WO2021237182A1

WO2021237182A1 - Shape-altered graphene nanobridge array, transfer-aligned for biomolecular sensing and information storage

Info

Publication number: WO2021237182A1
Application number: PCT/US2021/033784
Authority: WO
Inventors: Sungho Jin; Chulmin Choi; Paul MOLA; Barry Merriman
Original assignee: Roswell Biotechnologies, Inc.
Priority date: 2020-05-22
Filing date: 2021-05-21
Publication date: 2021-11-25
Also published as: WO2021237180A1

Abstract

The disclosed embodiments relate to a molecular detection device. An exemplary sensor includes an electrode array of conducting electrode pairs, each electrode pair having a source electrode and a drain electrode separated by a nanogap; a graphene bridge connecting the source electrode to the drain electrode of an electrode pair to form a Field Effect Transistor (FET); an enzyme biomolecule coupled to the graphene bridge; wherein the bridge is shaped to guide and limit the coupling of the biomolecule.

Description

SHAPE-ALTERED GRAPHENE NANOBRIDGE ARRAY, TRANSFER-ALIGNED FOR BIOMOLE CULAR SENSING AND INFORMATION STORAGE

[0001] The instant application claims priority to the Provisional Application No.

63/028,914 (filed May 22, 2020), and to the Provisional Application No. 63/028,796 (May 22, 2020), the specification of both of these applications is incorporated herein in its entirety.

FIELD

[0002] The instant disclosure relates to biomolecular sensing and memory storage devices, in particular, to the nanofabrication of molecular sensor bridge structures for analyzing DNA and related biomolecules.

BACKGROUND

[0003] Analysis of biomolecules such as DNAs, proteins, genomes has received much attention in recent years, with respect to potential applications in precision medicine or nanotechnology. The seminal work of Maclyn McCarty and Oswald T. Avery in 1946, (see, "Studies On The Chemical Nature Of The Substance Inducing Transformation Of Pneumococcal Types II. Effect Of Desoxyribonuclease On The Biological Activity Of The Transforming Substance," The Journal of Experimental Medicine 83(2), 89-96 (1946)), demonstrated that DNA was the material that determined traits of an organism. The molecular structure of DNA was then first described by James D. Watson and Francis HC Crick in 1953, (see a published article, "Molecular structure of nucleic acids.", Nature 777,737-738 (1953)), for which they received the 1962 Nobel Prize in Medicine. This work made it clear that the sequence of chemical letters (bases) of the DNA molecules encode the fundamental biological information. Since this discovery, there has been a concerted effort to develop means to actually experimentally measure this sequence. The first method for systematically sequencing DNA was introduced by Sanger, et al in 1978, for which he received the 1980 Nobel Prize in Chemistry. See an article, Sanger, Frederick, et al., "The nucleotide sequence of bacteriophage fC174." Journal of molecular biology 125, 225-246 (1978).

[0004] Sequencing techniques for genome analysis evolved into utilizing automated commercial instrument platform in the late 1980’s, which ultimately enabled the sequencing of the first human genome in 2001. This was the result of a massive public and private effort taking over a decade, at a cost of billions of dollars, and relying on the output of thousands of dedicated DNA sequencing instruments. The success of this effort motivated the development of a number of “massively parallel” sequencing platforms with the goal of dramatically reducing the cost and time required to sequence a human genome. Such massively parallel sequencing platforms generally rely on processing millions to billions of sequencing reactions at the same time in highly miniaturized microfluidic formats. The first of these was invented and commercialized by Jonathan M. Rothberg's group in 2005 as the 454 platform, which achieved thousand fold reductions in cost and instrument time. See, an article by Marcel Margulies, et al., "Genome Sequencing in Open Microfabricated High Density Picoliter Reactors," Nature 437, 376-380 (2005). However, the 454 platform still required approximately a million dollars and took over a month to sequence a genome.

[0005] The 454 platform was followed by a variety of other related techniques and commercial platforms. See, articles by M. L. Metzker, "Sequencing Technologies — the Next Generation," Nature reviews genetics 11(1), 31-46 (2010), and by C. W. Fuller et al, "The Challenges of Sequencing by Synthesis," Nature biotechnology 27(11), 1013-1023 (2009). This progress led to the realization of the long-sought “$1,000 genome” in 2014, in which the cost of sequencing a human genome at a service lab was reduced to approximately $1,000, and could be performed in several days. However, the highly sophisticated instrument for this sequencing cost nearly one million dollars, and the data was in the form of billions of short reads of approximately 100 bases in length. The billions of short reads often further contained errors so the data required interpretation relative to a standard reference genome with each base being sequenced multiple times to assess a new individual genome.

[0006] Thus, further improvements in quality and accuracy of sequencing, as well as reductions in cost and time are still needed. This is especially true to make genome sequencing practical for widespread use in precision medicine (see the aforementioned article by Fuller et al), where it is desirable to sequence the genomes of millions of individuals with a clinical grade of quality.

[0007] While many DNA sequencing techniques utilize optical means with fluorescence reporters, such methods can be cumbersome, slow in detection speed, and difficult to mass produce to further reduce costs. Label-free DNA or genome sequencing approaches provide advantages of not having to use fluorescent type labeling processes and associated optical systems, especially when combined with electronic signal detection that can be achieved rapidly and in an inexpensive way. [0008] In this regard, certain types of molecular electronic devices can detect single molecule, biomolecular analytes such as DNAs, RNAs, proteins, and nucleotides by measuring electronic signal changes when the analyte molecule is attached to a circuit. Such methods are label-free and thus avoid using complicated, bulky and expensive fluorescent type labeling apparatus. These methods can be useful for lower cost sequencing analysis of DNA, RNA and genome.

[0009] Certain types of molecular electronic devices can detect the biomolecular analytes such as DNAs, RNAs, proteins, and nucleotides by measuring electronic signal changes when the analyte molecule is attached to the circuit comprising a pair of conductive electrodes. Such methods are label-free and thus avoids using complicated, bulky and expensive fluorescent type labeling apparatus.

[0010] While current molecular electronic devices can electronically measure molecules for various applications, they lack the reproducibility as well as scalability and manufacturability needed for rapidly sensing many analytes at a scale of up to millions in a practical manner. Such highly scalable methods are particularly important for DNA sequencing applications, which often need to analyze millions to billions of independent DNA molecules. In addition, the manufacture of current molecular electronic devices is generally costly due to the high level of precision needed.

[0011] Disclosed herein are new and improved sequencing apparatus and associated sensor configurations and methods using advanced elongated bridge structures comprising shape-altered graphene nanoribbons, which can provide reliable DNA genome analysis performance and are amenable to scalable manufacturing. The disclosed structures are also useful for DNA-based information storage devices including archival or randomly accessible memory and logic devices.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The features and advantages of the embodiments of the present disclosure will become more apparent from the detailed description set forth below when taken in conjunction with the drawings. The drawings and the associated descriptions are provided to illustrate embodiments of the disclosure and not to limit the scope of what is claimed.

[0013] Fig. 1. Transfer of linearly aligned and width-reduced graphene nanoribbons

(on flat substrate) by a PDMS or PMMA (later removable by solvent dissolution) type soft stamp. The PDMS stamp optionally has protruding ridges for easier pick up of the aligned graphene ribbons.

[0014] Fig. 2. Transfer-aligned graphene nanoribbon with narrowed neck width (e.g.,

<5 nm) for bandgap opening. The graphene field effect transistor (FET) with a single enzyme polymerase attached becomes a molecular sensor for genome/DNA sequencing. A single enzyme polymerase is attached onto the bridge to form a biosensor structure, with electrical current signals measured via complimentary nucleotide attaching events (A-T or G-C as adenine is the complementary base of thymine, and guanine is the complimentary base of cytosine in DNA and of uracil in RNA) for sequencing analysis. The biosensor can be utilized for other protein or biomolecule sensing, or DNA memory reading.

[0015] Fig. 3. Top view of an array of Au electrode pairs (or other noble metals), with

PDMS or PMMA placed on them as stamp-transferred, ~5 nm wide and aligned graphene bridges. Additional redundant nanoribbons are provided to ensure at least one bridge connection occurs with the conducting metal electrode pair.

[0016] Fig. 4. Processing sequence for neck-width-reducing heat treatment, followed by stamp transfer of pre-pattemed, pre-aligned, and pre-narrowed graphene nanoribbons onto device Au electrode pairs (or other noble metals) using a PDMS or PMMA stamp.

[0017] Fig. 5. Design to enable a single polymerase attachment on graphene nanobridge by ~5 nm size guide or hole, comparable to the streptavidin or polymerase dimension (a) Tapered guiding channel prepared by micro/nano-fabrication or nanoimprinting, (b) Guided single polymerase attachment onto graphene nanoribbon bridge, followed by dissolution removal of the guide structure above (which was intentionally made to be removable (e.g., by using sacrificial materials like dextrin, polysaccharide, polyvinyl alcohol, etc. that can be dissolved in water, alcohol or some other solvent, or dissolvable resists such as PMMA). Alternatively, the structure as in (b) can be fabricated by itself and utilized to allow only a single polymerase attachment.

[0018] Fig. 6. Nanoimprinting-assisted formation of single polymerase guide structure

(a) Nanocone shaped Si, SiCh or metal is formed first, (b) Truncated nanocone is shaped by a combination of CMP polishing, planarization and RIE etch processing (c) Nanoimprinting a resist to shape a recessed cone space, (d) RIE to remove residual polymer, (e) provide silanetype linkage between the graphene and biotin, add streptavidin and then guided insertion of a single polymerase into the slot to form molecular binding of polymerase enzyme. A DNA template is added to the polymerase to detect the attachment of complimentary nucleotides onto ssDNA portion for electrical signal detection.

[0019] Fig. 7. (a) DNA bridge formation instead of graphene bridge by focused ion beam (FIB) slicing of the graphene nanoribbon (with a created gap spacing of 15-30 nm) followed by DNA attachment to the two inner ends of the sliced graphene ribbon across the gap, e.g., by electric field alignment, flow alignment, or transfer positioning of pre-aligned DNA array) (b) Biomolecular sensor comprising only a single (or at most a few) DNA bridge (due to the narrow graphene electrode serving as size-confinement structure) and a single enzyme polymerase for clear sequencing analysis, or clear DNA memory reading.

[0020] Fig. 8. Use of various gradient type guide structures to introduce graphene etching-gas concentration gradient, (a) Sloped laterally standing guide structure, (b) local concentrated graphene etching gas supply. Gradient-shaped graphene nanoribbon is formed with the tip radius <10 nm, preferably <5 nm, as a result of such processing. After connecting to lead wires (such as Au, Pd), the sharp-pointed ends of graphene allow size-exclusion to enable attachment of only one or two DNAs for clear signal detection on sequencing or DNA memory reading.

[0021] Fig. 9. Creation of pointed graphene nanoelectrodes (with the tip radius <5 nm, preferably <2 nm) by nanoimprinting of resist, mask metal deposit, and lift-off. Gradient shaped, sharp-tip graphene nanoribbon is formed by plasma etching of unmasked graphene region. Stamp transfer of the sharp-tip graphene array onto the metal electrode array, followed by DNA (or peptide) bridging (e.g., via di electrophoretic field alignment or stamp transfer of pre-aligned DNA) and enzyme polymerase attachment produces a label -free genome molecular biosensor array for sequencing, protein sensing or DNA memory information reader device. [0022] Fig. 10. Resultant gradient-shaped graphene after gradient etching. The sharp- pointed ends of graphene (either rounded cone shape or sharp needle cone shape) allow size- exclusion to enable attachment of only one or two DNAs for clear signal detection on sequencing or DNA memory reading. The graphene ends can be functionalized, e.g., by carboxylic (COOH), while the DNA ends can be modified by amine (NFh) functionality for DNA attachment to the graphene tips. Alternatively, the DNA nanobridges can be achieved by flow alignment in microfluidic environment, or by stamp transfer placement of pre-aligned parallel DNA array on another substrate, for example, using nano-imprinter type accurate position controller. [0023] Fig. 11. (a) Tethered array of encoded (memory written) DNA fragments periodically positioned on a substrate, (b) Polymerase-single-graphene, or polymerase- single- DNA/peptide nanobridge array approaching information stored DNA array being released, (c) DNA templates are taken up by polymerase array and the completed sensor array is moved away for DNA sequencing to read the recorded memory information. (Microfluidics chamber not shown). Massively parallel DNA written information array in combination with massively parallel single-DNA nanobridge reader array allows ultrafast, “random-access-enabled” DNA memory retrieval. Several different configuration of tethered DNA memory array, with various methods of tethering and releasing, or nano sensor access schemes can be made to maximize the DNA memory processing capability. DESCRIPTION

[0024] A bridge-configured sensor structure comprising an elongated nano-dimension semiconductor wire or ribbon is one way of producing label-free molecular sensors for genome sequencing without introducing complicated fluorescence imaging methodologies. Such semiconductor nanowires can be inorganic, or can be organic (such as DNA or peptide biomolecules). It is possible to attach a polymerase single molecule to such a nanobridge or other elongated biomolecules, e.g., using functionalities and ligands such as biotin- streptavidin, antibody-antigen or peptide complexes.

[0025] According to one embodiment of the disclosure, such biosensor bridges between a pair of conducting electrodes (such as Au, Pd, Pt, etc.), can be accomplished by an active attachment route using electrical field (electrophoretic alignment and attachment using various AC or DC mode of electric fields, often at a level of 0.2 to 10 volts). According to another embodiment of the disclosure, aligned DNA structures connecting the mating electrodes can also be achieved by flow alignment in a microfluidic chamber, or by stamp transfer of pre-aligned nanowire or nanoribbon arrays onto the device surface, using PDMS or PMMA stamps.

[0026] While a number of nanomaterials can be considered for such nanobridge structures, graphene films are useful for electronic devices such as electronic sensors because of very thin characteristics to allow nano-dimension fabrications, chemical inertness and a possibility of altering it to be a semiconductors. Two dimensional layer nanomaterials like graphene need to be processed into nano-dimension wires or ribbons by nanolithography or other treatments for sequencing sensor bridge applications, which tends to cause damage to crystallographic structures or disruption of atomic arrangements causing unintended changes or deterioration of physical or electronic properties. Such processed nanoribbons need to be annealed, e.g., 500 - 1,000°C to repair or reduce such damages. Also, graphene is essentially a metallic material with closed bandgap. In order to open up the bandgap to make graphene a semiconductor (for example, for field effect transistor sensors), the graphene geometry needs to be altered to have nano-dimension width (of less than <20 nm, preferably <10 nm) or an internally porous structure, which often requires high temperature processing.

[0027] According to the disclosed embodiments, since the use of high temperature processing damages electronic device structures, the best way is to perform the nanofabrication processing into parallelly aligned nanoribbon geometry, repair processing, as well as width- reducing wet chemical or thermal etching (e.g., using a gas -environment annealing containing ammonia or hydrogen together with oxygen gas) on a separate substrate, with the processed graphene transferred to the final device. The graphene nanoribbons array can be transferred, for example, by using PMMA (Polymethyl methacrylate), PDMS (Polydimethylsiloxane) or other polymer type stamp materials so that they can be to be integrated with the final electronic devices. The pre-made narrowed bridge can be used as a sensor bridge on which a single enzyme molecule such as polymerase can be attached.

[0028] Alternatively, a graphene nanoribbon strip can be split into two nanobridges between which another molecular bridge (such as a single DNA or peptide) can be attached for ease of polymerase bonding and nucleotide analysis. Such splitting of amorphous semiconductor into two separated parts with a nanogap in-between (e.g., 20 - 100 nm) can be accomplished by e.g., focused laser beam slicing, focused ion beam cutting or patterning and etching. The ends of the split ribbons facing each other can desirably be sharpened to a pointed- tip geometry of e.g., 2-5 nm radius of curvature, so as to facilitate an attachment of a single DNA or a single peptide molecular bridge, using either electric field alignment, flow alignment in a microfluidic chamber, or stamp transfer of pre-aligned DNA or peptide. Stamp transfer of pre-aligned DNA or peptide array can be made using PMMA, PDMS or other polymer type soft stamps.

[0029] For accurate signal detection on nucleotide attachment events (or other biomolecule attachment events, e.g., to polymerase, to enable electrical signal detection for genome sequencing), it is highly desirable to provide a single elongated bridge (made of inorganic or organic nanowires or nanoribbons) between mating electrodes (made of Au, Pd or other conducting lead wires). If multiple nanobridges are attached between the two mating electrodes, often clumped together, multiple signals may occur by the presence of parallel sensors, which makes the analysis of such complicated signals very difficult.

[0030] In certain embodiments, the disclosure provides novel approaches of producing a single nanobridge structure of graphene nanoribbon, by forcing a size exclusion via very small area of graphene electrodes or extremely sharp graphene tips. A unique pointed-tip graphene nanoribbon geometry is produced by gradient thermo-chemical etching. Using such a sharp tip in combination with electrical field alignment attachment, an essentially single DNA nanobridge can be constructed between two mating graphene electrodes with sharp nanotips (e.g., <5 nm, preferably < 3 nm tip radius).

[0031] Various graphene nanoribbon related bridge structures, processing methods and applications are disclosed as described in relations to the following exemplary figures.

[0032] Fig. 1. schematically illustrates an exemplary embodiment for transfer of linearly aligned and width-reduced graphene nanoribbons (on flat substrate) by a PDMS- or PMMA-type soft stamp. The PDMS or PMMA substrate may be later removed by solvent dissolution. The PDMS stamp optionally has protruding ridges for easier pick up of the aligned graphene ribbons.

[0033] More specifically, Fig. 1(a) shows aligned graphene array 110 (e.g., nanopattemed by electron beam (EB) or by nano-lithography. The graphene array is formed over Si substrate 108 and dielectric layer (SiC ) 109. Each graphene array 110 may be about 20 nm wide and 1 pm long. Additional mask layer may be added in the event that the graphene layer is not to be narrowed. Fig. 1(a) also shows exposed graphene 112 after width-reducing heat treatment in Eh - or NEE- containing atmosphere at about 600-900 °C for about 0.1 to about 10 hours.

[0034] Fig. 1(b) shows a stamp 120 aligned with substate 108 and 5nm-wide graphene nano-ribbon array (bandgap open). Stamp 120 is then lowered on to substrate 108 until contact is made.

[0035] Fig 1(c) illustrates the stamp pickup of the graphene ribbon array once the transfer is made. Fig. 1(d) illustrates the release of the graphene array on device surface 130. In one embodiment, each of the graphene layers may define a bridge between two mating electrodes. [0036] Fig. 1(e) illustrates the stamp-transferred aligned graphene ribbons with nano bridge FET formed by metal deposition. Electrodes 140 act as contact pad with lead wire (e.g., Au, Pt, Pd, etc.). An optional Ti bond layer may be added.

[0037] Fig. 2. schematically illustrates the transfer-aligned graphene nanoribbon of Fig.

1 with narrowed neck width (e.g., <5 nm) for bandgap opening. The graphene field effect transistor (FET) has a single enzyme polymerase 210 attached becomes a molecular sensor for genome/DNA sequencing. Electrodes 224 are on either sides of bridge 228. A single enzyme polymerase 210 is attached onto graphene bridge 228 to form a biosensor structure, with electrical current signals measured via complimentary nucleotide attaching events (A-T or G- C as adenine is the complementary base of thymine, and guanine is the complimentary base of cytosine in DNA and of uracil in RNA) 218 for sequencing analysis. The biosensor can be utilized for other protein or biomolecule sensing, or DNA memory reading. In Fig. 2, biotin- streptavidin other binding complex links or linker molecules are identified as 222.

[0038] Fig. 3. is a top view illustration of an array of Au electrode pairs (or other noble metals). The array may be formed through the PDMS or PMMA transfer process of Fig. 1. Each graphene nan-ribbon may be about 1-10 nm wide. In an exemplary embodiment, the nanoribbon is about 5 nm wide. In certain embodiments, the graphene bridge is aligned with the electrodes. Additional redundant nanoribbons are provided to ensure at least one bridge connection occurs with conducting metal electrode pair.

[0039] In Fig. 3, electrode pair 302, 304 is coupled to a graphene nanobridge. In order to ensure at least one coupling with electrode pair 302, 304, a plurality of pre-pattemed parallel array of graphene ribbons 306 (optionally, neck-width-reduced to about 5nm for BG opening) are formed. The array of conducting electrodes (and lead wires) 302, 304 may be used to detect a signal from molecular interaction discussed in relation to Fig. 2. The graphene bridges may be attached to the electrodes using van Der Waal forces. In another embodiment, a metallization layer may be added to firmly attach the graphene nanoribbons to the electrodes 302, 304. This is illustrated as metal layer 308 (Ti, Au, Ni, etc.) deposited over the graphene nanoribbons’ outer ends. Unit 310 in Fig. 3 illustrates a FET comprising a substantially aligned graphene nanobridge with a polymerase assembly 312. Enzyme polymerase 312 may comprise an associated DNA template, biotin, streptavidin linker, etc.

[0040] Fig. 4 schematically illustrates a processing sequence for neck-width-reducing heat treatment, followed by stamp transfer of pre-pattemed, pre-aligned, and pre-narrowed graphene nanoribbons onto device Au electrode pairs (or other noble metals) using a PDMS or PMMA stamp. As illustrates, at step 410 a graphene sheet is formed into a plurality of nanoribbons. Each ribbon may be about 20 nm wide. At step 412, metal mask is deposited over the ends of each nanoribbon. The metal mask may comprise Cu, Ni, allows or ceramic film. In one embodiment, the middle part of the nanoribbon may be narrowed by one or more of thermal reactive etching, RIE etching, focused ion-beam itching, etc. At step 414 an optional width-reducing anneal step may be implemented. The annealing may be at about 800 °C for about 20 minutes and in NEE and O2 environment. This will form a neck in the nanoribbons as illustrated in Fig. 4.

[0041] At step 416, the metal mask is etched away to expose necked graphene ribbon.

At step 418, the shape-altered and aligned graphene is transferred onto a sensor device having a plurality of electrodes. The end result is schematically illustrated as FET 420.

[0042] Fig. 5 provides schematic illustrations of different design to enable a single polymerase attachment on graphene nanobridge by using a guide or hole as compared to the streptavidin or polymerase dimension. In Fig. 5(a) a tapered guiding channel 510 is formed by micro/nano-fabrication or nanoimprinting. Guiding channel 510 may have formed thereon an optional blocking layer 512. The blocking layer may comprise PEG or Teflon. The guiding channel 510 may be formed by microfabrication and may have a width of about 1-10 nm, or 5 nm at its narrowest location. The guiding channel may comprise of conventional material 514 such as PMMA, HSQ, Silica or the like.

[0043] Guiding composition 514 may be formed on a dielectric layer. In one embodiment, the dielectric layer may comprise insoluble dielectric. Deposited metal electrode pair 518 are positioned on either side of nanobridge 520 which may act as the gate electrode. Finally, polymerase 522 may be attached to graphene nanobridge 520 as a single enzyme polymerase molecule.

[0044] Fig. 5(b) shows a vertical channel guide according to one embodiment of the disclosure. In Fig. 5(b), the opening has a constant width which is large enough to allow attachment of polymerase 522. In an exemplary application, guided single polymerase attachment onto graphene nanoribbon bridge, may be followed by dissolution removal of the guide structure (see Fig. 5(a), which may be made removable (e.g., by using sacrificial materials like dextrin, polysaccharide, polyvinyl alcohol, etc. that can be dissolved in water, alcohol or some other solvent, or dissolvable resists such as PMMA)). Alternatively, the structure as in Fig. 5(b) may be fabricated as shown and utilized to allow only a single polymerase attachment. [0045] Fig. 6 illustrates the nanoimprinting-assisted formation of single polymerase guide structure. In Fig. 6 (a), nanocone shaped Si, SiCh or metal is formed. In Fig. 6(b), a truncated nanocone is shaped by a combination of CMP polishing, planarization and RIE etch processing. Fig. 6(c) illustrates nanoimprinting a resist to shape a recessed cone space. Fig. 6(d) RIE is implemented to remove residual polymer. Fig. 6 (e) illustrates the steps of providing silanetype linkage between the graphene and biotin, adding streptavidin and then guiding insertion of a single polymerase into the slot to form molecular binding of polymerase enzyme. Finally, a DNA template may be added to the polymerase to detect the attachment of complimentary nucleotides onto ssDNA portion for electrical signal detection.

[0046] Fig. 7 (a) illustrates an exemplary DNA bridge formation instead of graphene bridge by focused ion beam (FIB) slicing of the graphene nanoribbon (with a created gap spacing of 15-30 nm) followed by DNA attachment to the two inner ends of the sliced graphene ribbon across the gap, e.g., by electric field alignment, flow alignment, or transfer positioning of pre-aligned DNA array). Specifically, in Fig. 7(a), the transfer neck-reduced graphene nanoribbon array (e.g., 3-5 nm wide) is transferred onto a device having surface electrodes. FIB is then used to cut the middle of the graphene nanoribbons to create a gap. The gap may be 15-30 nm wide. In another embodiment, the gap may be about 5-40 nm, 10-30 or 10-20 nm wide. Next, a single DNA bridge is attached across the two opposing nano-electrode ends of the narrowed graphene.

[0047] Fig. 7(b) illustrates biomolecular sensor comprising only a single (or at most a few) DNA bridge (due to the narrow graphene electrode serving as size-confinement structure) and a single enzyme polymerase for clear sequencing analysis, or clear DNA memory reading. Specifically, in Fig. 7(b) graphene ribbon with the middle gap created by FIB slicing, with the graphene neck width narrow enough (e.g., equal or less than about 3-5 nm to allow only a single sensor bridge (or at most 2 DNA bridges) to form for cleaner sequencing signals.

[0048] Fig. 8 illustrates the use of various gradient type guide structures to introduce graphene-etching-gas concentration gradient according to the disclosed embodiments. Specifically, Fig. 8(a) illustrates an oblique view of an exemplary process to reduce graphene shape-altering operation. Here, graphene sidewall etching gas (e.g., ¾, B and Ch gasses are introduced at about 600-900 °C for about 0.1-t hours as schematically illustrated by arrow 810. The resulting structure 812 is a gradient- wall -geometry that includes etching gas concentration gradient to produce gradient etching of graphene sidewall 814. [0049] Fig. 8 (b) illustrates another exemplary process for creating a pointed graphene nanoelectrode. Here, the locally concentrated graphene etching gas is supplied to the graphene nanoelectrodes to alter their geometry to pointed graphene nanoelectrodes. Gradient-shaped graphene nanoribbon is formed with the tip radius of about or less than 10 nm (or less than 5 nm) as a result of such processing. After connecting to lead wires (such as Au, Pd), the sharp- pointed ends of graphene allow size-exclusion to enable attachment of only one or two DNAs for clear signal detection on sequencing or DNA memory reading.

[0050] Fig. 9 schematically illustrates formation of pointed graphene nanoelectrodes

(with the tip radius of equal or less than 5 nm, (or equal or than 2 nm) by nanoimprinting of resist, mask metal deposit, and lift-off. Gradient-shaped, sharp-tip graphene nanoribbon may be formed by plasma etching of unmasked graphene region. Stamp transfer of the sharp-tip graphene array onto the metal electrode array is followed by DNA (or peptide) bridging (e.g., via dielectrophoretic field alignment or stamp transfer of pre-aligned DNA) and enzyme polymerase attachment produces a label-free genome molecular biosensor array for sequencing, protein sensing or DNA memory information reader device.

[0051] The process of Fig. 9 starts by depositing graphene sheet on a flat substrate surface. Next, pointed nano-mask is deposited over the graphene sheet by, for example, nano imprinting. In the following step, graphene is removed by plasma etch. The masked regions remain on the substrate. In the next step, the mask over the pointed graphene is removed to exposed the pointed-tip graphene nanoribbons. Next, the pointed graphene segments are stamped-transferred over metal electrodes. Finally, DNA or peptide bridge is attached between two pointed graphene electrodes. In an alternative implementation, all nano-patterning can be done directly on the final device surface without the use of stamp-transfer process.

[0052] Fig. 10 schematically shows a top view of pointed graphene nanoelectrodes

(single or multilayer graphene) with a DNA (or peptide) linear bridge array. In Fig. 10, single DNA bridge 110 may be placed by electric-field alignment. In one embodiment, surface functionalization may be done by adding COOH at the graphene ribbon end and NH2 at the DNA’s ends. In addition, flow-alignment and attachment or stamp transfer placement of pre aligned DNA array may be implemented. In Fig. 10, pointed graphene nanoribbons 1012 are illustrated in pairs. Electrodes 1014 may be optionally deposited by sputtering over the graphene nanoribbons. Graphene tips 1016 have even sharper tips than those of 1012.

[0053] The sharp-pointed ends of graphene (either rounded cone shape or sharp needle cone shape) allow size-exclusion to enable attachment of only one or two DNAs for clear signal detection on sequencing or DNA memory reading. The graphene ends can be functionalized, e.g., by carboxylic (COOH), while the DNA ends can be modified by amine (NFh) functionality for DNA atachment to the graphene tips. Alternatively, the DNA nanobridges can be achieved by flow alignment in microfluidic environment, or by stamp transfer placement of pre-aligned parallel DNA array on another substrate, for example, using nanoimprinter type accurate position controller.

[0054] Fig. 11 illustrates several embodiments for DNA memory applications according to the disclosed embodiments. Specifically, Fig. 11(a) shows tethered array of encoded (memory writen) DNA fragments periodically positioned on a substrate. Fig. 11(b) shows polymerase-single-graphene, or polymerase- single-DNA/peptide nanobridge array approaching information stored DNA array being released. In Fig. 11(c) DNA templates are taken up by polymerase array and the completed sensor array is moved away for DNA sequencing to read the recorded memory information (Microfluidics is chamber not shown). Massively parallel DNA writen information array in combination with massively parallel single-DNA nanobridge reader array allows fast, random-access-enabled DNA memory retrieval. Several different configuration of tethered DNA memory array, with various methods of tethering and releasing, or nano-sensor access schemes can be made to maximize the DNA memory processing capability.

[0055] The following provide some exemplary and non-limiting embodiments of the disclosure. Example 1 is directed to a molecular detection device, comprising: an electrode array of conducting electrode pairs, each electrode pair having a source electrode and a drain electrode separated by a nanogap; a graphene bridge connecting the source electrode to the drain electrode of an electrode pair to form a Field Effect Transistor (FET); an enzyme biomolecule coupled to the graphene bridge; wherein the nanobridge is shaped to guide and limit the coupling of the biomolecule.

[0056] Example 2 is directed to the device of example 1, wherein the graphene bridge defines a narrow neck region.

[0057] Example 3 is directed to the device of example 1, wherein the graphene bridge defines a gap therein.

[0058] Example 4 is directed to the device of example 2, wherein the gap is in the range of about 20 nm, 10 nm, 5 nm, or less than 5 nm to thereby provide a band gap. [0059] Example 5 is directed to the device of example 1, wherein the enzyme molecule comprises a polymerase.

[0060] Example 6 is directed to the device of example 1, wherein the graphene bridge is direction-aligned with the electrode pair.

[0061] Example 7 is directed to the device of example 1, wherein the graphene bridge is formed over the electrode pair by one or more of e-beam lithography or nanoimprint lithography.

[0062] Example 8 is directed to the device of example 1, wherein the graphene nanobridge is coupled to the electrode pair through van der Waals forces.

[0063] Example 9 is directed to the device of example 1, wherein the graphene bridge is coupled to the electrode pair by forming functionalized complexes or ligands therebetween.

[0064] Example 10 is directed to the device of example 1, further comprising a metallization layer formed over the coupling of graphene bridge and an electrode.

[0065] Example 10 is directed to the device of example 1, wherein a polymerase is coupled to the graphene bridge using molecular connection functionalities and ligands including biotin-streptavin complex, antibody-antigen complex, peptide complex.

[0066] Example 11 is directed to the device of example 1, further comprising a guiding structure formed over the graphene bridge to guide coupling of the enzyme biomolecule to the graphene bridge.

[0067] Example 12 is directed to the device of example 11, wherein the guiding structure is dissolvable.

[0068] Example 13 is directed to a biomolecular electronic sensor comprising an array of nanobridges made up with width-narrowed or pointed-tip graphene electrode pair having a gap produced by either nanopatteming, focused ion beam etching or focused laser beam etching;

[0069] Example 14 is directed to the sensor of example 13, wherein the pointed-tip graphene is made by graphene-etching-gas heat treatment under gradient geometry environment, by gradient gas supply environment, or by plasma etching of graphene under sharp-tip-shaped mask nanoimprinted processing.

[0070] Example 15 is directed to the sensor of example 13, wherein, the DNA array is attached onto the split gap between two nanowidth graphene electrodes by flow alignment in microfluidic chamber, or electric fi el d/di electrophoresis aligned.

[0071] Example 16 is directed to the sensor of example 13, wherein the DNA array is pre-aligned into a parallel geometry by flow alignment in microfluidic chamber, or electric field/dielectrophoresis aligned, and then stamp transferred and placed on graphene electrodes.

[0072] Example 17 is directed to the sensor of example 13, wherein the electrical connection of DNA array to the graphene electrodes is van der Waals force attached, metallization attached or focused ion beam writing attached.

[0073] Example 18 includes applications of the biosensor device for encoded DNA memory storage array devices including tethered memory block array or continuously feed memory wire DNA that can be sliced mechanically or chemically, and subsequently read by the graphene nanobridge biosensor or DNA bridge connected, split graphene electrode biosensor, both comprising a single molecule polymerase enzyme sensor on the bridge.

[0074] The embodiments and examples described above are only meant for exemplary purposes and are not meant to limit the scope of any of the embodiments described herein. Any equivalent modification or variation according to the spirit of any of the embodiments disclosed herein is to be also included within the scope of any of the embodiments disclosed herein.

Claims

What is claimed is:

1. A molecular detection device, comprising: an electrode array of conducting electrode pairs, each electrode pair having a source electrode and a drain electrode separated by a nanogap; a graphene bridge connecting the source electrode to the drain electrode of an electrode pair to form a Field Effect Transistor (FET); an enzyme biomolecule coupled to the graphene bridge; wherein the bridge is shaped to guide and limit the coupling of the biomolecule.

2. The device of claim 1, wherein the graphene bridge defines a narrow neck region.

3. The device of claim 1, wherein the graphene bridge defines a gap therein.

4. The device of claim 2, wherein the gap is in the range of about 20 nm, 10 nm, 5 nm, or less than 5 nm to thereby provide a band gap.

5. The device of claim 1, wherein the enzyme molecule comprises a polymerase.

6. The device of claim 1, wherein the graphene bridge is direction-aligned with the electrode pair.

7. The device of claim 1, wherein the graphene bridge is formed over the electrode pair by one or more of e-beam lithography or nanoimprint lithography.

8. The device of claim 1, wherein the graphene bridge is coupled to the electrode pair through van der Waals forces.

9. The device of claim 1, wherein the graphene bridge is coupled to the electrode pair by forming functionalized complexes or ligands therebetween.

10. The device of claim 1, further comprising a metallization layer formed over the coupling of graphene bridge and an electrode.

10. The device of claim 1, wherein a polymerase is coupled to the graphene bridge using molecular connection functionalities and ligands including biotin-streptavin complex, antibody-antigen complex, peptide complex.

11. The device of claim 1, further comprising a guiding structure formed over the graphene bridge to guide coupling of the enzyme biomolecule to the graphene bridge.

12. The device of claim 11, wherein the guiding structure is dissolvable.