WO2024234301A1

WO2024234301A1 - Improved flow cells, systems, and methods

Info

Publication number: WO2024234301A1
Application number: PCT/CN2023/094539
Authority: WO
Inventors: Jon BARTMAN; Fang-wei LIU; Victoria Ng; Xianyong Gu; Bing Du
Original assignee: MGI Tech Co Ltd
Current assignee: MGI Tech Co Ltd
Priority date: 2023-05-16
Filing date: 2023-05-16
Publication date: 2024-11-21
Anticipated expiration: 2025-11-16

Abstract

Variable-gap flow cells, and systems and methods using variable-gap flow cells. In one example, the variable-gap flow cell includes a substrate and a cover movably spaced from one another to define a variable fluid gap between them. An actuator may move one of the substrate and cover between a dispensing configuration and a processing configuration. In the dispensing configuration, the substrate and cover are spaced more widely apart such that a volume of reagent dispensed into the fluid gap is substantially less than the volume of the fluid gap. In the processing configuration, the substrate or cover has been moved by the actuator to narrow the fluid gap, such that the dispensed reagent spreads to substantially fill the narrowed fluid gap. In some implementations, variable-gap flow cells enable scaling of the flow cell for larger substrates while minimizing reagent consumption.

Description

IMPROVED FLOW CELLS, SYSTEMS, AND METHODS

BACKGROUND

Many current nucleic acid sequencing systems and processes are resource intensive, requiring significant amounts of reagents and a significant time to complete a sequencing process. Massively parallel systems and processes have been developed in an attempt to more efficiently use resources; however, there remains room for improvement.

One area for improvement is minimizing the flow gap in the flow cells used in the sequencing process. Minimizing the flow cell’s flow gap significantly reduces the reagents required to complete a sequencing process.

SUMMARY

This patent discloses several examples of improved flow cells, systems, and methods.

In one example, a variable-gap flow cell system includes a variable-gap flow cell. The variable-gap flow cell includes a substrate, a cover, a fluid inlet, and a fluid outlet. The substrate has an inner substrate surface. The cover has an inner cover surface facing the inner substrate surface. The substrate and the cover are movably spaced from one another to define a fluid gap between the inner cover surface and the inner substrate surface. The fluid inlet and the fluid outlet are in communication with the fluid gap between the inner cover surface and the inner substrate surface. In this example, at least one of the cover and substrate are movable between a first configuration of the variable-gap flow cell and a second configuration of the variable-gap flow cell, such that, in the second configuration, the fluid gap is narrower than in the first configuration. In this example, the system also includes an actuator configured to move at least one of the cover and the substrate between the first configuration and the second configuration.

In this example, the first configuration may be a reagent dispensing configuration and the second configuration may be a processing configuration.

In this example, when in the dispensing configuration, the system may be configured to dispense a volume of reagent into the fluid gap that is less than a volume of the fluid gap when in the dispensing configuration.

In this example, after dispensing the reagent into the fluid gap, the actuator may move at least one of the substrate and the cover from the dispensing configuration to the processing configuration such that the reagent substantially fills the fluid gap.

In this example, when in the dispensing configuration, the system may dispense a volume of reagent into a central area of the fluid gap; and the system may be configured to move at least one of the substrate and the cover from the dispensing configuration to the processing configuration such that the volume of reagent dispensed into the fluid gap spreads outward from the central area.

In this example, the actuator may be one of a pneumatic cylinder, a resilient element, or an expandable element.

In this example, the system may also include a substrate holder configured to receive and hold the substrate in a removable fashion.

In this example, the substrate holder may be a vacuum chuck.

In this example, the substrate holder may be a piston configured to move relative to the cover.

In this example, the holder may also include a fluid dispensing channel in fluidic communication with the fluid inlet of the flow cell.

In another example, a variable-gap flow cell includes a substrate, a cover, and a fluid inlet and outlet. The substrate includes an inner substrate surface. The cover includes an inner cover surface facing the inner substrate surface, the substrate and the cover movably spaced from one another to define a fluid gap between the inner cover surface and the inner substrate surface. The fluid inlet and the fluid outlet are in communication with the fluid gap between the inner cover surface and the inner substrate surface. In this example, at least one of the cover and substrate are movable between a first configuration of the variable-gap flow cell and a second configuration of the variable-gap flow cell, such that, in the second configuration, the fluid gap is narrower than in the first configuration.

In this example, the variable-gap flow cell may include a stop defining a minimum fluid gap height, the stop configured to limit movement of at least one of the substrate and the cover towards the other.

In this example, the stop may extend from one of the inner reaction surface or the inner cover surface.

In this example, the inner substrate surface may include an array of analyte binding sites.

In this example, the cover may be optically transparent to at least some wavelengths of light.

In another example, a method of using a variable-gap flow cell includes: (a) dispensing a volume v₁ of a first reagent into a fluid gap of a variable-gap flow cell; the variable- gap flow cell including a substrate, a cover, a fluid inlet, and a fluid outlet, the fluid gap extending between the substrate and the cover; and (b) moving at least one of the substrate and the cover to narrow the fluid gap of the variable-gap flow cell such that the volume v₁ of the first reagent spreads to substantially fill the narrowed fluid gap.

In this example, during dispensing of the first reagent, the fluid gap of the variable-gap flow cell may define a volume v₂ that is greater than the volume v₁ of the first reagent being dispensed.

In this example, after narrowing the fluid gap, the fluid gap may define a volume v₃ that is approximately the same as the volume v₁of the dispensed first reagent.

In this example, moving at least one of the substrate and the cover to narrow the fluid gap may include narrowing the fluid gap until a stop of the variable-gap flow cell is contacted.

In this example, the method may also include, after a processing time, washing the volume v₁of the first reagent from the fluid gap.

In this example, the method may also include moving at least one of the cover and the substrate to widen the fluid gap of the variable-gap flow cell, and also dispensing a volume v₄ of a second reagent into the widened fluid gap, the volume v₄ approximately the same as the volume v₁.

In this example, the method may also include, after dispensing the second reagent into the widened fluid gap, moving at least one of the cover and the substrate to narrow the fluid gap such that the dispensed volume v₄ of the second reagent spreads to substantially fill the narrowed fluid gap.

In this example, the method may also include imaging the substrate through the cover.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows an example of a flow cell with a flow gap.

FIG. 2 charts fluid surface tension versus flow gap height.

FIG. 3 charts fluid filling pressure versus filling time for different flow gap heights.

FIG. 4a-d show an example of a variable-gap flow cell.

FIG. 4e shows an example process flow for the variable-gap flow cell of FIGS. 4a-d.

FIGS. 5a-d illustrate an example of a variable-gap flow cell system using a pneumatic cylinder actuator.

FIGS. 6a-d illustrate an example of a variable-gap flow cell system using a resilient element actuator.

FIG. 7 illustrates an example of a variable-gap flow cell system using an expandable element actuator.

FIGS. 8a-e illustrate an example of a variable-gap flow cell system using a separable flow cell.

DETAILED DESCRIPTION OF FIGURES

Flow Cell Gap Minimization

Some of the examples of flow cells, systems, and methods described in this patent provide for minimization of the flow cell’s flow gap, which helps to minimize reagent consumption during sequencing processes.

FIG. 1 shows an example of a flow cell 10 usable in a sequencing system. The flow cell 10 includes a substrate 12 and a cover 14. The substrate 12 has an inner substrate surface 16 and the cover has an inner cover surface 18 facing the inner substrate surface 16, with surfaces 16, 18 spaced apart by a height h to define a fluid gap 20 between them. The flow cell 10 also includes a fluid inlet 22 and a fluid outlet 24.

The height h of fluid gap 20 has a significant impact on the operation and performance of a sequencing system. The greater the height h, the greater the volume of reagents that the system will require to operate. On the other hand, reducing the height h has the potential to significantly increase resistance to fluid flow (due to fluid surface tension and viscosity) and pressure on the flow cell 10. FIG. 2 shows that resistance to flow exponentially increases as the fluid gap height decreases between the two plates of a flow cell (e.g. the substrate 12 and cover 14 in FIG. 1) . FIG. 3 shows that filling pressure increases significantly as the fluid gap height decreases and as filling time is sped up. High pressures can cause failure of the flow cell, including due to failure of the adhesive used to assemble the flow cell. These increases in resistance to fluid flow and pressure place practical limitations on how narrow of a flow gap could be employed in prior art flow cells. This patent provides several examples of improved flow cells that allow for narrowing of a flow cell gap while limiting increases in resistance of fluid flow and pressure.

The flow cells described in the following examples may be used in a wide variety of implementations, including systems where larger surface area flow cells are desired while at the same time minimizing reagent consumption.

In some implementations, the flow cells may be used as part of a sequencing system for analyzing nucleic acid material such as DNA or RNA, or other biological or non-biological/synthetic material to be analyzed. The flow cell substrate may include an array of analyte attachment sites for attaching nucleic acid fragments or other analyte to the flow cell substrate (e.g. discrete attachment sites 26 schematically shown in FIG. 1–which may be positively charged features on the substrate separated and spaced apart by negatively charged or neutral areas of the substrate) . In some implementations, the number of discrete attachment sites may be arranged in arrays that include up to millions or billions of discrete sites, spaced at pitches that may be on the order of tens or hundreds of nanometers.

The nucleic acid fragments attached to the flow cell substrate may be imaged by an optical imaging system or otherwise analyzed. For example, DNA templates may be immobilized at greater than 10e7 attachment sites in an array on the flow cell substrate. In this example, a nucleic acid sequencing method may involve carrying out greater than 400 sequencing cycles. In each cycle, single nucleotides (e.g., adenine, guanine, thymine, and cytosine) may be flowed across the substrate through the fluid gap 20 and incorporated (into a growing strand) at each site where there is a complementary nucleotide base. In one approach, each of the four different nucleotides may be labeled with a different color fluorescent dye or bound by a dye-labeled antibody. In each sequencing cycle, a light source (e.g., a laser) may illuminate the spots (e.g., in series) , causing the dye to emit light corresponding to the respective colors. The color emitted at each spot from one of the four dyes may be detected by a camera (e.g., a time delay integration charge-coupled device (TDI-CCD) camera or a similar camera) , and the imaging system may thereby record, for each spot, the detection of a nucleotide corresponding to the detected color. Persons knowledgeable in the art will be aware of variations in sequencing methods including variations in template type (see, e.g., Huang et al., 2017, Gigascience 6: 1–9; Mardis et al., 2013, Annu Rev Anal Chem 6: 287–303) , labeling systems (see, e.g., WO2018129214) and labeling strategies (see, e.g., US 9,523,125) .

The attachment sites 26 on the substrate 12 of flow cell 10 may be fabricated by well-known lithography tools, such as 248-nm KrF (krypton fluoride) , 193-nm ArF (argon-fluoride) lithography systems, or e-beam lithography systems. The arrays are typically separated with spaces between each other in ultra-high density, high density, medium density, or low density. At ultra-high density, separation is less than 250nm. At high density, separation is 300 to 350nm. At medium density, separation is 400nm to 500nm. At low density, separation is 500nm or more. In some implementations (for example, some low density implementations) 2-dimensional patterning with photoresist is sufficient to sequester DNA nanoballs or other discrete nucleic acid samples. In some implementations (for example, some medium, high, or ultra-high density implementations) , to reduce risk that discrete samples will not remain in single locations, smaller samples may be required, which may require 3-dimensional patterning for more efficient capturing of fluorescence from tagged DNA nanoballs or other tagged nucleic acid samples. In such implementations, 3-dimensional patterned well nanostructures can be developed by non-binding material as a well wall and binding material for the well bottom surface for sequestering DNA nanoballs.

Variable-Gap Flow Cell–FIGS. 4a-e

FIGS. 4a-d shows one example of a variable-gap flow cell 100. The flow cell 100 includes a substrate 102 and a cover 104. The substrate 102 has an inner substrate surface 106 and the cover 104 has an inner cover surface 108 facing the inner substrate surface 106, with surfaces 106, 108 spaced apart to define a fluid gap 110 between them. The flow cell 100 also includes a fluid inlet 114 centrally located in the substrate and fluid outlets 116 for dispensing and removing fluid from the fluid gap 110.

As shown in FIGS. 4a-b, the substrate 102 can move relative to the cover 104, from the configuration shown in FIG. 4a where the fluid gap is of height h₁, to the configuration shown in FIG. 4b where the fluid gap 110 is of height h₂, h₂ being smaller than h₁. In this example, when the flow cell 100 is in the configuration shown in FIG. 4b, the fluid gap 110 is narrower and defines a smaller fluid volume than when the flow cell 100 is in the configuration shown in FIG. 4a.

In this example, the substrate 102 is mounted on an actuator 116 that moves the substrate 102 relative to the cover 104, although, in other examples, the cover 104 could be mounted on an actuator to move the cover 104 relative to the substrate 102. The substrate 102 includes stops 118 limiting movement of the substrate 102 towards the cover 104 and defining a minimum height (h₂) of the fluid gap 110. In other examples, stops could be incorporated into a cover or other parts of the flow cell.

FIG. 4a depicts the flow cell 100 in a fluid dispensing configuration and FIG. 4b depicts the flow cell 100 in a processing configuration. As shown in FIG. 4c, when in the fluid dispensing configuration, a predetermined volume of fluid 120 may be dispensed into the fluid gap 110. In this example, the pre-determined volume of fluid 120 may be determined as the volume of fluid that is less than the volume of the fluid gap 110 when at height h₁, but that will substantially fill the fluid gap 110 when the gap is reduced to height h₂ as shown in FIG. 4d. In some implementations, “substantially” filled may constitute, when the gap height is reduced to height h₂, approximately 120%-80%filled (percentages greater than 100%accounting for excess fluid that enters fluid outlets 114 or other fluid passageways outside of the fluid gap 110) , 110%-90%filled, 105%-95%filled, 102%-98%filled, 101%-99%filled, at least 100%filled, at least 99%filled, at least 98%filled, at least 97%filled, at least 96%filled, at least 95%filled, at least 90%filled, or at least 80%filled.

As already noted, the predetermined volume of fluid 120 is less than the volume of the fluid gap 110 when the fluid gap 110 is at height h₁. The larger gap height h₁ reduces fluidic resistance to flow during fluid dispensing. As shown in FIG. 4d, after the predetermined volume offluid 120 has been dispensed into the fluid gap 110, the substrate 102 may be moved towards the cover 104 until the stops 118 contact the cover inner surface 108, reducing the gap height to h₂. Reducing the gap height to h₂ causes the volume of fluid 120 to spread out, substantially filling the fluid gap 110. Once substantially filled, the stops 118 and the hydraulic force of fluid 120 supports the cover 104 in an equilibrium state. In some implementations, equilibrium may be further maintained by regulating system pressure.

The variable-gap flow cell 100 beneficially provides for initial dispensing of fluid into the flow cell at relatively low fluidic resistance, when the flow cell gap is at a relatively large height, while also providing for substantial filling of the flow cell fluid gap 110 when the gap height is reduced to its minimum height. This may be a particularly efficacious system for filling flow cells that have substrates with relatively large surface areas for reactions, while at the same time allowing for minimal fluid gap heights during processing, thereby minimizing fluid use in the process.

FIG. 4e shows an example process flow for the variable-gap flow cell 100 of FIGS. 4a-d, which in this example is part of a genetic sequencing process flow. At step 1002, the flow cell 100 is placed on an actuator 116. In this example, when the flow cell 100 is placed on the actuator 116, the flow cell 100 is in the configuration shown in FIG. 4b, with the flow cell fluid gap 110 at its minimum height h₂. Subsequently, at step 1004, actuator 116 lowers substrate 102 to increase the flow cell fluid gap 110 to height h₁. In this particular example, flow cell fluid gap height is increased by approximately 0.2 to 1 mm at step 1004. In other examples, flow cell fluid gap height is increased by less than 0.2 mm or greater than 1 mm. Subsequently, at step 1006, a predetermined volume of a first reagent is dispensed through the fluid inlet 112 of substrate 10 as shown in FIG. 4c, at a central location on the substrate 102. In this example, the predetermined volume of the reagent is a volume that does not substantially fill the flow cell fluid gap 110 when it is at height h₁, but will substantially fill the flow cell fluid gap 110 when it is narrowed to height h₂. Subsequently, at step 1008, the actuator 116 raises the substrate 102 until stops 118 contact the inner surface 108 of cover 104, thereby narrowing the fluid gap 110 to its minimum height h₂, changing the flow cell 100 from its dispensing configuration to its processing configuration. In this particular example, height h₂ is approximately 0.25 mm or smaller. In other examples, height h₂ is approximately 0.5 mm or smaller. Subsequently, at step 1010, the flow cell is ramped to a processing temperature, and at step 1012, the flow cell is maintained at the processing temperature for a given amount of time. Subsequently, at step 1012, fluid inlet 112 and outlets 118 facilitate introduction of a buffer fluid and washing of the reagent from the flow cell 100. Subsequently, at step 1014, steps 1004-1012 may be repeated for as many additional reagent and washing cycles as required for the particular analysis being carried out. Subsequently, at step 1016, a reagent to facilitate optical scanning may be dispensed into the flow cell 100, and flow cell 100 may be optically scanned.

Variable-Gap Flow Cell With Pneumatic Cylinder Actuation–FIGS. 5a-d

FIGS. 5a-d show an example of a variable-gap flow cell using a pneumatic cylinder to move the flow cell substrate between a dispensing configuration and a processing configuration. In this example, the flow cell substrate is held by a vacuum chuck piston. As shown in FIG. 5b, the vacuum chuck piston is held between top and bottom sealing plates, with the top plate including a recess for receiving a glass coverslip forming the flow cell cover.

As shown in FIG. 5c, the glass coverslip may be glued into the top sealing plate. Glue trenches in the top sealing plate allow for adequate fixation of the glass coverslip to the top sealing plate, while limiting the spread of glue to undesired areas. In this example, the glass coverslip is optically transparent to at least some wavelengths of light, facilitating imaging of the flow cell substrate and analyte and/or other particles attached thereto. In other implementations, a flow cell cover may be attached or otherwise secured in a holder in another fashion.

As also shown in FIG. 5c, the vacuum chuck piston may have a recessed top surface for receiving a silicon wafer flow cell substrate (the substrate not being shown in this figure) . Grooves formed in the recessed top surface communicate with vacuum ports, such that application of negative pressure to those ports will retain the flow cell substrate in the recessed surface. Flow cell gap-setting shims act as the stops in this example, and limit the vertical translation of the vacuum chuck piston, thereby defining a minimum height of the fluid gap between the substrate and cover.

A fluidic chamber sealing O-ring extends around the perimeter of the vacuum chuck piston, and seals against the inner cylindrical wall of the top sealing plate. In this fashion, the lower surface of the glass coverslip and the top surfaces of the vacuum chuck piston and substrate define a fluidically sealed flow cell (with the exception of the fluid inlet port in the piston and fluid outlets (not shown in the figures) ) . The gap-setting shims defining the height of the fluid gap between those surfaces when the vacuum chuck piston is vertically translated to its maximum height.

As shown in FIG. 5d, the bottom sealing plate includes ports for air cylinder actuation, such that the piston can be vertically translated in an upward direction relative to the sealing plates by increasing air pressure in a cavity under the back surface of the piston.

Variable-Gap Flow Cell With Spring Actuation–FIGS. 6a-d

FIGS. 6a-d show an example of a variable-gap flow cell system using spring actuation to move the flow cell substrate between a dispensing configuration and a processing configuration. FIGS. 6a-b show the system in an assembled state from the top and bottom respectively. FIG. 6c shows the system in a disassembled state. FIG. 6d shows the system in cross-section.

In this example, the variable-gap flow cell system includes upper and lower plates 430, 432, with the plates having cylindrical openings for receiving a cylindrical vacuum chuck holder 434. Vacuum chuck holder 434 can vertically translate in the cylindrical openings and uses vacuum pressure to hold flow cell substrate 402 on an upper surface of the holder 434. A seal 436 extends around the holder 434. Upper plate 430 has a recess in its top surface for receiving a glass cover (not shown) , which may be glued into the upper plate or secured in another manner. A fluid inlet 412 and a fluid outlet (not shown) allow dispensing fluids into and removing fluids from the fluid gap between the substrate 402 and cover.

In this example, resilient members (e.g. springs, one of which is shown in Fig. 4c at 438) extend between the vacuum chuck holder 434 and the lower plate 432. Screws (one of which is shown in Fig. 4c at 440) can be rotated to compress the resilient members, which causes the resilient members to exert an upward force on the vacuum chuck holder 434, resulting in the vacuum holder and substrate thereon vertically translating upwardly in the openings in plates 430, 432, narrowing the fluid gap between the substrate 402 and cover.

Variable-Gap Flow Cell With Expanding Seal–FIG. 7

FIG. 7 shows an example of a variable-gap flow cell system that uses an expanding seal to adjust the height of the fluid gap in the flow cell. In FIG. 7, a flow cell substrate 502 is mounted on top of a vacuum fixture 534. The vacuum fixture 534 extends through openings in lower and upper plates 530, 532. A flow cell cover 504 is mounted on top of plate assembly 530, 532 to define a flow cell fluid gap between the substrate 502 and cover 504. Fluid inlet 512 and fluid outlet 514 allow for dispensing fluid into and removing fluid from the fluid gap of the flow cell.

Expandable element 540 supports the vacuum fixture 534 in the openings of the lower and upper plates 530, 532. The expandable element 540 can be expanded to translate vacuum fixture 534 and flow cell substrate 502 vertically relative to the upper and lower plates 430, 432, to move flow cell substrate 502 closer to flow cell cover 504, narrowing the fluid gap of the flow cell. In this particular example, expandable element 540 is an expandable seal that seals a gap between an outer edge of the substrate 502 and the opening in upper plate 530. The expandable element 540 includes an interior cavity 542 in fluid communication with a pressure inlet 544. Increased pressure in the interior cavity 542 expands the element 540, resulting in the vacuum fixture 534 and substrate 502 thereon vertically translating upwardly in the openings through plates 530, 532, narrowing the fluid gap between the flow cell substrate 502 and cover 504.

Variable-Gap Flow Cell With Separable Flow Cell–FIGS. 8a-e

FIGS. 8a-e show an example of a separable variable-gap flow cell in which a substrate assembly can be separated from the rest of the flow cell, reducing the number of disposable parts of the system.

FIG. 8a shows the substrate assembly 650, which includes a ring-shaped substrate holder 652, seal 654, and flow cell substrate 602.

FIG. 8b shows the substrate assembly 650 in relation to the reusable parts of the system, shown in a disassembled condition. The reusable parts include a vacuum fixture 634 on which the substrate assembly 650 is mounted, a frame 656 with a flow cell cover 604 secured on top of the frame 656, and a vacuum platen 658 that can be moved to translate the frame 656 and flow cell cover 604 vertically relative to the substrate assembly 650, thereby adjusting the flow cell fluid gap between the substrate 602 and cover 604.

FIGS. 8c-e illustrate automation steps for the separable flow cell in this example. In FIG. 8c the substrate assembly 650 is placed on vacuum fixture 634. Negative pressure is applied to the underside of the substrate assembly 650/substrate 602 via vacuum supply 660, holding substrate assembly 650 in place on top of vacuum fixture 634. Placing substrate assembly 650 on vacuum fixture 634 also aligns the fluid inlet 662 of the vacuum fixture 634 (see FIG. 8b) with a central fluid inlet 612 extending through the substrate 602.

Next, as shown in FIG. 8d, the flow cell frame 656 and cover 604 are placed over the substrate assembly 650, thereby forming a flow cell fluid gap between substrate 602 and cover 604. As shown in FIG. 8b, seal 654 of the substrate assembly 650 seals against an inner wall of the frame 656, and fluid outlet 664 extending through frame 656 fluidically connects to the flow cell fluid gap between substrate 602 and cover 604.

Next, as shown in FIG. 8e, vacuum platen 658 is lowered into contact with cover 604, and negative pressure is applied to engage the cover 604 and frame 656 assembly with the vacuum platen 658. An actuator (not shown) associated with the vacuum platen 658 may be used to vertically translate the cover 604 and frame 656 assembly relative to the substrate assembly 650 on the fixture 634, thereby raising or lowering the flow cell fluid gap height between the substrate 602 and cover 604.

While the principles of the disclosure have been described above in connection with specific examples of flow cells, systems, and methods, it is to be understood that this description is made only by way of example and not as limitation on the scope of the present inventions. Examples were chosen and described in order to explain the principles of the invention and practical applications to enable others skilled in the art to utilize the invention in various implementations and with various modifications, as are suited to a particular use contemplated. It will be appreciated that the description is intended to cover modifications and equivalents.

Claims

A variable-gap flow cell system, the system comprising:

(a) a variable-gap flow cell comprising:

(i) a substrate, the substrate comprising an inner substrate surface;

(ii) a cover, the cover comprising an inner cover surface facing the inner substrate surface, the substrate and the cover movably spaced from one another to define a fluid gap between the inner cover surface and the inner substrate surface;

(iii) a fluid inlet and a fluid outlet in communication with the fluid gap between the inner cover surface and the inner substrate surface; and

(iv) at least one of the cover and substrate movable between a first configuration of the variable-gap flow cell and a second configuration of the variable-gap flow cell, wherein, in the second configuration, the fluid gap is narrower than in the first configuration;

(b) an actuator configured to move at least one of the cover and the substrate between the first configuration and the second configuration.
The variable-gap flow cell system of claim 1, wherein the first configuration comprises a reagent dispensing configuration and the second configuration comprises a processing configuration.
The variable-gap flow cell system of claim 2, wherein, when in the dispensing configuration, the system is configured to dispense a volume of reagent into the fluid gap that is less than a volume of the fluid gap when in the dispensing configuration.
The variable-gap flow cell system of claim 3, wherein, after dispensing the reagent into the fluid gap, the actuator is configured to move at least one of the substrate and the cover from the dispensing configuration to the processing configuration such that the reagent substantially fills the fluid gap.
The variable-gap flow cell system of claim 2, wherein, when in the dispensing configuration, the system is configured to dispense a volume of reagent into a central area of the fluid gap; and wherein the system is configured to move at least one of the substrate and the cover from the dispensing configuration to the processing configuration such that the volume of reagent dispensed into the fluid gap spreads outward from the central area.
The variable-gap flow cell system of claim 1, wherein the actuator comprises one of a pneumatic cylinder, aresilient element, or an expandable element.
The variable-gap flow cell system of claim 1, further comprising a substrate holder configured to receive and hold the substrate in a removable fashion.
The variable-gap flow cell system of claim 7, wherein the substrate holder is a vacuum chuck.
The variable-gap flow cell system of claim 7, wherein the substrate holder is a piston configured to move relative to the cover.
The variable-gap flow cell system of claim 7, wherein the holder further comprises a fluid dispensing channel in fluidic communication with the fluid inlet of the flow cell.
A variable-gap flow cell comprising:

(a) a substrate, the substrate comprising an inner substrate surface;

(b) a cover, the cover comprising an inner cover surface facing the inner substrate surface, the substrate and the cover movably spaced from one another to define a fluid gap between the inner cover surface and the inner substrate surface;

(c) a fluid inlet and a fluid outlet in communication with the fluid gap between the inner cover surface and the inner substrate surface; and

(d) at least one of the cover and substrate movable between a first configuration of the variable-gap flow cell and a second configuration of the variable-gap flow cell, wherein, in the second configuration, the fluid gap is narrower than in the first configuration.
The variable-gap flow cell of claim 11, further comprising a stop defining a minimum fluid gap height, the stop configured to limit movement of at least one of the substrate and the cover towards the other.
The variable-gap flow cell of claim 12, wherein the stop extends from one of the inner reaction surface or the inner cover surface.
The variable-gap flow cell of claim 12, wherein the inner substrate surface comprises an array of analyte binding sites.
The variable-gap flow cell of claim 11, wherein the cover is optically transparent to at least some wavelengths of light.
A method of using a variable-gap flow cell, the method comprising:

(a) dispensing a volume v₁ of a first reagent into a fluid gap of a variable-gap flow cell; the variable-gap flow cell comprising a substrate, acover, afluid inlet, and a fluid outlet, the fluid gap extending between the substrate and the cover;

(b) moving at least one of the substrate and the cover to narrow the fluid gap of the variable-gap flow cell such that the volume v₁ of the first reagent spreads to substantially fill the narrowed fluid gap.
The method of claim 16, wherein, during dispensing of the first reagent, the fluid gap of the variable-gap flow cell defines a volume v₂ that is greater than the volume v₁ of the first reagent being dispensed.
The method of claim 17, wherein, after narrowing the fluid gap, the fluid gap defines a volume v₃ that is approximately the same as the volume v₁ of the dispensed first reagent.
The method of claim 18, wherein moving at least one of the substrate and the cover to narrow the fluid gap comprises narrowing the fluid gap until a stop of the variable-gap flow cell is contacted.
The method of claim 16, further comprising, after a processing time, washing the volume v₁ of the first reagent from the fluid gap.
The method of claim 20, further comprising moving at least one of the cover and the substrate to widen the fluid gap of the variable-gap flow cell, and further comprising dispensing a volume v₄ of a second reagent into the widened fluid gap, the volume v₄ approximately the same as the volume v₁.
The method of claim 21, further comprising, after dispensing the second reagent into the widened fluid gap, moving at least one of the cover and the substrate to narrow the fluid gap such that the dispensed volume v₄ of the second reagent spreads to substantially fill the narrowed fluid gap.
The method of claim 20, further comprising, imaging the substrate through the cover.