Electronic label-free DNA and genome sequencing

Abstract

A method of manufacturing a device useable in DNA or genome sequencing comprises disposing pairs of electrodes on a substrate, the electrodes within each pair separated by a nanogap; depositing a resist layer over the electrodes; patterning the resist layer to create an exposed region on each electrode at or near each nanogap; roughening the electrode surface within each exposed region using various methods; and exposing the exposed regions to biomolecules, wherein one biomolecule bridges each nanogap of each electrode pair, with each end of each biomolecule bound to the electrodes at each exposed region.

Description

FIELD

This disclosure relates generally to nanotechnology, nanofabrication and nanoelectronics, and more particularly to systems, devices, and processes for electronic sensing and analyzing of individual biomolecules, including DNA and proteins.

BACKGROUND

Since the discovery of DNA, there has been a concerted effort to develop ways to actually experimentally determine the sequences of the constitutive chemical bases. The first method for systematically sequencing DNA was introduced by Sanger in 1978.

This basic method was automated in a commercial instrument platform in the late 1980's, enabling the sequencing of the first human genome. 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. These automated methods generally rely on processing millions to billions of sequencing reactions at the same time, in highly miniaturized microfluidic formats.

Although a variety of other related techniques and commercial platforms followed, further improvements in quality and accuracy of sequencing, as well as reductions in cost and time, remain highly desirable. This is especially true to make genome sequencing practical for widespread use in precision medicine, where it is desirable to sequence the genomes of millions of individuals with a clinical grade of quality. Further, many DNA sequencing techniques utilize optical means with fluorescence reporters. Such methods can be cumbersome, slow in detection speed, and difficult to mass produce or make affordable. Label-free DNA or genome sequencing approaches would have the advantages of not having to use fluorescent type labeling processes and associated optical systems, and are thus particularly needed.

SUMMARY

This invention discloses new, manufacturable and highly-scalable techniques of sub-10 nm nano-electrode design and fabrication, which comprise parts of label-free DNA or genome sequencing methods, apparatus, and applications.

This invention provides unique structures, compositions and means of manufacturing the same, for extremely small and high-density nano-electrode arrays for use in electronic DNA sequencing systems. Such nano-electrode systems may also be used in analyzing other types of biomolecules, such as proteins, depending on how the nano-electrodes are functionalized to interact with biomolecule sensing targets. In general, the nano-electrode systems disclosed herein may comprise part of a system for such biomolecule analysis, wherein the nano-electrode system is coupled to biomolecules to constitute a molecular electronics sensor with specific application to sensing and characterizing a biomolecule target, in particular applications to sequencing of a DNA molecule, or a collection of such molecules constituting an entire genome.

In various aspects of the present disclosure, a method of manufacturing a device for DNA or genome sequencing is disclosed. The method includes: disposing a pair of electrodes on a substrate with the electrodes separated by a nanogap; depositing a resist layer over the pair of electrodes; patterning the resist layer to create an exposed region on each electrode at or near the nanogap; exposing the electrodes to plasma etching or gold (Au) ion beam implantation to roughen the surface within each exposed region; and exposing the exposed regions to a biomolecule, wherein the biomolecule has at least first and second ends, with each end including a functionalization for bonding to the pair of electrodes, wherein the biomolecule bridges the nanogap, with the first and second ends of the biomolecule being bound to the exposed regions.

In certain aspects, the resist layer may be an electrically insulating polymer or an oxide coating measuring from about 3 nm to about 20 nm in thickness, and the electrodes may be gold (Au) electrodes. In some examples, the patterning used is e-beam or nano-lithography.

In various embodiments, plasma etching includes RF plasma, DC plasma or sputter etching processes. The plasma etching or the Au ion beam implantation step results in an increase in the surface area of the electrode within each exposed region by at least 50% from the surface area of the exposed region prior to plasma etching or Au ion beam implantation.

In various embodiments of the present invention, a method of manufacturing a device for DNA or genome sequencing is disclosed. The method includes: disposing a pair of electrodes on a substrate, with the electrodes separated by a nanogap; depositing a resist layer over the pair of electrodes; patterning the resist layer to create an exposed region on each electrode at or near the nanogap; exposing the electrodes to Au nanoparticles, wherein the Au nanoparticles attach to the electrode surfaces within each exposed region; annealing the array at from about 200° to about 500° C. to bond the Au nanoparticles to the electrode surface; and exposing the exposed regions to a biomolecule, wherein the biomolecule has at least first and second ends, with each end including a functionalization for bonding to the pair of electrodes, wherein each biomolecule bridges the nanogap, with the first and second ends of the biomolecule being bound to the exposed regions. In certain examples, the resist layer is an electrically insulating ceramic layer such as Al₂O₃ or SiO₂ and the electrodes are gold (Au). The patterning step may include e-beam or nano-lithography.

In various embodiments of the present disclosure, a method of manufacturing a device for DNA or genome sequencing is disclosed. The method includes: disposing a pair of Au metal alloy electrodes on a substrate with the electrodes separated by a nanogap; depositing a resist layer over the electrode pair; patterning the resist layer to create an exposed region on each electrode at or near the nanogap; exposing the electrodes to an etchant solution to selectively remove the non-Au metal from the alloy within each exposed region; and exposing the exposed regions to a biomolecule, wherein the biomolecule has at least first and second ends, with each end including a functionalization for bonding to the pair of electrodes, wherein each biomolecule bridges the nanogap, with the first and second ends of the biomolecule being bound to the exposed regions. The Au metal alloy may be selected from the group consisting of Au—Si, Au—Ge, Au—Bi, Au—Co, Au—Mo, Au—Rh, Au—Ru, and Au—W. In certain aspects, the resist layer is an electrically insulating ceramic layer such as Al₂O₃ or SiO₂.

In certain examples, the method may further include a step of annealing at from about 200° to about 600° C. for about 10 minutes to about 12 hours to promote phase separation between the Au and non-Au metal.

In various embodiments of the present disclosure, a method of manufacturing a device for DNA or genome sequencing is disclosed. The method includes: disposing a pair of electrodes on a substrate, the electrodes separated by a nanogap; depositing a resist layer over the electrode pair; patterning the resist layer to create an exposed region on each electrode at or near the nanogap; depositing Au—Ag or Au—Cu alloy onto the electrode surface within each exposed region; exposing the electrodes to an etchant solution to selectively remove the non-Au metal from the alloy deposited within each exposed region; and exposing the exposed regions to a biomolecule, wherein the biomolecule has at least first and second ends, with each end including a functionalization for bonding to the pair of electrodes, wherein each biomolecule bridges the nanogap, with the first and second ends of the biomolecule being bound to the exposed regions. In certain examples, the resist layer is an electrically insulating ceramic layer such as Al₂O₃ or SiO₂. Also, the electrodes can be gold (Au) electrodes. In various examples, the patterning process includes e-beam or nano-lithography.

In various embodiments of the present disclosure, a method of manufacturing a device for DNA or genome sequencing is disclosed. The method includes: disposing a pair of electrodes on a substrate, the electrodes separated by a nanogap; depositing a latchable magnetic layer over the electrodes; depositing a resist layer over the latchable magnetic layer; patterning the resist layer to create an exposed region at or near the nanogap in which the latchable magnetic layer is exposed; exposing the electrodes to an external magnetic field or to an applied voltage to magnetize the latchable magnetic layer; and exposing the exposed regions to a biomolecule, wherein the biomolecule has at least first and second ends with each end tagged with a magnetic nanoparticle for attraction to the magnetized latchable magnetic layer, wherein the biomolecule bridges the nanogap, with each magnetic nanoparticle held to each electrode at the exposed regions. In certain examples, the latchable magnetic layer is a FeCrCo or FeCuNi spinodal alloy with Hc higher than 10 Oe. This method may further include a step of depositing a layer of Au over the latchable magnetic layer prior to the step of patterning the resist layer. Further, the magnetic layer can be magnetized or demagnetized to enable magnetically attracted attachment of magnetic nanoparticle-tagged biomolecules or removal and cleaning-out of attached magnetic nanoparticle-tagged biomolecules with a permanent magnet sweep, allowing multiple repeat operations of the device.

In various embodiments of the present disclosure, a method of manufacturing a device for DNA or genome sequencing is disclosed. The method includes: disposing a pair of platinum (Pt) or rhenium (Rh) electrodes on a substrate, the electrodes separated by a nanogap; depositing a gold (Au) film measuring from about 1 nm to about 10 nm in thickness over the electrode pair; patterning the resist layer to create an exposed region on each electrode at or near the nanogap; annealing the electrodes to induce spheroidization of the Au film into Au spheres, with one sphere disposed within each exposed region; etching the spheres to reduce the height of the spheres; repeating the annealing and etching steps at least two more times; and exposing the Au spheres to a biomolecule, wherein the biomolecule has at least first and second ends, with each end including a functionalization for bonding to the Au spheres, wherein the biomolecule bridges the nanogap, with the first and second ends of the biomolecule being bound to the Au spheres. In this method, the annealing can be from about 200° to about 400° C. for about 10 minutes to about 12 hours, or the time/temperature sufficient to cause spheroidization. The etching step may include at least one of ion milling, sputter etching, and masking and chemical dissolution.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter of the present disclosure is particularly pointed out and distinctly claimed in the concluding portion of the specification. A more complete understanding of the present disclosure, however, may best be obtained by referring to the detailed description and claims when considered in connection with the drawing figures.

FIG. 1 illustrates genome sequencing compatible electrodes having a structure comprising a 5-20 nm nanogap, with a pair of Au islands for attaching or immobilizing biomolecules such as proteins or fragmented DNA for the purpose of fluorescence-free (label-free) detection of nucleotide attachments via electrical measurements;

FIG. 2 illustrates an embodiment of nano-patterning usable to obtain local-area-defined, massively parallel, Au island electrode arrays;

FIG. 3 is a schematic illustration of microstructures having high surface area Au islands, introduced through particle-induced porous structure or etch-induced porous structure methods;

FIG. 4 illustrates a high surface area Au island for stronger, reduced-error enhanced biomolecule adhesion;

FIG. 5 illustrates methods to obtain Au islands on electrodes (Pt, Rh or Au);

FIG. 6 illustrates progressive Au island diameter reduction by repeated spheroidization and sequential top-etching steps;

FIG. 7(a) illustrates a magnetic attraction method to securely adhere a biomolecule (protein or DNA segment) to the selected position on all the electrodes surfaces simultaneously. 7(a) illustrates magnetic attraction for secure adhesion of a biomolecule (protein or DNA segment) to the selected positions on all the Au base electrode surfaces simultaneously;

FIG. 7(b) illustrates magnetic attraction for secure adhesion of biomolecule. The magnetic layer is deposited on Pt, Pd, or Rh type electrode surface;

FIG. 8 illustrates a massively parallel gold-tip-exposed electrode array: (a) Tapered or sharp-tip Au electrode regions made by nano-imprinting or other lithography; (b) A structure that masks most of the Au electrode surfaces except an island position near the protruding tip to avoid unwanted biomolecule attachments on other regions of Au electrode surface;

FIG. 9 illustrates an array of electrode pairs for strong adhesion of biomolecules for massively parallel, label-free detection of nucleotide attachment or detachment events (e.g., 100×100 device array or 1,000×1,000 array);

FIG. 10 illustrates a configuration for sequential interrogation of electrodes by using a common lead wire on one side of the array and wherein the left side electrodes are interrogated one at a time, sequentially;

FIG. 11 illustrates a three-dimensional array of molecular electronics genome-sequencing platform. An electrically insulating top coating such as a polymer or oxide layer is applied except over the very tip gold islands; and

FIG. 12 illustrates a flowchart showing various aspects of a genome or DNA sequencing system according to the disclosure.

It is to be understood that the drawings are for purposes of illustrating the concepts of the invention and are not to scale.

DETAILED DESCRIPTION

The detailed description of embodiments herein makes reference to the accompanying drawings, which show particular embodiments by way of illustration. While these embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, it should be understood that other embodiments may be realized and that logical, chemical, and mechanical changes may be made without departing from the spirit and scope of the inventions. Thus, the detailed description herein is presented for purposes of illustration and not as a source of limitation. For example, unless otherwise noted, the steps recited in any of the method or process descriptions may be executed in any order and are not necessarily limited to the order presented. Furthermore, any reference to singular includes plural embodiments, and any reference to more than one component or step may include a singular embodiment or step. Also, any reference to attached, fixed, connected or the like may include permanent, removable, temporary, partial, full and/or any other possible attachment option. Additionally, any reference to without contact (or similar phrases) may also include reduced contact or minimal contact.

Definitions

Any term not expressly defined should be given the common meaning known to a person skilled in the art.

As used herein, the term “sub-10 nm” refers to the size of devices and components therein, and the scale at which methods can be executed. In particular, the term refers to physical structures measuring less than about 10 nm in size, and the ability to manipulate steps in various processes on a scale of less than about 10 nm.

As used herein, the term “nanogap” refers to a space, or “gap,” between elements, such as between two electrodes or two conductive islands, measuring in the nanometer range. Similarly, the term “nanogapped” refers to two elements having a gap between them on a nanometer scale.

As used herein, the term “label-free” refers to a process that does not require fluorescence labeling, or is in other words, is “fluorescence-free.”

As used herein, symbols for atomic elements and the associated names of the elements may be used interchangeably, or together. For example, the element gold may be denoted herein “Au,” “gold,” or “gold (Au).”

As used herein, the term “latchable magnetic stripe” (or “latchable magnetic layer”) refers to a thin film or layer of magnetizable material having a capability to change magnetization direction in response to an external magnetic field, such as a layer having magnetic properties that are programmable and reconfigurable as an applied field or an applied voltage is altered.

DNA or genome sequencing by electronic conductance measurements often entails using devices comprising a pair of electrically separated, conductive islands onto which a biomolecule is bridged, and through which electronic current or voltage signals are detected. Referring to the drawings, FIG. 1 illustrates electrodes having a 5-20 nm nanogap and a pair of gold (Au) islands for attaching or otherwise immobilizing biomolecules such as proteins or fragmented DNA for use in devices capable of label-free detection of nucleotide attachments via electronic conductance measurements. FIG. 1(a) shows a top view, and FIG. 1(b) shows a side view, of an embodiment of a nanogapped electrode pair usable for genome/DNA sequencing by electronic conductance measurements. The electrodes may comprise a stable and inert metal having high conductivity. The nanogap 10 between the electrodes may be from about 5-20 nm in width. Exemplary electrode metals include, but are not limited to, platinum (Pt), palladium (Pd), rhenium (Rh), titanium (Ti), silver (Ag), and gold (Au), or their alloys, with gold (Au) being the most widely used electrode material. However, in accordance to various embodiments of the present disclosure, alternatives to Au, such Pt, Pd, Rh and their alloys, can be utilized so as to prevent the non-specific, random attachment of biomolecules, proteins or DNAs on the electrode surface. For example, by using Pt base electrodes, biomolecules are much less prone to attach directly on the Pt surface. Thus, with an addition of pair of Au islands measuring several nanometers in size on to the Pt, the attachment of a biomolecule, especially a single biomolecule, can be confined to the gold islands rather than on the Pt electrode surface itself.

Nanoscale particles, such as, for example, ˜5 nm diameter gold nanoparticles, are difficult to accurately position and place directly on an electrode surface, such as on to a Pt electrode. In certain instances, Atomic Force Microscopy (AFM) or Scanning Probe Microscopy (SPM) methods may be used to pick-up, move, and release an individual Au nanoparticle onto an electrode surface, whereby van der Walls forces assist in holding the nanoparticle at its intended location, such as illustrated in FIG. 1(c), depicting Au nanoparticles 11 sitting indiscriminately on top of the Pt base electrodes, (see, for e.g., Huang, et al., U.S. Patent Application Publication No. 2014/0048776, published on Feb. 20, 2014). However, such delicate movement and placement of nanoparticles can lead to nanoparticles that are not particularly well adhered onto the electrode surface. Further, the positioning of individual nanoparticles at desired locations is not reproducible. These nanoparticles 11, which are not-so-strongly adhered, as illustrated in FIG. 1(c), will cause high contact resistance and reduced electrical conductivity. Further, these Au nanoparticles can be easily moved laterally to a different location, or detached entirely during handling, for example, during washing or microfluidic processing of biomolecules. Accordingly, such AFM guided placement of a nanoparticle, one by one, is laborious, time consuming and not scalable for low-cost manufacturing.

Therefore, in accordance with an embodiment of the present disclosure, the Au nanoparticle is more strongly bonded onto the electrode surface such that the contact area of the Au particle used in the bonding with the electrode surface comprises at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably up to 100% of the Au particle diameter, as illustrated in the top example in FIG. 1(d), which shows hemispherically shaped islands. In this example of hemispherical islands, a single biomolecule 12 is shown in contact with the islands and bridging the gap between the electrodes. The second example in FIG. 1(d) illustrates an embodiment comprising “pillar” shaped islands on the Pt base electrodes, which are available for bonding with a biomolecule bridge.

Another embodiment according to the disclosure avoids fabricating Au islands on Pt electrodes entirely, relying instead on exposed portions of Au electrodes for biomolecule bonding and bridging. As illustrated in the third example in FIG. 1(d), a nano-patterned mask is used to block almost the entire Au electrode surface except for a selected region left exposed and available for selective biomolecule attachment. In a variation of this concept, a resist layer, such as for example, PMMA (polymethylmethacrylate), methacrylate-based co-polymers, hydrogen silsesquioxane (HSQ), and various siloxane polymers, can be utilized to cover the majority of the Au electrodes except for just the tips of the electrodes, as illustrated in the fourth example of FIG. 1(d). These two methods that utilize a resist layer are discussed in more detail herein.

FIG. 2 illustrates an embodiment of a nano-patterning process usable to obtain local-area defined electrodes having exposed regions on Au electrodes and single biomolecule bonding and bridging. The process begins with a pair of Au electrodes as illustrated in FIG. 2(a). This cross-sectional side view illustration shows two Au electrodes as a pair of electrodes on a substrate comprising Si with a SiO₂ insulator surface. The nanogap 20 in each electrode pair is from about 5 nm to about 20 nm. The electrodes are labeled “array” to emphasize that in a device, there can be a plurality of such electrode pairs, from less than a hundred, to hundreds, or thousands, or millions or more electrode pairs. The goal of the process illustrated in FIG. 2 is to obtain a single nano-defined exposed region on each Au electrode, measuring about 3-10 nm in diameter, which will allow bonding of only one end of one biomolecule in each exposed region. In other words, the exposed region on each electrode needs to be sub-10 nm. For this purpose, and as illustrated in FIG. 2(b), a positive or negative resist layer 23 is added over the electrode pair. A positive resist (e.g., polymethylmethacrylate (PMMA)), or a negative resist, (e.g., hydrogen silsesquioxane (HSQ) or SU-8 epoxy resist), can be used, and any of these are compatible with subsequent e-beam or nano-imprint lithography. In certain examples, the resist layer 23 comprises a negative resist. The e-beam or nano-imprint lithography is then used to pattern the exposed regions 24 as illustrated in FIG. 2(c).

In alternative embodiments, Al₂O₃, SiO₂ or Si₃N₄, or other oxide or nitride layers, may be deposited on the Au electrodes and used as an insulating layer instead of using a polymer resist layer. In this case, a very thin adhesion layer, such as a 1-3 nm thick Ti layer, may be deposited on the Au electrode surface before the oxide or nitride coating is applied. This protective coating, whether polymeric or ceramic, serves as an electrical insulator and also as a coating that prevents or minimizes the adhesion of biomolecules at unwanted locations.

Referring to FIG. 2(c), the configuration of an exposed region 24 may be any shape, such as circular, oval, square, rectangular, or any other geometry. The size of each exposed region 24 on an Au electrode is about 3 nm to about 15 nm in diameter on average (or the equivalent dimension across and parallel to the electrode surface if not circular in shape). In certain embodiments, each exposed region 24 is about 5 nm to about 10 nm in diameter (or equivalent dimension across the region if not circular).

The process depicted sequentially in FIG. 2 is amenable to the processing of massively parallel array sequencing devices. Nano-patterning approaches such as nano-imprinting, electron beam lithography, shadow mask patterning, EUV (extreme UV) lithography, x-ray lithography, and other known patterning methods, allow fabrication of at least 1,000, preferably at least 10,000 or more, single electrode pair devices simultaneously. Such a large array of electrodes in a sequencing device greatly speeds up sequencing speed and reduces sequencing cost. FIG. 2(d) illustrates an attached biomolecule 22 (e.g., a protein, a DNA segment, etc.) attached at each of its ends to the exposed region on each electrode to form a bridge. In various embodiments, the biomolecule 22 may be immobilized into the position shown by functionalized ends using antibody-antigen, biotin-streptavidin, peptide bonding, functionalized ligands, surface charges, or other biomolecule immobilizing techniques. A complete electrode pair device illustrated in FIG. 2(d) is shown comprising electrical connections from each electrode, used for electrical interrogation (“i”) of sequencing events that comprise interactions of the biomolecule 22 with other molecules.

For molecular electronics devices, including those for protein analysis or DNA/genome sequencing, parallel electronic sensing using an array of many electrode pair devices is desirable. In order to package more electrical measurement devices and circuits within a given space, the electrode dimensions must be reduced to micro- or nano-dimensions. An array of nano-electrodes having the geometry such as shown in FIG. 2 can be utilized. Such an array can be made by using convenient and scalable processing methods such as nano-imprint lithography. Alternative methods such as e-beam lithography, self-assembly and other means may also be utilized.

In DNA and genome sequencing comprising label-free electronic detection, as well as molecular electronics devices in general, a strong and reliable biomolecule-to-electrode (or biomolecule-to-conductive island) attachment is essential, so as to obtain reproducibly strong, noise-reduced signals during electronic interrogation of nucleotide interaction with the biomolecule. Attachment of a biomolecule to a surface can be enhanced if the surface is “roughened,” meaning that the surface is modified in a way that increases the surface area onto which the biomolecule is to attach. Ordinarily it is difficult to make branched or porous structures on what are already nano-dimensioned (e.g., 5-10 nm area) structures. However, in accordance with the present disclosure, this difficulty has been overcome, whereby further subdivided nanostructures for enhanced adhesion of biomolecules onto the electrode surface has been accomplished. An embodiment comprising subdivided nanostructures on an electrode surface is where an Au pillar, deposited on an electrode surface, is surface damaged in a way to make at least the top surface rough or porous.

Au surfaces allow for the opportunity to use thiol (—SH) groups on a biomolecule for adhesion (immobilization) of the biomolecule to the Au surface, through Au-sulfur bonding. Nonetheless, if biomolecule adhesion can be enhanced beyond the basic Au-sulfur bonding afforded by thiol groups, the reliability and signal-to-noise ratio in electronic, label-free sequencing can be significantly improved. According to the present disclosure, increasing the surface area of a nano-island of Au increases biomolecule binding to the Au. Herein, further surface nano-structuring of a sub-10 nm island of Au is disclosed.

Referring now to FIG. 3, a schematic illustration of exemplary microstructures having large-surface-area Au islands is provided. In accordance with the disclosure, the increase in surface area can be the result of a particle-induced porous structure (illustrated in FIG. 3(a)) or an etch-induced porous structure (illustrated in FIG. 3(b)), or from other means. Methods to produce a particle-induced porous structure and an etch-induced porous structure are detailed herein. According to certain embodiments, the increase in the surface area at or near the top surfaces of Au islands where biomolecules are to be adhered is at least 50%, and preferably at least 100% over a flat surfaced Au islands. The biomolecule adhesion strength is improved by at least 30%, and preferably by at least 60% over adhesion on to plain, unmodified Au islands.

Referring now to FIG. 4, exemplary large surface area Au island structures and methods of producing them are shown. As mentioned, increased surface area for bonding biomolecules results in stronger, reproducible, enhanced biomolecule adhesion. In FIGS. 4(a) through 4(f), only the left side each electrode pair is illustrated for the purposes of clarity. FIG. 4(a) schematically illustrates an embodiment of a base electrode structure, comprising electrode 49 and resist layer 48, on which an exposed island location 44. It is inside this region 44 where the surface area of the island is to be increased. The process begins by forming a pattern-defined Au island region on a conductive electrode (e.g., Au, Pd, Pt, Rh, Ag or their alloys) surface, by utilizing insulating coating 48 such as PMMA and other polymers, or an oxide layer such as sputter-deposited SiO₂ or Al₂O₃. As discussed, e-beam or nano-lithography can be used to create each of the exposed regions 44 on the electrodes. The nature of the coating is chosen based on desirable electrical insulation properties and the resistance the coating has to biomolecule adhesion.

Once the resist layer 48 is patterned, the exposed regions 44 are processed to increase the surface area within the exposed region 44. FIG. 4(b) is an example of a porous surface structure 401 created by plasma etching. In certain embodiments, rough surface structure, such as a porous or vertical nanowire forest type surface structure, can be formed by RF plasma or DC plasma etching (or sputter etching) processing, more selectively on the exposed Au island region. The intensity and duration of plasma etching can be adjusted so as to minimize complete plasma etching of the insulation mask layer material. The thickness of the resist layer may be from about 3 nm to about 20 nm, and this thickness can be adjusted to mitigate complete plasma etching of the mask layer.

A rough surface having an increased surface area can also be created by ion implantation, e.g., by using an Au ion beam. An example of a rough surface formed by ion beam implantation is illustrated as surface 441 in FIG. 4(c). However, ion implantation damage can occur on the Au electrode base underneath the insulating coating, depending on the penetration depth of ions as dictated by the accelerating field and total dose of ion implantation. As such, the insulating resist coating does not necessarily protect the Au surface beneath from ion implantation. However, a resultant roughened surface in areas other than the exposed island region does not affect the sequencing operation because the insulation layer remains covering the roughened electrode surface. In various embodiments of the present disclosure, a post-annealing step may follow the ion implantation.

Another embodiment used to increase the surface area of an Au island comprises attaching Au nanoparticles 421 to the exposed Au region, as shown in FIG. 4(d). Au nanoparticles attached to the Au electrode increases the surface area which results in enhanced biomolecule attachment. In various examples, Au nanoparticles measuring about 1 nm to about 2 nm in diameter, made by known methods, tend to preferentially attach naturally to the Au electrode within the exposed region. The Au nanoparticles thus deposited may then be optionally annealed at from about 200 to about 500° C. Higher temperatures can only be used if the insulating layer 48 is ceramic based, (e.g., if the insulating layer comprises Al₂O₃ or SiO₂). Post-annealing at these temperatures improves the adherence of the Au nanoparticles to the exposed Au surface. An example of particle-induced porous structure is illustrated, for example, in FIG. 3(a).

FIG. 4(e) represents another embodiment of a process to increase the surface area of an exposed Au island surface, leveraging the immiscibility that Au and Fe exhibit at or near room temperature. For this process, an electrode base layer may be made, for example, from an Au—Fe alloy having about 50 wt. % Au and 50 wt. % Fe by sputtering or evaporation deposition. Toward room temperature, the alloy phase segregates into an Au matrix embedded with Fe nanoparticles that have precipitated out. The Fe nanoparticles can then be etched away to leave behind a porous Au matrix. An example of this type of etch-induced porous structure is illustrated in FIG. 3(b). Other Au base alloys, such as Au—Si, Au—Ge, Au—Bi, Au—Co, Au—Mo, Au—Rh, Au—Ru, Au—W, can be used as they behave in a similar way as the Au—Fe alloy. Annealing at high temperatures (e.g., at 200-600° C. for 10 min to 12 hrs) enhances the phase segregation. Another way of enhancing phase segregation is to perform sputter deposition or evaporation deposition at warm substrate temperatures, such as 150-400° C., optionally with a bias voltage applied.

FIG. 4(f) illustrates another embodiment of a process used to increase the surface area of an Au island surface comprises de-alloying. The process begins by first depositing Au—Ag, Au—Cu, or other Au base alloy layer having an Au content of about 20 to 80 wt. %, such as by sputtering or evaporation deposition, into each exposed region 44. This is followed by strong chemical etching of the non-Au metal, such as by using 30% HNO₃ or other etchant. Such de-alloying process removes Ag or Cu from the surface to leave behind a highly porous structure. Such de-alloying techniques, when applied to Au nano-islands in the electronic sequencing devices, results in significant surface area increases and associated improvement of biomolecule adhesion.

Shown in FIG. 4(g) is an embodiment of how Au islands having increased surface areas (such as obtained by the processes exemplified in FIGS. 4(b)-(f)) can be utilized for enhanced attachment of a biomolecule 42 such as a protein or DNA.

In various embodiments, the volume of porosity within an Au island region, characterized as branched or porous, is at least 10%, preferably at least 30%, and even more preferably at least 50%, so as to increase the surface area of the Au island at or near its top surface by at least by 20%, preferably by at least 40%, and even more preferably by at least 60% over a flat and smooth Au island structure.

With continued reference to the drawings, FIG. 5 shows three embodiments of methods to produce Au islands on electrodes such as Pt, Rh or Au. The process begins with an electrode pair separated by a nanogap 50. In each of these illustrations, the underlying substrate of the device, such as Si with SiO₂ insulator surface, is not shown for the purposes of clarity. A first embodiment (1) utilizes a Pt base electrode incapable of biomolecule (e.g. protein or DNA segment) attachment, so that a biomolecule adheres only to an Au island region rather than to the electrode. To obtain smaller diameter Au 501, patterned Au island 51 is heated to cause it to ball up into the smaller diameter sphere 501. An Rh electrode is also a useful option by virtue of having a lower electrical resistivity (˜4.5 uΩ·cm for Rh, vs 9.8 uΩ·cm for Pt and 2.1 uΩ·cm for Au), and a reduced propensity for undesired biomolecule attachment. Also, Rh has no (or very small) mutual solubility with Au, unlike Pt, so alloy mixing is minimized.

A second embodiment (2) uses an all Au electrode configuration, with the Au electrode disposed in a rectangular shape but with much of the surface masked by insulator coating 58, except for an island-like region at the end of the electrode. The insulator coating may comprise polymethylmethacrylate (PMMA), polyethylene glycol (PEG), polydimethylsiloxane (PDMS), or their combinations, hydrogen silsesquioxane (HSQ), or various oxide coatings such as Al₂O₃, SiO₂ or nitride coatings such as Si₃N₄ coating.

A third embodiment (3) comprises the fabrication of the Au electrodes in tapered shapes having sharp tip geometry, and then masking the majority of the surface of the electrode by insulator layer 580 at all other regions except for the sharp tips of the electrodes. PEG and PDMS have remarkably non-adhesive characteristics toward proteins and cells, which can be an additional advantage for electronic sequencing devices where protein adhesion at places other than on the Au island is undesirable.

As the adhesion of biomolecules is dependent on the size of an Au island, controlling the size of Au island is critical for securing the biomolecule in sequencing devices. In various embodiments, an optimal Au island size is from about 5 to about 9 nm in diameter. Too large a diameter or too small a diameter Au island does not enable reproducible and strong adhesion of biomolecules. Thus, in various aspects of the present disclosure, techniques are shown that are used to conveniently control the Au island diameter.

With continuing reference to the drawings, FIG. 6 illustrates a progressive Au island diameter reduction process. The method comprises repeated “spheroidization” (a balling up) and top-etching, progressing from FIG. 6(a) through FIG. 6(f). In the illustrations, only the left side electrode of each electrode pair is shown for the purposes of clarity. Further, the underlying device substrate is not shown for clarity. By employing repeated spheroidization and island top etch away processes, much smaller diameter Au islands 641 having diameters of about 5 nm to about 9 nm can be achieved more easily. In the first step of the process, a thin Au film (e.g., 1-10 nm thickness, not illustrated) is first deposited on a Pt or Rh electrode 69 by sputtering, ion beam deposition, evaporation, pulsed laser deposition, or other method, which is then nano-patterned (e.g. by nano-imprinting or other lithographic methods) to create a defined circular or oval film geometry 61 illustrated in FIG. 6(a). Subsequent annealing induces a spheroidization ball-up of the Au region into a smaller diameter Au island 601. Annealing may be at about 200 to about 400° C. for a period of time of about 10 min to 12 hrs in an atmosphere such as argon, nitrogen, forming gas type reducing atmosphere. The balled-up Au island 601 is then subjected to chemical or plasma etching for height-reduction (e.g., by ion milling, sputter etch, masking and chemical dissolution etching, etc.) to produce the flatter Au island geometry 611. Subsequent spheroidization annealing reduces the Au island diameter further and results in Au spherical islands 621. The steps of etching and spheroidization annealing are repeated again to convert the Au islands 621 to 631 and then to 641.

Referring to the drawings, FIG. 7(a) illustrates an embodiment of a process for forming sequencing devices in which magnetic attraction provides a more secure adhesion of a biomolecule (protein or DNA segment) to selected positions on all of the electrode surfaces simultaneously. As shown at the top of the figure, the process begins with an electrode pair having electrodes separated by a nanogap 70. A latchable magnetic stripe 785 (such as a thin film stainless magnetic alloy based on FeCrCo or FeCuNi spinodal alloys, or other corrosion-resistant magnet alloys) is deposited on the Au electrode surface 790. The magnetic hardness with coercivity (Hc) is selected to be higher than 10 Oersted (Oe) in order to guard against accidental magnetizing by a stray magnetic field, but less than 100 Oe for easier magnetization or demagnetization to attract or not attract magnetic nanoparticles. Nano-patterning of insulator spacer layer 780 is then used to create an exposed area 74 on which magnetic nanoparticles can be strongly and reliably adhered with good electrical connection.

Once the electronic sequencing device is in place, the magnetic layer is magnetized by an externally applied magnetic field, e.g., using a ferrite magnet or an electromagnet, at a field of e.g., 100-1,000 Oe. Then, using a surrounding microfluidic chamber, a liquid medium comprising biomolecules 72 already tagged with magnetic nanoparticles 71 is introduced. Biomolecules with magnetic nanoparticles bonded thereto may comprise antibody-antigen binding, streptavidin-biotin binding, peptide bonding, or electrical charge attraction, or other binding approach. A magnetic particle 71 with biomolecule 72 attached will then be attracted onto the exposed area of the magnetic stripe 791 where it will be magnetically and electrically anchored as illustrated in the lower drawing in FIG. 7(a). Optionally an intermediate Au nanoparticle may also be inserted to the biomolecule for various control purposes.

The magnetic nanoparticles to be tagged onto the biomolecules can be selected from Fe₂O₃, Fe₃O₄, or surface-protected or surface-functionalized metallic magnetic particles having higher magnetic moment than iron oxide, including Fe, Co, Ni and their alloys. The oxide-based magnetic nanoparticles are chemically stable, but the metal-based magnetic nanoparticles, while having 2-4 times higher magnetic moment, may lack the chemical stability in a liquid environment of genome sequencing. A slight surface oxidation as a protective coat against oxidation is desirable. Alternatively, the surface of magnetic nanoparticles can be Au-coated to provide chemical stability, as well as enhanced electrical conductivity and more affinity to biomolecules.

Once the desired magnetic attraction and biomolecule attachment has occurred, the liquid medium in the microfluidic chamber is washed away to remove unbonded biomolecules, and then the sequencing electronic measurements can be conducted and completed. A feature of such magnetic attachment technique is the reversibility of biomolecule attachment, i.e., easy detaching of biomolecules if needed. As the magnetic stripe (the portion of the magnetic layer on an electrode) can easily be demagnetized (e.g., by using gradually diminishing magnetic field of from 200 Oe to near zero field using a 60 Hz AC field in a few seconds), all the magnetically attached biomolecules can be thoroughly removed once the desired sequencing measurement is completed, e.g., by employing a sweeping permanent magnet to detach and collect all the magnetic particles and associated biomolecules and discarding them. In this way, the sequencing device is reusable, for example, the device can be reused at least 10 times, at least 100 times, or even at least 1,000 times.

A modified method, utilizing Pt, Pd, or Rh base electrode pairs separated by nanogap 700 instead of Au base electrodes, is illustrated in FIG. 7(b). In this method, a thin Au coating 785 is added on top of the surface of the magnetic layer 790 to enable better attachment of biomolecules. The Au layer does not noticeably affect magnetic attraction between the nanoparticle and the underlying magnetic stripe because the magnetic layer underneath is positioned very close to the exposed surface. As per the embodiment shown in FIG. 7(a), the exposed regions 740 are created by e-beam or nano-patterning of a resist layer 780. Then the device is flooded with a solution of biomolecules 720 tagged with magnetic nanoparticles 710, wherein the magnetic nanoparticle 710 bonds to the Au layer 791 and is magnetically attracted to the underlying magnetic stripe.

Shown in FIG. 8 is a massively parallel Au-tip-exposed electrode array. Tapered or sharp-tip Au electrode regions 89 are first made by nano-imprinting or other lithography, with many electrode pairs in the array, as shown in FIG. 8(a). Substrate material such as Si with SiO₂ surface insulator is not shown for the purposes of clarity. As illustrated in FIG. 8(b), the Au electrodes 89 are then masked by a deposited mask layer 880 which is then patterned to cover the majority of the electrode surface areas except for the very tips 890 of the electrodes, in order to mitigate unwanted biomolecule attachments on other regions of the electrodes. An insulator film for this purpose may comprise, for example, PMMA, PEG, PDMS, Al₂O₃, SiO₂ or Si₃N₄. The desirable dimension of an Au electrode tip 890 thus left exposed is about 5-9 nm in diameter. Each electrode further comprises lead wires 802 and 820. Nano-imprinting, for the simultaneous fabrication of a large number of devices as detailed in FIG. 9, for example, more than 10,000 devices, is possible. In various embodiments, a massively parallel electrode array may comprise at least 1,000, preferably at least 10,000, and even more preferably at least 1 million devices.

Illustrated in FIG. 9 is an exemplary array of Au nano-tip structured electrode pairs 90 for massively parallel, label-free detection of nucleotide attachment or detachment events (e.g., a 100×100 device array or a 1,000×1,000 array), with pattern-defined exposed Au electrode tips 990 (or other conductor island activated for immobilization of biomolecules) near the electrode pair gap area. The lead wires 920 can be Au or any other conductor (e.g., Ag, Cu, Pt, Pd, or Rh), insulated with a patterned top coating 98. All of the electrode pairs for label-free detection of nucleotide attachment or detachment are masked by an insulator film 98 (e.g., PMMA, PEG, PDMS, or SiO₂, Al₂O₃ or Si₃N₄) to cover the Au electrode surface except the electrode tips 990 as illustrated in the drawing figure. The interrogation of nucleotide attachment can be performed in real time using the array structural configuration exemplified in FIG. 9, comprising the individual lead wires 920 as shown.

An alternative embodiment of electronic signal interrogation is possible using the array configuration illustrated in FIG. 10. In this configuration, all the lead wires from the right side of the electrode pairs of the array of devices 1000 is shorted into a common lead wire 1020. The array also includes the insulator layer 1008 covering the majority of the electrode array except for the exposed Au tips 1009. Using the configuration depicted, interrogation of nucleotide attachment comprises performing sequential interrogation with the right side of all the lead wires shorted to a common lead 1020 and then taking turns with the individual electrical lead wires 1021 present on the left side of the pairs, one at a time, e.g., every millisecond. This method results in fewer electronic measurement complications than handling many thousands of parallel signals all at once.

Shown in FIG. 11 is an exemplary three-dimensional array of molecular electronics genome-sequencing platform. As per the embodiments in the previous two figures, an electrically insulating top coating 1108 such as polymer or oxide layer (e.g., PMMA, PEG, PDMS, aluminum oxide, Si oxide, Si nitride, etc.) is applied and patterned to cover all the electrode surfaces in the electrode pairs 1100 except for the very tips 1109 of the electrodes. This protective coating serves as electrical insulator but also as a coating that prevents or minimizes the adhesion of biomolecules at unwanted locations. As per the configuration in FIG. 10, the right side electrode leads may be shorted into a common lead wire 1120 whilst the left side lead wires 1121 may be left singly for sequential interrogation.

Even larger data can be obtained if the FIG. 9 or FIG. 10 type aerial array sequencing structure is stacked in three dimensions, such as illustrated in FIG. 11, representing a three-dimensional array of molecular electronics genome-sequencing platform. The 3D array further comprises an accompanying microfluidic chamber for each two-dimensional array (as shown in FIG. 11(a), wherein 100 to 1,000 layers, each comprising about 10,000 devices and a microfluidic chamber), or in alternative embodiments, comprising one or more common microfluidic chambers encasing the entire three-dimensional array (as shown in FIG. 11(b), wherein 100 to 1,000 layers of sensing layers, each having about 10,000 devices, all encased in one microfluidic chamber). In such 3D configurations, at least one million to one billion devices can be operated simultaneously for extremely rapid DNA or genome sequencing. The desirable packing density of electronic sequencing pair devices is at least 10,000/cm², preferably at least 100,000/cm², more preferably at least 1,000,000/cm², and even more preferably at least 10,000,000/cm².

The genome or DNA sequencing structures and systems according to the disclosure is described in the flowchart of FIG. 12. Appropriate control and signal analysis/integration software and computer system are incorporated, and microfluidic system is connected to the massively parallel electronic detection system using biomolecules and coupling agents, together with DNAs and nucleotides to be sequenced.

Additional Embodiments

In various embodiments, a DNA or genome sequencing structure comprises an array of electrode pairs, each electrode in a pair separated by a nanogap and comprising a strongly adherent and highly conductive island with reduced contact resistance, and a biomolecule securely attached with each of its ends on one conductive island to bridge the nanogap, wherein the conductive islands comprise nano-pattern defined Au on a Au electrode, with the Au island dimension being sub-10 nm, and further comprising electronic means of DNA or genome sequencing using DC, RF or pulse signal on nucleotide attachment, but without using fluorescent element.

In various embodiments, a non-Au electrode based DNA or genome sequencing structure comprises an array of electrodes and strongly adherent and highly conductive Au or other conductive islands having reduced contact resistance, with a biomolecule securely attached on a pair of conductive islands, with the Au island dimension being sub-10 nm, and the electrode base material selected from Pt, Pd, Rh, and Ag, with electronic means of DNA or genome sequencing using DC, RF or pulse signal on nucleotide attachment, but without using fluorescent element.

In various embodiments, the adherent conductor is made of Au, having a contact area to the electrode surface of at least 50%, and preferably %100 of the Au island diameter.

In various embodiments, the adherent gold nano-island is branched or porous with a porosity of at least 10%, preferably at least 30%, and even more preferably at least 50%, so as to increase the surface area of gold island top surface by at least by 20%, preferably by at least 40%, and even more preferably by at least 60%, over a flat and smooth Au nano-island structure.

In various embodiments, an increase in the surface area of the Au islands is obtained by one or more of the methods selected from a list of: i) plasma etching; ii) ion implantation and optional annealing heat treatment; iii) gold (Au) nanoparticles attaching and bonding; iv) selective dissolving non-Au component from a nanocomposite structure; or v) de-alloying process.

In various embodiments, the structure comprises parallel devices with at least 1,000, preferably at least 10,000, even more preferably at least 1 million devices, fabricated by one or more methods selected from nano-patterning approaches such as nano-imprinting lithography, shadow mask patterning, electron beam lithography, extreme UV lithography, or X-ray lithography.

In various embodiments, the structure comprises multilayers of devices with at least 10,000, preferably at least 1 million, even more preferably at least 100 million devices, in combination with microfluidic systems.

In various embodiments, a sequential interrogation of electrodes is enabled by a connected structure wherein one side of the device array lead wires are ganged into a common lead wire.

In various embodiments, a biomolecule is attached to an Au island using antibody-antigen coupling, streptavidin-biotin coupling, peptide bonding, thiol-gold binding, gold binding proteins, or other coupling configurations.

In various embodiments, a genome or DNA sequencing system comprises a chamber containing a multitude of biomolecule sensing devices described herein, and a microfluidic subsystem that supplies or maintains biomolecules, nucleotides, PBS or water solution, and other needed materials.

In various embodiments, the Au island diameter is progressively reduced by repeated spheroidization annealing and top-etching, with the process repeated by at least 2 cycles of spheroidization and part of the top etching to reduce the height and diameter.

In various embodiments, the height reducing etching comprises chemical etching or plasma etching.

In various embodiments, the structure comprises an added magnetic layer to enhance the attachment of magnetic-particle-tagged biomolecules, with the magnetic particles based on oxide material having natural chemical stability or metallic alloy material with surface coated with thin oxide or gold for anti-corrosion protection by a factor of at least 50% reduced corrosion rate, and/or enhanced affinity to biomolecules with at least 50% increased probability of biomolecule attachment.

In various embodiments, magnetic particles are selected from Fe₂O₃, Fe₃O₄, surface-protected or surface-functionalized metallic magnetic particles having higher magnetic moment than iron oxide including Fe, Co, Ni and their alloys.

In various embodiments, the magnetic layer is magnetized or demagnetized to enable magnetically attracted attachment of biomolecules or removal and cleaning-out of attached biomolecules with a permanent magnet sweep, allowing multiple repeat operations of the sequencing devices by at least 10 times, preferably at least 1,000 times.

In various embodiments, methods of assembly, methods of operation associated with FIGS. 1-12 and specifications therein are disclosed.

In various embodiments, these device structures for DNA sequencing can be used for cancer detection and other medical uses.

In various embodiments, these devices are usable for human genome sequencing.

The embodiments described above are only to exemplify the present invention but not to limit the scope of the present invention. Any equivalent modification or variation according to the spirit of the present invention is to be also included within the scope of the present invention.

Claims

1. A method of manufacturing a device for DNA or genome sequencing, the method comprising: depositing a resist layer over a pair of electrodes disposed on a substrate, wherein each of the electrodes in the pair are separated by a nanogap;patterning the resist layer to create an exposed region on each electrode at or near the nanogap;increasing a surface area of the exposed region on each electrode; andintroducing a biomolecule to the exposed regions, wherein the biomolecule has at least first and second ends, with each end comprising a functionalization for immobilizing the biomolecule to the pair of electrodes,wherein the biomolecule bridges the nanogap, with the first and second ends of the biomolecule being bound to the exposed regions.

2. The method of claim 1, wherein the resist layer comprises an electrically insulating polymer or oxide coating of from about 3 nm to about 20 nm in thickness.

3. The method of claim 1, wherein the patterning comprises e-beam or nanolithography.

4. The method of claim 1, wherein each electrode comprises an alloy of a metal selected from the group consisting of platinum (Pt), palladium (Pd), rhenium (Rh), titanium (Ti), silver (Ag), and gold (Au).

5. The method of claim 4, wherein the alloy is an Au metal alloy selected from the group consisting of Au—Fe, Au—Si, Au—Ge, Au—Bi, Au—Co, Au—Mo, Au—Rh, Au—Ru, and Au—W.

6. The method of claim 5, wherein the alloy is an Au—Fe alloy comprising about 50 wt. % Au and 50 wt. % Fe, and wherein at room temperature the alloy segregates into an Au matrix embedded with Fe nanoparticles.

7. The method of claim 6, wherein increasing a surface area of the electrode comprises etching the Fe nanoparticles out of the exposed region.

8. The method of claim 7, further comprising annealing the alloy at 200 to 600° C. for 10 to 12 hours.

9. A method of manufacturing a device for DNA or genome sequencing, said method comprising: disposing a pair of electrodes on a substrate, wherein each of the electrodes in the pair are separated by a nanogap;depositing a resist layer over the pair of electrodes;patterning the resist layer to create an exposed region on each electrode at or near the nanogap;increasing a surface area of the exposed region on each electrode and introducing a biomolecule to the exposed regions, wherein the biomolecule has at least first and second ends, with each end comprising a functionalization for immobilizing the biomolecule to the pair of electrodes,wherein the biomolecule bridges the nanogap, with the first and second ends of the biomolecule being bound to the exposed regions.

10. The method of claim 9, wherein the resist layer comprises an electrically insulating polymer or oxide coating of from about 3 nm to about 20 nm in thickness.

11. The method of claim 9, wherein the patterning comprises e-beam or nanolithography.

12. The method of claim 9, wherein each electrode comprises an alloy of a metal selected from the group consisting of platinum (Pt), palladium (Pd), rhenium (Rh), titanium (Ti), silver (Ag), and gold (Au).

13. The method of claim 12, wherein disposing the pair of electrodes comprises depositing the electrodes by sputtering or evaporation deposition.

14. The method of claim 13, wherein the sputtering or evaporation deposition is performed from about 150° C. to about 400° C.

15. The method of claim 14, wherein a bias voltage is applied during the sputtering or evaporation deposition.

16. The method of claim 13, wherein the alloy is an Au metal alloy selected from the group consisting of Au—Fe, Au—Si, Au—Ge, Au—Bi, Au—Co, Au—Mo, Au—Rh, Au—Ru, and Au—W.

17. The method of claim 16, wherein the alloy is an Au—Fe alloy comprising about 50 wt. % Au and 50 wt. % Fe, and wherein at room temperature the alloy segregates into an Au matrix embedded with Fe nanoparticles.

18. The method of claim 17, wherein increasing a surface area of the electrode comprises etching the Fe nanoparticles out of the exposed region.

19. The method of claim 18, further comprising annealing the alloy from about 200° C. to about 600° C. for about 10 minutes to about 12 hours.

20. The method of claim 9, wherein the nanogap is from about 5 nm to about 20 nm in width.