Patent Description:
Advances in micro-miniaturization within the semiconductor industry in recent years have enabled biotechnologists to begin packing traditionally bulky sensing tools into smaller and smaller form factors, onto so-called biochips. It would be desirable to develop techniques for biochips that make them more robust, efficient, and cost-effective. <CIT>, <CIT>, <CIT>, <CIT> and <CIT> disclose consumable devices similar where the connection to a dispensing device is not satisfying.

The present invention relates to a consumable device for use with a sequencing system according to claim <NUM> and a method of delivering fluid to a consumable device for sequencing according to claim <NUM>.

In some embodiments, a consumable device for use with a sequencing system is provided. The consumable device can include a sequencing chip, the sequencing chip can include a plurality of wells, each well including a working electrode; a flow cell including at least one flow channel, wherein the flow cell is configured to be disposed over sequencing chip such that the at least one flow channel is disposed over the plurality of wells of the sequencing chip; at least one inlet boss having a lumen in fluid communication with the at least one flow channel; and at least one counter electrode disposed over at least a portion of the at least one flow channel; and a flow cell cover including at least one inlet boss receptacle for receiving the at least one inlet boss of the flow cell; and at least one dispense tip receptacle configured to receive a dispense tip, wherein the at least one dispense tip receptacle is in fluid communication the at least one inlet boss receptacle.

In some embodiments, the sequencing chip includes at least about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> million wells.

In some embodiments, the at least one dispense tip receptacle is funnel shaped.

In some embodiments, the at least one dispense tip receptacle is connected to the at least one inlet boss receptacle via a hole or lumen sized to pass the dispense tip.

In some embodiments, the lumen of the at least one inlet boss has a constant diameter.

In some embodiments, the lumen of the at least one inlet boss is tapered.

In some embodiments, the lumen of the at least one inlet boss includes a chamfered inlet opening.

In some embodiments, the at least one inlet boss is made of a deformable material capable of conforming to the dispense tip.

In some embodiments, the at least one inlet boss receptacle has a bowed surface configured to form a gap with the at least one inlet boss when the at least one inlet boss is inserted into the at least one inlet boss receptacle.

In some embodiments, the at least one inlet boss receptacle has a chamfered opening.

In some embodiments, the at least one inlet boss comprises a plurality of inlet lumens that are combined into a single inlet boss.

In some embodiments, each inlet lumen has a conical opening, wherein the at least one dispense tip receptacles includes a plurality of dispense tip receptacles in fluid communication with a single inlet boss receptacle configured to receive the single inlet boss, wherein the single inlet boss receptacle comprises a plurality of conical sealing surfaces configured to mate with each conical opening of each inlet lumen.

In some embodiments, the at least one dispense tip receptacle is sealed with a pierceable material.

In some embodiments, a method of delivering fluid to a consumable device for sequencing is provided. The method can include inserting a piercing tool through a seal that is covering a dispense tip receptacle of a consumable device. The consumable device can include a sequencing chip, the sequencing chip including a plurality of wells, each well including a working electrode; a flow cell including at least one flow channel, wherein the flow cell is configured to be disposed over sequencing chip such that the at least one flow channel is disposed over the plurality of wells of the sequencing chip; at least one inlet boss having a lumen in fluid communication with the at least one flow channel; and at least one counter electrode disposed over at least a portion of the at least one flow channel; and a flow cell cover including at least one inlet boss receptacle for receiving the at least one inlet boss of the flow cell; and at least one dispense tip receptacle configured to receive a dispense tip, wherein the at least one dispense tip receptacle is in fluid communication the at least one inlet boss receptacle. The method can further include removing the piercing tool from the dispense tip receptacle and leaving one or more seal fragments that extend into the dispense tip receptacle; inserting a distal end of a dispense tip past the one or more seal fragments; after the step of inserting the distal end of the dispense tip past the one or more seal fragments, partially dispensing a first fluid from the distal end of the dispense tip so that the first fluid extends distally from the distal end as a partial droplet; and advancing the dispense tip so that the partial droplet makes a liquid-liquid connection with a second fluid in the dispense tip receptacle.

In some embodiments, the method further includes advancing the distal end of the dispense tip into the lumen of the at least one inlet boss receptacle to make a fluid tight seal between the dispense tip and the at least one inlet boss.

In some embodiments, the method further includes delivering fluid through the dispense tip into the at least one flow channel.

In some embodiments, the method further includes piercing a seal of a reagent reservoir with the piercing tool, wherein the first fluid is stored in the reagent reservoir.

In some embodiments, the seal is a pierceable cap.

In some embodiments, the reagent reservoir is selected from the group consisting of a bottle and a trough.

The scope of the invention is limited only by the claims.

Nanopore membrane devices having pore sizes on the order of one nanometer in internal diameter have shown promise in rapid nucleotide sequencing. When a voltage potential is applied across a nanopore immersed in a conducting fluid, a small ion current attributed to the conduction of ions across the nanopore can be observed. The size of the current is sensitive to the pore size.

A nanopore based sequencing chip may be used for DNA sequencing. A nanopore based sequencing chip incorporates a large number of sensor cells configured as an array. For example, an array of one million cells may include <NUM> rows by <NUM> columns of cells.

<FIG> illustrates an embodiment of a cell <NUM> in a nanopore based sequencing chip. A membrane <NUM> is formed over the surface of the cell. In some embodiments, membrane <NUM> is a lipid bilayer. The bulk electrolyte <NUM> containing protein nanopore transmembrane molecular complexes (PNTMC) and the analyte of interest is placed directly onto the surface of the cell. A single PNTMC <NUM> is inserted into membrane <NUM> by electroporation. The individual membranes in the array are neither chemically nor electrically connected to each other. Thus, each cell in the array is an independent sequencing machine, producing data unique to the single polymer molecule associated with the PNTMC. PNTMC <NUM> operates on the analytes and modulates the ionic current through the otherwise impermeable bilayer.

With continued reference to <FIG>, analog measurement circuitry <NUM> is connected to a metal electrode <NUM> covered by a thin film of electrolyte <NUM>. The thin film of electrolyte <NUM> is isolated from the bulk electrolyte <NUM> by the ion-impermeable membrane <NUM>. PNTMC <NUM> crosses membrane <NUM> and provides the only path for ionic current to flow from the bulk liquid to working electrode <NUM>. The cell also includes a counter electrode (CE) <NUM>, which is an electrochemical potential sensor. The cell also includes a reference electrode <NUM>.

In some embodiments, a nanopore array enables parallel sequencing using the single molecule nanopore based sequencing by synthesis (Nano-SBS) technique. <FIG> illustrates an embodiment of a cell <NUM> performing nucleotide sequencing with the Nano-SBS technique. In the Nano-SBS technique, a template <NUM> to be sequenced and a primer are introduced to cell <NUM>. To this template-primer complex, four differently tagged nucleotides <NUM> are added to the bulk aqueous phase. As the correctly tagged nucleotide is complexed with the polymerase <NUM>, the tail of the tag is positioned in the barrel of nanopore <NUM>. The tag held in the barrel of nanopore <NUM> generates a unique ionic blockade signal <NUM>, thereby electronically identifying the added base due to the tags' distinct chemical structures.

<FIG> illustrates an embodiment of a cell about to perform nucleotide sequencing with pre-loaded tags. A nanopore <NUM> is formed in a membrane <NUM>. An enzyme <NUM> (e.g., a polymerase, such as a DNA polymerase) is associated with the nanopore. In some cases, polymerase <NUM> is covalently attached to nanopore <NUM>. Polymerase <NUM> is associated with a nucleic acid molecule <NUM> to be sequenced. In some embodiments, the nucleic acid molecule <NUM> is circular. In some cases, nucleic acid molecule <NUM> is linear. In some embodiments, a nucleic acid primer <NUM> is hybridized to a portion of nucleic acid molecule <NUM>. Polymerase <NUM> catalyzes the incorporation of nucleotides <NUM> onto primer <NUM> using single stranded nucleic acid molecule <NUM> as a template. Nucleotides <NUM> comprise tag species ("tags") <NUM>.

<FIG> illustrates an embodiment of a process <NUM> for nucleic acid sequencing with pre-loaded tags. At stage A, a tagged nucleotide (one of four different types: A, T, G, or C) is not associated with the polymerase. At stage B, a tagged nucleotide is associated with the polymerase. At stage C, the polymerase is in close proximity to the nanopore. The tag is pulled into the nanopore by an electrical field generated by a voltage applied across the membrane and/or the nanopore.

Some of the associated tagged nucleotides are not base paired with the nucleic acid molecule. These non-paired nucleotides typically are rejected by the polymerase within a time scale that is shorter than the time scale for which correctly paired nucleotides remain associated with the polymerase. Since the non-paired nucleotides are only transiently associated with the polymerase, process <NUM> as shown in <FIG> typically does not proceed beyond stage B.

Before the polymerase is docked to the nanopore, the conductance of the nanopore is ~<NUM> pico Siemens (<NUM> pS). At stage C, the conductance of the nanopore is about <NUM> pS, <NUM> pS, <NUM> pS, or <NUM> pS corresponding to one of the four types of tagged nucleotides. The polymerase undergoes an isomerization and a transphosphorylation reaction to incorporate the nucleotide into the growing nucleic acid molecule and release the tag molecule. In particular, as the tag is held in the nanopore, a unique conductance signal (e.g., see signal <NUM> in <FIG>) is generated due to the tag's distinct chemical structures, thereby identifying the added base electronically. Repeating the cycle (i.e., stage A through E or stage A through F) allows for the sequencing of the nucleic acid molecule. At stage D, the released tag passes through the nanopore.

In some cases, tagged nucleotides that are not incorporated into the growing nucleic acid molecule will also pass through the nanopore, as seen in stage F of <FIG>. The unincorporated nucleotide can be detected by the nanopore in some instances, but the method provides a means for distinguishing between an incorporated nucleotide and an unincorporated nucleotide based at least in part on the time for which the nucleotide is detected in the nanopore. Tags bound to unincorporated nucleotides pass through the nanopore quickly and are detected for a short period of time (e.g., less than <NUM>), while tags bound to incorporated nucleotides are loaded into the nanopore and detected for a long period of time (e.g., at least <NUM>).

<FIG> illustrates an embodiment of a fluidic workflow process <NUM> for flowing different types of fluids (liquids or gases) through the cells of a nanopore based sequencing chip during different phases of the chip's operation. The nanopore based sequencing chip operates in different phases, including an initialization and calibration phase (phase <NUM>), a membrane formation phase (phase <NUM>), a nanopore formation phase (phase <NUM>), a sequencing phase (phase <NUM>), and a cleaning and reset phase (phase <NUM>).

At the initialization and calibration phase <NUM>, a salt buffer is flowed through the cells of the nanopore based sequencing chip at <NUM>. The salt buffer may be potassium choloride (KCl), potassium acetate (KAc), sodium trifluoroacetate (NaTFA), and the like.

At the membrane formation phase <NUM>, a membrane, such as a lipid bilayer, is formed over each of the cells. At <NUM>, a lipid and decane mixture is flowed over the cells. At <NUM>, a salt buffer is flowed over the cells. At <NUM>, voltage measurements across the lipid bilayers are made to determine whether the lipid bilayers are properly formed. If it is determined that the lipid bilayers are not properly formed, then step <NUM> is repeated; otherwise, the process proceeds to step <NUM>. At <NUM>, a salt buffer is again introduced.

At the nanopore formation phase <NUM>, a nanopore is formed in the bilayer over each of the cells. At <NUM>, a sample and a pore/polymerase mixture are flowed over the cells.

At the sequencing phase <NUM>, DNA sequencing is performed. At <NUM>, StartMix is flowed over the cells, and the sequencing information is collected and stored. StartMix is a reagent that initiates the sequencing process. After the sequencing phase, one cycle of the process is completed at <NUM>.

At the cleaning and reset phase <NUM>, the nanopore based sequencing chip is cleaned and reset such that the chip can be recycled for additional uses. At <NUM>, a surfactant is flowed over the cells. At <NUM>, ethanol is flowed over the cells. In this example, a surfactant and ethanol are used for cleaning the chip. However, alternative fluids may be used. Steps <NUM> and <NUM> may also be repeated a plurality of times to ensure that the chip is properly cleaned. After step <NUM>, the lipid bilayers and pores have been removed and the fluidic workflow process <NUM> can be repeated at the initialization and calibration phase <NUM> again.

As shown in process <NUM> described above, multiple fluids with significantly different properties (e.g., compressibility, hydrophobicity, and viscosity) are flowed over an array of sensors on the surface of the nanopore based sequencing chip. For improved efficiency, each of the sensors in the array should be exposed to the fluids or gases in a consistent manner. For example, each of the different types of fluids should be flowed over the nanopore based sequencing chip such that the fluid or gas may be delivered to the chip, evenly coating and contacting each of the cells' surface, and then delivered out of the chip. As described above, a nanopore based sequencing chip incorporates a large number of sensor cells configured as an array. As the nanopore based sequencing chip is scaled to include more and more cells, achieving an even flow of the different types of fluids or gases across the cells of the chip becomes more challenging.

<FIG> illustrates an exemplary flow of a fluid across the nanopore based sequencing chip. In <FIG>, an inlet (e.g., a tube) <NUM> delivers a fluid to a nanopore based sequencing chip <NUM>, and an outlet <NUM> delivers the fluid or gas out of the chip. Due to the difference in width between the inlet and the nanopore based sequencing chip, as the fluid or gas enters chip <NUM>, the fluid or gas flows through paths that cover the cells that are close to the outer perimeter but not the cells in the center portion of the chip.

<FIG> illustrates another exemplary flow of a fluid across the nanopore based sequencing chip. In <FIG>, an inlet <NUM> delivers a fluid to a nanopore based sequencing chip <NUM>, and an outlet <NUM> delivers the fluid or gas out of the chip. As the fluid or gas enters chip <NUM>, the fluid or gas flows through paths that cover the cells that are close to the center portion of the chip but not the cells that are close to the outer perimeter of the chip.

As shown in <FIG> above, the nanopore based sequencing chip has one or more "dead" zones in the flow chamber. In the embodiment shown in <FIG>, the dead zones are distributed close to the center of the chip. In the embodiment shown in <FIG>, the dead zones are distributed close to the outer perimeter of the chip. The sensors in the chip array beneath the dead zones are exposed to a small amount of the fluid or a slow flow of the fluid, while the sensors outside of the dead zones are exposed to an excess or fast flow of the fluid.

Furthermore, the introduction of a second fluid may not displace the first fluid in the dead zones effectively. <FIG> illustrates an exemplary flow of a first type of fluid across the nanopore based sequencing chip. In this example, since the dead zones are located at the corners of the nanopore base sequencing chip, the corners of the chip are exposed to the first fluid later than other portions of the chip, but eventually the corners are finally filled up with the first fluid. <FIG> illustrates that a second fluid is flowed through the chip after a first fluid has been flowed through the chip at an earlier time. Because the dead zones are located at the corners of the chip, the second fluid fails to displace the first fluid at the corners within a short period of time. As a result, the sensors in the array are not exposed to the right amount of fluid in a consistent manner.

The design of the flow chamber may also affect the formation of lipid bilayers with the appropriate thickness. With reference to step <NUM> of process <NUM> in <FIG>, a lipid and decane mixture is flowed over the cells, creating a thick lipid layer on top of each of the cells. In order to reduce the thickness of a lipid layer, in some embodiments, one or more air bubbles are flowed over the sensor to scrape the lipid layer into a thinner layer at step <NUM> of process <NUM>. The design of the flow chamber should be optimized to control the scraping boundary between the air and the lipid layers, such that an even wiping action is performed over all of the sensors. In addition, the design of the flow chamber may be optimized to prevent the air bubbles from collapsing mid-way across the flow chamber; otherwise, only a portion of the lipid layers in the chip are scraped or "thinned.

With continued reference to <FIG> and <FIG>, when the flow chamber flows the fluid from one end to the opposite end of the chip, the size of the dead zones within the chip and the collapsing of the air bubbles, in some embodiments, may be reduced by controlling the flow of the fluids and the air bubbles using different pressure and velocity. However, the improvement is limited.

<FIG> illustrates the top view of a nanopore based sequencing system <NUM> with a flow chamber enclosing a silicon chip that allows liquids and gases to pass over and contact sensors on the chip surface. In this example, the nanopore array chip <NUM> includes <NUM> sensor banks (<NUM>) in a <NUM> X <NUM> row-column arrangement. However, other arrangements of the sensors cells may be used as well. System <NUM> includes a counter electrode <NUM> positioned above the flow chamber. Fluids are directed from an inlet <NUM> to the flow chamber atop chip <NUM>, and the fluids are directed out of the flow chamber via an outlet <NUM>. The inlet and the outlet may be tubes or needles. Inlet <NUM> and outlet <NUM> are each positioned at one of two corners of the nanopore array chip <NUM> diagonally across from each other. Because the chamber is considerably wider than the inlet's width, as the fluid or gas enters the chamber, the fluid or gas flows through different paths <NUM> that cover more cells that are close to the center portion of the chip than cells that are close to the remaining two corners of the chip. The fluid or gas travels from one corner to another diagonal corner, leaving trapped fluids in dead zones in the remaining corners.

<FIG> illustrates the various components that are assembled together to form the nanopore based sequencing system <NUM> as shown in <FIG>. System <NUM> includes various components, including a printed circuit board <NUM>, a nanopore array chip <NUM>, a gasket <NUM>, counter and reference electrodes <NUM> connected by a flexible flat circuit <NUM> to a connector <NUM>, a top cover <NUM>, an inlet/outlet guide <NUM>, an inlet <NUM>, and an outlet <NUM>.

<FIG> illustrates another exemplary view of nanopore based sequencing system <NUM>. The flow chamber is the space formed between the top cover <NUM>, the gasket <NUM>, and the nanopore array chip <NUM>. The chamber volume is shown as <NUM> in <FIG>.

<FIG> illustrates the top view of a nanopore based sequencing system <NUM> with an improved flow chamber enclosing a silicon chip that allows liquids and gases to pass over and contact sensors on the chip surface. <FIG> illustrates the cross sectional view of system <NUM> from the position of a plane <NUM> through the system.

A fluid is directed into system <NUM> through an inlet <NUM>. Inlet <NUM> may be a tube or a needle. For example, the tube or needle may have a diameter of one millimeter. Instead of feeding the fluid or gas directly into the flow chamber, inlet <NUM> feeds the fluid or gas to a fan-out plenum space or reservoir <NUM>. As shown in the top view of system <NUM> (<FIG>), fan-out plenum <NUM> directs the fluid or gas outwardly from a central point, a small orifice <NUM> of inlet <NUM> that intersects (see <FIG>) with the fan-out plenum <NUM>. Fan-out plenum <NUM> spreads out from orifice <NUM> into a fanlike shape. For example, the fanlike shape as shown in <FIG> is a substantially triangular shape. However, other similar shapes that direct the fluid or gas outwardly from the small orifice <NUM> may be used as well. In one example, orifice <NUM> is one millimeter wide, and fan-out plenum <NUM> fans out to seven millimeters, the width of one row of four sensor banks <NUM>.

With reference to the cross sectional view of system <NUM> (<FIG>), the fluid or gas fills fan-out plenum <NUM> first and then spills over and drains down a narrow slit or slot <NUM> that intersects with a flow chamber <NUM>, like a waterfall. Flow chamber <NUM> allows the fluid or gas to pass over and contact sensors on the surface of nanopore array chip <NUM>. Because slit <NUM> spans across a row of sensor banks <NUM>, the fluid or gas is flowed more evenly across the sensor cells, reducing the number and areas of the dead zones within the chip. As the fluid or gas sweeps across the chip, the fluid or gas reaches a second narrow slit <NUM> at the opposite end of the chip, and the fluid or gas is directed through slit <NUM> up to a reverse fan-out plenum <NUM>. Reverse fan-out plenum <NUM> directs the fluid or gas towards a central point, a small orifice <NUM> of outlet <NUM> that intersects (see <FIG>) with the reverse fan-out plenum <NUM>. The fluid or gas is then directed out of system <NUM> via an outlet <NUM>.

<FIG> illustrates another exemplary view of nanopore based sequencing system <NUM> with a fan-out plenum. <FIG> illustrates the various components that are assembled together to form nanopore based sequencing system <NUM> as shown in <FIG>. System <NUM> includes various components, including a printed circuit board <NUM>, a nanopore array chip <NUM>, a gasket <NUM>, a gasket cover <NUM>, a middle plate <NUM>, a middle plate <NUM>, a reference electrode <NUM>, a middle plate <NUM>, a counter electrode <NUM>, a reference electrode <NUM>, a top plate <NUM>, an inlet <NUM>, and an outlet <NUM>.

The fan-out plenum is the space formed between top plate <NUM>, a fan-out void <NUM> on the middle layer <NUM>, and middle layer <NUM>. Slit <NUM> is the space formed by aligning a slit 1108A on middle plate <NUM>, a slit 1108B on middle plate <NUM>, and a slit 1108C on gasket cover <NUM>, and stacking middle plate <NUM>, middle plate <NUM>, and gasket cover <NUM> on top of each other. The flow chamber is the space formed between gasket cover <NUM>, gasket <NUM>, and the nanopore array chip <NUM>.

<FIG> illustrates the paths that are followed by a fluid as it flows through the nanopore based sequencing system <NUM> with a fan-out plenum. A fluid flows down inlet <NUM> (see path 1302A), fills fan-out plenum <NUM> first (see path 1302B) and then spills over and drains down slit <NUM> (see path 1302C) that intersects with the flow chamber. The flow chamber allows the fluid or gas to pass over and contact sensors on the surface of the nanopore array chip as shown in path 1302D. Because slit <NUM> spans across a row of sensor banks, the fluid or gas is flowed more evenly across the sensor cells, reducing the number and areas of the dead zones within the chip. As the fluid or gas sweeps across the chip, the fluid or gas reaches slit <NUM> at the opposite end of the chip, and the fluid or gas is directed up through slit <NUM> (see path 1302E) to a reverse fan-out plenum <NUM>. Reverse fan-out plenum <NUM> converges the fluid or gas towards a central point (see path 1302F), a small orifice of outlet <NUM> that intersects with reverse fan-out plenum <NUM>. The fluid or gas is then directed out of system <NUM> via an outlet <NUM> as shown in path <NUM>.

<FIG> illustrates the top view of a nanopore based sequencing system <NUM> with another improved flow chamber enclosing a silicon chip that allows liquids and gases to pass over and contact sensors on the chip surface. The flow chamber is divided into multiple channels <NUM>, each channel <NUM> directing the fluids to flow directly above a single column (or a single row) of sensor banks <NUM>. As shown in <FIG>, system <NUM> includes four inlets <NUM> and four outlets <NUM>.

With reference to <FIG>, a fluid is directed into system <NUM> in parallel through the four inlets <NUM>. Inlet <NUM> may be a tube or a needle. For example, the tube or needle may have a diameter of one millimeter. Instead of feeding the fluid or gas directly into a wide flow chamber with a single continuous space, each of the inlets <NUM> feeds the fluid or gas into a separate channel <NUM> that directs the fluid or gas to flow directly above a single column of sensor banks <NUM>. The channels <NUM> may be formed by stacking together a top plate and a gasket with dividers <NUM> that divide the chamber into channels, and then mounting them on top of the chip. Once the fluid or gas flows through the channels <NUM> to the opposite side of the chip, the fluid or gas is directed up in parallel through the four outlets <NUM> and out of system <NUM>.

<FIG> illustrates the top view of a nanopore based sequencing system <NUM> with another improved flow chamber enclosing a silicon chip that allows liquids and gases to pass over and contact sensors on the chip surface. Similar to system <NUM>, the flow chamber in system <NUM> is divided into multiple channels <NUM>, but each channel <NUM> directs the fluids to flow directly above two columns (or two rows) of sensor banks <NUM>. The width of the channels is about <NUM> millimeters. As shown in <FIG>, system <NUM> includes two inlets <NUM> and two outlets <NUM>.

Both system <NUM> and system <NUM> allow the fluids to flow more evenly on top of all the sensors on the chip surface. The channel width is configured to be narrow enough such that capillary action has an effect. More particularly, the surface tension (which is caused by cohesion within the fluid) and adhesive forces between the fluid and the enclosing surfaces act to hold the fluid together, thereby preventing the fluid or the air bubbles from breaking up and creating dead zones. Therefore, when the width of a sensor bank is narrow enough, each of the flow channels may flow the fluids directly above two or more columns (or two or more rows) of sensor banks. In this case, system <NUM> may be used. When the width of a sensor bank is not narrow enough, then each of the flow channels may flow the fluids directly above one column (or one row) of sensor banks only. In this case, system <NUM> may be used.

<FIG> illustrates the top view of a nanopore based sequencing system <NUM> with another improved flow chamber enclosing a silicon chip that allows liquids and gases to pass over and contact sensors on the chip surface. The flow chamber is divided into two horseshoe-shaped flow channels <NUM>, each channel <NUM> directing the fluids to flow directly above a single column (or a single row) of sensor banks <NUM> from one end of the chip to the opposite end and then directing the fluids to loop back and flow directly above a second adjacent column of sensor banks to the original end of the chip. As shown in <FIG>, system <NUM> includes two inlets <NUM> and two outlets <NUM>.

With reference to <FIG>, a fluid is directed into system <NUM> in parallel through the two inlets <NUM>. Inlet <NUM> may be a tube or a needle. For example, the tube or needle may have a diameter of one millimeter. Instead of feeding the fluid or gas directly into a wide flow chamber with a single continuous space, each of the inlets <NUM> feeds the fluid or gas into a separate channel <NUM> that directs the fluid or gas to flow directly above a single column of sensor banks <NUM>. The channels <NUM> may be formed by stacking together a top plate and a gasket with dividers <NUM> that divide the chamber into channels, and then mounting them on top of the chip. Once the fluid or gas flows through the channels <NUM>, the fluid or gas is directed up in parallel through the two outlets <NUM> and out of system <NUM>.

<FIG> illustrates the top view of a nanopore based sequencing system <NUM> with another improved flow chamber enclosing a silicon chip that allows liquids and gases to pass over and contact sensors on the chip surface. Similar to system <NUM>, the flow chamber in system <NUM> includes a horseshoe-shaped flow channel <NUM>, but horseshoe-shaped flow channel <NUM> directs the fluids to flow directly above two columns (or two rows) of sensor banks <NUM>. The width of the channel is about <NUM> millimeters. As shown in <FIG>, system <NUM> includes an inlet <NUM> and an outlet <NUM>.

Both system <NUM> and system <NUM> allow the fluids to flow more evenly on top of all the sensors on the chip surface. The channel width is configured to be narrow enough such that capillary action has an effect. More particularly, the surface tension (which is caused by cohesion within the fluid) and adhesive forces between the fluid and the enclosing surfaces act to hold the fluid together, thereby preventing the fluid or the air bubbles from breaking up and creating dead zones. Therefore, when the width of a sensor bank is narrow enough, each of the horseshoe-shaped flow channels may flow the fluids directly above two or more columns (or two or more rows) of sensor banks. In this case, system <NUM> may be used. When the width of a sensor bank is not narrow enough, then each of horseshoe-shaped flow channels may flow the fluids directly above one column (or one row) of sensor banks only. In this case, system <NUM> may be used.

In some embodiments, the nanopore based sequencing system includes an improved flow chamber having a serpentine fluid flow channel that directs the fluids to traverse over different sensors of the chip along the length of the channel. <FIG> illustrates the top view of a nanopore based sequencing system <NUM> with an improved flow chamber enclosing a silicon chip that allows liquids and gases to pass over and contact sensors on the chip surface. The flow chamber includes a serpentine or winding flow channel <NUM> that directs the fluids to flow directly above a single column (or a single row) of sensor banks <NUM> from one end of the chip to the opposite end and then directs the fluids to repeatedly loop back and flow directly above other adjacent columns of sensor banks until all of the sensor banks have been traversed at least once. As shown in <FIG>, system <NUM> includes an inlet <NUM> and an outlet <NUM> and the serpentine or winding flow channel <NUM> directs a fluid to flow from the inlet <NUM> to the outlet <NUM> over all <NUM> sensor banks <NUM>.

With reference to <FIG>, a fluid is directed into system <NUM> through inlet <NUM>. Inlet <NUM> may be a tube or a needle. For example, the tube or needle may have a diameter of one millimeter. Instead of feeding the fluid or gas directly into a wide flow chamber with a single continuous space, inlet <NUM> feeds the fluid or gas into a serpentine flow channel <NUM> that directs the fluid or gas to flow directly above the columns of sensor banks <NUM> serially connected together through serpentine flow channel <NUM>. The serpentine channel <NUM> may be formed by stacking together a top plate and a gasket with dividers <NUM> that divide the chamber into the serpentine channel, and then mounting them on top of the chip. Once the fluid or gas flows through the serpentine channel <NUM>, the fluid or gas is directed up through outlet <NUM> and out of system <NUM>.

System <NUM> allows the fluids to flow more evenly on top of all the sensors on the chip surface. The channel width is configured to be narrow enough such that capillary action has an effect. More particularly, the surface tension (which is caused by cohesion within the fluid) and adhesive forces between the fluid and the enclosing surfaces act to hold the fluid together, thereby preventing the fluid or the air bubbles from breaking up and creating dead zones. For example, the channel may have a width of <NUM> millimeter or less. The narrow channel enables controlled flow of the fluids and minimizes the amount of remnants from a previous flow of fluids or gases.

<FIG> illustrates an exemplary view of one embodiment of a nanopore based sequencing system <NUM> with a serpentine flow channel. <FIG> illustrates the various components that are laminated together to form nanopore based sequencing system <NUM>. System <NUM> includes various components, including a printed circuit board <NUM>, a nanopore array chip <NUM>, a gasket <NUM> with dividers <NUM>, a backing plate <NUM>, a counter electrode <NUM> on the underside of backing plate <NUM>, a flexible flat circuit <NUM> connecting to counter electrode <NUM>, an inlet <NUM>, an outlet <NUM>, a spring plate <NUM>, and a plurality of fastening hardware <NUM>. The serpentine flow channel is the space formed between backing plate <NUM>, gasket <NUM>, and nanopore array chip <NUM>.

<FIG> illustrates the top side view of a backing plate and a flexible flat circuit that is connected to the counter electrode (not visible) located on the bottom side of the backing plate. <FIG> illustrates the same unit <NUM> as shown in <FIG> when the backing plate is flipped upside down. As shown in this figure, the counter or common electrode <NUM> has a serpentine, spiral, or winding shape. Referring back to <FIG>, the counter electrode's serpentine shape matches with the serpentine channel of gasket <NUM>, such that the counter electrode is positioned directly above the sensor banks without being blocked by the dividers <NUM> of the gasket. The dividers <NUM> are disposed between the sensor banks so that the dividers do not block the flow of the fluids or gases over the sensor banks.

<FIG> illustrates the various components of unit <NUM> that are laminated together. Unit <NUM> includes a dielectric layer <NUM>, a counter electrode <NUM> on a film <NUM>, a reference electrode <NUM>, a reference electrode <NUM>, a flexible flat circuit <NUM>, and a backing plate <NUM>.

<FIG> and <FIG> illustrate that the flow channel is formed by laminating a backing plate with the counter electrode, a gasket, and the silicon chip together. However, the backing plate with the counter electrode and the gasket may be integrated together as a single unit made of the electrode material, and the unit is machined to form the serpentine flow channel.

Besides the geometry and dimensions of the flow chamber, other features may also facilitate a more even flow of the fluids on top of all the sensors on the chip surface. <FIG> illustrates a cross sectional view of a flow channel <NUM> with sharp edges or sharp corners that may trap fluids more easily. <FIG> illustrates a cross sectional view of a flow channel <NUM> that has a curved roof <NUM> and a D-shaped cross-sectional geometry. The sharp edges or sharp corners are replaced by round and smooth surfaces. <FIG> illustrates a cross sectional view of another flow channel <NUM> that has a curved roof <NUM>. <FIG> illustrates a side view of a nanopore based sequencing system <NUM> with flow channels having a D-shaped cross sectional geometry.

Another factor that affects the flow of the fluids on top of all the sensors on the chip surface is the height of the flow channel. For example, the height of the flow channel should be limited to one millimeter or below. In one embodiment, the height of the flow channel is <NUM> millimeters. Other factors that affect the flow of the fluids on top of all the sensors on the chip surface include the surface characteristics of the surfaces defining the flow channel, the flow rate of the fluids, the pressure of the fluid and the gases, and the like.

<FIG> is a diagram illustrating an embodiment of a molded flow channel component. In some embodiments, the component shown in <FIG> forms at least a portion of a flow channel shown in <FIG>. As shown in <FIG>, gasket <NUM> with dividers <NUM> must be carefully aligned with counter electrode <NUM> on the underside of backing plate <NUM> during assembly to correctly assemble the flow channel. Additionally, as shown in <FIG>, dielectric layer <NUM>, counter electrode <NUM>, film <NUM>, and backing plate <NUM> must be carefully aligned during assembly. Requiring such a large number of components to be aligned together during assembly introduces complexities that may increase manufacturing cost and risk of error. Therefore, there exists a need for an efficient and reliable way to form the flow channel/chamber of a nanopore system.

Molded flow channel component <NUM> includes molded portion <NUM> and counter electrode portion <NUM>. When molded flow channel component <NUM> is placed on a nanopore array chip (e.g., chip <NUM>) and secured, the serpentine void shown in molded flow channel component <NUM> (e.g., counter electrode portion <NUM> is exposed through the serpentine void of the molded portion <NUM>) becomes a flow chamber of a fluid flow channel that directs fluids to traverse over different sensors of the nanopore array chip. Molded portion <NUM> includes shown raised dividers that guide the fluid flow along the length of the serpentine channel. Molded flow channel component <NUM> includes inlet <NUM> and outlet <NUM>. Both inlet <NUM> and outlet <NUM> include a tubular channel that provides fluid/gas flow in or out of the fluid flow chamber/channel. The inlet/outlet tubular channels are formed at least in part by molded portion <NUM> and pass through counter electrode portion <NUM>.

Molded portion <NUM> has been molded over counter electrode portion <NUM>. For example, counter electrode portion <NUM> was placed inside a mold and molding material is injected into the mold to form molded portion <NUM> over counter electrode portion <NUM> to create molded flow channel component <NUM>. An example material of molded portion <NUM> is an elastomer. An example of counter electrode portion <NUM> is a metal (e.g., titanium nitride sputtered/coated stainless steel).

<FIG> is a diagram illustrating an embodiment of a counter electrode insert. In some embodiments, counter electrode insert <NUM> becomes counter electrode portion <NUM> of <FIG> after assembly. Counter electrode insert <NUM> may be made of metal (e.g., stainless steel, steel, aluminum, etc.), semiconductor material (e.g., doped silicon) or other conductive material that may be rigid or flexible (e.g., foil). In some embodiments, counter electrode insert <NUM> has been coated and/or sputtered with an electrically conductive material. For example, titanium nitride (TiN) has been sputtered on to a base material (e.g., glass, stainless steel, metal, silicon, etc.) to coat counter electrode insert <NUM>. This allows the portion of the counter electrode insert <NUM> that is exposed inside a flow channel of molded flow channel component <NUM> (e.g., portion exposed to chemical reagents) to be titanium nitride. In some embodiments, TiN is a preferred material because of its certain beneficial electrochemical properties, its general availability in and compatibility with existing standard semiconductor manufacturing processes, its compatibility with the biological and chemical reagents, and its relative low cost.

Counter electrode insert <NUM> includes cutouts <NUM> and <NUM> that allow counter electrode insert <NUM> to be aligned and stabilized inside an injection mold. Cutouts <NUM> and <NUM> are on tab ends of counter electrode insert <NUM> that are removed (e.g., snapped off) after molding and discarded from counter electrode insert <NUM> to form counter electrode portion <NUM> of <FIG>. Cutouts <NUM> and <NUM> allow the tab ends to be more easily removed (e.g., snapped off) in the direction of the length of the cutouts <NUM> and <NUM>. The tab ends allow easier alignment and stabilization inside a mold as well as easier handling of the component during molding. Cutout <NUM> corresponds to inlet <NUM> of <FIG> and cutout <NUM> corresponds to outlet <NUM> of <FIG> that allows a fluid/gas to pass through counter electrode insert <NUM> via tubular channels and in/out of a fluid flow chamber/channel. The other cutouts shown in counter electrode insert <NUM> couple counter electrode insert <NUM> with a molding material by allowing the molding material to flow through and remain molded in the cutouts to secure the coupling between the counter electrode insert <NUM> and the molding material. These other cutouts are placed in locations on the counter electrode insert <NUM> to avoid surface portions that are to be exposed inside the fluid chamber/channel. A laser cutter may be utilized as well to produce the shown cutouts. In various embodiments, insert <NUM> may have been stamped, chemically etched or cut using a water-jet to produce the shown cutouts.

<FIG> is a diagram illustrating an embodiment of a mold for a molded flow channel component. In some embodiments, mold <NUM> is utilized to produce molded flow channel component <NUM> of <FIG>. A transparent view of mold <NUM> is shown to illustrate the internal structure of mold <NUM>. Mold <NUM> includes a plurality of sectional pieces that are joined together when molding a component. These pieces may be separated after molding to extract the molded component. Counter electrode insert <NUM> has been placed inside mold <NUM>. Mold <NUM> includes tube features that are inserted in cutouts <NUM> and <NUM> of counter electrode insert <NUM> to produce inlet/outlet tubular channels of molded flow channel component <NUM>. Counter electrode insert <NUM> is secured inside mold <NUM> via its tab ends, one or more cutouts and features of mold <NUM> contacting a surface of counter electrode insert <NUM>. A molding material is injected into mold <NUM> to injection mold the molding material around counter electrode insert <NUM> and in the shape of mold <NUM>. In various embodiments, molding material may be an elastomer, rubber, silicone, polymer, thermoplastics, plastics, or any other injection moldable material. One or more cutouts/holes on counter electrode insert <NUM> may be filled with the molding material to couple the molding material with counter electrode insert <NUM>. In some embodiments, the molding material injected into mold <NUM> becomes molded portion <NUM> of <FIG>. In various other embodiments, other molding techniques such as liquid injection molding (LIM), transfer molding, or compression molding are utilized to produce molded flow channel component <NUM> of <FIG>.

<FIG> is a diagram illustrating an embodiment of a molded flow channel component removed from a mold. Component <NUM> has been removed from mold <NUM> after injection molding. After removing (e.g., bending/snapping off) shown end tabs of counter electrode insert <NUM>, component <NUM> becomes molded flow channel component <NUM> of <FIG>. The four by three array of openings/holes on top of molded portion <NUM> exposes surfaces of counter electrode insert <NUM>. In some embodiments, this array of openings/holes is an artifact resulting from the twelve features/pins of mold <NUM> that are used to hold and stabilize counter electrode insert <NUM> in place in the mold during molding. In some embodiments, at least some of these openings/holes of the array are utilized to make electrical contact with a counter electrode portion. For example, a spring contact is placed in some of the openings/holes of the array and connected to an electrical potential source of a circuit board (e.g., shown in <FIG>).

<FIG> is a diagram illustrating a portion of an embodiment of a nanopore based sequencing system utilizing a molded flow channel component. Molded flow channel component <NUM> has been placed on top of a nanopore array chip that is mounted on circuit board <NUM>. Spring contact wire component <NUM> is to be placed in the array of openings/holes on top of molded flow channel component <NUM> to make electrical and physical contact with its counter electrode portion. The other end of spring contact wire component <NUM> is to be connected to circuit board <NUM> to provide an electrical potential source for its counter electrode. In some embodiments, the molded flow channel component <NUM> and electrical contact of spring contact wire component <NUM> of the counter electrode are secured and clamped via a clamp plate (e.g., without use of adhesives) that clamps spring contact wire component <NUM> to molded flow channel component <NUM>. In some embodiments, the clamp plate also clamps molded flow channel component <NUM> to the nanopore array chip to provide the compression required to seal and couple them together in creating a flow channel/chamber between molded flow channel component <NUM> and the nanopore array chip. Encapsulate berm <NUM> encapsulates wire bonds between electrical terminals of the nanopore array chip and circuit board <NUM>.

<FIG> is a diagram illustrating an embodiment of a clamping of nanopore based sequencing system components. The views shown in <FIG> have been simplified to illustrate the embodiment clearly. View <NUM> shows an overview of the components of <FIG> that have been clamped together.

Clamping plate <NUM> clamps together molded flow channel component <NUM> over a nanopore array chip. Clamping plate <NUM> may be fastened and clamped to circuit board <NUM> via screws or other coupling mechanisms. Clamping plate <NUM> includes holes to accommodate and allow inlet <NUM> and outlet <NUM> to pass through clamping plate <NUM>. Clamping plate <NUM> clamps the molded flow channel component to the nanopore array chip to provide the compression required to seal and couple them together in creating a flow channel/chamber without the use of adhesives and/or permanent/physical bonding to couple together the molded flow channel component to the nanopore array chip. Spring contact wire component <NUM> is not shown to simplify the diagram but exists in the embodiment under clamping plate <NUM>. Clamping plate <NUM> also secures the spring contact wire component to the counter electrode of the molded flow channel component via compression.

View <NUM> shows a cutaway view of the embodiment shown in view <NUM>. Clamping plate <NUM> is secured to circuit board <NUM> and clamps together nanopore array chip <NUM> with counter electrode portion <NUM> and molded portion <NUM> of molded flow channel component <NUM>. The gap between counter electrode portion <NUM> and nanopore array chip <NUM> is a part of a flow chamber/channel that holds and directs the fluids to traverse over different sensors of nanopore array chip <NUM>. The clamping force/pressure seals contact between molded portion <NUM> and nanopore array chip <NUM>. In some embodiments, the material of molded portion <NUM> is compliant to provide the seal under clamping force/pressure.

<FIG> is a diagram illustrating an embodiment of encapsulated wire bonds. Wire bonds <NUM> electrically connect nanopore based sequencing chip <NUM> to circuit board <NUM>. Wire bonds <NUM> are protected by encapsulant <NUM>. Although only two wire bonds have been shown to simplify the diagram, other wire bonds <NUM> all around the perimeter of chip <NUM> may be utilized to electrically connect chip <NUM> to circuit board <NUM>. In order to protect wire bonds <NUM> from damage and wetting, encapsulant <NUM> (e.g., adhesive) is utilized to encapsulate wire bonds <NUM> by applying a ring of encapsulant around the perimeter of chip <NUM>. In some embodiments, encapsulant <NUM> is the encapsulant shown as surrounding a nanopore chip in other figures. However, the applied encapsulant cannot encroach on portions of the chip where one or more flow channel components are to be coupled to the chip.

Thus care must be taken to minimize the encroachment of the encapsulant onto the surface of the chip in order to maximize the area available on the chip surface for coupling the one or more flow channel components onto the chip. In various embodiments, the one or more flow channel components may be coupled to the chip surface by room temperature bonding, adhesive bonding, and/or compression bonding. During manufacturing, two distinct steps may need to be performed - first, encapsulating the wire bonds with careful attention paid to preventing undesired encroachment of the encapsulant onto the chip and then coupling and sealing the one or more flow channel components onto the chip.

<FIG> is a diagram illustrating an embodiment of encapsulating wire bonds together with one or more flow channel components in a single manufacturing step. For example, rather than encapsulating wire bonds between a nanopore chip and a circuit board in a separate step from securing one or more flow channel components to the nanopore chip, a single application of encapsulant <NUM> is utilized to both encapsulate the wire bonds and couple/secure the one or more flow channel components to the chip. In some embodiments, without first encapsulating wire bonds, flow channel component <NUM> is placed on the nanopore chip. Then, a single application of encapsulant (e.g., adhesive) is applied to simultaneously encapsulate the wire bonds and seal the perimeter of flow channel component <NUM> to the nanopore based sequencing chip. By combining two manufacturing steps into one encapsulation step, manufacturing becomes more efficient and simplified. Additionally, the chip encroachment restriction on the application of the encapsulant is eliminated because the flow channel component is already placed on the chip and limits the flow of the encapsulant on undesired portions of the chip. Examples of flow channel component <NUM> include various components placed on a nanopore cell to form a chamber/flow channel over the sensors of the chip. For example, various gaskets, plates, and reference electrodes described in various embodiments herein may be included in flow channel component <NUM>. In some embodiments, flow channel component <NUM> includes molded flow channel component <NUM> of <FIG>.

<FIG> illustrate an embodiment of an adapter <NUM> that helps form a fluidic seal between the flow cell <NUM> and the dispense tip <NUM>. The adapter <NUM> can have a lumen <NUM> for receiving the dispense tip <NUM> and providing a fluidic connection with the flow cell <NUM>. The lumen <NUM> can have a constricted region <NUM> with a diameter that is smaller than the diameter of the other portions of the lumen <NUM>. The <NUM> constricted region <NUM> is designed to form a seal with the dispense tip <NUM> when the dispense tip <NUM> is inserted into the lumen <NUM> of the adapter <NUM>. The lumen <NUM> can have an inlet <NUM> for receiving the dispense tip <NUM> that is conical or tapered outwards such that the inlet diameter is greater than the internal diameter of the lumen <NUM>. A conical or tapered inlet <NUM> facilitates both insertion and alignment of the dispense tip <NUM> into the lumen <NUM> of the adapter <NUM>. The outlet <NUM> of the adapter <NUM> can be sized and shaped to be complementary to a receptacle <NUM> in the flow cell backer 3160a.

The adapter <NUM> can be made of a material with a durometer that allows the constricted region <NUM> of the lumen <NUM> to deform and conform to the dispense tip <NUM> to provide a fluidic seal, while also being sufficiently rigid to not undergo excessive deformation when assembled between the flow cell <NUM> and flow cell backer 3160a, 3160b, and subjected to various pressures (i.e., from mechanical compression between the flow cell and flow cell cover, and from fluid pressurization during use), which could result in leaks. In some embodiments, the Shore durometer can be between about <NUM> to 80A. In some embodiments, the durometer can be between about <NUM> to 70A. In some embodiments, the durometer can be between about <NUM> to 90A.

One embodiment of a disposable consumable device that can be used with the sequencer is formed by the assembly of the flow cell <NUM> over the nanopore chip, with the adapter <NUM> and flow cell backer 3160a, 3160b. The flow cell <NUM> can have one or more bosses <NUM> for each fluid channel the flow cell <NUM> forms over the nanopore chip array. The bosses <NUM> can fit into corresponding receptacles <NUM> in the flow cell backer 3160a. In some embodiments, a cannula <NUM> can extend from the bosses <NUM> and through a channel <NUM> in the flow cell backer 3160a that connects the two sets of receptacles <NUM>, <NUM>. The cannula <NUM> can extend into the receptacle <NUM> in which the adapter <NUM> is inserted. The adapter <NUM> can be inserted into the receptacle <NUM> such that the cannula <NUM> extends into the lumen <NUM> of the adapter <NUM>. For each fluid channel in the flow cell <NUM> that is to be used, an adapter <NUM> can be inserted into the corresponding receptacle <NUM>. A second portion of the flow cell backer 3160b can then be placed over the adapters <NUM> to secure the adapters <NUM> in place over the flow cell <NUM>. The second portion of the flow cell backer 3160b can have a recess <NUM> for receiving the adapters <NUM> and an opening <NUM> that provides access to the inlet <NUM> of the adapter <NUM>.

<FIG> illustrate various embodiments of direct injection fluidic interfaces that can be integrated into the flow cell and that eliminate the use of an adapter. For example, <FIG> illustrate one embodiment of a direct injection flow cell <NUM>. The flow cell <NUM> can have an inlet boss <NUM> for each flow channel in the flow cell. The inlet boss <NUM> can have a lumen <NUM> in fluid communication with the fluid channels over the nanopore chip array. In some embodiments, as shown in <FIG>, the lumen <NUM> can have a constant diameter that is slightly less than the diameter of the dispense tip <NUM> so that a tight fluidic seal can be formed between the dispense tip <NUM> and inlet boss <NUM> when the dispense tip <NUM> is inserted into the lumen <NUM>. As described above in connection with <FIG>, the durometer for the inlet boss <NUM> can be soft enough to allow the inlet boss <NUM> to deform and conform against the dispense tip <NUM>, but also hard enough to maintain the fluidic seal when the flow channels are pressurized with fluids.

<FIG> illustrates another embodiment of an inlet boss <NUM>'. In this embodiment, the inlet boss <NUM>' can have a tapered lumen <NUM>' having a wider diameter at the inlet that receives the dispose tip and a narrower diameter at the outlet that leads to the interior of the flow cell. Such a configuration facilitates insertion of the dispense tip into the inlet boss and formation of a fluid tight seal around the dispense tip. <FIG> illustrates yet another embodiment of an inlet boss <NUM>" with a lumen <NUM>" with a chamfered inlet opening <NUM>‴ with a wider diameter than the lumen <NUM>". Similar to the tapered lumen embodiment described above, the wider diameter at the inlet opening <NUM>" facilitates insertion of the dispense tip into the lumen <NUM>", while the narrower portion of the lumen <NUM>" forms a tight fluidic seal with the dispense tip.

<FIG> illustrates a cross-section of an inlet boss <NUM> with a high parting line 3208a and an inlet boss <NUM> with a low parting line 3208b. <FIG> illustrate a cross-section of an inlet boss <NUM> with a high parting line 3208a, an inlet boss <NUM> with a low parting line 3208b, an inlet boss <NUM>" with a chamfered inlet opening <NUM>" and a constant diameter lumen <NUM>", and an inlet boss <NUM> with a constant diameter lumen <NUM>. <FIG> also illustrates a flow cell backer <NUM> placed directly over the flow cell <NUM> as shown in <FIG>. In the inlet boss <NUM> with the high parting line 3208a, the dispense tip <NUM> is inserted past the high parting line 3208a, such that the depth of the dispense tip <NUM> is below the high parting line 3208a when the dispense tip <NUM> is fully inserted into the lumen of the inlet boss <NUM>. In the inlet boss <NUM> with the low parting line 3208b, the dispense tip <NUM> is not inserted past the low parting line 3208b, and instead, the depth of the dispense tip <NUM> is above low parting line 3208b when the dispense tip <NUM> is fully inserted into the lumen of the inlet boss <NUM>. With a high parting line, the dispense tip crosses the parting line with full insertion, which may be riskier for sealing. The benefit, however, is that the fluid flow could be cleaner because the fluid does not need to cross the parting line. With a low parting line, the dispense tip does not cross the parting line, so the seal is more reliable. However, the fluid flow must now cross the parting line and may be not as ideal as a result.

As shown in <FIG>, the flow cell backer <NUM> can have receptacles <NUM> sized and shaped for receiving the inlet bosses <NUM> and connected receptacles <NUM> for receiving the dispense tip <NUM>. The receptacles <NUM> for receiving the dispense tip <NUM> can be conical or funnel shaped to help guide the dispense tip <NUM> into the lumen of the inlet boss <NUM>. The diameter of inlet of the receptacles <NUM> for receiving the dispense tip <NUM> is greater than the diameter of the inlet opening of the inlet boss <NUM>, and the diameter of the outlet of the receptacles <NUM> is greater than or equal to the diameter of the inlet opening of the inlet boss <NUM>. In <FIG>, the dispense tip <NUM> is shown inserted into the lumen of the inlet boss <NUM> of the flow channel to different depths. As shown, the diameter of the lumen of the inlet boss <NUM> is less than the diameter of the dispense tip <NUM>. The inlet boss <NUM> can be made of a material that deforms and/or compresses to form a fluidic seal against the dispense tip <NUM>.

<FIG> illustrate additional embodiments of inlet boss receptacles <NUM> that are designed to reduce overcompression of the inlet bosses <NUM>. Radial overcompression of the inlet boss <NUM> can cause excessive deformation of the walls and lumen of the inlet boss <NUM> such that an inadequate seal is formed around the dispense tip, which can result in fluid leakage. By oversizing the receptacle relative to the inlet boss, a space between the receptacle and inlet boss can be provided that accommodates deformation of the inlet boss upon insertion, thereby preserving the lumen of the lumen boss. Without the extra space, deformation of the inlet boss would result in inward deformation that would tend to collapse the lumen of the inlet boss. For example, as shown in <FIG>, the receptacles <NUM> of the flow cell backer <NUM> can be made larger, by increasing the diameter of the receptacles for example, in order to apply less radial compressive force to the inlet bosses <NUM>. In some embodiments, the diameter of the receptacles is greater than the diameter of the inlet bosses. For example, the diameter of the receptacles can be about <NUM> to <NUM> greater, or about <NUM> to <NUM> greater, or about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> greater, or at least about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> greater, or at most <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> greater than the diameter of the inlet bosses. In some embodiments, the shape and diameter of the receptacles <NUM> and the inlet bosses <NUM> are equal or about equal. In some embodiments, the diameter of the receptacles <NUM> is between about <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, or <NUM> to <NUM>% larger than the diameter of the inlet bosses <NUM>. In other embodiments, the diameter of the receptacles <NUM> is between about <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, or <NUM> to <NUM>% larger than the diameter of the inlet bosses <NUM>. In some embodiments, the degree or magnitude of oversizing of the receptacle depends of the hardness/softness (i.e. durometer) of the inlet boss material. A softer material would tend to undergo more deformation, and therefore, the receptacle can be oversized to a larger degree. Conversely, an inlet boss made of a harder material would tend to deform less, and therefore, a smaller degree of oversizing of the receptacle would be sufficient to prevent collapse of the lumen.

<FIG> illustrates another embodiment of a receptacle <NUM>' that has a bowed sidewall that leaves a gap <NUM> between the inlet boss <NUM> and the sidewall receptacle <NUM>'. The gap <NUM> prevents undue pressure from being applied to inlet boss <NUM>. As shown, the gap <NUM> can have a maximum width at an intermediate location along the inlet boss <NUM> and receptacle <NUM>' and taper away to about zero at the top and bottom of the inlet boss <NUM> and receptacle <NUM>'. This configuration allows the flow cell cover to be precisely aligned with the flow cell cover, while also providing a gap to relieve compression to the inlet bosses. Ins some embodiments, the bowed wall can be curved. In other embodiments, the bowed wall can be formed from two or more angled surfaces. In other embodiments, the receptacle can have straight walls (in a cross-sectional view) while the outer wall of the inlet boss can have a bowed surface to form the gap. In some embodiments, a gap between the receptacle and the inlet wall can be provided along an intermediate portion of the inlet boss and receptacle by removing material around an intermediate portion of the receptacle and/or inlet boss.

<FIG> illustrates yet another embodiment of a receptacle <NUM>" with a chamfered edge <NUM> around the opening of the receptacle <NUM>". The chamfered edge <NUM> provides a gap that functions to relieve compression, particularly around the base of the inlet boss <NUM>. In some embodiments, the receptacle and or inlet boss can be modified using any combination of the features recited herein to reduce overcompression.

<FIG> illustrate additional embodiments of fluidic interfaces that can be integrated into the flow cell <NUM>. In this embodiment, the inlet bosses <NUM> can be combined together into a single structure, such as a wall structure with a plurality of receptacles. Combining the inlet bosses <NUM> into a single structure provides additional support to the inlet bosses and reduces the likelihood of buckling when the dispense tip <NUM> is inserted into the inlet boss <NUM>. In some embodiments, the inlet bosses <NUM> can have conical sealing surfaces <NUM> that are similar to the chamfer inlet opening as described above, except that the conical sealing surfaces may be larger to take advantage of the additional material of the wall structure between each receptacle/fluidic interface. The angle of the conical sealing surface can be adjusted to increase or decrease a radially directed sealing force that develops when the flow cell backer <NUM> is pressed against the flow cell <NUM>. In some embodiments, the angle of the conical sealing surface can be between about <NUM> to <NUM> degrees relative to an axis that extends through the lumen of the flow cell <NUM>. In other embodiments, the angle of the conical sealing surface can be relatively shallow and can be between about <NUM> to <NUM> degrees, or between about <NUM> to <NUM> degrees, or between about <NUM> to <NUM> degrees, or between about <NUM> to <NUM> degrees, or between about <NUM> to <NUM> degrees, relative to an axis that extends through the lumen of the flow cell <NUM>. The conical sealing surface feature can be used in other embodiments, such as the individual bosses shown in <FIG>, for example. The flow cell backer <NUM> can have receptacles <NUM> with a complementary shape to the inlet boss <NUM> structure, including complementary conical sealing surfaces <NUM> that are configured to abut against the conical sealing surfaces <NUM> of the inlet bosses <NUM>.

The flow cell backer <NUM> can have one or more alignment pins <NUM> and one or more fastening mechanisms <NUM>, such as screw holes located at the corners of the flow cell backer <NUM>, that allow the flow cell backer <NUM> to be fastened securely and evenly over the flow cell <NUM> and to a substrate, such as a printed circuit board (PCB). Other fastening mechanisms include snaps or clips.

<FIG> illustrate an embodiment of a dispense tip <NUM> that can be inserted into the fluidic interface of the flow cell to form a fluidic seal. The dispense tip <NUM> can be made of stainless steel or another metal or metal alloy and can be coated with a low friction, hydrophobic material, such as a fluoropolymer (i.e., polytetrafluoroethylene (PTFE) or fluorinated ethylene propylene (FEP)). Both the outer surface and the interior surface of the dispense tip <NUM> can be coated. In some embodiments, the coating is at least <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> thick. Use of metal for the dispense tip over other materials, such as plastic, provides the dispense tip with increased structural strength and allows the dispense tip to be used repeatedly before needing to be replaced. Use of metal also allows the dispense tip to function as a probe, such as a liquid level probe using capacitive sensing. The length of the dispense tip <NUM> can be sufficiently long to ensure that the dispense tip can reach the bottom of the reagent reservoirs that are used. For example, the length can be between about <NUM> to <NUM>, or between about <NUM> to <NUM>, or between about <NUM> to <NUM>, or at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> in length. In some embodiments, the volume of the lumen of the dispense tip <NUM> can be sufficiently large to ensure that the entire reagent and/or sample volume that is to be dispensed can be held within the lumen of the dispense tip <NUM> in order to prevent the reagents and/or sample from being aspirated into the tubing, which may cause the waste of precious reagents. For example, the swept volume of the dispense tip <NUM> can be between <NUM> and <NUM> ul, or at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> ul. The outer diameter of the dispense tip <NUM> can be slightly greater than the diameter of the lumen of the inlet boss of the flow cell. The tip <NUM> of dispense tip <NUM> may be gently tapered to facilitate insertion into the lumen of the inlet boss and formation of a fluidic seal. In some embodiments, the taper can be between about <NUM> to <NUM> degrees, or be about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> degrees. A ferrule <NUM> can be placed on the proximal end of dispense tip in order to provide an attachment feature that can be used to attached the dispense tip to the dispensing head of the instrument. In addition, the ferrule also acts as: (<NUM>) a fluidic sealing surface between the instrument and the dispense tip, and an electrical connection between the instrument and the dispense tip so that the dispense tip can function as a sensor in a capacitive liquid level detection circuit. The ferrule may be uncoated and can be either straight or slightly tapered. In some embodiments, the dispense tip <NUM> may also be used to puncture a seal covering the inlet boss.

<FIG> illustrate an embodiment of a disposable dispense tip <NUM> that can be made of a plastic material, for example. The swept volume and/or the dimensions of the disposable dispense tip <NUM> can be the same or similar to that described above for the reusable dispense tip <NUM>. In some embodiments, the plastic material can be made of a conductive polymer or contain an embedded electrode in order for the dispense tip to function as a probe, such as a capacitive liquid level probe. In some embodiments, the disposable dispense tip <NUM> can be coated with a hydrophobic material, such as a fluoropolymer, similar to that as described above for metal dispense tips.

<FIG> illustrate an embodiment of a piercing tool <NUM> that can be used to create an opening in a seal covering the fluidic interface. In some embodiments, the piercing tool <NUM> can be made of metal or metal alloy, such as stainless steel. In other embodiments, the piercing tool <NUM> can be made of a polymer or ceramic material. The piercing tool <NUM> can have a distal end <NUM> with a sharp tip for piecing the seal and a proximal end <NUM> with an attachment feature <NUM> or mechanism, such as internal screw threads, for attaching the piercing tool <NUM> to an actuator.

In some embodiments, the distal end <NUM> can have a plurality of sharp cutting edges <NUM> and cutting faces <NUM> that can be angled between about <NUM> degrees to about <NUM> degrees from a longitudinal axis <NUM> that extends through an elongate shaft <NUM> of the piercing tool <NUM>. The angle of the cutting edges <NUM> and cutting faces <NUM> can be set along with the seal material and thickness such that the force required to pierce the seal can be between about <NUM> to <NUM> N. In some embodiments, the distal end <NUM> can have a two stage taper, with the first taper as described above with respect to the cutting edges <NUM> and cutting faces <NUM> located at the very end of the piercing tool. In some embodiments, a second tapering section <NUM> can extend proximally from the cutting edges <NUM> and cutting faces <NUM> at an angle that is more acute than the angle of the cutting edges <NUM> and cutting faces <NUM>. For example, the second tapering section <NUM> can have an angle that is about <NUM> to <NUM> degrees less the angle of the cutting edges <NUM> and cutting faces <NUM>. The second taper is designed to dilate the opening in the foil during the piercing step to a maximum diameter and minimize any `foil flaps' that might interfere with the dispensing of liquid from the dispense tip. To achieve these functions, the second taper can have a maximum diameter that is greater than the diameter of the receptacle to ensure that the opening in the foil is created all the way to the outer edges of the receptacle.

In some embodiments, the piercing tool <NUM> can have an elongate shaft <NUM> with at least two sections, a distal section <NUM> and a proximal section <NUM>, of different diameters, with the distal section <NUM> having a smaller diameter than the proximal section <NUM>. A third tapering section <NUM> can provide a transition between distal section <NUM> and the proximal section <NUM>. In some embodiments, the third tapering section <NUM> can have an angle between about <NUM> and <NUM> degrees. In some embodiments, the third tapering section <NUM> has an angle that is equal to or less than the second tapering section <NUM>.

<FIG> illustrate a method of piercing the seal of the flow cell and inserting the dispense tip into the fluidic interface in a manner that does not introduce air bubbles into the flow cell. In some embodiments, the diameter of the proximal section <NUM> can be greater than the opening of the receptacle <NUM> of the consumable device backer <NUM> into which the piercing device <NUM> is inserted, such that a location along the third tapering section <NUM> has a diameter equal to the diameter of the opening and functions as a stop to limit further advancement of the piercing tool <NUM> into the consumable device. In some embodiments, the piercing tool is driven to a set, target (i.e. predetermined) force to ensure that the tip of the piercing tool bottoms out against the stop in order to create the largest opening possible. In contrast, driving or inserting the piercing tool to a set depth past the seal, may create different sized openings in the foil due to positioning tolerances of the tip of the piercing tool and to differences in the conical shape of the piercing tool. In some embodiments, the length of the piecing tool <NUM> from the distal end <NUM> to the stop location <NUM> on the third tapering section <NUM> can be approximately equal to or less than the distance between the opening in the consumable device backer <NUM> to the top of the inlet boss <NUM> of the flow cell interface, in order to prevent the piercing tool <NUM> from being inserted into the inlet boss <NUM> of the flow cell interface.

<FIG> illustrate a reagent bottle cap <NUM> that can be also pierced by the piercing tool <NUM>. In some embodiments, the reagent bottle cap <NUM> can have a receptacle <NUM> for receiving the piercing tool <NUM>. In some embodiments, the receptacle <NUM> can be tapered and can be wider at the opening and narrower at the bottom <NUM>. The piercing tool <NUM> can have a length and diameter that allows the distal end <NUM> of the piercing tool <NUM> to be inserted into the receptacle far enough to pierce the bottom <NUM> of the receptacle <NUM>. In some embodiments, one of the tapering portions of the piercing tool <NUM>, such as the third tapering portion <NUM> has a diameter at a portion of the third tapering portion <NUM> that is equal to a diameter of an intermediate portion of the tapered receptacle <NUM>. The portion of the third tapering portion <NUM> with a matching diameter as the intermediate portion of the tapered receptacle functions as a stop, and the length of the piercing tool <NUM> from the portion of the third tapering portion <NUM> to the distal end <NUM> of the piercing tool <NUM> is greater than the length between the intermediate portion of tapered receptacle <NUM> to the bottom of the tapered receptacle <NUM> so that the piercing tool <NUM> penetrates through the bottom <NUM> of the tapered receptacle <NUM> when fully inserted into the receptacle <NUM>.

<FIG> illustrate a system and method of making a reliable fluid to fluid connection between the fluid in the dispense tip <NUM> and the fluid in the receptacle <NUM> of the consumable device backer, thereby preventing the inadvertent introduction of a bubble into the fluidics. As shown in <FIG>, the dispense tip <NUM> can have lumen that his filled with a fluid ready to be dispensed. However, a small amount of gas <NUM> (i.e. air) can be trapped at the distal end of the lumen of the dispense tip <NUM>. If the dispense tip <NUM> is inserted into the fluid in receptacle <NUM> and inserted into the inlet boss <NUM> when gas is trapped in the dispense tip <NUM>, gas <NUM> can be introduced into the fluidics when fluid is dispensed from the dispense tip <NUM>.

As shown in <FIG>, a small amount of fluid can be partially dispensed from the dispense tip so that a partial droplet <NUM> of fluid can extend from the distal end of the dispense tip <NUM>. This ensures that no gas is trapped within the lumen of the dispense tip <NUM> so that a fluid to fluid connection can be properly made between the droplet <NUM> and the fluid in the receptacle <NUM> when the dispense tip <NUM> is inserted into the receptacle <NUM>.

As shown in <FIG>, a covering, such as a foil or plastic sheet, can be used to seal the receptacles <NUM> before use. A piercing tool as described herein can be used to pierce the covering. In some embodiments, a flap <NUM> (i.e., a foil or plastic flap) can be formed from the covering after the covering has been pierced by the piercing tool. In some embodiments, this flap <NUM> can extend into the lumen of the receptacle <NUM> and can make contact with the distal end of the dispense tip <NUM> when the dispense tip <NUM> is inserted into the receptacle <NUM>. If fluid has been partially dispensed from the dispense tip <NUM> as a partial droplet <NUM> before the distal end of the dispense tip <NUM> has been inserted past the flap <NUM>, then the droplet <NUM> may be wicked from the distal end of the dispense tip <NUM>, which can result in a small amount of gas <NUM> at the distal end of the dispense tip <NUM> as shown in <FIG>. If the dispense tip <NUM> is then inserted into the fluid in the receptacle <NUM>, the gas <NUM> in the dispense tip <NUM> can be transferred into the inlet boss <NUM> and into the fluidic system. In some embodiments, to reduce the likelihood of wicking, the flaps <NUM> are pushed against the sidewalls of the receptacle <NUM> by a proximal portion of the piercing tool that can at least partially match the geometry of the upper portion of the receptacle adjacent to the foil cover.

As shown in <FIG>, to reduce the risk of wicking away the droplet <NUM> from the dispense tip <NUM>, the distal end of the dispense tip <NUM> can be inserted past the flap <NUM> before forming the partial droplet <NUM> at the distal end of the dispense tip <NUM>. For example, the distal end of the dispense tip <NUM> can be inserted into the receptacle <NUM> past the flap <NUM>. Then fluid can be partially dispensed from the distal end of the dispense tip <NUM> to expel any trapped gas in the lumen and to form a partial droplet <NUM> at the distal end of the dispense tip <NUM>. The dispense tip <NUM> with the partial droplet <NUM> can then be lowered to make the fluid to fluid connection with the fluid in the receptacle.

Although the foregoing embodiments have been described in some detail for purposes of clarity of understanding, the invention is not limited to the details provided. There are many alternative ways of implementing the invention. The disclosed embodiments are illustrative and not restrictive.

Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as defined by the claims.

The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of the invention. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claim 1:
A consumable device for use with a sequencing system, the consumable device comprising:
a sequencing chip, the sequencing chip comprising a plurality of wells, whereby each well comprises a working electrode and is a nanopore based sequencing cell;
a flow cell comprising:
at least one flow channel, wherein the flow cell is configured to be disposed over the sequencing chip (<NUM>, <NUM>) such that the at least one flow channel (<NUM>, <NUM>) is disposed over the plurality of wells of the sequencing chip (<NUM>, <NUM>);
at least one inlet boss (<NUM>) having a lumen (<NUM>) in fluid communication with the at least one flow channel (<NUM>, <NUM>) and
at least one counter electrode (<NUM>, <NUM>, <NUM>) disposed over at least a portion of the at least one flow channel (<NUM>, <NUM>), and characterized by
a flow cell cover comprising:
at least one inlet boss receptacle (<NUM>) for receiving the at least one inlet boss (<NUM>) of the flow cell; and
at least one dispense tip receptacle (<NUM>) configured to receive a dispense tip (<NUM>), wherein the at least one dispense tip receptacle (<NUM>) is in fluid communication with the at least one inlet boss receptacle (<NUM>).