阅读说明：本技术 纳米孔设备以及使用所述设备检测带电粒子的方法 (Nanopore device and method of detecting charged particles using the same ) 是由 B·卡里米拉德 韩景晙 R·R·亚兹迪 W·J·柳 于 2019-07-27 设计创作，主要内容包括：一种用于检测带电生物聚合物分子和限定纳米通道的纳米孔设备包括定址纳米通道的第一末端的第一门控纳米电极。该设备还包括定址与第一末端相对的纳米通道第二末端的第二门控纳米电极。该设备进一步包括定址第一和第二末端之间的纳米通道中的第一位置的第一传感纳米电极。(A nanopore device for detecting charged biopolymer molecules and defining a nanochannel includes a first gated nanoelectrode addressing a first end of the nanochannel. The device also includes a second gated nanoelectrode addressing a second end of the nanochannel opposite the first end. The device further includes a first sensing nanoelectrode addressing a first location in the nanochannel between the first and second ends.)

1. A nanopore device for detecting charged biopolymer molecules, wherein the device defines a nanochannel, the device comprising: addressing a first gated nanoelectrode at a first end of the nanochannel; addressing a second gated nanoelectrode at a second end of the nanochannel opposite the first end; addressing a first sensing nanoelectrode at a first location in the nanochannel between the first and second ends; and

2. the device of claim 1, wherein the charged biopolymer molecules are negatively charged or defined based on isoelectric point and zeta potential.

3. The device of claim 1, wherein the first potential directs the flow of the charged biopolymer molecules from the first gated nanoelectrode to the second gated nanoelectrode.

4. The device of claim 3, wherein the first and second gated nanoelectrodes are also operable to generate a second electrical potential across the nanochannel to direct the flow of the charged biopolymer molecule through the nanochannel.

5. The apparatus of claim 4, wherein the second potential is opposite the first potential.

6. The device of claim 4, wherein the second potential directs the flow of charged biopolymer molecules from the second gated electrode to the first gated electrode.

7. The device of claim 6, wherein the first and second gated-nanoelectrodes are further operable to alternately generate first and second electrical potentials across the nanochannel to direct the alternating flow of charged biopolymer molecules through the nanochannel between the first and second gated-nanoelectrodes.

8. The apparatus of claim 1, wherein the inner surface of the apparatus comprises Al₂O₃、HfO₂、SiO₂Or ZnO.

9. The device of claim 1, further comprising a buffer selected from the group consisting of KCl, LiCl, and Deionized (DI) water.

10. The device of claim 1, wherein the nanopore device is configured to detect a point mutation in the biopolymer using an endonuclease.

11. The device of claim 1, wherein the nanopore device is configured to perform target sequencing of biopolymers using immobilized dCas9 protein and target-associated guide rna (grna).

12. The device of claim 1, wherein the nanopore device is integrated into a microfluidic device, a nanofluidic device, a nanodevice, or a lab-on-a-chip device.

13. The device of claim 1, wherein the nanopore device is integrated into an integrated device for extracting and sensing targeted biopolymers.

14. The device of claim 13, wherein the targeted biopolymer is selected from the group consisting of DNA, RNA, mRNA, miRNA, cDNA, peptide, protein immobilized antigen, and antibody.

15. The device of claim 1, wherein the nanopore device is integrated into a liquid biopsy panel platform for detection without biomolecule amplification or use of PCR.

16. The device of claim 1, wherein the nanopore device is configured to detect hybridization of the charged biopolymer molecule to the first biopolymer probe based on a tunneling current, a lateral tunneling current, or a change in capacitance.

17. A method for detecting a charged biopolymer molecule, comprising:

providing a nanopore device defining a nanochannel, the device comprising a first gated nanoelectrode addressing a first end of the nanochannel, a second gated nanoelectrode addressing a second end of the nanochannel opposite the first end, a first sensing nanoelectrode addressing a first location in the nanochannel between the first and second ends, and a first biopolymer probe coupled to an interior surface of the device defining the nanochannel; the first and second gated nanoelectrodes generate a first electrical potential across the nanochannel to direct the flow of the charged biopolymer molecules through the nanochannel; and

18. the method of claim 17, further comprising alternately generating a first potential and a second potential across the nanochannel to direct the charged biopolymer molecules to alternately flow through the nanochannel between the first and second gated nanoelectrodes.

19. The method of claim 17, wherein the nanopore device further comprises a second sensing nanoelectrode addressing a second location in the nanochannel between the first and second ends.

20. The method of claim 17, wherein the nanopore device is integrated into a microfluidic device, a nanofluidic device, a nanodevice, or a lab-on-a-chip device.

21. The method of claim 17, wherein the nanopore device is integrated into an integrated ASIC platform system for extracting and sensing targeted biopolymers.

22. The method of claim 17, further comprising a nanopore device detecting hybridization of a charged biopolymer molecule to a first biopolymer probe, the charged biopolymer molecule being at a minimum concentration of about 10 femtomoles (detection limit).

23. The method of claim 22, further comprising the nanopore device detecting hybridization of the first charged biopolymer molecule to the first biopolymer probe without amplification of the first charged biopolymer molecule or using PCR.

24. The method of claim 22, wherein the nanopore device is integrated into a liquid biopsy panel platform for detection without biomolecule amplification or use of PCR.

Technical Field

The present invention relates generally to systems and devices for characterizing biopolymer molecules, and methods of detecting charged biopolymer molecules using the same. In particular, the present invention relates to nanopore sensors for detecting charged biopolymer molecules.

Background

Many diseases, such as cancer, are curable if detected early before disease progression. However, millions of people die each year from such "curable" diseases. An affordable and rapid in-the-field detection (point of care) device for accurate and early diagnosis of low-grade stage cancer would allow early treatment, reducing morbidity and mortality.

As a result of the human genomic project, many disease-associated mutations (e.g., cancer-associated) in the human genome can now be detected by detecting genetic polymorphisms. Similarly, many pathogenic organisms (e.g., viruses) can also be detected by probing their specific gene sequences. Detection of the target gene/sequence of interest for about 10 minutes can facilitate rapid detection in situ for disease diagnosis, determining disease progression, and/or disease monitoring.

Mutations are common in nucleic acid (e.g., DNA, RNA, etc.) replication. In fact, mutation is the driving force behind natural selection and evolution. The mutations result in genetic/genetic polymorphisms in the population that can be associated with blood type, genetic disease, and the like. Detection of genetic polymorphisms is one of the most effective methods for identifying genetic variations at the molecular level. Many characteristics of genetic diseases (signatures) can be detected by information gathered through detection of genetic polymorphisms such as single nucleotide polymorphisms ("SNPs"), small scale insertions/deletions ("Indels"), gene fusions, transposable elements, microsatellites, and the like. Many gene polymorphism detection techniques require nucleic acid amplification (e.g., using PCR) and/or tagging/labeling of gene probes (e.g., using enzymes/radioisotopes). These molecular biology techniques are expensive and time consuming.

Whole genome sequencing can also be used to "detect" gene polymorphisms, which is another expensive and time consuming technique. Current techniques for sequencing nucleic acids at the single molecule level include nanopore sequencing technologies, which have advantages over previous sequencing technologies because nanopore sequencing technologies feature tag-free, label-free, and amplification-free technologies, which also have improved read lengths and improved system throughput. Thus, nanopore sequencing technology has been incorporated into high quality gene sequencing applications.

Early experimental systems for nanopore-based DNA sequencing detected the electrical behavior of ssDNA passing through the nanopore of alpha-hemolysin (α HL) protein. Since then, nanopore-based nucleic acid sequencing technologies have improved. For example, solid-state nanopore-based nucleic acid sequencing replaces biological/protein-based nanopores with solid-state (e.g., semiconductor, metal gate) nanopores, as described below.

A nanopore is a small hole (e.g., having a diameter in the nanometer range) through which the flow of charged particles through the hole can be detected by changes in ion current and/or tunneling current. Since each nucleotide of the nucleic acid (e.g., adenine, cytosine, guanine, thymine in DNA, uracil in RNA) affects the current density across the nanopore in a specific manner as it physically passes through the nanopore, measuring the change in current flowing through the nanopore during translocation yields data that can be used to directly sequence nucleic acid molecules that pass through the nanopore. Nanopore technology is therefore based on electrical sensing, which is capable of detecting nucleic acid molecules at much lower concentrations and volumes than required by other conventional sequencing methods. Advantages of nanopore-based nucleic acid sequencing include long read length, plug-and-play capability, and scalability. With advances in semiconductor manufacturing technology, solid-state nanopores have become a cheap and superior alternative to biological nanopores, in part because of their superior mechanical, chemical, and thermal properties and compatibility with semiconductor technology, allowing integration with other sensing circuits and nanodevices.

Figure 1 schematically depicts a state of the art solid-state based two-dimensional ("2D") nanopore sequencing apparatus 100. Although the device 100 is referred to as "two-dimensional," the device 100 has a thickness along the Z-axis. To address some of the shortcomings (sensitivity and some manufacturing costs) of the current state-of-the-art nanopore technology, label-free, amplification-free, and rapid sequencing can be achieved using multi-channel nanopore arrays that allow parallel processing of biomolecule sequencing. Examples of such multi-channel nanopore arrays are described in U.S. provisional patent application serial nos. 62/566,313 and 62/593,840, the contents of which have been previously incorporated by reference.

Although nanopore devices have been used to sequence nucleic acid polymers with increased efficiency and effectiveness, whole genome sequencing is overly complex for the detection of specific genetic polymorphisms and other charged biopolymers. For example, target gene detection of interest involves much smaller and easier to manage than conventional amplification-based methods, such as whole genome sequencing involving large data sets. There is a need for more efficient detection of gene polymorphisms and other charged biopolymers. In particular, there is a need for label-free, amplification-free and rapid detection of gene polymorphisms and other charged biopolymers.

Summary of The Invention

Embodiments described herein relate to nanopore-based electrically-assisted charged biopolymer detection systems and methods of using the same to detect charged biopolymers (e.g., genetic polymorphisms). In particular, the embodiments relate to charged biopolymer detection systems based on various types (2D or 3D) of nanopores, methods of using nanopore array devices, and methods of detecting charged polymers using the devices.

In one embodiment, a nanopore device for detecting charged biopolymer molecules and defining a nanochannel includes a first gated nanoelectrode addressing a first end of the nanochannel. The device also includes a second gated nanoelectrode addressing a second end of the nanochannel opposite the first end. The device further includes a first sensing nanoelectrode addressing a first location in the nanochannel between the first end and the second end. In addition, the device includes a first biopolymer probe coupled to the interior surface of the device defining the nanochannel.

In one or more embodiments, the device further includes a second sensing nanoelectrode addressing a second location in the nanochannel between the first end and the second end. The charged biopolymer molecules may be negatively charged.

In one or more embodiments, the first and second gated nanoelectrodes can be operated to generate a first electrical potential across the nanochannel to direct the flow of charged biopolymer molecules through the nanochannel. The first potential may direct the flow of the charged biopolymer molecules from the first gated nanoelectrode to the second gated nanoelectrode. The first and second gated nanoelectrodes are also operable to generate a second electrical potential across the nanochannel to direct the flow of the charged biopolymer molecule through the nanochannel. The second potential may be opposite to the first potential. The second potential may direct the flow of the charged biopolymer molecules from the second gated nanoelectrode to the first gated nanoelectrode. The first and second gated nanoelectrodes are also operable to alternately generate first and second electrical potentials across the nanochannel to direct the charged biopolymer molecules to alternately flow through the nanochannel between the first and second gated nanoelectrodes.

In one or more embodiments, the first sensing nanoelectrode is configured to detect hybridization of a first charged polymer molecule to a first biopolymer probe. The first biopolymer probe may have a first predetermined length.

In one or more embodiments, the device further comprises a buffer, wherein the first and second gated nanoelectrodes and the first sensing nanoelectrode are disposed. The buffer may be selected from KCl, LiCl and Deionized (DI) water. The buffer may be DI water, and the first sensing nanoelectrode may be configured to detect hybridization of the charged biopolymer molecules to the first biopolymer probes using a DNA hybridization mechanism.

In one or more embodiments, the nanopore device comprises a three-dimensional (3D) array. The nanopore device may be configured to detect point mutations in a biopolymer using an endonuclease-based SNP detection array. The nanopore device may be configured to use immobilized dCas9 protein and guide rna (grna) for target sequencing of DNA biopolymers. Each of the first and second gated nano-electrodes and the first sensing nano-electrode may comprise a respective band surrounding the nanochannel. The nanopore device can be integrated into a small ultra-sensitive sensor. The nanopore device may be integrated into a microfluidic device, a nanofluidic device, a nanodevice, or a lab-on-a-chip device. The nanopore device may be integrated into an integrated device for extracting and sensing targeted biopolymers. The targeted biopolymer may be selected from the group consisting of DNA, RNA, mRNA, miRNA, cDNA, peptide, protein immobilized antigen, and antibody.

In one or more embodiments, the nanopore device is configured to detect hybridization of a charged biopolymer molecule to a first biopolymer probe, the charged biopolymer molecule being at a minimum concentration (detection limit) of about 10 femtomoles. The nanopore device may be configured to detect hybridization of a charged biopolymer molecule to a first biopolymer probe without amplification of the first charged biopolymer molecule or use of PCR.

In one or more embodiments, the nanopore device is integrated into a liquid biopsy panel (panel) platform for detection without biomolecule amplification or the use of PCR. The nanopore device may be integrated into a label-free sensor platform. The nanopore device may be configured to detect hybridization of the charged biopolymer molecule to the first biopolymer probe based on a negative charge of the charged biopolymer molecule. The first biopolymer molecules may be coupled to the interior surface of the nanochannel-defining device using Plasma Enhanced Chemical Vapor Deposition (PECVD) or Molecular Layer Deposition (MLD). The nanopore device may be configured to detect hybridization of a charged biopolymer molecule to a first biopolymer probe based on a tunneling current, a lateral tunneling current, or a change in capacitance.

In another embodiment, a method for detecting a charged biopolymer molecule includes providing a nanopore device defining a nanochannel. The device includes a first gated nanoelectrode addressing a first end of the nanochannel, a second gated nanoelectrode addressing a second end of the nanochannel opposite the first end, a first sensing nanoelectrode addressing a first location in the nanochannel between the first end and the second end, and a first biopolymer probe coupled to an interior surface of the device defining the nanochannel. The method also includes first and second gated nanoelectrodes that generate a first electrical potential across the nanochannel that directs the flow of charged biopolymer molecules through the nanochannel.

In one or more embodiments, the method further comprises generating a first electrical potential across the nanochannel to direct the flow of the charged biopolymer molecule through the nanochannel from the first gated nanoelectrode to the second gated nanoelectrode. The method further includes generating a second electrical potential across the nanochannel to direct the flow of the charged biopolymer molecule through the nanochannel. The second potential may be opposite to the first potential. The method may further comprise generating a second electrical potential across the nanochannel to direct the charged biopolymer molecule to flow through the nanochannel from the second gated nanoelectrode to the first gated nanoelectrode. Further, the method may include alternately generating the first and second electrical potentials across the nanochannel to direct the charged biopolymer molecules to alternately flow between the first and second gated nanoelectrodes through the nanochannel.

In one or more embodiments, the nanodevice further includes a second sensing nanoelectrode addressing a second location in the nanochannel between the first end and the second end. The charged biopolymer molecules may be negatively charged.

In one or more embodiments, the first biopolymer probe has a first predetermined length. The device may also include a second biopolymer probe coupled to the inner surface of the device defining the nanochannel. In one or more embodiments, the nanopore device further comprises a buffer, wherein the first and second gated nanoelectrodes and the first sensing nanoelectrode are disposed. The buffer may be selected from KCl, LiCl and Deionized (DI) water. The buffer may be DI water and the first sensing nanoelectrode may detect hybridization of the first charged biopolymer molecule to the first biopolymer using a DNA hybridization mechanism.

In one or more embodiments, the nanopore device comprises a three-dimensional (3D) array. The method can further include a nanopore device that detects point mutations in the biopolymer using an endonuclease. The method may further include a nanopore device for target sequencing of biopolymers using immobilized dCas9 protein and guide rna (grna). The first and second gated nano-electrodes and the first sensing nano-electrode may each comprise a band that each surrounds a nanochannel.

In one or more embodiments, the method further comprises a nanopore device detecting hybridization of a first charged biopolymer molecule to a first biopolymer probe, the first charged biopolymer molecule having a minimum concentration of about 10 femtomoles (detection limit). The method can further include detecting a nanopore device of hybridization of the first charged biopolymer molecule to the first biopolymer probe without amplification of the first charged biopolymer molecule or using PCR. The method may further comprise integrating a nanopore device into a liquid biopsy panel platform for detection without biomolecule amplification or use of PCR. The nanopore device may be integrated into a label-free sensor platform. The method may further comprise detecting hybridized nanopore devices.

The above and other embodiments of the invention are described in the following detailed description.

Brief Description of Drawings

Aspects of the above and other embodiments are described in more detail, with reference to the accompanying drawings, wherein like elements in different figures are represented by common reference numerals, and wherein:

FIG. 1 schematically illustrates a solid state 2D nanopore device of the prior art;

fig. 2 to 4 schematically illustrate a 3D nanopore device according to various embodiments;

fig. 5 to 11 schematically depict methods of detecting charged biomolecules using a 3D nanopore device according to some embodiments.

Fig. 12A and 12B schematically depict a method for fabricating a nanopore device according to some embodiments.

Fig. 13 is a graph illustrating the relationship between target biomolecule concentration and current in a nanopore charged biopolymer detection device according to some embodiments.

Fig. 14 to 17B are graphs illustrating a relationship between time and current after addition of a target charged biopolymer in a nanopore charged biopolymer detection device according to various embodiments.

In order to better appreciate how the above-recited and other advantages and objects of various embodiments are obtained, a more detailed description of the embodiments is provided with reference to the accompanying drawings. It should be noted that the figures are not drawn to scale and elements of similar structure or function are represented by like reference numerals throughout. It is to be understood that these drawings depict only certain exemplary embodiments and are therefore not to be considered limiting of the scope of the embodiments.

Detailed description of illustrative embodiments

Described herein are methods of achieving label-free, amplification-free, and rapid (e.g., less than 10 minutes) detection of charged biopolymers. Described below is a nanopore electrically assisted charged biopolymer detection device that efficiently and effectively detects charged biopolymers by manipulating the electrical potential to increase hybridization of charged biomolecules and detecting the electrical characteristics resulting from the hybridization of charged biomolecules. Such detection devices and methods may be used in a variety of biomolecule arrays, including microarrays, CMOS arrays, and nanopore arrays (e.g., solid state and hybrid nanopore arrays). Such detection apparatus and methods may also be used with various multi-channel nanopore arrays, including the 3D multi-channel nanopore arrays and planar multi-channel nanopore arrays described above.

Multi-channel nanopore arrays that allow parallel processing of charged biomolecule detection can be used to achieve label-free, amplification-free, and rapid biomolecule detection. Examples of such multi-channel nanopore arrays are described in U.S. provisional patent application serial nos. 62/566,313 and 62/593,840, the contents of which have been previously incorporated by reference. Such multi-channel nanohole arrays can be electrically addressed to direct charged particles (e.g., biomolecules) into specific channels in these multi-channel nanoarrays. Other arrays are coupled to the microfluidic channels outside the array. Electrical addressing and sensing of individual nanopores within a multi-channel nanopore array, as described in U.S. provisional patent application serial No.62/612,534 (the contents of which have been previously incorporated by reference), can facilitate more efficient and effective use of multi-channel nanopore arrays for low cost, high throughput, label-free, amplification-free detection of charged particles (e.g., biomolecules).

Exemplary nanopore device

Fig. 2 schematically depicts a nanopore device 200 having a three-dimensional ("3D") array structure according to one embodiment. The apparatus 200 includes a plurality of 2D arrays or layers 202A-202D stacked along a Z-axis 204. Although the 2D arrays 202A-202D are referred to as "two-dimensional," the 2D arrays 2020A-202D each have a thickness along the Z-axis.

The top 2D array 202A includes first and second selective (inhibitory nanoelectrode) layers 206, 208 configured to direct movement of charged particles (e.g., biopolymers) through nanopores 210 (pillars, nanochannels) formed in the first and second selective layers 206, 208. The first selection layer 206 is configured to select from a plurality of rows (R1-R3) in the 2D array 202A. The second selection layer 208 is configured to select from a plurality of columns (C1-C3) in the 2D array 202A. In one embodiment, the first and second selection layers 206, 208 are selected from rows and columns, respectively, by changing the charge adjacent to selected rows and columns and/or adjacent to unselected rows and columns. Other 2D arrays 202B-202D include rate control/current sensing nanoelectrodes. The rate control/sensing nanoelectrodes can be made of highly conductive metals and polysilicon such as Au-Cr, TiN, TaN, Ta, Pt, Cr, graphene, Al-Cu, etc. The rate controlling/sensing nanoelectrodes may have a thickness of about 0.3 to about 1000 nm. Rate controlling/sensing nanoelectrodes can also be made in the bio-layer in hybrid nanopores. Each sensing nanoelectrode can be operatively coupled/addressed to a nanopore 210 column such that each nanopore 210 column can be operatively coupled to a particular memory cell. Electrical addressing in nanopore devices is described in U.S. provisional application serial No.62/612,534, the contents of which have been previously incorporated by reference.

Hybrid nanopores include a stable biological/biochemical component and a solid-state component to form a semi-synthetic porin to enhance the stability of the nanopore. For example, the biological component may be an α HL molecule. The α HL molecule can be inserted into a SiN-based 3D nanopore. The α HL molecules can be induced to adopt a structure to ensure alignment with the SiN-based 3D nanopores by biasing the nanoelectrodes (e.g., in the top 2D array 202A).

The nanopore device 200 has a 3D vertical pillar stack array structure that provides a larger surface area for charge detection than conventional nanopore devices having a planar structure. As charged particles (e.g., biopolymers) travel through each 2D array 202A-202E in the apparatus, their charge can be detected with a detector (e.g., a nanoelectrode) in some of the 2D arrays 202B-202E. Thus, the 3D array structure of device 200 facilitates higher sensitivity, which can compensate for low signal detectors/nanoelectrodes. The integration of memory cells into a 3D array structure minimizes any memory-related performance limitations (e.g., using external memory devices). In addition, the highly integrated small-sized 3D array structure provides a high-density nanopore array while minimizing the manufacturing cost.

In use, the nanopore device 200 is placed between and separates a top chamber and a bottom chamber (not shown), such that the top chamber and the bottom chamber are fluidically coupled through the nanopore column 210. The top and bottom compartments include nanoelectrodes (e.g., Ag/AgCl2, etc.) and a buffer (DI water or electrolyte solution containing KCl) containing the charged particles (e.g., DNA) to be detected. Different nanoelectrodes and electrolyte solutions can be used to detect different charged particles.

Charged particle electrophoretic translocation may be driven by applying a bias voltage to nanoelectrodes placed in the top chamber (not shown) adjacent the top 2D array 202A of the nanopore device 200 and the bottom chamber (not shown) adjacent the bottom 2D array 202E of the nanopore device 200. In some embodiments, the nanopore device is placed between a top chamber and a bottom chamber (not shown) such that the top chamber and the bottom chamber are fluidically and electrically coupled through a nanopore 210 in the nanopore device 200. The top and bottom compartments may contain an electrolyte solution.

Fig. 3 schematically depicts a nanopore device 300 according to one embodiment. Nanopore device 300 includes a layer of insulating membrane (Si3N4) followed by row and column select (inhibitory nanoelectrodes) 306 and 308 (e.g., metal or doped polysilicon) respectively, and a plurality of (1 st through nth) cell nanoelectrodes 310 (e.g., metal or doped polysilicon). The nanoelectrodes 306, 308, 310 of the nanopore device are covered by an insulating dielectric film 312 (e.g., Al2O3, HfO2, SiO2, ZnO).

When the translocation rate control bias signals 410 for the column and row voltages (e.g., Vd) are applied to the 3D nanopore sensing array 400, the row and column inhibit voltage/bias pulses are followed by verify (sense) voltage/bias pulses (e.g., Vg1, Vg2), as described below. Vg3 and the subsequent electrodes (Vg 4-VgN) are sensing and easy-to-position electrodes. An exemplary signal 410 is depicted in fig. 4, overlaid on top of the 3D nanopore sensor array 400. A suppression bias is applied to deselect each column and row nanopore pillar channel/nanochannel, respectively. During the sensing operation, the column and row (inhibit) select nanoelectrodes are selected. The resulting surface charge 412 may be detected as a change in an electrical characteristic, such as current.

In some embodiments, the nanoelectrodes can detect current modulation using various principles including ion blocking, tunneling, capacitive sensing, piezoelectric, and microwave sensing. It is also possible to amplify and accurately sense the so-called ion current change or ion concentration in the electrode (detected by the reference electrode) by means of a connected CMOS transistor, as shown in fig. 4.

Exemplary nanopore electrically assisted charged biopolymer detection apparatus and methods

Fig. 5 depicts a nanopore electrically assisted charged biopolymer detection device according to some embodiments. Although a portion of a nanopore detection device 500 including a single nanochannel 510 is depicted in fig. 5, a nanopore electrically assisted charged biopolymer (e.g., genetic polymorphism) detection device may include a 3D array having a plurality of nanochannels. Charged biopolymer sensing structures, such as nanopore detection device 500 depicted in fig. 5, take advantage of the charge sensitivity of nanochannels and the large surface area obtained from parallel processing and 3D arrays to facilitate rapid amplification-free, label-free, and label-free detection of charged biopolymers.

Nanopore detection device 500 includes nanoelectrodes 522, 524, 526, 528. These nanoelectrodes 522, 524, 526, 528 are independently electrically addressed to control flow through the nanochannel 510 (first and second gated nanoelectrodes 522, 524) and to detect charge in the nanochannel 510 (first and second sensing nanoelectrodes 526, 528).

The nanopore detection device 500 further comprises a neutral probe (PNA, DNA morpholino oligomer) 532 coupled to the inner surface 530 of the nanochannel 510. The inner surface 530 may include Al2O 3. Al2O3 includes a large number of hydroxyl groups to facilitate functionalization of the immobilization of neutral probes 532 on the inner surface 530 of nanochannel 510. Neutral probes 532 can be generated using known molecular biology techniques to complement target regions within a gene. The peer probes 532 can be of various lengths (e.g., 24 base pairs, 40 base pairs, etc.).

The neutral probes 532 can be coupled/covalently bonded to the inner surface using gas phase silylation. The thickness of the organic coating of the neutral probe 532 can also be adjusted by changing the time of the gas phase silanization.

In some embodiments, the nanopore device is first plasma treated with O2 to generate-OH groups on the Al2O3 substrate, thereby activating the substrate for attachment of target functional groups. Subsequently, 3-Aminopropyltriethoxysilane (APTES) was used for silanization because it was effective on a variety of possible surface structures and because it was very reactive. Prior to covalent attachment of neutral probes 532, nanopore device 510 is exposed to silane in the gas phase (e.g., a 1:3 ratio of APTES and OTMS in ethanol) at ambient temperature and a base pressure of about 30kPa by placing it in a low vacuum chamber under dynamic suction adjacent to a glass container containing 50 μ Ι of APTES (from Sigma-Aldrich). Subsequently, the nanopore device 510 was removed from the vacuum chamber and immersed in a 2.5% glutaraldehyde solution (Sigma-Aldrich) for one hour. Next, the nanopore device 510 is removed from the crosslinker and washed twice in IPI and twice in double distilled water. Finally, nanopore device 510 was treated with 100nM amino-modified neutral probe (e.g., by immersion) overnight at 37 ℃. After each step, the nanopore device was washed in Ultrapure DNase/RNase-free distilled water (used as wash buffer). Using this method, covalent attachment/immobilization of the neutral probe 532 can be accomplished within about 24 hours, or within eight hours at 45 ℃.

The hybridization sensitivity of the charged biomolecule 540 (e.g., a negatively charged nucleic acid) to the nanopore detection device 500 of neutral probes 532 covalently bound to the inner surface 530 of the nanochannel 510 is such that: single base mismatches can be detected based on the resulting charge difference. The parallel processing brought about by the 3D array structure of the nanopore device significantly increases the interfacial area between the nanopore device and the charged biomolecule to be detected, thereby increasing the sensitivity to a level sufficient for rapid in-situ diagnosis and determination of prognosis of various diseases (e.g., genetic diseases).

The first and second gated nanoelectrodes 522, 524 are independently addressed and thus can rapidly generate a "ping-pong" like movement of the charged biomolecule 540 by electrical modification, which increases hybridization of the charged biomolecule 540 and the neutral probe 532. The potential across the first and second gated nano-electrodes 522, 524 in the nanochannel 510 can be rapidly reversed by applying a current to the first and second gated nano-electrodes 522, 524. The first and second gated nanoelectrodes 522, 524 can also be addressed to control translocation of the charged biomolecule 540 through the nanochannel 510.

The target charged biomolecule 540 can be any number of nucleic acids, such as DNA, cDNA, mRNA, and the like. The neutral probes 532 can be complementary DNA strands, Locked Nucleic Acid (LNA) oligomers, neutral backbone oligo-like Peptide Nucleic Acids (PNA), DNA morpholino oligomers, or any type of complementary strand that can hybridize to the target charged biomolecule 540.

As shown in fig. 5, the charged biomolecule 540 is not adsorbed to the nanopore 510 prior to any current/potential applied to the nanopore detection device 500. Fig. 6 depicts the application of a current to create a positive potential in the first and second gated nano-electrodes 522, 524. This positive potential attracts negatively charged biomolecules 540 towards nanochannel 510.

Fig. 7 depicts the continuous application of current to create a positive potential in the first and second gated nano-electrodes 522, 524. Over time, some of the negatively charged biomolecules 540 enter the nanochannel 510 and interact with neutral probes 532 covalently bonded to the inner surface 530 of the nanochannel 510. This interaction between the negatively charged biomolecule 540 and the neutral probe 532 results in hybridization between the two molecules. This electrically connects the negatively charged biomolecule 540 to first and second sensing nanoelectrodes 526, 528, which can detect the negative charge 534 bound to the negatively charged biomolecule 540.

Fig. 5 depicts the change in electrical potential in the first and second gated nano-electrodes 522, 524. In fig. 5, current is no longer applied to the first gated nano-electrode 522, eliminating the positive potential therein. However, the current through the second gated nano-electrode 524 is maintained to maintain a positive potential therein. This change in potential directs the negatively charged biomolecule 540 in the nanochannel 510 to the second gated nanoelectrode 524 as indicated by flow arrow 550. Fig. 5 also shows that more negatively charged biomolecules 540 hybridize to neutral probes 532 in nanochannels 510.

Fig. 9 depicts another change in electrical potential in the first and second gated nano-electrodes 522, 524. In fig. 9, the current is no longer applied to the second gated nano-electrode 524, eliminating the positive potential therein. However, a current is applied through the first gated nano-electrode 522 to maintain a positive potential therein. This change in potential pulls the negatively charged biomolecule 540 in the nanochannel 510 back to the first gated nanoelectrode 522 as indicated by flow arrow 552. Fig. 9 also shows that as more charged biomolecules 540 are exposed to neutral probes 532 in nanochannels 510, even more negatively charged biomolecules 540 hybridize to central probes 532.

Fig. 10 depicts yet another change in electrical potential in the first and second gated nano-electrodes 522, 524. In fig. 9, current is no longer applied to the first gated nano-electrode 522, eliminating the positive potential therein. However, a current is applied through the second gated nano-electrode 524 to maintain a positive potential therein. This change in potential pulls negatively charged biomolecules 540 in nanochannel 510 back to second gated nanoelectrode 524 as indicated by flow arrow 550. Fig. 10 also shows that as even more charged biomolecules 540 are exposed to neutral probes 532 in nanochannels 510, still more negatively charged biomolecules 540 hybridize to central probes 532.

The directional changes depicted in flow arrows 550, 552 in fig. 5-10 depict the first two directional changes in the "ping-pong" movement of the charged biomolecule 540, which increases hybridization of the charged biomolecule 540 and the neutral probe 532. The direction change is controlled by changing the electrical potential in the first and second gated nano-electrodes 522, 524, which are modified by the alternating current applied thereto. Because under processor control, current can be applied to the first and second gated nanoelectrodes 522, 524, which are independently electrically addressed, alternation of current and potential can be rapidly implemented. The "ping-pong" movement of the charged biomolecule 540 increases the amount of time the charged biomolecule 540 is exposed to the neutral probe 532 in the nanochannel 510, thereby increasing the amount of hybridization between the two molecules. Only two changes of orientation are depicted in fig. 5 to 10, but the biomolecule detection method may comprise many more changes of orientation to improve the hybridization of the target charged biomolecule 540.

FIG. 11 depicts the end of a series of "ping-pong" movements in a biomolecule detection method. At the end of the detection method, a plurality of negatively charged biomolecules 540 have hybridized to the neutral probes 532, which are themselves covalently bonded to the inner surface 530 of the nanochannel 510. As each negatively charged biomolecule 540 hybridizes to the central probe 532, its additional negative charge 534 is detected by the first and/or second sensing nanoelectrodes 526, 528. Sensing nanoelectrodes 526, 528 are sensitive enough to distinguish single base pair mismatches. Thus, sensing nanoelectrodes 524, 528 can detect negative charges 534 associated with hybridization of each charged biomolecule 540. Thus, the nanopore detection device 500 can rapidly (e.g., within 10 minutes) detect and quantify a target charged biomolecule in a solution without the need to label or label the probe. This method also allows detection of target charged biomolecules with GC-rich regions that are difficult to sequence in conventional methods.

Although the nanopore detection device depicted in fig. 5-11 is configured to detect only a single negatively charged target biomolecule 540 in a particular procedure, nanopore detection devices according to other embodiments may be configured to detect multiple negatively charged target biomolecules. Such nanopore detection devices include a plurality of neutral probes that (1) hybridize to different negatively charged target biomolecules and (2) have different lengths. Because the neutral probes have different lengths, hybridization of target biomolecules of different negative charges will result in different amounts of negative charge being added to the inner surface of the nanochannel by the electricity. The sensing nanoelectrodes are sensitive enough to distinguish these different amounts of negative charge and thus to distinguish hybridization of different negatively charged target biomolecules.

Exemplary nanopore device fabrication methods

Fig. 12A and 12B schematically depict a method 1210 for fabricating a nanopore device (such as nanopore detection device 500, 600 described above) according to some embodiments.

At step 1212, the inner surface of the nanopore device (in the nanochannel) is subjected to O₂Plasma treatment, cleaning and activation. The surface is functionalized by treating it with (3-aminopropyl) triethoxysilane (APTES) to silanize the surface at step 1214. At step 1216, an aldehyde linker is attached to the functionalized surface. In step 1218 (fig. 12B), Probes (PNAs) are attached to the surface through the aldehyde. At step 1220, negatively charged target biomolecules (e.g., DNA) are attached to the probes on the surface and the charge of the surface is altered for electrically detecting the negatively charged target biomolecules as described above.

Exemplary DNA sensing embodiments

The following description relates to embodiments of sensing DNA as a charged biopolymer/target molecule.

The first embodiment relates to sensing artificial DNA as a target molecule. A second embodiment relates to sensing lambda phage DNA as a target molecule. These embodiments demonstrate the ability of nanopore sensing/detection systems to sense/detect genomic DNA as well as synthetic DNA.

Fig. 13 depicts a graph illustrating target biomolecule concentration ("molarity") and current (I) in a nanopore electrically assisted charged biopolymer detection device, such as devices 500, 600 described above, according to some embodiments_d) A graph of the relationship between. The relationship is approximately inverse log (inverse logarithm), at about 10^-9molar and-1000 pA have a single inflection point. Thus, this relationship can be used to quantify targets in solution based on the detected currentThe concentration of the biomolecule.

In some embodiments, the lowest detection limit is femtomole (10)^-15) A range of target biomolecules. Fig. 14-17B depict various relationships between a concentration (10 femtomoles) of target biomolecule (DNA) and time (sample) before and after current (nA) addition in a device according to some embodiments (such as devices 500, 600 described above).

In the embodiments depicted in fig. 14-17B, the sensitivity is measured to determine the detection limit based on the output signal from the nanopore detection device. Amino-modified PNA probes (quantified by hplc (agilent technology) and MALDI-ms (shimadzu biotech)) with 99.9% purity, NH2 ends as ligation sites and capped C-ends were used in a nanopore detection device. The C-terminus of the PNA probes was capped with a CONH2 group to prevent undesirable binding between the probes. An O-junction is used to prevent interference between the probe and the surface. According to some embodiments, two different types of probes are utilized: one for detecting synthetic DNA and the other for detecting lambda DNA (genomic DNA of lambda phage). The detection probe for the 17bp synthetic DNA (COSMO GENECECH Korea) was:

NH2-O-GGGGCAGTGCCTCACAA-CONH2, having a target sequence of 5 'TTGTGAGGCACTGCCCC 3'.

The 20bp lambda DNA detection probe (Thermo Fisher Scientific) was:

NH2-O-CGTAACCTGTCGGATCACCG-CONH2 has a complementary target sequence in lambda DNA.

As shown in the graphs in fig. 14 to 17B, the lowest detection limit is about 10 femtomoles.

Based on the findings of Maniatis in 1982, the 17bp length of the probe was one bp longer than the 16bp minimum probe length, which was calculated based on the number of positions on the genome to which the oligonucleotide probe would hybridize with a certain complexity using the following formula,

P0＝[1/4]L×2C

where P0 is the number of independent perfect matches, L is the length of the oligonucleotide probe, and C is the complexity of the target genome (this value multiplied by 2 represents the two complementary strands of DNA, either of which may hybridize to the probe). Based on this formula, an oligonucleotide with a length of 16bp is expected to bind to the human genome at one position. Thus, such probes may be used to detect specific sites associated with a particular type of cancer or infectious disease. Thus, a 17bp probe is the smallest size of probe used in a detection system for sequencing a target of interest in a human genome or a liquid biopsy platform.

FIG. 14 illustrates that the addition of the above-described target DNA at a concentration of about 10 femtomolar increases the current in the sensing nanoelectrode from about 10.58nA to about 11.35 nA. FIG. 15 illustrates that the addition of approximately 1 picomolar concentration of the target DNA, which is 100-fold higher than 10 femtomoles. Thus, a detectable current increase of 0.77nA in the sensing nanoelectrode obtained from the addition of approximately 10 femtomoles of target DNA represents the lowest detection limit.

Fig. 16A to 16C illustrate that in the top-gated nanoelectrode, the current increased from about 1.72nA to about 2nA 20 minutes after addition of DNA, and to about 3nA 30 minutes after addition of DNA. Fig. 17A to 17B illustrate that in the bottom-gated nanoelectrodes, the current increased from about 1nA to about 1.6nA after DNA addition. The current in all of these nanoelectrodes in the nanopore detection system increases to a detectable level with the addition of target DNA.

The nanopore detection system described herein is a 3D sensor working with DI water as a buffer. The function and exact mechanism of action of water molecules within small spaces on a nanometer scale have not been previously studied and understood, but the high sensitivity and clear resolution of the 3D arrays described herein can demonstrate the benefit of using DI water instead of electrolytes or other buffers, which increases the noise level within such sensitive sensors.

The mechanism of reaction and signal generation in the nanopore detection system described herein is based on changes in the charge distribution in the surface due to hydration of the DNA molecule attached to the central probe described above. This hydration results in a change in the electrode, the charge density on the gate electrode being redistributed. The nanoelectrodes inside the nanopore have an all-around (all-around) or stripe-like morphology around the nanopore, which improves the sensitivity of the nanopore sensor.

By using a different potential gradient at each nanopore, the user can control the speed of travel of the charged biomolecule internally and through each nanopore. The use of low concentrations of buffer/electrolyte or DI water to increase the debye length of the sensing area in a nanopore is one of the unique characteristics of the 3D nanopore detection system described herein. The user electrophoretically controls the movement of the charged target biopolymer described above and the same ping-pong-like motion between the nanoelectrodes by varying the amount and duration of the electrical potential of each nanoelectrode, thereby having broad control over the nanopore detection system. As described above, the time required for the charged target biopolymer to attach to the neutral probe will be reduced to less than 10 minutes as the charged target biopolymer moves back and forth between the nanoelectrodes with changing/alternating nanoelectrode potentials. This reduction in ligation time is due to the enhanced interaction between the target and probe such that they can bond to each other in a shorter time.

In some embodiments of nanopore detection systems, the size, shape, and depth of the nanopore structure can be varied based on the size of the neutral probe, such as those described herein. E.g. having a wavelength of 50nmThe pore size of the diameter can be used to sense the target biopolymer using a 40bp neutral probe. In other embodiments, a pore size with a diameter of 100nm can be used to sense target biopolymers using more than 100bp neutral probes. In still other embodiments, a pore size with a diameter of 200nm can be used to sense a target biopolymer using a neutral probe that is still longer.

The 3D nanopore array sensor described herein is more sensitive and more compact than a 2D or planar structure sensor because the 3D array of nanopores increases the surface to volume ratio, which makes it possible to minimize the intelligent (smart) surface area inside the nanochannels of the nanopore array. The high surface to volume ratio allows sensing of very low concentrations (e.g., 10 femtomoles) of charged biopolymers.

The 3D nanopore array sensor described herein provides better control than charge perturbation or electrochemical based sensor systems because the dielectric layer insulates the inner surface of each nanochannel, thereby enhancing control of the capacitive and electric field effects of each nanochannel.

The 3D nanopore array sensor described herein may use a change in capacitance to sense charged biomolecules (e.g., DNA) using immobilized probes. As the target DNA molecule passes within the nanopore of the array structure (electrophoresis driven by an external voltage), the top and bottom electrodes register the potential change created by the passage of DNA within the nanopore structure, polarizing the nanopore like a capacitor. The resulting change in capacitance can be measured electrically to detect the passage of the target DNA molecule. The speed of the DNA molecules can be controlled by controlling the applied positive gating bias, so that the 3D nanopore array sensor can be used in single nucleotide sequencing. The 3D nanopore array sensor described herein can detect the passage of charged biomolecules by detecting both tunneling current and capacitance changes. Previously available biological nanopores were unable to detect tunneling currents and capacitance changes because there were no embedded nanoelectrodes in their structure.

The neutral probes used in the 3D nanopore array sensors described herein can be modified to change their surface chemistry, allowing for more system control and design options. For example, thiol group modification can be used for gold thiol binding. Using structural and chemical improvements of the immobilization technique, avidin/biotin and EDC crosslinker/N-hydroxysuccinimide (NHS) are other probe modification and target pairs that may be used with the 3D nanopore array sensors described herein.

The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below are intended to include any structure, material, act, and equivalent for performing the function in combination with other claimed elements as specifically claimed. It is to be understood that while the invention has been described in conjunction with the above-described embodiments, the foregoing description and claims are not intended to limit the scope of the invention. Other advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Various exemplary embodiments of the present invention are described herein. Reference is made to these examples in a non-limiting sense. They are provided to illustrate more broadly applicable aspects of the present invention. Various changes may be made to the described aspects and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process operation or steps, to the objective, spirit or scope of the present invention. In addition, those skilled in the art will recognize that the various modifications described and illustrated herein have discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. All such modifications are intended to be within the scope of the claims associated with this disclosure.

Any of the devices described for performing the present diagnostic or interventional procedures may be provided in a packaged combination for performing these interventional procedures. These supply "kits" may further include instructions for use and packaged in sterile trays or containers as are commonly used for these purposes.

The invention includes methods that can be practiced using the present apparatus. The method may comprise the operation of providing such suitable apparatus. Such provisioning may be performed by the end user. In other words, the "provide" operation only requires the end user to obtain, obtain (access), approach (approach), position (position), set-up (set-up), activate, power-up (power-up), or otherwise operate to provide the necessary equipment in the present method. The methods recited herein can be performed in any order of the listed events that is logically possible, as well as in the listed order of events.

Exemplary aspects of the invention, as well as details regarding material selection and fabrication, have been set forth above. Other details of the invention can be understood in connection with the above-referenced patents and publications and as generally known or understood by those skilled in the art. This applies equally to the method-based aspects of the invention in terms of additional operations as commonly or logically used.

Furthermore, while the invention has been described with reference to several examples that optionally include various features, the invention is not limited to those described or indicated with respect to the various variations of the disclosure. Various changes may be made to the invention described and equivalents may be substituted (whether listed herein or not included for the sake of brevity) without departing from the true spirit and scope of the invention. Further, if a range of values is provided, it is understood that each intervening value, to the extent that there is no such stated, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention.

It is also contemplated that any optional feature of the described inventive variations may be set forth and claimed independently or in combination with any one or more of the features described herein. Reference to a singular item includes the possibility that there are plural of the same items present. More specifically, as used herein and in the claims related thereto, the singular forms "a," "an," "the," and "the" include plural referents unless the context clearly dictates otherwise. In other words, use of the article allows for "at least one" object item (item) in the above description and in the claims related to the present disclosure. It is also to be understood that such claims may be drafted without including any optional elements. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

The term "comprising" in the claims relating to the present disclosure should be allowed to include any additional elements, whether or not a given number of elements are listed in these claims, or the addition of a feature may be considered to transform the nature of the elements as set forth in these claims, if such exclusive term is not used. Unless expressly defined herein, all technical and scientific terms used herein are to be given the broadest possible commonly understood meaning, while maintaining the validity of the claims.

The breadth of protection of the present invention is not limited by the examples provided and/or the present description, but is only limited by the scope of the claims associated with the present disclosure.