Reference is now made to the following drawings. Note that the components in the drawings are not necessarily to scale.
As will be described in greater detail here, nanopore analysis systems incorporating nanopore flow cell systems, are provided. By way of example, some embodiments provide for a plurality of structures that include openings for fluid to flow once the structures are secured to one another. The openings can include, but are not limited to, fluid flow channels, reservoirs, and the like. The fluid flow channels can be used to introduce fluids to the reservoirs from a fluid source within or outside of the nanopore analysis system. In one embodiment, the reservoir is in fluid communication with a nanopore aperture, where molecules (e.g., nucleotides, peptides, and the like) can, under proper conditions, interact with the nanopore aperture. In another embodiment, the fluid flow channels can be positioned so that the reservoir is filled from the bottom of the reservoir, which reduces the probability of air bubbles blocking a part or all of the nanopore aperture.
The nanopore flow cell systems can be used in nanopore sequencing of polynucleotides, which has been described in U.S. Pat. No. 5,795,782 to Church et al.; and U.S. Pat. No. 6,015,714 to Baldarelli et al., the teachings of which are both incorporated herein by reference. In general, nanopore sequencing involves the use of two separate pools of a medium and an interface between the pools. The interface between the pools is capable of interacting sequentially with the individual monomer residues of a polynucleotide present in one of the pools. Interface dependent measurements are continued over time, as individual monomer residues of the polynucleotide interact sequentially with the interface, yielding data suitable to infer a monomer-dependent characteristic of the polynucleotide. The monomer-dependent characterization achieved by nanopore sequencing may include identifying physical characteristics such as, but not limited to, the number and composition of monomers that make up each individual polynucleotide, in sequential order.
The term “sequencing” as used herein means determining the sequential order of nucleotides in a polynucleotide molecule. Sequencing, as used herein, includes in the scope of its definition, determining the nucleotide sequence of a polynucleotide in a de novo manner in which the sequence was previously unknown. Sequencing, as used herein, also includes in the scope of its definition, determining the nucleotide sequence of a polynucleotide wherein the sequence was previously known. Sequencing polynucleotides, the sequences of which were previously known, may be used to identify a polynucleotide, to confirm a polynucleotide, or to search for polymorphisms and genetic mutations.
The nanopore detection system 14 includes, but is not limited to, electronic equipment capable of measuring characteristics of the polynucleotide as it interacts with the nanopore aperture, a computer system capable of controlling the measurement of the characteristics and storing the corresponding data, control equipment capable of controlling the conditions of the nanopore flow cell 12, control equipment capable controlling the flow of fluids into and out of the nanopore flow cell 12, and components that are included in the nanopore flow cell 12 that are used to perform the measurements as described below.
The nanopore detection system 14 can measure characteristics such as, but not limited to, the amplitude or duration of individual conductance or electron tunneling current changes across the nanopore aperture. Such changes can identify the monomers in sequence, as each monomer has a characteristic conductance change signature. For instance, the volume, shape, or charges on each monomer can affect conductance in a characteristic way. Likewise, the size of the entire polynucleotide can be determined by observing the length of time (duration) that monomer-dependent conductance changes occur. Alternatively, the number of nucleotides in a polynucleotide (also a measure of size) can be determined as a function of the number of nucleotide-dependent conductance changes for a given nucleic acid traversing the nanopore aperture. The number of nucleotides may not correspond exactly to the number of conductance changes, because there may be more than one conductance level change as each nucleotide of the nucleic acid passes sequentially through the nanopore aperture. However, there is a proportional relationship between the two values that can be determined by preparing a standard with a polynucleotide having a known sequence.
Exemplary detection components have been described in WO 00/79257 and can include, but are not limited to, electrodes directly associated with the structure 22 at or near the pore aperture 24, and electrodes placed within the cis and trans pools. The electrodes may be capable of, but are not limited to, detecting ionic current differences across the two pools or electron tunneling currents across the pore aperture.
As the polynucleotide 26 translocates through or passes sufficiently close to the nanopore aperture 24, measurements (e.g., ionic flow measurements, including measuring duration or amplitude of ionic flow blockage) can be taken by the nanopore detection system 14 as each of the nucleotide monomers of the polynucleotide passes through or sufficiently close to the nanopore aperture 24. The measurements can be used to identify the sequence and length of the polynucleotide.
The medium 28 disposed in the pools on either side of the substrate 22 may be any fluid that permits adequate polynucleotide mobility for substrate interaction.
The target polynucleotide being characterized may remain in its original pool, or it may cross the nanopore aperture 24 into the other pool. In either situation, the target polynucleotide moves in relation to the nanopore aperture 24, individual nucleotides interact sequentially with the nanopore aperture 24 to induce a change in the conductance of the nanopore aperture 24. The nanopore aperture 24 can be traversed either by a polynucleotide translocation through the nanopore aperture 24 so that the polynucleotide passes from one of the pools into the other, or by the polynucleotide traversing across the nanopore aperture 24 without crossing into the other pool. In the latter situation, the polynucleotide is close enough to the nanopore aperture 24 for its nucleotides to interact with the nanopore aperture 24 passage and bring about the conductance changes, which are indicative of polynucleotide characteristics.
Now having described the nanopore flow cell 12 in general,
The nanopore flow cell 12a includes, but is not limited to, a first structure 30, a second structure 40, a spacer structure 50, and a third structure 60. The first structure 30 includes the nanopore aperture 32 (e.g., about 2 to 5 nanometers in diameter). The second structure 40 is adjacent the first structure 30. The second structure 40 includes a first opening (42a) that defines a portion of the cell reservoir (42a and 42b) and a second opening (44) that defines a portion of the fluid flow channel 44. The spacer structure 50 is disposed between the second structure 40 and a third structure 60. The spacer structure 50 includes an opening 52a in fluid communication with the fluid flow channel 44 and an opening (42b) for the cell reservoir 42a and 42b. The electrode 62 is disposed on the surface of the third structure 60 and in-line (e.g., all or a portion of the electrode 62 is exposed to the openings of the cell reservoir 42a and 42b) with the cell reservoir 42a and 42b. In addition, the third structure 60 includes an opening 52b for the fluid flow channel 44. In another embodiment, the nanopore flow cell 12a does not include the spacer structure 50 and the third structure 60 is adjacent the second structure 40.
In other words, the first structure 30, the second structure 40, the spacer structure 50, and the third structure 60, are aligned and secured against one another to form a part of the nanopore flow cell 12a. The openings form the flow channels and reservoirs of the nanopore flow cells in which fluids and samples flow. The first structure 30, the second structure 40, the spacer structure 50, and the third structure 60, can be secured against one another by physical (e.g., mechanical (e.g., screws), heat and/or pressure, and the like) and/or chemical (e.g., adhesives and the like) securing mechanisms.
In particular, a portion of the cell reservoir 42a and 42b is defined on a first side by the first structure 30, and another portion of the cell reservoir is defined on a second side by the third structure 60 and the electrode 62. In addition, the spacer structure 50 defines a portion of the cell reservoir 42a and 42b.
A portion of the fluid flow channel 44 is defined on a first side by the first structure 30 and on a second side by the spacer structure 50. In addition, the fluid flow channel 44 flows through openings 52a in the spacer structure 50 and the openings 52b of the third structure 60 from an appropriate fluid or sample introduction system (not shown). A sample or other fluid can flow into and/or out of the cell reservoir 42a and 42b via one or more of the openings 52b and 52a and one or more of the fluid flow channels 44. The openings 52a and 52b and openings for the fluid flow channels 44 can be reversibly opened and closed to facilitate proper flow into and out of the cell reservoir 42a and 42b. In another embodiment, some of the openings 52a and 52b may not be present to facilitate proper flow into and out of the cell reservoir 42a and 42b.
The nanopore aperture 32 can be dimensioned so that only a single stranded polynucleotide can translocate through the nanopore aperture 32 at a time or so that a double or single stranded polynucletide can translocate through the nanopore aperture 32. The nanopore aperture 32 can have a diameter of about 3 to 5 nanometers (for analysis of single or double stranded polynucleotides) and from about 2 to 4 nanometers (for analysis of single stranded polynucleotides).
The first structure 30, the second structure 40, the spacer structure 50, and the third structure 60, can each have similar lengths and/or heights (e.g., about 3 to 12 mm). Each structure can have a width (narrowest dimension) of about 50 to 5000 nm. In addition, the width can vary across the structure. Each of the first structure 30, the second structure 40, the spacer structure 50, and the third structure 60, can have a different width. For example the spacer structure 50 may have a minimum width to account for the electrode 62 that is slightly raised on the third structure 60. In another example, the second structure 40 and/or the spacer structure 50 can each have a width to define a specific volume of the cell reservoir 42a and 42b. In this way, the nanopore flow cell 12 can be reconfigured or modified by the addition or removal of substrates to produce nanopore flow cells with different dimensions, fluid flow channels 44, and the like. The widths for each of the first structure 30, the second structure 40, the spacer structure 50, and the third structure 60, can be selected based on the configuration needed for a particular application.
The first structure 30, the second structure 40, the spacer structure 50, and the third structure 60 can be made of materials such as, but not limited to, silicon nitride, silicon oxide, mica, polyimide, stainless steel, polymer, various glasses, ceramics, and the like.
The fluid flow channels 44 and the cell reservoir 42a and 42b can be configured to enhance the operation of the nanopore flow cell 12a. For example, the fluid flow channels 44 and the cell reservoir 42a and 42b can be configured (e.g., one or more of the openings 52a and 52b or the opening to one side of the fluid flow channel 44 is reversibly closed) so that the cell reservoir 42a and 42b is filled from the bottom-up. In other words, one of the fluid flow channels 44 that introduces the sample fluid is positioned at the bottom of the cell reservoir 42a and 42b, for example. By filling the cell reservoir 42a and 42b from the bottom-up, the probability of having an air bubble block the nanopore aperture 32 or a portion thereof is reduced.
The nanopore flow cell 12b includes, but is not limited to, a first structure 30, a second structure 40, a third structure 60, a first mixing structure 70, and a second mixing structure 80. The first structure 30, the second 40, and the third structure 60, include the same components and may have the same characteristics as those same structures described in
In addition, the first mixing structure 70 includes a first opening (72) that defines a portion of the mixing cell reservoir 72 and second opening (74) that defines a portion of the mixing fluid flow channel 74. The first mixing structure 70 is adjacent the third structure 60. The second mixing structure 80 includes an opening/channel 82 to flow fluid into the mixing cell reservoir 72, which is in fluid communication with mixing fluid flow channel 74. The second mixing structure 80 is adjacent the first mixing structure 70.
In other words, the first structure 30, the second structure 40, the third structure 60, the first mixing structure 70, and the second mixing structure 80 are aligned and secured against one another to form a part of the nanopore flow cell 12b. The openings form the flow channels and reservoirs of the nanopore flow cells in which fluids and samples flow and are mixed. The first structure 30, the second structure 40, the third structure 60, the first mixing structure 70, and the second mixing structure 80 can be secured against one another by physical (e.g., mechanical (e.g., screws), heat and/or pressure, and the like) and/or chemical (e.g., adhesives and the like) securing mechanisms.
In particular, a portion of the cell reservoir 42 is defined on a first side by the first structure 30, and another portion of the cell reservoir is defined on a second side by the third structure 60 and the electrode 62.
A portion of the fluid flow channel 44 is defined on a first side by the first structure 30 and on a second side by the third structure 60.
A portion of the mixing cell reservoir 72 is defined on a first side by the third structure 60, and another portion of the mixing cell reservoir 72 is defined on a second side by the second mixing structure 80.
A portion of the mixing fluid flow channel 74 is defined on a first side by the third structure 60, and another portion of the mixing cell reservoir 72 is defined on a second side by the second mixing structure 80.
Fluid flows from the openings/channels 82 in the second mixing structure 80 from an appropriate fluid or sample introduction system (not shown) into the mixing cell reservoir 72. Once the fluid is mixed, the fluid can flow out of the mixing cell reservoir 72 through the mixing fluid flow channel 74. Then fluid flows to the fluid flow channel 44 and the cell reservoir 42 via openings 64 in the third structure 60.
The openings 64 and 82 and openings for the fluid flow channels 44 and the mixing fluid flow channels 74, can be reversibly opened and closed to facilitate proper flow into and out of the cell reservoir 42 and/or into and out of the mixing cell reservoir 72. In another embodiment, some of the openings 64 and 82 may not be present to facilitate proper flow into and out of the cell reservoir 42 and/or into and out of the mixing cell reservoir 72.
The first structure 30, the second structure 40, the third structure 60, the first mixing structure 70, and the second mixing structure 80, can each have similar lengths and heights (e.g., about 3 to 12 mm). Each structure can have a width (narrowest dimension) of about 50 to 5000 nm. In addition, the width can vary across the structure. Each of the first structure 30, the second structure 40, the third structure 60, the first mixing structure 70, and the second mixing structure 80, can have a different width. For example the second structure 40 and/or the first mixing structure 70 can each have a width to define a specific volume of the cell reservoir 42 and mixing cell reservoir 72, respectively. In this way, the nanopore flow cell 12b can be reconfigured or modified by the addition or removal of substrates to produce nanopore flow cells with different dimensions, fluid flow channels 44, and the like. The widths for each of the first structure 30, the second structure 40, the third structure 60, the first mixing structure 70, and the second mixing structure 80, can be selected based on the configuration needed for a particular application.
It should be emphasized that many variations and modifications may be made to the above-described embodiments. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.