordance with the present disclosure and is not intended to represent the only forms in which the present disclosure may be constructed or utilized. The description sets forth the features of the present disclosure in connection with the illustrated embodiments. It is to be understood, however, that the same or equivalent functions and structures may be accomplished by different embodiments that are also intended to be encompassed within the scope of the disclosure. As denoted elsewhere herein, like element numbers are intended to indicate like elements or features.

[0036] Over the past two decades, significant advancements have been made in genomics, enabled by next-generation sequencing (NGS). With the introduction of single-cell DNA and RNA sequencing, the understanding of biological diversity and heterogeneity has been transformed. These achievements have led to an explosion of genomic data, laying down a foundation for multi-omics. Besides genomics, multi-omics includes biological information from epigenomics (epigenetic modifications on deoxyribonucleic acid (DNA)), transcriptom ics (ribonucleic acid (RNA)), proteomics (proteins), metabolomics (metabolites), and others to collectively study the underlying biology of an organism or disease, overcoming the limits of a single-omics analysis. For example, genomic sequencing only provides information about a DNA sequence and does not reveal the functional roles of the genes and their interactions with other molecules.

[0037] The integration of multiple omics levels of data may benefit the understanding of the complexity of biological systems and the identification of new targets for diagnosis and treatment. Several years ago, the National Heart, Lung, and Blood Institute (NHLBI) launched a Trans-Omics for Precision Medicine (TOPMed) program that integrates multi-omics data with environmental, imaging, clinical, and other data. The study of trans-omics in a clinical setting may lead to new diagnostic and therapeutic strategies. Such applications may require multi-omics analysis with high accuracy at a low cost. Multi-omics data may be acquired by NGS and mass spectroscopy (MS). As used herein, “multi-omics” refers to the use of a plurality of analytical techniques or to the analysis (e.g., sequencing) of different kinds of molecules (e.g., DNA, RNA, proteins, or metabolites), or both. As used herein, “trans-omics” refers to integrating data fromdifferent multi-omics characterizations as a method for reconstructing a global biochemical network by connecting multi-omic layers.

[0038] NGS sequences DNA by reading fluorescence signals and is a technology for obtaining genomics and transcriptom ics data. Since NGS is based on the Watson-Crick- based pairing principle, it may be effective for genomics, but not for epigenomics. In addition, due to its short read length, NGS relies on the reference genome for the sequence assembly. Given that approximately 50% of the human genome is composed of repeats with 2 to 100,000 bases in length 10, a longer reading length is urgently needed. Third-generation sequencing, such as single-molecule real-time sequencing (SMRT) and protein nanopore sequencing, increases the read length tremendously, but at the expense of reading accuracy. In addition, RNA sequencing is done by reverse transcription of RNA to complementary DNA (cDNA), by which the position and identity of RNA modifications are all lost.

[0039] Although a protein nanopore sequencer may be successful in rapid singlemolecule DNA sequencing with a very long read length, and may potentially sequence RNA directly, it remains extremely challenging, if not impossible, to identify modified ribonucleotides.

[0040] On the other hand, for proteomics and metabolomics, MS is the dominant technology, which identifies molecules and their fragments by measuring their molecular weights. However, protein sequencing by MS is compounded by isoforms. In addition, single-cell proteomics provides a practical approach to identifying rare cell types and gaining insights into cellular processes often overlooked in ensemble analysis. It may address the misleading effects of Simpson's Paradox, which may be prevalent in ensemble assays. However, single-cell proteomics encounters several persistent challenges.

[0041] A first challenge of single-cell proteomics is high complexity. Protein expression undergoes transcription, alternative splicing, and various post-translational modifications (PTM), such as phosphorylation, glycosylation, and acetylation. As a result, a proteome consists of proteoforms rather than "canonical proteins". The complexity of the human proteome is estimated to range from 98,000 to 6 millionproteoforms, potentially exceeding 1 billion species. Nonetheless, determining the exact upper limit of proteomic complexity remains challenging.

[0042] A second challenge of single-cell proteomics is dark matter. Of the 21637 protein-encoding genes reported in the human gene database (as of Jan. 2023), only 90% of the predicted proteins have been detected. The remaining proteins may be unexpressed or only be expressed in limited quantities, making them difficult to detect using conventional analytic techniques. It is also possible that these proteins lack essential features for current techniques to see.

[0043] A third challenge of single-cell proteomics is little material: the small amount of protein present in a single cell poses a challenge for the current proteomic analysis tools. A yeast cell may contain approximately 42 million protein molecules accurately. Based on this data, a representative human cell may contain around 2 x 109protein molecules. The cellular proteome exhibits a dynamic range spanning from one copy per cell to ten million copies per cell. Some methods may only capture the high-abundance proteins, missing those low-abundance proteins referred to as "the dark proteome".

[0044] Significant efforts have been put to single-molecule detection methods in response to the growing need for higher dynamic range and a lower detection limit. Two main technological paths have emerged. The first approach utilizes enzyme chemistry and fluorescent labeling, following the design logic of NGS.

[0045] Promising results have been seen, but the specificity and dynamic range of detection need considerable optimization due to the much wider range of variations that need to be differentiated (compared to only four bases for genomics). The second approach is based on nanoelectronic transducers, mainly nanopore sensors, for label- free single-molecule characterization. These sensors detect ion current fluctuations as individual biomolecules (e.g., biopolymers) are driven through a confined nanoscale orifice. While nanopores are very useful at confining, manipulating and investigating a single molecule, the ionic current readout is fundamentally limited by a spatial resolution of approximately 3 nm due to the extension of the electric field on either side of the pore being comparable to its diameter. As a result, significant data processing is required to distinguish combinations of at least 4-5 bases, which correspond to approximately 256 or 1024 possible signals. Although other translocating biopolymers like RNA or proteinshave characteristic ionic current fluctuations, the exponentially increasing combinations of possible signals may cause the corresponding data processing to be challenging. Overall, resolution and specificity still require significant improvement for practical use.

[0046] Overall, new developments in sequencing technology may rely on a single type of readout signal (either fluorescence or ionic current). Advanced algorithms may be used to exploit the kinetic characteristics of the readout from a single-molecule event to better identify specific information about the biomolecule. However, it remains an open question what multidimensional data clusterization capacity is needed to differentiate larger structural arrangements, such as the 20 amino acids in proteomes, as required for direct sequencing. As such, further increasing the parameter space may involve integrating additional types of readout signals correlated to the same event.

[0047] Two candidate readout mechanisms, quantum tunneling conductance of a molecule and plasmon-enhanced Raman detection, may potentially reach extremely high spatial resolution.

[0048] Quantum tunneling conductance of a molecule may be employed as follows. Charge transport across a metal nanopore bridged by a single molecule is a quantum process. The construction of the metal-molecule-metal structure may be achieved using several methods. In a first method, a fixed metal nanopore is fabricated by high- resolution lithography, then the molecule is placed onto the gap by absorption. For such a fixed metal gap, it may be difficult to match the size of a biomolecule (typically a few nm) by fabrication, and surface contamination of the electrodes may make the quality of molecular bridging unreliable. A second method uses a scanning tunneling microscope, in which a sharp metal tip is pushed into and pulled out of a metal substrate with very accurate distance control, such that a fresh breaking junction is repetitively constructed due to the elastic deformation and eventual breaking of the metal contact. Such a method may be referred to as a scanning tunneling microscope breaking junction (STM-BJ) method. Meanwhile a single molecule may randomly enter such a break junction by diffusion and the conductance of the molecule may be detected statistically out of the exponentially decaying current background. In a third method, which may be referred to as a mechanical break junction (MBJ) method, and which is similar to STM-BJ, a mechanically bent substrate with a thin metal film suspended with a bottleneck shape onthe top may be used to create fresh break junctions repetitively (MBJ), to probe molecules that randomly diffuse in between.

[0049] The tunneling conductance strongly depends on the molecule's structure and the interface between the molecule and the metal surface. For example, when a DNA molecule is sandwiched between the electrodes of a metal nanopore, the current across the gap (which may be referred to as a the “transverse” current) may be used to identify the sequence with very high spatial resolution and chemical specificity. This transverse current may include a contribution due to a “hopping” process, which may arise when electrons move from a first electrode of the two electrodes to a second electrode of the two electrodes by tunneling across the gap between the first electrode and the biopolymer molecule being characterized, and then by tunneling across the gap between the biopolymer molecule and the second electrode. In embodiments in which capture molecules are present on the surfaces of the electrodes (as discussed in further detail below), the hopping process may correspond to electrons tunneling (i) from the first electrode to a first capture molecule on the surface of the first electrode, then (ii) from the first capture molecule to the biopolymer molecule, then (iii) from the biopolymer molecule to a second capture molecule on the surface of the second electrode, and then (iv) from the second capture molecule to the second electrode. Or in embodiments in which capture molecules are present on the surfaces of the electrodes, the capture molecule can serve to change the tunneling probability between the first electrode and the biopolymer molecule and between the biopolymer and the second electrode by forming non-covalent interactions between the capture molecule and the biopolymer, such as hydrogen bonds, pi-pi stacking, or hydrophobic interactions. As used herein, a “tunneling” current is a current the flow of which is due at least in part to quantum tunneling across a junction between an electrode and a molecule. Various biological molecules may be investigated by their characteristic transverse tunneling current signals, including DNA nucleoside monophosphates, RNA nucleoside and modified nucleosides monophosphates, amino acids, and short peptides, stereoisomers of carbohydrates and glycosylated peptides. The transverse tunneling current may be used to distinguish different molecular structures. However, in some apparatuses, hundreds to thousands of repetitive operations may be needed to generate one meaningful result.There may be no control of the position or the states of the molecule for each run, and it may be generally assumed that all configurations will be sampled. Significant postprocessing of the data may be required to extract useful information from the strong background.

[0050] Plasmon-enhanced Raman detection, including surface-enhanced and tip- enhanced Raman scattering (SERS and TERS), may be employed to detect vibrational fingerprint spectra of individual molecules. For example, the target molecules can be trapped within metal nanopores or cavities that are created by roughened metal surfaces, closely packed metal nanoparticles, or a sharp metal tip near a substrate. The highly focused electric field within the gap or cavity (electromagnetic enhancement) and the interaction of trapped molecules with the metal surface (chemical enhancement) may give more than eight orders of magnitude increase in Raman scattering signals, which would otherwise be undetectable for a single molecule. However, Raman measurements may be limited to quasi-static mapping or recording of random single-molecule events, possibly due to the difficulty in the integration with definite control of the position and motion of a single molecule. In addition, the time resolution may be low for moving molecules due to a relatively long acquisition time of the spectrum for some systems.

[0051] In some embodiments, a bowtie shaped metal nanopore may enhance Raman scattering of the trapped molecule by a factor of 1010or more when the gap size is less than 5 nm.

[0052] In some embodiments, these two types of signals, quantum tunneling conductance of a molecule and plasmon-enhanced Raman scattering, may be detected together for a multi-modal detection of the same molecule. For example, cysteine molecules may randomly diffuse into a fresh Au nanopore created by a microelectromechanical break junction (MEMS-BJ). The tunneling current and Raman spectrum may be acquired simultaneously. A correlated change in the Raman spectrum, with the fingerprint peaks of cysteine, may occur when an increase in the tunneling conductance occurs, which marks the brief formation of a single molecule bridge between the two electrodes. Although in such an apparatus the molecules may not be controllably delivered, and the measurement may be performed in a dry state, the Raman spectrum may be highly localized at the tunneling junction, which may act like astrong plasmonic structure, to identify the functional groups unambiguously, and the tunneling conductance may be directly associated with the structure of the molecular bridge.

[0053] Such techniques may respectively address the sensitivity, specificity and controllability requirements for single-molecule analysis. However, various challenges may remain in some approaches that make them impractical for actual sequencing, including the requirement of repetitive operations (e.g., the inability to characterize a single molecule at a time), lack of time resolution, and lack of controlled sample delivery. Some methods rely on special instruments such as a STM and only work in a dry state. Some embodiments, however, combine advantages of different techniques in a device design that is easy to make and use. FIG. 1 shows a conceptual schematic such a system. Included in FIG. 1 is a top view of a device on a microchip. The biomolecule 205 is guided through a pair of transverse electrodes 210 that are perpendicular to its translocation pathway on the surface of the chip. The molecule only has one degree of freedom to pass through the electrodes 210 as the whole device is enclosed inside a nanofluidic channel from the top, which allows translocation control by the translocation control circuit 215 shown on the right side (in purple). The electrodes 210 form a nanopore (with the top seal cover), a tunneling junction and a plasmonic structure in one place to allow conductance and Raman measurements on the same molecule. Some embodiments integrate plasmon-enhanced Raman scattering, capture molecule tunneling (which may contribute to a hopping process), and nanopore technology into a microchip-based universal single-molecule sequencing platform, featuring amplification- free sample preparation, multiplexed operation, and multi-modal high-resolution characterization for high-throughput multi-omics analysis and beyond.

[0054] Some embodiments achieve feasibility and cost of fabrication, feasibility and scaling-up of multi-modal detection, and a workflow and methodology for practical sequencing applications.

[0055] To achieve feasibility at a relatively low cost of fabrication, some embodiments use reversible electrochemical deposition with feedback control to fill the gap between two metal electrodes, which are sandwiched between two dielectric layers, as shown in a cross-section view in FIG. 2. N1 and N2 are two Au electrodes with a thicknessbetween 1 nm and 100 nm fabricated on the substrate, and the initial gap between them is between 100 nm and 1000 nm. A dielectric layer seals both the electrodes and the gap from the top and forms a cavity. Metal ions are introduced and electrodeposition may be employed to reversibly add or remove metal inside this cavity on the sidewall of the metal electrodes as the impedance between N1 and N2 is monitored.

[0056] When the distance between N1 and N2 enters the tunneling distance of a few nm, the nanopore may be finely tuned based on a desired tunneling conductance or AC impedance. The result, after such tuning, may be a tunneling gap in the middle, which simultaneously also forms a nanopore channel in this cross-section plane with a height defined by the original thickness of the cavity (1 nm -100 nm), and a width defined by the tunneling gap. The substrate material and the layout of the electrodes may be selected to give optical access to all devices.

[0057] The narrowest part of the gap between the electrodes may serve three functions, providing (i) the nanopore bottleneck along the path of molecule translocation, (ii) the tunneling gap, and (iii) the strongest plasmon-enhancement volume for the Raman scattering detection. The size of this detection volume may be approximately 10 nm x 1 nm x 100 nm (based on the estimated sharpness of the tips of electrodes), i.e. , 0.001 zeptoliters (10-24L). A three-dimensional schematic of this structure is shown in FIG. 3, highlighting how the molecules may be guided through this detection volume and the multi-modal characterization enabled by this structure.

[0058] In some embodiments, a chip design including electrodes forming a nanopore may be fabricated in a manner making it possible to make the chip available to various users at low cost and with high reproducibility. The initial gap between electrodes may be easily fabricated with standard top-down photolithography, and every pair of devices may have about 100 pm spacing between them to allow wiring. Based on this constraint, it may be possible to prepare up to thousands of "initial" electrode pairs on a precursor chip having an area or approximately one square inch, at low cost. Such precursor chips may be stable in the dry state and the initial cavities may also be stabilized by leaving in place, until just prior to use, the sacrificial layer that may be etched away to form the nanofluidic channel. Once the user receives one such low-cost chip, the sacrificial layer may be selectively etched away, and the electrodeposition process may be used toprepare each device on the chip. This has two advantages: first, each tunneling gap will have a freshly prepared metal surface before actual recording experiments, which may be advantageous for the formation of reproducible molecule bridges; second, feedback control (based on the tunnelling current measured during the electrodeposition process) may be employed to control the electrodeposition process, which may ensure that the gap distance and nanopore cross-section is reproducible from device to device. The macroscopic shapes of the electrodes may have differences, but the measurement may be primarily determined by the smallest bottleneck. The chip may have no moving parts and the deposition process may be automated in a multiplexed manner.

[0059] The time resolution of the Raman detection may be determined by the number of photons collected from each device. If each device receives 1 pW of laser power with a wavelength corresponding to green light, then the molecule may receive approximately 3 million (M) photons / s. The Raman scattering process may produce one photon for every 10 M photons received, which may mean that at most 0.3 photons / s may be collected. This low rate may make single-molecule Raman scattering challenging without enhancement. For a nanopore of approximately 2 nm, an enhancement factor between 106to 108may be expected, resulting in a total of 3 x 105to 3 x 106photons / s for collection. For an enhancement factor of 106, a camera with a detection limit of 100 photons, and a collection efficiency of at least 30% (which may be achieved with a high numerical aperture (NA) objective 305), an exposure time of 1 ms may be sufficient for detection of the Raman scattering signal.

[0060] In some embodiments, multiplexed Raman detection may be performed using optical fibers. As shown in FIG. 4, the device array 405 may be arranged in a close- packed array, and laser light from a Raman probe laser 410 may be introduced to the device plane with a total internal reflection configuration (using a total internal reflection fluorescence (TIRF) objective 305). Light from a laser 415 (which may be referred to as a “gradient force” laser and which may be used to reduce the translocation speed, as discussed in further detail below) may be combined with the light from the Raman probe laser 410 (which may be a probe laser for measuring Raman scattering) by a first dichroic mirror 420. A second dichroic mirror 425 may reflect the light from both lasers into the objective 305, which may focus the light on the device array 405. The lasers 410, 415may share the same objective as shown in FIG. 4. The lasers need not both work in TIRF mode. For example, the gradient force laser 415 may be scanned with a focused point through the same objective lens 305. Different lasers may be combined to serve different purposes, and the light from all of the lasers may come out of the same objective 305 to interact with the sample. Light that scatters (e.g., by Raman scattering) from the device array 405 may be transmitted through the second dichroic mirror 425, and transmitted through one or more optical filters (which may suppress the wavelength of the Raman probe laser 410 or the wavelength of the gradient force laser 415, or both), to a tube lens 430. These filters may be or include one or more notch filters (e.g., notch filters having notches at the laser wavelengths (e.g., two notch filters, each configured to suppress the wavelength of a respective one of the lasers 410, 415)) or one or more low-pass or high-pass or band-pass filters (configured to suppress the laser wavelengths and only allow the Raman scattering signals that are of interest to pass). The filtered optical signals from the device array 405 may form, after the tube lens 430, an image in which each nanogap in the device array 405 matches the size and position of a respective fiber in a close-packed optic fiber bundle 435. Since each effective Raman signal may originate in a corresponding 0.001 zeptoliter detection volume, each device may appear to be a single point light source with minimum background. For a numerical aperture (NA) 0.95 objective 305, the collection yield may be approximately 34% for each device, and the coupling efficiency to the optic fibers may be close to 100%. As such, it may be possible to capture the Raman spectra for all devices on a millisecond time scale based on the estimate described above. A spectrometer 440 connected to the fibers, may be employed to obtain spectra simultaneously, from the fibers, for all of the devices.

[0061] In some nanopore devices, the nanopore is directly exposed to bulk solution, which means that when a cross-membrane potential is applied, almost the entire potential drop between the chambers is concentrated within a very small distance (e.g., a few nm) at the nanopore, as shown by a first curve 605 in FIG. 5B (which illustrates how, with nano-fluidic guides, the potential profile between the first chamber 505 and the second chamber 510 (FIG. 5A) may significantly increase capture rate). As used herein, the first chamber 505 is the chamber from which the molecule being characterized moves (by translocation) into the second chamber 510. Outside this radius, the chargedmolecules may essentially move randomly. This significantly limits the capture rate especially at high ionic strength, and limits the detection sensitivity (e.g., to at least 10 picomoles / liter (pM)).

[0062] As such, some embodiments increase the detection sensitivity for low-copy- number species by including a thin lateral nanofluidic channel between the nanopore and each of the chambers (e.g., the first chamber 505 and the second chamber 510), as illustrated in FIG. 5A. The potential profile (shown, in one example, as a second curve 610 in FIG. 5B) may be engineered to have different gradients through the translocation path of the DNA by tuning the geometry and the dimensions of different sections of the nanofluidic channels. For example, at 100 mM electrolyte concentration, the potential drops at the outer channel (the fan-shaped region, approximately 100 nm thick), the inner channel (the center region, approximately 10 nm thick), and the nanopore, may be estimated to be around 10%, 40% and 50% of the total applied bias, respectively. This not only creates a much larger region for 'capturing' the DNA molecules, as the entire outer rim of the nanofluidic channel may attract DNA molecules into the guided path with a much larger cross-sectional area, but also enables preparing the DNA molecules for a more uniform translocation as the potential gradient may be able to straighten the molecule when the height of the channel is close to the persistent length of DNA.

[0063] A solid state nanopore may have a translocation speed of over 1 Mbases / s, which may be too fast for most detection methods. In some embodiments, methods for translocation control by local fields, may be employed. FIG. 6A illustrates the use of a transverse electric field, which may slow or stall the translocation, by promoting stronger interaction between the biopolymer molecule 205 and capture molecules 620 (discussed in further detail below) on the surface of the electrode 210 toward which the biopolymer molecule 205 is driven by the transverse electric field. Other embodiments may use optical modulation of the metal gap, which may apply optical force to trap the molecule (as discussed in further detail below). In some embodiments, the biopolymer-nanopore interaction may be modulated by surface functionalization and the local electric field or optical field inside the nanopore. For example, surface functionalization of a metal surface inside a solid-state nanopore using a capture molecule may significantly slow down the translocation of DNA. In addition, a uniform transverse electric field of 180mV / nm may completely stop the translocation of a DNA molecule based on the van-der- Waals interaction with the side wall. A 400-fold decrease of translocation speed may be observed with a nominal transverse field of 10 mV / nm in a 60 nm high nanochannel. Since a sharp tip with a radius of less than 50 nm may cause a field enhancement of more than a factor of 10 to the field at the gap, the nanopore (with a width of approximately 2 nm) may produce a local field between 125 mV / nm and 1250 mV / nm in physiological solutions that in principle may effectively retard translocation. In addition, in some embodiments, single molecules are trapped and released by optically modulating a plasmonic nanopore. As such, in some embodiments, the transverse nanopore in the device is employed to find the proper combination of surface modification, local electric field, and optical plasmon modulation to slow down, and potentially control, the translocation of biopolymers.

[0064] As a first example, the nanofluidic channel and the metal nanopore and nanopore may be chemically modified independently. The surface charge of the nanofluidic channels may determine the direction and strength of the electroosmotic force which is generated by the moving opposite ions near the surface when driving bias is applied. Meanwhile, the electrostatic force acting on the translocating molecule is only determined by its charge under the same driving bias. Therefore, the electroosmotic force may be tuned to be in the same direction or against the electrostatic force on the same translocating molecule. Different silane coupling reagents may be used to change the surface charge from negative, to neutral and positive with different charge density. This may make it possible to find an optimal total electrohydraulic force to reduce the speed of molecules moving inside the channel. In addition, for the gold surface of the nanopore, which is the smallest constriction point, thiol molecules with different functional groups, including polyethylene glycol (PEG), 4(5)-(2- mercaptoethyl)-1 H- imidazole-2-carboxamide (ICA), and alkyl chains may be introduced; these functional groups may affect (e.g., increase or decrease) the translocation speed of the molecule. Molecules or functional groups for such an application, or for increasing the contrast in the tunneling conductivity, may be selected based in part on their ability to interact with the molecules to be characterized. Such interactions may include a hydrogen bonding interaction, a pi-pi stacking interaction, and a hydrophobic interaction. A suitablemolecule may include an anchoring portion to anchor the molecule to the electrode, and an exposed functional group for interacting with the molecule to be characterized.

[0065] As a second example, the electric field may be modulated. The transverse electrodes may switch between two bias modes (as illustrated in FIGs. 6A and 6B). First, as shown in FIG. 6B, both electrodes may be held at the same potential (zero transverse field), and the translocation may be controlled by the joint electroosmotic and electrostatic forces; second, a shown in FIG. 6A, a high transverse electric field, focused at the tip of the gap, may be formed by applying different potentials at the electrodes, where the charged molecule, for example, a DNA chain, may be pushed toward one side of the nanopore, resulting in an appreciable reduction in the translocation speed of the molecule.

[0066] As a third example, optical modulation of the metal nanopore, produced by illuminating the gap with a focused laser beam from a gradient force laser may be used to change the translocation speed of the molecule, using a single-beam gradient force trap effect, which may produce a force, on the portion of the biopolymer that is in the gap, proportional to the gradient in the magnitude of the electric field to the laser light (and either in the direction of the gradient, or in the opposite direction, depending on the refractive index of the portion of the biopolymer relative to the surrounding medium). For example, laser sources at 440 nm, 530 nm, 561 nm, 640 nm, or 780 nm may be used, depending on the gap size. Laser power between 1 mW and 100 mW may be used to adjust the optical force strength and translocation modulation. This laser may operate at a wavelength that is sufficiently different from any wavelengths being used for Raman measurements to avoid interfering with such measurements; as such, the gradient force laser may operate while Raman measurements are performed.

[0067] As another example, a molecular motor may be integrated inside the nanopore channel by being chemically bonded with a linker molecule to the nanofluidic surface or the electrodes so that the biopolymer can be first captured, and the translocation of the molecule may be further regulated and slowed down. Biochemical engineering may be applied to the motor so that it may be positioned properly.

[0068] In some embodiments, the nanopore device has the first chamber 505 and second chamber 510 on the same plane and the molecules flow through the device onthe same side of the substrate. In such an embodiment, a large surface area, for the microfluidic channels, may be used; this may limit the density of the nanopore devices. As such, in some embodiments, both sides of the substrate may be used, by creating one or more through-holes next to each nanopore device, as illustrated in FIG. 7. As a result, the molecule may be introduced from one side of the substrate, flow laterally through the nanopore, and then be guided to the through-hole and exit on the other side of the substrate. As shown in FIG. 7, the first chamber 505 includes the continuous space from the top side of the substrate to the entry of the nanofluidic channel (which opens, on one side, onto the first chamber 505 and, on the other side, onto the second chamber 510) The second chamber 510 includes the continuous space between the confined nanofluidic channel connected to the via through the substrate, and the space on the bottom side of the substrate. The arrow of FIG. 7 marks the path that a molecule may follow as it enters the channel, moves through the tunneling gap between the first and the second electrodes 210, and exits to the other side of the substrate. This design may be able to significantly increase the density of devices, and reduce the complexity of fluidic channel structures required for multiplexed delivery of molecules to different regions of the chip.

[0069] In a recognition-tunneling system, the charge is transported through the target molecule, with orders of magnitude of conductance changes when a molecular bridge is present. The quantum process may observe profound changes in electrical conductance due to small changes in the chemical structure of the molecule. Therefore, recognition tunneling may identify specific bases and amino acids. However, there may be several ways in which a sub-molecular moiety (base, amino acid, etc.) may bind between the two electrodes, resulting in a resistance dispersion for each moiety that overlaps with other substituents. Alternatively, Raman-based measurements may provide a direct fingerprint of specific moieties, but due to the optical resolution limit of an unenhanced Raman measurement, it may not be possible to identify sub-molecular substituents. Therefore, in some embodiments, a system that provides a clear indication of when a sub-molecular component is bound between two electrodes in a plasmonic nanopore and allows a Raman fingerprint to be obtained from the specific molecule involved in thebinding / charge transport process provides a unique, multi-dimensional capability for sub- molecular characterization.

[0070] To use multi-dimensional spectroscopy measurements for identifying many residues and their known modifications, a database of combined recognition tunneling and junction-based Raman spectra may be used to identify specific targets. A database that includes a large sampling of both the conductance values and the Raman spectra of all of the monomers of interest (DNA / RNA bases, modified nucleotides, and amino acids) may be used for this purpose. This database may be leveraged for machine- learning-based approaches for identifying the substituents. Each substituent may be characterized in an isolated case to understand the variation in conductance and spectroscopic features that occur in each case. Using such a database, and machine learning models, some embodiments may differentiate two sequences with a single point difference, and achieve reading length exceeding 50,000 nuclear bases or 50,000 amino acid residues.

[0071] Some embodiments include a new device design and fabrication strategy, which enables multi-modal characterization. Such embodiments may include a plasmonic quantum tunneling structure precisely enclosed in a guided nanopore channel that may be mass-fabricated on silicon or sapphire substrates as a multi-channel microchip device. The chip may be mounted on a compact hyper-Raman-spectrum imaging system, such that in each channel, individual molecules may be accurately delivered through a detection volume of 0.001 zeptoliters (10’24L) at the nanopore channel, where highly localized plasmon-enhanced Raman signals and quantum tunneling conductance may be acquired simultaneously.

[0072] Some embodiments combine top-down lithography and reversible electrochemical deposition in a confined nanoscale cavity. Such an approach enables integration of electrodes or plasmonic structures with a nanopore device, and may exhibit several advantages: (i) the geometry of the device ensures that all molecules that translocate through the nanopore pass through the tunneling junction; (ii) the metal nanopore also serves as the plasmonic structure for generating an enhanced Raman signal localized within the same detection volume as the tunneling current; (iii) the type of metal, gap distance, and surface functional groups of the tunneling electrodes may allbe tuned by electrochemical control and chemical modification, for optimal recognition tunneling detection and Raman spectroscopy; and (iv) the chips containing such nanopore devices may be mass-produced at low cost.

[0073] Some embodiments further include a multi-modal analysis paradigm. Such an embodiment may combine the correlated Raman signal and quantum tunneling conductance, which carry the intrinsic molecular information, to differentiate a wide range of structures with high chemical specificity. Such a multi-modal analysis may open a much bigger parameter space for a universal sequencing framework and may be used to build and train machine-learning models that provide real-time sequencing results with improved accuracy. Some embodiments incorporate the integration of tightly coupled plasmon-enhanced Raman spectroscopy, recognition tunneling, and nanopore technology into a single platform. Such an embodiment may enable comprehensive, multi-modal characterization at the single-molecule level, with amplification-free sample preparation and long reading length, and may reach a spatial resolution of less than 1 nm and a time resolution of less than 1 ms.

[0074] The term “alkyl” or “alkyl group”, as used herein, means a straight-chain (i.e. , unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation. Unless otherwise specified, alkyl groups contain 1 -100 carbon atoms. In other embodiments, alkyl groups contain 1 -50 carbon atoms. In still other embodiments, alkyl groups contain 1 -25 carbon atoms. In yet other embodiments, alkyl groups contain 1-10 carbon atoms. In further embodiments, alkyl groups contain 1 -6 carbon atoms. In even further embodiments, alkyl groups contain 1 -5 carbon atoms. In yet even further embodiments, alkyl groups contain 1-4 carbon atoms. In other embodiments, alkyl groups contain 1 -3 carbon atoms. In some embodiments, alkyl groups contain 1 -2 carbon atoms.

[0075] As used herein, “a portion of” something means “at least some of’ the thing, and as such may mean less than all of, or all of, the thing. As such, “a portion of” a thing includes the entire thing as a special case, i.e., the entire thing is an example of a portion of the thing. As used herein, when a second quantity is “within Y” of a first quantity X, it means that the second quantity is at least X-Y and the second quantity is at most X+Y. As used herein, when a second number is “within Y%” of a first number, it means thatthe second number is at least (1 -Y / 100) times the first number and the second number is at most (1 +Y / 100) times the first number. As used herein, the word “or” is inclusive, so that, for example, “A or B” means any one of (i) A, (ii) B, and (iii) A and B.

[0076] As used herein, when a method (e.g., an adjustment) or a first quantity (e.g., a first variable) is referred to as being “based on” a second quantity (e.g., a second variable) it means that the second quantity is an input to the method or influences the first quantity, e.g., the second quantity may be an input (e.g., the only input, or one of several inputs) to a function that calculates the first quantity, or the first quantity may be equal to the second quantity, or the first quantity may be the same as (e.g., stored at the same location or locations in memory as) the second quantity.

[0077] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the inventive concept. As used herein, the terms “substantially,” “about,” and similar terms are used as terms of approximation and not as terms of degree, and are intended to account for the inherent deviations in measured or calculated values that would be recognized by those of ordinary skill in the art.

[0078] As used herein, the term “major component” refers to a component that is present in a composition, polymer, or product in an amount greater than an amount of any other single component in the composition or product. In contrast, the term “primary component” refers to a component that makes up at least 50% by weight or more of the composition, polymer, or product. As used herein, the term “major portion”, when applied to a plurality of items, means at least half of the items. As used herein, any structure or layer that is described as being “made of” or “composed of” a substance should be understood (i) in some embodiments, to contain that substance as the primary component or (ii) in some embodiments, to contain that substance as the major component.

[0079] Any numerical range recited herein is intended to include all sub-ranges of the same numerical precision subsumed within the recited range. For example, a range of "1.0 to 10.0" or “between 1.0 and 10.0” is intended to include all subranges between (and including) the recited minimum value of 1 .0 and the recited maximum value of 10.0, that is, having a minimum value equal to or greater than 1 .0 and a maximum value equalto or less than 10.0, such as, for example, 2.4 to 7.6. Similarly, a range described as “within 35% of 10” is intended to include all subranges between (and including) the recited minimum value of 6.5 (i.e. , (1 - 35 / 100) times 10) and the recited maximum value of 13.5 (i.e., (1 + 35 / 100) times 10), that is, having a minimum value equal to or greater than 6.5 and a maximum value equal to or less than 13.5, such as, for example, 7.4 to 10.6. Any maximum numerical limitation recited herein is intended to include all lower numerical limitations subsumed therein and any minimum numerical limitation recited in this specification is intended to include all higher numerical limitations subsumed therein.

[0080] Although exemplary embodiments of a nanopore system have been specifically described and illustrated herein, many modifications and variations will be apparent to those skilled in the art. Accordingly, it is to be understood that a nanopore system constructed according to principles of this disclosure may be embodied other than as specifically described herein. The invention is also defined in the following claims, and equivalents thereof.

Claims

WHAT IS CLAIMED IS:1 . A system, comprising: a first nanopore device, the first nanopore device comprising: a first chamber and a second chamber in a planar substrate; a nano-fluidic channel connecting the first chamber and the second chamber; a first electrode and a second electrode forming a nanopore between the first electrode and the second electrode; an optical coupling element configured to couple an electromagnetic beam with the nanopore; a first bias circuit for applying a bias to the first electrode and the second electrode, to form a transverse electric field in the nanopore; and a current-measuring circuit configured to measure a current between the first electrode and the second electrode.

2. The system of claim 1 , further comprising: a third electrode and a fourth electrode in the first chamber and the second chamber, respectively; and a second bias circuit, for generating an electroosmotic flow, a longitudinal electric field, or the combination of both, for causing a molecule to translocate from the first chamber to the second chamber.

3. The system of claim 2, wherein: the system comprises one or more surfaces functionalized to reduce a translocation speed of the molecule, or the system is configured to apply an electromagnetic field to slow the translocation speed of the molecule, or the system comprises one or more molecular motors to control the translocation speed of the molecule.

4. The system of claim 3, wherein the system comprises one or more surfaces functionalized to reduce a translocation speed of the molecule.

5. The system of claim 4, wherein a surface of the one or more surfaces is a surface of the first electrode.

6. The system of claim 5, wherein the surface is functionalized with a thiol containing molecule further comprising a functional group selected from a polyethylene glycol, a 4(5)-(2- mercaptoethyl)-1 H-imidazole-2-carboxamide (ICA), and an alkyl group.

7. The system of claim 3, wherein the system is configured to apply an electromagnetic field to slow the translocation speed of the molecule.

8. The system of claim 7, wherein the electromagnetic field is an optical electromagnetic field.

9. The system of claim 8, wherein the optical electromagnetic field is an optical electromagnetic field of a laser directed at the nanopore.

10. The system of claim 7, wherein the electromagnetic field is an electric field.11 . The system of claim 10, wherein the electric field is a field resulting from a bias applied by the first bias circuit.

12. The system of claim 1 , wherein the optical coupling element comprises a total internal reflection objective having a focus at the nanopore.

13. The system of claim 1 , comprising: a nanopore device array comprising the first nanopore device, anda group of lenses for relaying and projecting an image of the nanopore device array onto the ends of a plurality of optical fibers.

14. The system of claim 13, wherein the group of lenses images each of the nanopores of the nanopore device array onto a respective one of the ends of the fibers.

15. The system of claim 14, further comprising a spectrometer connected to the optical fibers.

16. A method, comprising: operating a system comprising a first nanopore device, the first nanopore device comprising: a first chamber and a second chamber in a planar substrate; a nano-fluidic channel connecting the first chamber and the second chamber; a first electrode and a second electrode forming a nanopore between the first electrode and the second electrode; an optical coupling element configured to couple an electromagnetic beam with the nanopore, the operating comprising: applying a first bias to the first electrode and the second electrode, to form a transverse electric field in the nanopore; and measuring a current between the first electrode and the second electrode.

17. The method of claim 16, further comprising applying a second bias to a third electrode in the first chamber and a fourth electrode and the second chamber, to cause a molecule to translocate from the first chamber to the second chamber.

18. The method of claim 17, further comprising measuring a Raman scattering signal from the molecule.

19. The method of claim 16, further comprising etching a sacrificial layer from within a precursor chip, to form the nano-fluidic channel and the cavity between the first electrode and the second electrode.

20. The method of claim 16, further comprising adjusting a gap between the first electrode and the second electrode by a reversible electrodeposition process.

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