HK1171783B - Method and apparatus for the analysis and identification of molecules - Google Patents

Abstract

An apparatus and method for performing analysis and identification of molecules have been presented. In one embodiment, a portable molecule analyzer includes a sample input/output connection to receive a sample, a nanopore-based sequencing chip to perform analysis on the sample substantially in real-time, and an output interface to output result of the analysis.

Description

Method and apparatus for analyzing and identifying molecules

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application No.61/177,553 filed on 12.5.2009, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates generally to analyzing and identifying molecules, and more particularly to providing a real-time, portable nanopore (nanopore) -based molecular analysis device.

Background

Nucleic acids, deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) are present and have unique sequences in every living organism. It naturally treats itself as a definitive identification of various biological subjects (agents). Therefore, analysis of nucleic acids, DNA and/or RNA (which is herein referred to broadly as genetic analysis) is very useful in the study of living organisms. However, currently commercially available nucleic acid sequencing technologies, such as microarrays, pyrosequencing, sequencing by integration, and sequencing by ligation, are very limited in various respects. For example, some or all of these techniques are unable to perform real-time analysis, require long sample nucleic acid amplification processes and protocols (such as polymerase chain reaction), have long turnaround times (typically taking days to weeks to analyze small pieces of sample nucleic acid), have high operating costs (some of which use expensive chemical reagents), have high false-positive (false-positive) error rates, and are non-portable.

Due to the above limitations of current nucleic acid sequencing technologies, people working in this field (such as medical workers, security personnel, scientists, etc.) cannot perform on-site genetic analysis locally. Instead, the field worker has to collect and transport the sample to a specialized laboratory for several days or even weeks of analysis to identify the nucleic acids present in the sample. This long and tedious process makes it difficult to meet the needs of today's genetic analysis, especially during epidemic outbreaks (such as foot and mouth disease epidemic in the uk, Severe Acute Respiratory Syndrome (SARS) in asia, and recently H1N1 influenza (also commonly referred to as swine flu) in mexico and the united states by using current nucleic acid sequencing technologies, it is difficult for the authorities to make rapid and informed decisions (if not impossible) that would have a tremendous safety and economic impact on society.

To address the shortcomings of current nucleic acid sequencing technologies, scientists have developed various nanopore-based sequencing technologies. Recently, professor hagan bayey at university of foe and his colleagues demonstrated in the bio-nanopore assay that long reads (longread) with an accuracy of 99.8% could be achieved by using α -hemolysin. Based on established detection rates, an array of 256 × 256 nanopores is generally sufficient for analyzing the entire human genome in about 30 minutes. This would be a success of turning significance if one could successfully implement a biological nanopore array. However, one drawback of biological nanopores is the relatively short lifetime of the proteins and enzymes used in forming the biological nanopores, which is typically several hours to several days.

Solid-state nanopores are a more robust alternative to biological nanopores because no biological agents are involved in constructing solid-state nanopores. However, conventional photolithographic techniques employed in the semiconductor industry cannot define the 2nm feature size required for solid-state nanopore-based sequencing technologies. To date, different fabrication techniques (e.g., electron/ion milling) have been used to sequentially cut nanopores at a time. But these techniques cannot be scaled (sacle) to produce 256 x 256 arrays at a less expensive cost and reasonable production time.

Drawings

The present invention is illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which:

FIG. 1 illustrates one embodiment of a nanopore based sequencer (sequencer) and related nanopore based sequencing biochip;

FIG. 2A illustrates one embodiment of a translocation process of a molecule during detection and analysis of nanopore-based nucleic acid sequencing;

FIG. 2B shows an exemplary electrical readout of a nucleic acid sequence compared to a background signal of a void;

FIG. 3 illustrates a side view and a top view of one embodiment of an singlet lens used in a nanoscaler for both etching and deposition;

FIGS. 4A-4E illustrate one embodiment of a subtractive process (subtractive method) for fabricating a nanopore and/or nanopore array;

FIGS. 5A-5I illustrate one embodiment of an additive method (additive method) for fabricating a nanopore and/or nanopore array;

FIG. 6 illustrates one embodiment of a nanoring and one embodiment of a nanogap;

FIG. 7 illustrates one embodiment of a bonded nanopore array wafer and integrated circuit wafer for forming a bottom cavity of a measurement chamber;

FIG. 8 illustrates one embodiment of a bonded top wafer and composite wafer for forming a top cavity of a measurement chamber;

FIG. 9 illustrates one embodiment of a voltage biasing scheme and current sensing circuit capable of operating with a nanopore-based sequencer;

FIG. 10A shows one embodiment of a nanopore based sequencer;

FIG. 10B illustrates one embodiment of a multi-measurement chamber;

FIG. 11 shows a cross-sectional view of one embodiment of a nanopore based sequencer along a selected path from a sample inlet, along a microfluidic channel and a nanofluidic channel, through a measurement chamber, and to a sample outlet;

FIG. 12 shows one embodiment of a three-layer biochip structure with embedded electrodes;

FIG. 13A illustrates one embodiment of a biasing and sensing scheme for nanopore detection;

FIG. 13B illustrates one embodiment of a planar electrode implementation;

FIG. 13C illustrates a top view of one embodiment of a sensing electrode and a nanobelt in a planar electrode implementation;

FIG. 14 shows a high-level hardware architecture of one embodiment of a nanopore based sequencer; and

FIG. 15 illustrates a high-level structure of software and related hardware components for an operating system and genetic analysis software in one embodiment of a nanopore-based sequencer.

Detailed Description

In the following description, numerous specific details are set forth, such as examples of specific components, devices, methods, etc., in order to provide a thorough understanding of embodiments of the present invention. It will be apparent, however, to one skilled in the art that these specific details need not be employed to practice embodiments of the present invention. In other instances, well-known materials or methods have not been described in detail in order to avoid unnecessarily obscuring the embodiments of the invention.

Various embodiments of devices and methods for performing analysis and identification of molecules (such as nucleic acids, amino acids, peptides, proteins, and polymers) and nano-sized particles (such as carbon nanotubes, silicon nanorods, and coated/uncoated gold nanoparticles) are described below. Note that the following discussion focuses on one example of molecular analysis (i.e., genetic analysis) in order to illustrate the idea. One skilled in the art will readily recognize that the techniques disclosed herein can be applied to analysis and identification of molecules in general. In one embodiment, the nanopore based sequencer is a portable gene analysis system. FIG. 1 illustrates one embodiment of a portable nanopore based sequencer 110 and an associated nanopore based sequencing biochip 120. During detection and analysis, molecules in a sample to be tested are electrophoretically driven through a nanoscale pore (also referred to as a nanopore) in solution, as shown in fig. 2A. In some embodiments, the nanopore is about 2nm in size. Note that the size of the nanopores may vary in different embodiments. For example, the nanopores in some embodiments have fixed, identical dimensions. In some alternative embodiments, the nanopores have different sizes. In addition, the shape of the nanopore may also vary, such as circular, elliptical, elongated slit, etc., in the same or different embodiments. Depending on the relative size and speed of movement of the molecules in the space defined by the nanopore, various electrical characteristics (such as current pulses of different amplitudes and durations as represented by fig. 2B) can be observed and used to identify the molecules. As a result, direct reading of the nucleic acid sequence can be achieved without destroying the molecule to be detected. In other words, the measurement of the nucleic acid sequence can be performed while maintaining the integrity of the nucleic acid sequence.

Without fundamental limitation, nanopore arrays, which in some embodiments include arrays with pores greater than 2nm, may be mass produced using a number of fabrication techniques. Details of some exemplary fabrication techniques are described in detail below to illustrate the idea. One skilled in the art will appreciate that other compatible fabrication techniques or variations of these techniques may also be employed to produce the nanopore array. By incorporating a network of microfluidic/nanofluidic channels, nanopore-based sequencers are able to accurately decipher genomes at unprecedented speeds and without human intervention.

In addition to small form factors (form-factors) and speed in gene analysis, some embodiments of nanopore-based sequencers provide other advantages as described below. One of the advantages is that it can readily provide further evidence of any variation in a biological subject. This is possible because nanopore-based sequencing technology is a direct-read technology, the result of which does not require prior information of the genome to be tested. In addition, some embodiments of nanopore-based sequencers can operate in extreme and unknown environments, as sterility and cleanliness of the nanopore can always be guaranteed, as the nanopore is always enclosed within the nanopore-based sequencing biochip and not exposed to any undesirable foreign substances during the entire analysis process.

Some embodiments of nanopore-based sequencers, as handheld portable devices, can accelerate many different industrial and scientific advances. For example, in commerce and research and development, some embodiments of nanopore-based sequencers are useful in basic research, pharmacogenomics, bacterial diagnostics, pathogen detection, agriculture, food industry, biofuels, and the like. As a further example, some embodiments of nanopore-based sequencers are useful in rapid DNA forensics, port of entry bio-screening, and the like.

Nanopore array for nanopore-based sequencing

In the 70's of the 20 th century, the resistive pulse technology based on coulter counter, debois and his colleagues succeeded in demonstrating the use of a single sub-micron diameter hole in characterizing particles by particle size and electrophoretic mobility. Later, Deamer proposed the idea of using nano-sized pores for gene sequencing. He and his colleagues demonstrated that single stranded DNA (ssDNA) and RNA molecules can be driven through pore-forming proteins and detected by their effect on the ionic current through the nanopore. Given the recently demonstrated high sequencing speeds, the progress of nanopore-based sequencing has been largely limited by the lack of an inexpensive and parallel-written manufacturing process to create large arrays of nanopores for rapid gene analysis. Many conventional lithographic methods (e.g., electron milling, ion milling, and silicon etch back) are not a viable way to fabricate the nanopore arrays needed for real-time genetic analysis. Until recently, Donnelly and his colleagues at houston university developed some embodiments of a nanoscale pantograph that could produce a 2nm array of nanopores in large quantities without much restriction. From their simulation results, the nanoscale pantograph can define holes or dots having a size as small as 1 nm. By incorporating microfluidic/nanofluidic technology, nanoscale zoom devices open the possibility of implementing real-time or near real-time gene analysis systems.

In a nano-scale, a broad, collimated, monoenergetic ion beam is directed at an array of sub-micron diameter electrostatic lenses (also referred to as einzels, as shown in fig. 3) fabricated on a conductive substrate (e.g., a silicon (Si) doped wafer). By applying appropriate voltages to the lens electrodes, the "beamlet" entering the lens is focused to a point that can be 100 times smaller than the diameter of the lens. This means that 1nm features can be defined by a 100nm lens, which can be processed by the photolithographic techniques used in current semiconductor processing. Furthermore, each lens writes its own nanofeatures on the substrate, so the nanoscale pantograph is a parallel writing process that is very different from sequential writing of focused electron or ion beams. Since the lens array is part of the substrate, the method is substantially (substitially) immune to misalignment caused by vibration or thermal expansion. The size of the nanofeatures is controlled by the size of the predefined lenses, which may be the same or different in diameter, i.e., the same or different arrays of nanofeatures can be processed substantially simultaneously. In the presence of Cl₂With an Ar + beam in the case of gas, etched Si pores with a diameter of 10nm and a depth of 100nm were obtained. Etching may occur only in the holes where a voltage is applied to the upper metal layer so that the ion beamlets are focused. The remaining holes will not have a voltage applied to the upper metal layer, the beamlets will not be focused, and the current density will be too small to cause any significant etching.

By using a nanoscale pantograph, there are two methods, the subtractive method and the additive method, to make nanopores for genetic analysis. One embodiment of the direct etching method will be discussed first, followed by one embodiment of the indirect etching method.

A. One embodiment of a subtractive process for making nanopores and/or nanopore arrays

Figures 4A-4E illustrate one embodiment of a subtractive process for fabricating a nanopore and/or nanopore array. Referring to fig. 4A, nitride 420 is deposited onto a Si (silicon) (100) wafer 410. In fig. 4B, a bottom conductive material 422 (doped silicon or metal) is deposited, followed by dielectric separators 424 and an upper metal layer 426. Thereafter, a plurality of einzel lenses are defined. Referring to fig. 4C, a nano-scale etch is performed on conductive material 422 to define a nanopore 430, which nanopore 430 is used as a hard mask for the nanopore etch in nitride layer 420. In fig. 4D, the singlet lens is removed. Oxide 435 is then coated onto the nanopores 430 for protection. Finally, in fig. 4E, the bottom cavity 440 of the measurement chamber is formed by Chemical Mechanical Polishing (CMP), photolithography and hydroxide (KOH) etching of the backside of the silicon substrate 410. Oxide layer 435 is then removed to expose nanopore 430.

B. One embodiment of an additive process for fabricating nanopores and/or nanopore arrays

Figures 5A-5I illustrate one embodiment of an additive process for fabricating nanopores and/or nanopore arrays. Referring to fig. 5A, nitride 520 is deposited onto a Si (100) wafer 510. In fig. 5B, a bottom conductive material 522 (doped silicon or metal), dielectric separators 524, and an upper metal layer 526 are deposited. Thereafter, a single lens is defined. In fig. 5C, a nano-seed 530 for nanorod or nanotube growth is deposited. In fig. 5D, nanorods or nanotubes 535 are formed. In fig. 5E, an oxide 540 is deposited. In fig. 5F, the nanorods or nanotubes 535 are removed. The remaining oxide nanopores 550 are used as a hard mask for the conductive or nitride layer. In fig. 5G, the pattern is transferred from oxide layer 540 onto conductive layer 522, followed by transfer to nitride layer 520. In fig. 5H, the einzel lens is removed, followed by removal of the oxide layer 540. The nanopores are coated with an oxide for protection 552. Finally, in FIG. 5I, the bottom cavity 560 of the measurement chamber is formed by CMP, photolithography and KOH etch on the backside of the silicon substrate 510. Oxide layer 552 is then removed to expose nanopores 565.

Figure 6 illustrates one embodiment of a nanoaperture and one embodiment of a nanoring used in some embodiments of the invention. Elongated nano-sized nanoaperture 610 and nanoring 620 may be defined by some embodiments of subtractive method with a single lens (which have been discussed above) in which the openings are rectangular and semi-circular, respectively, as shown in fig. 6. Based on the disclosure made herein, one of ordinary skill in the art will recognize that any two-dimensional shape can be defined by using similar patterning techniques. Since the wafer stage is substantially stationary throughout the process in some embodiments, this non-circular patterning approach solves at least three major technical problems faced by conventional approaches to tilting wafer stages. First, there is no need to precisely control the tilt angle and speed of the wafer stage. Second, it generally overcomes the line broadening effect and line width non-uniformity introduced by tilting the wafer relative to the incident beam of ions. Third, it allows different shapes and sizes of patterns to be defined at about the same time.

Nanopore-based sequencing biochip

After formation of the nanopore array by subtractive or additive methods, a nanopore array wafer 750 may be bonded to a wafer with pre-fabricated integrated circuits 720 and microfluidic channels 730, as shown in fig. 7. This completes the formation of the bottom cavity of the measurement chamber. If desired, the nucleic acid sample can be extracted from the biochip through microfluidic channels on the bottom wafer.

Similarly, in some embodiments, the top cavity of the measurement chamber is formed by bonding a top wafer 810 having integrated circuits 820 and/or fluidic channels 830 to a composite wafer comprising a bonded nanopore wafer 840 and a bottom wafer 850. The tri-layer wafer structure 800 is shown in fig. 8. One embodiment of a voltage biasing scheme 910 and associated current sensing circuit 920 is shown in fig. 9. The three-layer composite wafer 800 is then mounted onto a support frame for wire bonding or bell grid (bellgrid) wire bonding using appropriate fluid I/O connections, such as those with minimal dead volume. Typical packaging techniques (e.g., epoxy packaging and ceramic packaging) can be used to enclose the entire assembly to form a nanopore-based sequencing biochip. Alternatively, integrated circuits (such as those associated with sensing and biasing) may be fabricated on a nanopore wafer 840, which nanopore wafer 840 may be bonded to a blank substrate rather than another wafer with integrated circuits thereon.

Additionally, in some embodiments, there are two or more features embedded on the top wafer 810, namely a sample-directing electrode 1015 along the microfluidic channel 1010 and a nanofluidic channel 1013 to a measurement chamber 1030, as shown in fig. 10A and 11. To further illustrate sample flow through a nanopore-based sequencing biochip, one example will be discussed in detail below.

The buffer inlet 1025 and buffer outlet 1027 are used to pre-wet and pre-fill the network of microfluidic channels 1010, nanofluidic channels 1013, and measurement chambers 1030 prior to loading with a sample for detection. During detection, fluid flow in the microfluidic channel can be regulated by using on-chip or off-chip micropumps or microvalves to regulate flow rates through the buffer inlets and outlets.

In one example, the phosphate-deoxyribose backbone (backbone) of a single-stranded nucleic acid molecule is charged with a negative charge for each base fragment, and there are two negative charges at the 5' end of the molecule. Pulsing a positive voltage along sample-directing electrode chain 1015 from receiving vessel 1020 through sample inlet connector 1023 to target measurement chamber 1030 can extract nucleic acid molecules from receiving vessel 1020 and deliver the molecules to pre-dispensed measurement chamber 1030. Similarly, sample-conducting electrodes 1017 are also embedded on the bottom wafer along the microfluidic channel 1010 for extracting samples from the nanopore-based sequencing biochip in a similar manner.

By using a similar scheme of sample-directing electrodes along the fluidic channel network, one can extend the number of measurement chambers 1040 to more than one. An example of measurement chambers arranged in a tree structure is shown in fig. 10B. In addition to the ability to perform multiple independent analyses, the order in which DNA fragments are extracted from the sample receiving container 1020 is pre-assigned to each measurement chamber in some embodiments. As shown in fig. 10B, the measurement chamber 1040 is labeled with 2 decimal numbers. The first decimal number indicates the branch number and the second decimal number indicates the position of the measurement chamber in the branch. The dispensing order of the measurement chambers 1040 may simply be in ascending order, i.e., using the chamber with the lowest number first followed by the chamber with the next higher number. In this way all measurement chambers in a branch can be used before moving to the next branch. Alternatively, in the present example, the sample may be assigned to the chamber with the lowest number in each branch, where the lowest branch number is used first, i.e. 11, 21, 31, 41, followed by 12, 22, 32, 42, etc. According to the distribution method, the measurement chamber of each branch having the furthest distance from the central microfluidic channel may be distributed first, followed by the next measurement chamber of each branch. The method may reduce interference of the electrical signal of the sample-guiding electrode in the central microfluidic channel with the measured signal. When the entire measurement is complete, the sequence of the entire DNA sample can be systematically assembled according to the order of extraction of the fragments. This can eliminate the time consuming post-detection analysis required by other traditional sequencing techniques, such as microarrays, where the hybridization process randomizes the original sequence of the sample and requires computational intensive and error-prone post-detection analysis to patch the correct sequence. In addition, since the order of extraction of the fragments is recorded, the fragments can be recombined to form the original DNA sample. With other conventional sequencing techniques, the original sample is typically corrupted and cannot be recovered for subsequent use.

Referring back to fig. 10A, nanofluidic channel 1013 formed by wafer bonding of a top wafer and a composite wafer may be used as a filter, a sample flow rate controller, and a molecular stretcher (stretcher). In some embodiments, the nanofluidic channel acts as a filter by turning on and off the top electrode on the top wafer, one can selectively pull (pull) into the sample from the microfluidic channel. In some embodiments, the nanofluidic channel serves as a sample flow rate controller by adjusting the voltage of the top and bottom electrodes, one can control the flow rate of the sample through the nanofluidic channel. The nanofluidic channel 1013 may also serve as a molecular stretcher because the single-stranded nucleic acid molecule 1101 is stretched from a natural bent state when passing through the nanofluidic channel 1013, as shown in fig. 11.

In some embodiments, the velocity control properties of nanofluidic channels are exploited to allow more accurate analysis of molecules. As shown in fig. 12, sensing electrode 1205 is embedded in nanopore 1225, which becomes the main part of the microchannel/nanochannel network in controlling the flow of molecules through nanopore 1225. In addition to nanofluidic channels, the applied DC voltage is also designed to fine tune the speed and direction of the molecules as they change position through nanopore 1225. By alternating the magnitude of the DC bias voltage applied to the top 1230 and bottom 1235 drive electrodes, respectively, and the embedded sense electrode 1205, the molecule under test can be dragged back and forth through the nanopore 1225 multiple times for repeated analysis, so as to increase the accuracy of identifying the molecule by eliminating statistical errors, i.e., to reduce the false positive error rate.

Unlike some conventional methods (where the sensing electrodes are integrated into the nanopore), each base in the DNA may only take a few nanometers to pass through the nanopore. The transit time is too short for any meaningful measurement. In view of this shortcoming, other conventional methods have been developed to slow the movement of molecules through nanopores. One conventional approach proposes a voltage trapping scheme for controlling the velocity of molecules by embedding additional electrodes into the nanopore. The proposed voltage trapping scheme is difficult to implement because it requires four or more conductive electrodes stacked on top of each other and electrically insulated from each other by sandwiching a dielectric material between the conductive electrodes. The desired 2-3nm nanopores formed in the multilayer film may have an aspect ratio greater than 30: 1, which is difficult, if not impossible, to achieve with current integrated circuit fabrication techniques.

As shown in fig. 13A, an applied AC sensing voltage 1310 is used to interrogate molecules as they change position through the nanopore 1325. Thus, various sensing mechanisms can be employed to identify molecules, such as resistance changes, capacitance changes, phase changes, and/or tunneling currents. The signal-to-noise ratio can be improved due to the close proximity of the embedded sensing electrodes 1305 to the molecules to be detected.

The above exemplary sample delivery and filtering mechanisms serve as examples to illustrate how the nanopore measurement chamber array may be implemented. Those skilled in the art will appreciate that variations of the above delivery and filtering mechanisms may be employed in different embodiments. In addition, arrays with different sized holes can be realized by using the illustrated method. Combining protein wells (such as alpha-hemolysin) with the solid-state nanopore arrays mentioned above also enables the realization of biological nanopore arrays. In some embodiments, both sensing electrodes may be arranged on the same conductive layer instead of the stacked electrodes on different conductive layers described above. Fig. 13B illustrates one embodiment of a planar electrode implementation. In this planar electrode implementation, both sense electrodes 1307 are disposed on the same conductive layer, with a nano-slot 1327 defined between the sense electrodes 1307. A top view of the sensing electrode 1307 and the nano-slot 1327 is shown in figure 13C. As described above, both nanopores and nanogaps are defined using a nanoscale pantograph.

In some embodiments, the ability to perform molecular detection in substantially real-time, the ability to perform single molecular detection without pre-detection sample amplification, the ability to perform multiple and substantially simultaneous detections, the ability to perform multi-channel detection, the ability to identify samples without computational post-reinforcement detection analysis, and/or the ability to retain samples for subsequent use after detection is critical in providing low-cost, rapid, and accurate genetic analysis (e.g., in identifying single nucleotide polymorphisms).

Nanopore-based sequencer

Nanopore-based sequencers provide a portable gene detection and analysis system. In some embodiments, the nanopore based sequencer includes two main components, namely hardware and software. Some embodiments of high-level architectures and subunits are discussed below.

A. Hardware system

In some embodiments, the hardware system of the nanopore based sequencer includes two main units, namely a computing, communication, and control unit and a nanopore based sequencing biochip interface unit, as well as various modules. One embodiment of the high-level architecture is shown in FIG. 14. The details of the various elements will be further explained below.

I. Computing, communication and control unit

In one implementation, the hardware system 1400 includes a portable computing system 1410 having a display device. This may be accomplished using a tablet, laptop, netbook, all-in-one desktop computer, smart phone, Personal Digital Assistant (PDA), or any handheld computing device, etc. It serves as a central unit for running an Operating System (OS), executing data analysis software, storing data, controlling the operation of the nanopore based sequencing biochip, and collecting data from the nanopore based sequencing biochip.

In one embodiment, hardware system 1400 further includes a network communication module 1420. The network communication module 1420 includes one or more of WiFi, WiMAX, ethernet, bluetooth, telephony capabilities, satellite links, and Global Positioning System (GPS), among others. The network communication module 1420 functions as a communication hub for communicating with a central computing system for data sharing, program updating, data analysis, etc., for communicating with other computing devices so that data can be shared and data analysis can be run in parallel among multiple computing devices, for communicating with other bluetooth-enabled devices (e.g., cellular phones, printers, etc.), data sharing, program updating, etc., and for sending and receiving GPS signals.

In one embodiment, hardware system 1400 also includes input device 1430. Input devices 1430 may include one or more of a keyboard, touch screen, mouse, trackball, Infrared (IR) pointing device, and voice device, among others. The input device 1430 serves as a human-machine interface for command input and data input.

In one embodiment, hardware device 1400 also includes input/output ports 1440, which may include flash memory card interfaces, IEEE1394 interfaces, and Universal Serial Bus (USB) interfaces, among others. I/O port 1440 serves as a serial interface to other electronics, a secondary data storage interface, and measurement data I/O for nanopore-based sequencing biochips.

II. Nanopore-based sequencing biochip interface unit

In some embodiments, nanopore based sequencing (nSeq) biochip interface unit 1450 is coupled to nSeq electronics module 1460, fluidic control module 1470, chemical storage and fluidic I/O connection module 1480, and nSeq fluidic control and sample I/O connection module 1490. The nSeq electronics module 1460 controls the distribution of nucleic acid modules, controls the flow rate of nanofluidic channels, collects measurement data, and outputs data to the computation, communication, and control unit.

In some embodiments, the fluid control module 1470 controls fluid flow between the chemical storage and the nSeq biochip via buffer inlet/outlet connectors and the use of on-chip or off-chip micropumps and microvalves. Chemical storage and fluidic I/O connection module 1480 is capable of supplying chemicals to the nSeq biochip when needed, and is also capable of serving as a chemical and/or biological waste storage. The nSeq fluid control and sample I/O connection module 1490 is capable of controlling the fluid and sample flow in the nSeq biochip, as well as controlling the sample inlet and outlet of the nSeq biochip. For example, referring back to fig. 10B, if one wishes to perform a test at measurement chamber #11, the buffer outlet for branch #1 will be opened while all other buffer outlets are closed. As a result, fluid will flow from the sample inlet to branch #1 and work in synchronism with the sample directing electrodes along the microfluidic channel to provide the sample to measurement chamber # 11.

B. Software architecture

FIG. 15 illustrates a high-level structure of software and related hardware components for an operating system and genetic analysis software in one embodiment of a nanopore-based sequencer. The various logical processing modules in the illustrated software architecture can be implemented by a processing device (such as portable computing system 1410 in fig. 14) executing instructions embedded in a computer-readable medium. A computer-readable medium includes any mechanism for providing (i.e., storing and/or transmitting) information in a form accessible by a computer (e.g., a server, a personal computer, a network device, a personal digital assistant, a manufacturing tool, any device with a set of one or more processors, etc.). For example, computer readable media includes recordable/non-recordable media (e.g., Read Only Memory (ROM), Random Access Memory (RAM), magnetic disk storage media, optical storage media, flash memory devices, etc.), among others.

As mentioned above, the operating system 1510 is installed in the computing, communication, and control unit. Operating system 1510 may include Windows, Linux, or any operating system suitable for computing devices. In addition to the operating system 1510 installed in the calculation, communication and control unit, the gene analysis software includes five processing modules, i.e., a Graphic User Interface (GUI)1520, a data viewer 1530, a gene data analyzer interface 1540, a gene data analyzer 1550 and a gene database 1560. Some embodiments of the interaction between the operating system 1510, the above processing modules 1520 and 1560, and other hardware components are discussed below with reference to FIG. 15.

In some embodiments, the genetic data analyzer interface 1540 serves as a data flow control unit in the genetic analysis software architecture. After obtaining commands and/or input data from the input device, operating system 1510 communicates this information to gene data analyzer 1550 through gene data analyzer interface 1540. Genetic data analyzer 1550 then acts accordingly. By employing appropriate commands (e.g., GET, ADD, etc.), genetic data analyzer interface 1540 controls the flow of data between I/O port 1570 and genetic database 1560 so that data stored in database 1560 can be sent out or updated. Similarly, analyzer software can be periodically updated via the I/O port 1570 and/or the input device 1580. The genetic data analyzer interface 1540 is also coupled to a nSeq biochip interface 1590 to monitor the nSeq biochip. The status of the nSeq biochip is monitored and displayed in a display unit (such as the display device of portable computing system 1410 in fig. 14) via analyzer interface 1540. The gene data analyzer interface 1540 also acquires the results from the gene data analyzer 1550 and displays them in the display unit.

In some embodiments, genetic data analyzer 1550 is the primary data analysis unit of the genetic analysis software. It takes measurements from the nSeq biochip, performs analysis and then compares these results to data stored in database 1560 to identify the biological subject. The analysis results can be displayed in a display unit and stored in the database 1560 for subsequent reference.

The gene database 1560 is a data repository for storing existing biological subjects and newly discovered nucleic acid sequences. The data viewer 1530 includes software routines that retrieve data and information from some or all of the other units and display them on a display identification.

Thus, a method and apparatus for portable real-time analysis and identification of molecules has been described. It should be apparent from the foregoing that aspects of the invention may be embodied, at least in part, in software. That is, the techniques may be implemented in a computer system or other data processing system in response to its processor executing sequences of instructions contained in memory. In various embodiments, hard-wired circuitry may be used in combination with software instructions to implement the present invention. Thus, the techniques are not limited to any specific combination of hardware circuitry and software or to any particular source for the instructions executed by the data processing system. In addition, throughout the specification, various functions and operations are described as being performed by or caused by software code to simplify description. However, those skilled in the art will recognize what is meant by such expressions is that the functions result from execution of the code by a processor or controller.

It should be appreciated that reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, it is emphasized or should be appreciated that two or more references to "some embodiments" or "one embodiment" or "an alternative embodiment" in various portions of this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined as suitable in one or more embodiments of the invention. In addition, while the invention has been described in terms of several embodiments, those skilled in the art will recognize that the invention is not limited to the embodiments described. Embodiments of the invention can be practiced with modification and alteration within the scope of the appended claims. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

Claims (13)

1. A method of fabricating a nanopore based sequencing chip usable in a portable molecular analyzer, the method comprising:

applying a nano-scale etch to a bottom layer of conductive material deposited onto a silicon substrate of a nanopore array wafer to define a plurality of nanopores on the nanopore array wafer;

bonding the nanopore array wafer to a bottom of a top wafer to define one or more nanofluidic channels between the nanopore array wafer and the top wafer; and

bonding the nanopore array wafer to a top of a bottom wafer to define one or more microfluidic channels between the nanopore array wafer and the bottom wafer, wherein each of the nanofluidic channels is connected to a respective one of the microfluidic channels via a respective nanopore to allow molecules to move from the nanofluidic channel to the microfluidic channel via the nanopore for analysis and identification of the molecules,

wherein applying the nano-scale etching to the bottom conductive material layer comprises directing a collimated monoenergetic ion beam toward a sub-micron diameter electrostatic lens array to define a plurality of nanofeatures on the bottom conductive material layer on the silicon substrate of the nanopore array wafer.

2. The method of claim 1, further comprising:

fabricating a top drive electrode and a set of sense and bias circuits on the top wafer prior to bonding the top wafer to the nanopore array wafer.

3. The method of claim 1, further comprising:

fabricating a bottom drive electrode and a set of sense and bias circuits on the bottom wafer prior to bonding the bottom wafer to the nanopore array wafer.

4. The method of claim 1, wherein the size of each nanopore in the plurality of nanopores is in the range of 1nm to 200 nm.

5. The method of claim 1, further comprising:

depositing a dielectric separator on the bottom conductive material layer;

depositing a metal layer on the dielectric separator; and

forming a plurality of singlet lenses through the upper metal layer and the dielectric separators, wherein the nano-scale etching is applied onto the bottom conductive material layer to define a plurality of nanopores.

6. The method of claim 1, further comprising:

depositing the bottom conductive material layer on the nitride layer;

depositing a dielectric separator on the bottom conductive material layer;

depositing a metal layer on the dielectric separator; and

forming a plurality of singlet lenses through the upper metal layer and the dielectric separators, wherein applying a nano-scale etch comprises applying a voltage to the bottom conductive material layer on which the plurality of nanofeatures is defined such that the plurality of nanopores are formed at locations where the plurality of nanofeatures are defined.

7. The method of claim 6, wherein the nanopore is used as a hard mask for nanopore etching in the nitride layer.

8. The method of claim 6, further comprising:

removing the plurality of singlet lenses after applying the nano-scale etch;

coating an oxide onto the nanopore to protect the nanopore;

forming a cavity for each of the nanopores in the back of the silicon substrate using a chemical mechanical polishing process, a photolithography process, and a potassium hydroxide process; and

removing the oxide to expose the nanopore after the cavity of the backside of the silicon substrate has been formed.

9. The method of claim 6, further comprising:

depositing a nano-seed in each of the single lenses to grow a nanorod or nanotube;

depositing an oxide layer on the nanorods or nanotubes and the bottom conductive material layer in each singlet lens;

removing the nanorods or nanotubes to form nanopores through the oxide layer in each singlet lens; and

extending the nanopore from the oxide layer to the bottom conductive material layer and the nitride layer of each singlet lens by using the remaining oxide layer as a mask.

10. The method of claim 1, wherein the method further comprises embedding two sensing electrodes into the nanopore, wherein each electrode is separated by a dielectric material.

11. The method of claim 1, further comprising disposing two sensing electrodes on the same conductive layer, wherein the nanopore is defined between the sensing electrodes.

12. The method of claim 1, wherein the molecule comprises at least one of a nucleotide, an amino acid, a protein, a peptide, and a polymer.

13. The method of claim 1, wherein the shape of the nanopore comprises at least one of a circle, an ellipse, and an elongated slit shape.