CN116867906A

CN116867906A - Semiconductor nanosensor device with multi-layered graphene sheets for sequencing or sensing nucleic acids

Info

Publication number: CN116867906A
Application number: CN202180091216.1A
Authority: CN
Inventors: 克里斯塔·弗雷特斯
Original assignee: Gentroma Co ltd
Current assignee: Gentroma Co ltd
Priority date: 2020-11-30
Filing date: 2021-11-29
Publication date: 2023-10-10

Abstract

The application discloses a nano-sensing device for high-throughput nucleic acid sequencing or sensing. The device is a silicon chip with a top layer of silicon nitride, openings through the chip, holes in the top layer, and graphene sheets over the top layer and holes. A graphene layer with p-type layer pleats is placed over the holes, and one or more nanopores in the graphene layer may be provided over the holes. The nucleic acid strands transported onto the chip can be displaced through the nanopore and the well under the influence of the microfluidic forces. The electromotive force across the nanopore can be measured with a patch clamp amplifier for nucleobase assignment and DNA sequencing. In addition, no nanopore and interaction with the layer pleats are used to sense the presence of the nucleic acid polymer.

Description

Semiconductor nanosensor device with multi-layered graphene sheets for sequencing or sensing nucleic acids

Technical Field

The present application relates to nucleic acid sequencing and sensing using a chip employing a multi-layer graphene nanostructure and a p-type layer pleat (p-type crinkle ruga) over at least one well in the chip.

Background

One and the next generation of DNA sequencing technologies, ranging from Sanger sequencing to nanopore sequencing, have been developed to address the important applications of gene sequencing, the most important of which are personalized medicine for cancer, genetic diseases and complex disease treatments. Other applications that also require efficient DNA sequencing platforms include vaccine research, epidemic prevention, food monitoring, forensic sample analysis, gene drug development, and consumer gene testing, among others. While most current sequencing methods involve chemical alterations, amplification and labeling of DNA, certain applications in personalized medicine and single cell sequencing require label-free real-time sequencing to preserve the original DNA sample and complete faster turnaround times in hours.

Nanopore sequencing is the current technology [1-3], and is the only method currently available that is label-free and widely studied in the field of DNA sequencing. The DNA or RNA sample is guided into biological or solid state nanopores embedded in a membrane matrix by an electric field in solution [4]. When DNA bases are shifted through the nanopore under the action of an electric field, the ion current through the membrane will change in real time and be measured by a sensitive amperometric detector. Nanopore sequencing allows real-time sequencing without the need to label DNA, and the read length in a current run can even reach 1 to 2mb [5].

While this approach has reasonably long-read (long-read) DNA sequencing capabilities, there are problems that prevent it from becoming a realistic efficiency and capability for personalized medicine and other long-expected applications. First, while commercially available devices can provide speeds up to 250 bases/second, such speeds are not suitable for personalized medical applications, in which large numbers of genomes must be sequenced more quickly. Second, base accuracy of less than 99% is reasonable for many applications, but insufficient for haplotype analysis and personalized medicine. Thus, there is still a need for greater progress in this technology in order to make some of the most basic genomic sequencing applications widespread.

The present invention provides an alternative apparatus and method for detecting detectable changes in ion current due to the displacement of bases through nanopores within a novel nanostructure made of graphene, to make sequencing faster, more accurate, and high throughput. Graphene is attracting attention as a nanopore base material because it is capable of forming atomic pores and different structures [6]. The main structures of graphene nanostructures used in the sequencing field include graphene nanopores, lamellae, nanotubes, and nanogaps. Recently, a study by Kim et al has described a new shape related to the "crinkle ruga" structure, i.e. graphene is bent to form grooves on rectangular grooves [7]. The corrugations have a saw tooth profile with a perfectly flat surface and highly curved peaks and troughs at the ends. The accordion structure has a flexoelectric effect, or electromechanical coupling of polarization and strain gradients. As shown in silicon dioxide or silicon substrates, when a strain is applied, the graphene bends inward, creating a negative or positive charge gradient, respectively, that builds up at the fold channels, which is the polarization density effect of the flexoelectric effect. The type of charge accumulated depends on the material with the grooves, as van der Waals forces present at the edges of the grooves of the material will directly result in the flexoelectric coupling effect of graphene with the material. Control of structural properties and localization using substrate selection and radius of curvature for bending graphene is also described. Kim et al further studied the molecular effects at folds, for example, observing DNA strand linearization, indicating the possibility of using graphene folds to study and control polar molecular position. This is one of the many advantages of graphene in many biomolecular studies in this area.

For DNA sequencing, graphene has been demonstrated to be capable of ultra-fast sequencing, and one study of NIST has shown that the rate of translocation can be as fast as 6600 kilobases per second [8]. Because of these speeds, electrical detection requires a method of controlling DNA translocation, requiring a special electrolyte solution that can add difficulty while successfully slowing DNA translocation, e.g., adding more interference to signal detection. In addition, another feature that hampers the effectiveness of current nanopore sequencing is that the orientation of the bases within the pore affects the ion current signal, and thus becomes one of the reasons for the <99% accuracy of the method. Nanopore sequencing probes the structural properties of nucleobases and creates an electronic fingerprint in the current signal based on structural features, resulting in signal overlap between structurally similar guanines and adenines, as demonstrated by Derrington et al [9] and Manrao et al [10]. Nanopore sequencing can be improved if it can incorporate another physical assay method in addition to electronic assays, and a method that uses other features (such as molecular interactions) to distinguish each base (except for the sample-only base range).

Molecules having a size slightly smaller than the pore size pass through the pores by electrostatic potential by applying an external voltage. Nano-sized pores are typically embedded in biological membranes, typically protein nanopores, which present a number of problems in translocation control and accuracy [5]. They are also formed in solid thin films such as silicon or graphene that divide two reservoirs containing conductive electrolyte into cis and trans compartments. Electrodes immersed in each chamber generate a magnetic field and help detect the electrical signal. Under bias, electrolyte ions in the solution pass through the pores electrophoretically, thereby producing an ion current signal. When the pore is blocked by an analyte (e.g., a negatively charged DNA molecule added to the cis-chamber), the current flowing through the nanopore will be blocked, interrupting the current signal [5]. The physicochemical properties of the target molecule can be calculated by statistically analyzing the magnitude and duration of transient current blockages resulting from shift events (Venkatesan et al, [11 ]). Solid state nanopores have many advantages over biological nanopores, including higher stability. In particular, graphene nanopores exhibit extremely high DNA sequencing potential, exhibiting higher spatial resolution.

Rapid translocation of DNA bases is one of the problems with current sequencing using nanopores [12]. The nucleotides provide unique electronic signatures regarding orientation and charge properties in the pores [5]. Controlling the position of the nucleotide in the nanopore will help to improve the accuracy and practicality of nanopore sequencing.

The present invention addresses the continuing need for a rapid, inexpensive, and high throughput nanopore device for molecular detection that can be used to perform real-time and label-free sequencing of nucleic acid samples on site. Rapid, inexpensive, but accurate DNA sequencing is a continuing challenge. The personalized medicine field can benefit greatly from a real-time sequencing device that matches droplet-based DNA transfer methods, which can rapidly sequence many genomes in a short time, especially in neonatal health analysis and epidemic risk fields. The nanopore of the current device still requires specific electrolyte solutions and DNA environments, thus impeding progress in this field, slowing down the transition of nanopore sequencing to certain fields.

In view of these prior art, there is a need for a technique that improves on existing nanopores that can directly and variously determine changes in electronic signals to match the shift rate of nucleobases in the pore. Also necessary are nanopores with higher sensing capability for better identification by detecting molecular characteristics and interaction behavior of each base and directly controlling molecular orientation within the nanopore to obtain reproducible results with different quality of medium used. The methods can be flexibly used with various solutions and methods to meet different requirements and purposes of different industries and research fields.

Disclosure of Invention

Accordingly, provided herein is a chip for sequencing nucleic acid strands. The chip may have a substrate made of silicon having a width of about 1.0mm to about 10mm, a length of about 1.0mm to about 10mm, and a thickness of about 50 μm to 500 μm (preferably 200 μm), wherein the substrate has a thickness of silicon nitride (Si) of 20nm to 500nm (preferably 200 nm) ₃ N ₄ ) Optionally with a silicon nitride bottom layer having a thickness of 20nm to 500nm, preferably 200 nm.

In one embodiment, the chip may have an opening on the bottom side leading to a square (preferably 20 μm) window of 5 to 50 μm in the center of the chip, which opening penetrates into the substrate of the chip from an optional bottom layer, wherein the opening does not penetrate the top layer of silicon nitride. In one embodiment a hole is provided in the top layer of the silicon nitride centered on the window, wherein the hole has a width of 50nm to 1000nm (preferably 350 to 400 nm) and a length of 50nm to 1000nm (preferably 500 to 600 nm), wherein the hole has a circular or simple polygonal shape (preferably an hourglass shape).

In an alternative embodiment, the chip is drilled using a TEM or electron beam, in which case there is no window, but a straight axis through the chip.

In one embodiment, a multi-layered graphene sheet having a thickness of about 1 to 60nm is attached to the SiN layer on the top substrate in a lateral direction, and wherein the graphene sheet is laterally compressed to create a p-type layer pleat over the at least one hole, the p-type layer pleat having a pleat formed thereon, and the graphene sheet may have one or more nanopores having a diameter of 0.3 to 3.0nm centered on the hole.

In one embodiment, the chip may have a pair of electrodes on opposite ends of the hole, wherein the electrodes are connected to patch clamp amplifiers capable of measuring a charge on the graphene sheet.

In one embodiment, a method of detecting nucleobases in a nucleic acid strand is provided, the method comprising: a chip having a graphene sheet, directing an aqueous salt solution of a nucleic acid comprising a nucleobase strand to a nanopore in the graphene sheet, wherein the nucleobase strand is displaced through the pore and interacts with a layer of the multi-layer graphene sheet, and the patch clamp amplifier measures the change in the ion current and detects each nucleobase in the nucleic acid strand displaced through the nanopore and specifies the nucleic acid sequence.

Drawings

Fig. 1A is a perspective view of the top of a silicon/silicon nitride chip according to an embodiment.

Fig. 1B is a perspective view of the bottom of a silicon/silicon nitride chip according to one embodiment.

Fig. 2 is a top view of a silicon/silicon nitride chip according to an embodiment. Graphene sheets are shown.

Fig. 3A is a cross-section of a silicon/silicon nitride chip shown by the line labeled A-A' in fig. 2. In this embodiment, windows are formed from the bottom of the chip by chemical etching, which creates truncated pyramid-shaped cavities.

Fig. 3B is a cross-section of a silicon/silicon nitride chip in which the holes are drilled through the chip, according to an alternative embodiment.

Fig. 3C is a cross-section of a silicon/silicon nitride chip in which multiple holes are drilled through the chip according to an alternative embodiment. In this embodiment, there may be a grid of holes. Four wells are shown.

Fig. 4 is a schematic view of an hourglass-shaped opening in the top of the chip of the present invention.

Fig. 5 is a perspective view of the top of a chip according to the present invention with a graphene sheet across the top, a crease in the graphene over the opening, a nanopore in the crease, and two electrodes.

Fig. 6A is a front view of the bottom of a chip with a grid showing a plurality of windows formed by chemical etching, each window having a hole therein, according to an embodiment of the invention.

Fig. 6B is an elevation view of the top of a chip with larger grid of holes (122) formed by drilling in accordance with the present invention.

Fig. 7 is a schematic view of a circular silicon chip engraved with a grid to be cut to form a silicon/silicon nitride chip according to an embodiment of the present invention.

Fig. 8 is a schematic diagram of an embodiment of the invention in which DNA probes are attached to graphene sheets 140.

Detailed Description

Disclosed herein is a nanosensor device capable of high throughput nucleic acid sequencing, wherein a polarized graphene sheet or flake interacts with a nucleic acid strand or polymer to detect and identify nucleobases in the nucleic acid strand. Alternatively, the device may sense nucleic acid polymers. The device includes a semiconductor die having one or more holes drilled or etched therein, and a multilayer graphene (MLG) sheet laminated over a top surface of the die, wherein the graphene is subjected to compressive pressure to form a p-type layer pleat over each hole. The electromechanical effect of the folds may attract the nucleic acid polymer into the folds.

The nanosensor semiconductor chip has a "top side" and a "bottom side". The top orientation represents (1) the side to which the MLG is attached, and (2) the side of the nucleic acid polymer that is in proximity to the chip for sequencing or sensing. The nucleic acid polymer will interact with graphene on the top surface of the chip of the present invention for use in the sensing and sequencing embodiments of the present invention.

In one embodiment, at least one nanopore is provided in the graphene sheet over the pore that forces the nucleic acid strand to translocate through the nanopore and be in close alignment with a sensor capable of detecting and identifying nucleobases in the nucleic acid strand. In addition, nanopores may not be used.

When nucleobases comprising nucleic acid strands are interacted or displaced by graphene, detection or sensing of nucleobases can be performed by several methods. In one embodiment, the ion current may be used with an ion current detector that can determine an electrical characteristic of a particular nucleobase as it is displaced through a nanopore in a graphene sheet and assign the nucleobase based on the electrical characteristic (A, C, T/U, G). The ion current may be measured on a pair of electrodes on either side of the aperture of the chip.

The nucleic acid strand may comprise a nucleotide or nucleoside, and may be derived from a natural source, for example isolated from a cell (e.g., a bacterial, plant or animal cell), or extracted from a viral source. The nucleic acid polymer may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As used herein, "nucleic acid strand (nucleic acid strand)" corresponds to "nucleic acid polymer (nucleic acid polymer)". The term "sensing" refers to detecting the presence of a nucleic acid, whether generally or having a particular sequence, without the need to elucidate the sequence of the nucleic acid.

In one embodiment, a device with nanopores is used to elucidate nucleic acid sequences wherein the nucleic acid polymer translocates through the nanopores. In one embodiment, a device without a nanopore for sensing a nucleic acid based on an electrical characteristic measured on a pair of electrodes on either side of the pore, wherein a layer above the pore interacts with the nucleic acid and provides a characteristic feature of an electrical current through the pore caused by the interaction with a nucleic acid strand.

In one embodiment, the invention discloses a high throughput single cell sequencing device that is capable of label-free sequencing without the need for chemical modification or cell culture of the single cells to be analyzed. The disclosed sequencing instrument will enable massively parallel and label-free sequencing that can be accomplished faster (less than 2 hours) and at lower cost than traditional single-cell nucleic acid sequencing techniques used in the pharmaceutical industry, biotechnology research and healthcare. In one embodiment, a platform utilizing nanopore sequencing technology is disclosed that uses graphene nanopores with optical or electrical properties that can detect and distinguish nucleobases in a nucleic acid strand. The inventors have demonstrated that interactions with DNA are theoretical verification data for label-free and cost-effective sequencing. The invention can integrate the droplet transfer of DNA fragments into a sequencer, and directly read nucleotides through electric or optical interaction, thereby enabling single-cell sequencing for personalized medicine and genetic and immunotherapeutic applications to be fast, high-throughput, economical and efficient.

As used herein, the term "about" means one imprecise dimension and the term "about" means ±20% of the value.

As shown, one embodiment of the present application discloses a high throughput nanopore biosensing device, comprising a chip 100, a silicon substrate 114 having a width (FIG. 1A, dimension M) of about 1.0mm to about 10mm, and a length (FIG. 1A, dimension N) of about 1.0mm to about 10 mm. In one embodiment, the chip 100 has a width of about 1.5mm to about 3.0mm and a length of 1.5mm to 3.0mm, or a width of about 1.5mm or 2.0mm and a length of about 1.5mm or 2.0mm. In one embodiment, the total thickness of the chip 100 may be about 50 μm to 500 μm (fig. 1A, dimension Q). In one embodiment, the total thickness of the chip 100 may be about 150 μm to 300 μm, or about 200 μm.

The term "chip" refers to a semiconductor chip fabricated using semiconductor processes. The substrate may be cut into square or rectangular shapes or may be circular. For example, chips having a width and length of 1.5mm to 10.0mm may be made from a 2 inch diameter (5 cm) wafer 150, with a grid of grooves 152 cut or etched into the wafer 150. As depicted in fig. 7. The grid 152 is then cut to obtain chips of a size suitable for the present application.

In the drawings or text of this patent application, the term "SiN" refers to silicon nitride (Si ₃ N ₄ )。

In one embodiment, the chip 100 has an upper surface (116) and a lower surface (118). The upper and lower surfaces are interchangeably referred to herein as "top" and "bottom" surfaces.

In one embodiment, a layer of silicon nitride (110) may be provided that forms the top surface (116) of the chip 100. The silicon nitride 110 may have a thickness of about 20nm to about 500 nm. Alternatively, the thickness of the silicon nitride 110 may be about 100nm to 300nm. Alternatively, the silicon nitride 100 may have a thickness of 200 nm. Optionally, the chip may have an underlayer (112) of silicon nitride forming the bottom surface 118. Alternatively, the bottom surface 118 may be the bottom of the silicon substrate 114. Layer 112, if present, may have a thickness of 20nm to 500 nm. In one embodiment, the thickness of layer 112 may be about 100nm to 300nm. Alternatively, silicon nitride 112 may have a thickness of 200 nm.

There may be a square window 130 (fig. 1B) in the center of the bottom of the chip. Alternatively, window 130 may be referred to as having an "opening". The cross section of window 130 is shown in fig. 3A. If an underlayer 114 of silicon nitride is provided, the window 130 penetrates the underlayer. The opening may pass through the silicon substrate from the bottom surface of the chip to the top silicon nitride layer and not penetrate the top silicon nitride layer. In one embodiment, the upper boundary of the opening is a square window at the top of the substrate at about 5 μm to 50 μm (preferably 20 μm) (dimension P). The window 130 may be formed by potassium hydroxide (KOH) etching. KOH etching will create walls in the silicon substrate that are at an angle of 54.7, so the walls will not be vertical [13]. Thus, the opening just described will be a truncated square pyramid (truncated square pyramid), also called truncated pyramid, the truncated top of which (the "window") forms a square of 5 μm to 50 μm (preferably 20 μm) (dimension P) on one side. The base of the pyramid has edges of dimension T. For a 20 μm truncated pyramid top and 200 μm thick substrate, the square truncated pyramid bottom on each side is about 302 μm due to the angle of KOH etching in silicon. Shown in fig. 3A is a multilayer graphene sheet 140 and optional nanopore 146.

In one embodiment, a hole 120 (fig. 2 and 4) is provided in the top layer of window-centered silicon nitride, wherein the hole has a width of about 50nm to about 1000nm (preferably 350 to 400 nm) (fig. 4, dimension S) and a length of about 50nm to 1000nm (preferably about 500 to 600 nm) (fig. 4, dimension U), wherein the hole is circular or a simple polygon. In one embodiment, as shown in FIG. 4, the aperture may have an hourglass shape (120). In another embodiment, the aperture has a width of 350 to 500nm and a length of 350 to 500nm and comprises a simple polygon selected from the group consisting of a circle, triangle, square, rectangle, and hexagon.

Alternatively, the holes 120 may be formed by drilling with a microscopic method, such as with a Transmission Electron Microscope (TEM) or electron beam technique (fig. 3B and 3C). A hole made by drilling will not have a window with inclined walls as in 130. Instead, the straight axis 132 will extend through the entire thickness of the chip. Graphene sheets 140 or 141 without nanopores are also shown.

In another embodiment, a plurality of holes 122 may be provided, wherein, for example, a grid or array of holes and windows are formed in the chip 100. As shown in fig. 6A and 6B. Fig. 6A shows the bottom 118 of the chip 104 with a grid of etched windows 130. Also shown is an aperture 121 in each window 130. Hole 121 is the bottom of hole 120. In the case of etching window 130, the grid may be up to about 10 x 10, and may be smaller, such as 6 x 6, 5 x 5, or 4 x 4.

Another embodiment of the plurality of holes is shown in fig. 6B, where fig. 6B is a top view of the chip 102 having the plurality of holes 122. In the embodiment shown, the borehole is a straight shaft 132. The grid or array in this embodiment may have more holes than the etching method, up to a 100 x 100 grid. The grid may also be smaller, for example, a 30 x 30 grid, a 20 x 20 grid, or a 10 x 10 grid. Fig. 6B shows an MLG sheet 141 laid on a grid. In one embodiment, after holes 122 are drilled, the sheet is secured to surface 116 and then nanopores 146 (not shown) are drilled. An embodiment with a bore is also shown in fig. 3C, which shows a cross section of the chip 102 with a plurality of bores 122.

In the array embodiment of fig. 6A and 6B, an electrode array may be used to measure the potential on each well in the grid. [14, 15]

In one embodiment, a graphene sheet 140 having a thickness of about 1 to 60nm is placed over the top surface of the silicon die in a lateral direction (fig. 2, 3, and 5), wherein the graphene sheet is laterally compressed to form a p-type layer pleat over each hole 120 in the biosensing die, the p-type layer pleat having a pleat formed thereon to form a p-type layer pleat having a pleat over each hole 120 in the biosensing die. The graphene may be a multilayer graphene (MLG) sheet placed over the surface of the chip, which may be 2 to 7 sheets (or more) of graphene. Graphene will naturally adhere to the silicon nitride surface. Lateral pressure may be applied to form a p-type layer pleat having a crease or crease over the aperture. Optionally, one or more nanopores 146 (fig. 2) in the graphene sheets may be disposed over the pores, wherein the nanopores have a diameter of 0.3 to 3.0nm. Transmission Electron Microscopy (TEM) or electron beam techniques can be used to drill the desired nanopores in the graphene corrugations.

In one embodiment, nanopores 146 drilled in the MLG have microfluidic capabilities for capturing and transporting DNA using small droplets (beads). "microfluidic" refers to the behavior, precise control and manipulation of fluids that are geometrically constrained to small dimensions (typically sub-millimeters) where surface forces dominate volumetric forces. The advantage of this design is that the droplets can transport the DNA, effectively speeding up the single cell sequencing process, and by using the positive charge located in the folds to easily attract the DNA, the DNA is pulled into the graphene layer folds within the Si. The layer pleats also physically attract droplets into the pleat, in combination with positive charges in the graphene layer, making nanopore transition and translocation easier. The nanopore technology (e.g., [16 ]) of the conventional planar substrates currently in use is unable to bind droplets because large droplets must be perfectly aligned to sub-2 nanopores and reliably direct DNA to the nanopores in solution. The design of the present invention enables droplets to be first trapped in the structure and then positive charges pull DNA into and align the nanopore, enabling DNA to more easily translocate into a nanopore of width 2nm or less. The integrated microfluidic system may flush the nanopore with an electrolyte solution to allow electrophoretic translocation.

In one embodiment, gel beads (gel beads) may be used to transport DNA molecules to the sequencing chip of the present invention. Gel beads containing barcode oligonucleotides are mixed with a sample, which may be High Molecular Weight (HMW) DNA. The gel beads and sample are then added to an oil surfactant solution to form "multiple gel beads in emulsion" (GEMs) as individual reaction vesicles, where the gel beads are solubilized and the sample is bar-coded (see [17 ]). In some cases, the bar code labeled products are pooled for downstream reactions to create short-read sequencer-compatible libraries. In one embodiment of the invention, these fragments will be sequenced directly to read the base differences between cells of similar DNA fragments in similar regions of interest in their genome, and also mapped directly back to the original cells by direct reading of the barcode sequence, since unlabeled nanopore sequencing can capture these droplets (then separate between bead and DNA) and sequence single cell transcriptomes in a highly parallel fashion. After sequencing, the resulting short read sequence of the barcode may be analyzed using bioinformatics, which uses the barcode information to map the reads back to their original DNA sequence.

In one embodiment, one or more nucleic acid probes 160 can be covalently bound to the MLG 140 (FIG. 8) [3]. During a sensing or sequencing operation of the biosensing chip of the present invention, probes can hybridize to complementary nucleic acid strand 162. FIG. 8 shows three nucleic acid probes 160, one of which hybridizes to a complementary strand 162. In embodiments of DNA/RNA sensing, the probe hybridizes to a complementary nucleic acid sequence, which causes a characteristic change in the potential across the well 120 that can be detected to confirm that the appropriate DNA/RNA sequence is present in the sample passing through the sensor. In embodiments of nucleic acid sequencing, the probes 160 may tend to increase the interaction of the MLG with the nucleic acid sample, which may increase the sensitivity of the sequencing process. In one embodiment, the probes 160 are bonded to the MLG as close as possible to the fold 142 or within the fold 142, which is closest to the electrical hot spot to increase the sensitivity of the hybridization event.

In one embodiment, a pair of electrodes 144 (fig. 5) are provided on the graphene sheets at opposite ends of each aperture 120, wherein the electrodes are connected to patch clamp amplifiers capable of measuring the charge on the graphene sheets.

In one embodiment, a method of detecting nucleobases in a nucleic acid strand is provided, the method comprising: a chip having a graphene sheet connected to a patch clamp amplifier and directing an aqueous salt solution of nucleic acids comprising nucleobase strands to nanopores in the graphene sheet, wherein the nucleobase strands are displaced through the pores and interact with layers of the multilayer graphene sheet, and the patch clamp amplifier measures changes in ion current and detects each nucleobase in the nucleic acid strand displaced through the nanopores and specifies a nucleic acid sequence.

In one embodiment, the present invention may improve the assay by using the positive charge within graphene folds 142 as a means of electronically detecting and electrostatically locating each nucleotide within a well. The nucleotide chains directed to the pores on the graphene will separate and shift through the pores and holes on the chip. Attracting the positive charge of the negatively charged DNA molecule will also slow down the translocation, thereby providing a means to control translocation while using the charge in graphene 1) to position the nucleotide within the pore with a more reliable orientation within the pore while 2) forming a temporary electrostatic interaction with the nucleotide to improve single base resolution, 3) analyzing the charge characteristics of specific bases, the determination of ionic current in 0.3 to 1M KCl solutions can be simplified to single base resolution.

By means of a patch clamp amplifier or the like, e.g. Axon ^TM Axopatch ^TM 200B, can be effectively measured at a transmembrane potential of 120 to 180mVAnd (5) determining ion current. During the displacement of DNA through the nanopore, the electrical signal of the ion current fluctuation due to the blockage in the nanopore will be converted to a nucleotide specific reading by a circuit that records the amplifier or patch device information [18 ]]Thereby specifying a particular nucleobase that passes through a nanopore or hole in the biosensing chip. Fluctuations in ion current, which are directly related to the polarization within the graphene layer and the nanoscale electronic interactions between nucleotides, will reflect the charge in the graphene corrugations over a millisecond time scale. The information will be obtained and converted to base assignments (assignments) in readable format by specialized software that uses conversion algorithms of known electronic signatures in ion current measurements made during sequencing runs. The possibility of reducing interference and improving signal quality is apparent because positive charges within graphene wrinkles will slow down translocation and potentially improve signal quality, eliminating the need to add electrolyte solution to slow down DNA translocation in traditional nanopore approaches [19 ] ]. Compatibility of the droplet delivery method is also implied because the natural curvature of the folds and charge polarization will work together to introduce DNA into the nanopore without the need for elaborate enzymes or complex microfluidic processes. The advantage is to add multiple real-time single-cell sequencing technology to the field, the technology can also keep sample quality, and directly analyze physical, structural and apparent genome characteristics of DNA through an electronic probe, thus providing a new data layer for single-cell analysis of each operation, greatly reducing chemical agents and shortening time.

List of reference numbers

Digital description

100. Semiconductor biological sensing chip

102. Semiconductor biosensing chip with multiple drilling windows

104. Semiconductor biosensing chip with multiple etching windows

105. Round silicon chip

106. Circular silicon chip with grid

110. Upper (top) silicon nitride (Si ₃ N ₄ ) Layer(s)

112. Lower part(s)(bottom) silicon nitride (Si ₃ N ₄ ) Layer(s)

114. Silicon substrate layer

116. Upper (top) surface of the biosensing chip 100

118. Lower (bottom) surface of the biosensing chip 100

120. Hole(s)

121. Holes (bottom view)

122. Multiple drill holes

130. Window-formed by etching inclined walls

132. Shaft through chip formed by drilling

140. Multilayer graphene (MLG) sheets

141. MLG laminated on multiple holes

142 Folds or wrinkles in MLG

144. Electrode

146 Nanopores in MLG

150 5cm chip with a grid of biosensing chips etched on it

152. Grid etching of biosensing chip on 5cm diameter chip

160. Nucleic acid strand probe

162. Complementary nucleic acid polymers hybridized to nucleic acid probes

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Claims

1. A chip for sequencing a nucleic acid strand, characterized in that: the chip comprises:

(a) A chip formed from a substrate made of silicon having a width of about 1.0mm to about 10mm, a length of about 1.0mm to about 10mm, and a thickness of about 50 μm to 500 μm;

(b) Wherein the substrate has a silicon nitride Si with a thickness of 20nm to 500nm ₃ N ₄ Is a top layer of (2);

(c) Wherein the substrate optionally has an underlayer of silicon nitride having a thickness of 20nm to 500 nm;

(d) Wherein the chip has at least one hole drilled by a microscopic method, the hole penetrating the entire thickness of the chip, and wherein the hole has a width of 50nm to 1000nm and a length of 50nm to 1000nm, wherein the hole is circular or simple polygonal in shape;

(e) Wherein a multi-layered graphene sheet having a thickness of about 1 to 60nm is attached to the Si on the top substrate in a lateral direction ₃ N ₄ A layer, and wherein the graphene sheet is laterally compressed to create a p-type layer pleat over the at least one aperture, the p-type layer pleat having a pleat formed thereon; and

(f) Wherein a pair of electrodes are provided on opposite ends of each hole on the graphene sheet, wherein the electrodes are connected to an electrical detection device capable of measuring an ion current on the graphene sheet.

2. The chip of claim 1, wherein: the graphene sheet includes one or more nanopores having a diameter of 0.3 to 3.0nm centered on the pore such that a nucleic acid polymer chain may be displaced through at least one nanopore.

3. The chip of claim 2, wherein: fluctuations in the ion current of a nucleic acid strand displaced through at least one nanopore are measured by the electrical detection device and designated by a switching nucleobase.

4. The chip of claim 1, wherein: the graphene sheets do not have nanopores.

5. The chip of claim 4, wherein: fluctuations in the ion current through a hole caused by a nucleic acid polymer are measured by the electrical detection device and converted into inductive information.

6. The chip of claim 1, wherein: the width of the substrate is 1.5mm or 3.0mm and the length is 1.5mm or 3.0mm.

7. The chip of claim 1, wherein: the substrate has silicon nitride Si with thickness of 200nm ₃ N ₄ Is a top layer of (c).

8. The chip of claim 1, wherein: the substrate has a bottom layer of silicon nitride with a thickness of 100nm to 300 nm.

9. The chip of claim 1, wherein: the substrate has a bottom layer of silicon nitride with a thickness of 200 nm.

10. The chip of claim 1, wherein: at least one hole in the top layer of silicon nitride centered on the hole is hourglass shaped.

11. The chip of claim 1, wherein: the chip further comprises a grid of holes, wherein the grid is a 100 x 100 grid at most.

12. The chip of claim 1, wherein: the chip further comprises a grid of holes, wherein the grid is a 20 x 20 grid at most.

13. The chip of claim 1, wherein: the chip further comprises a grid of holes, wherein the grid is a 10 x 10 grid at most.

14. The chip of claim 11, wherein: the chip further includes an electrode array.

15. The chip of claim 1, wherein: the chip further includes one or more nucleic acid probes bound to the multilayer graphene sheet.

16. A chip for sequencing a nucleic acid strand, characterized in that: the chip comprises:

(d) Wherein the chip has at least one opening defining a base of a square frustum pyramid penetrating from a bottom surface of the substrate to a top surface of the substrate of the chip, wherein the opening does not penetrate the top layer of silicon nitride, and wherein the top of the square frustum pyramid is a square window of 5 to 50 μm per side;

(e) Wherein a hole is provided in the top layer of silicon nitride centered on the window, wherein the hole has a width of 50nm to 1000nm and a length of 50nm to 1000nm, wherein the hole has a shape of a circle or a simple polygon;

(f) Wherein a multi-layered graphene sheet having a thickness of about 1 to 60nm is attached to the Si on the top substrate in a lateral direction ₃ N ₄ A layer, and wherein the graphene sheet is laterally compressed to create a p-type layer pleat over the at least one aperture, the p-type layer pleat having a pleat formed thereon; and

(g) Wherein a pair of electrodes are provided on opposite ends of each hole on the graphene sheet, wherein the electrodes are connected to an electrical detection device capable of measuring an ion current on the graphene sheet.

17. The chip of claim 1, wherein: the graphene sheet includes one or more nanopores having a diameter of 0.3 to 3.0nm centered on the aperture such that a nucleic acid strand may be displaced through at least one nanopore.

18. The chip of claim 17, wherein: fluctuations in the ion current of a nucleic acid strand displaced through at least one nanopore are measured by the electrical detection device and designated by a switching nucleobase.

19. The chip of claim 1, wherein: the substrate has a width of 1.5mm to 3.0mm and a length of 1.5mm to 3.0mm.

20. The chip of claim 1, wherein: the thickness of the substrate is 150 μm to 300 μm.

21. The chip of claim 1, wherein: the thickness of the substrate was 200 μm.

22. The chip of claim 1, wherein: the substrate has a silicon nitride Si with a thickness of 100nm to 300nm ₃ N ₄ Is a top layer of (c).

23. The chip of claim 1, wherein: the substrate has silicon nitride Si with thickness of 200nm ₃ N ₄ Is a top layer of (c).

24. The chip of claim 1, wherein: the substrate has a bottom layer of silicon nitride with a thickness of 100nm to 300 nm.

25. The chip of claim 1, wherein: the substrate has a bottom layer of silicon nitride with a thickness of 200 nm.

26. The chip of claim 1, wherein: the at least one hole in the top layer of silicon nitride centered on the window has a width of 350 to 400nm and a length of 500 to 600nm.

27. The chip of claim 1, wherein: the at least one hole in the top layer of silicon nitride centered on the window has a width of 350 to 500nm and a length of 350 to 500nm and comprises a simple polygon selected from the group consisting of a circle, a triangle, a square, a rectangle, and a hexagon.

28. The chip of claim 1, wherein: at least one hole in the top layer of silicon nitride centered on the window is hourglass shaped.

29. The chip of claim 1, wherein: the chip further comprises a grid of holes, wherein the grid is a 10 x 10 grid at most.

30. A method for detecting nucleobases in a nucleic acid strand, characterized by: the method comprises the following steps: the chip of claim 2 or 17 having a graphene sheet and directing an aqueous salt solution of a nucleic acid comprising a nucleobase strand to a nanopore in the graphene sheet, wherein the nucleobase strand is displaced through the pore and interacts with a layer of the multilayer graphene sheet, and wherein a patch clamp amplifier attached to the electrode of each pore measures the change in ion current and detects each nucleobase in the nucleic acid strand displaced through the nanopore and specifies the nucleic acid sequence.

31. A chip for sensing a nucleic acid strand, characterized in that: the chip comprises:

(e) Wherein a multi-layered graphene sheet having a thickness of about 1 to 60nm is attached to the Si on the top substrate in a lateral direction ₃ N ₄ A layer, and wherein the graphene sheet is laterally compressed to create a p-type layer pleat over the at least one aperture, the p-type layer pleat having a pleat formed thereon, and wherein no nanopores are present in the multi-layer graphene sheet; and

(f) Wherein a pair of electrodes are provided on opposite ends of each well on the graphene sheet, wherein the electrodes are connected to a patch clamp amplifier capable of measuring an ion current on the graphene sheet, and wherein fluctuations in the ion current are measured and converted to nucleobase designations.

32. A method for sensing nucleobases in a nucleic acid strand, characterized by: the method comprises the following steps: the chip of claim 31 having a graphene sheet and directing an aqueous salt solution of a nucleic acid comprising a nucleobase strand to a layer of pleats on the chip, wherein the nucleobase strand interacts with the layers of the multi-layer graphene sheet, and wherein a patch clamp amplifier connected to the electrode at each well measures the change in ion current and the characteristic electrical properties of the nucleic acid strand.