Nanofluidic flow cell and method of loading same

A flow cell for confining molecules in a fluid. The flow cell includes an upper substrate, an upper support member, a center substrate, a membrane, a lower support member and a lower substrate. The lower support member comprises an imaging chamber it is positioned below the membrane and above the lower substrate. In one embodiment the membrane comprises a nanopore and nanoscale groove extending through the membrane. In another embodiment the lower substrate comprises an upper face in communication with the imaging chamber, and the upper face comprises a plurality of nanoscale grooves extending partially through the lower substrate. In both embodiments the upper substrate, upper support member, center substrate and membrane are displaceable through the imaging chamber, thereby causing molecules in the imaging chamber to be confined or trapped through the nanoscale groove(s) of the membrane or of the lower substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

TECHNICAL FIELD

The application relates generally to molecular analysis and, more particularly, to a flow cell for same.

BACKGROUND

The direct visualization, manipulation, and quantification of long, delicate biopolymers is a challenge faced by emerging biotechnologies. Establishing long-range structural information when analyzing genomic DNA, protein-DNA complexes, or other biopolymers can be limited by polymer breakage within devices during handling.

SUMMARY

In one aspect, there is provided a flow cell for confining molecules in a fluid, comprising: a first substrate and a second substrate being spaced apart by support members, the first and second substrates and the support members defining a fluidic chamber to receive the fluid, at least one of the first and second substrates having a nanoscale surface topography including at least one nanoscale groove, at least one of the first and second substrates being displaceable through the fluidic chamber to contact the first substrate against the second substrate, contact between the first substrate and the second substrate causing displacement of the molecules into the at least one nanoscale groove.

In another aspect, there is provided a method of loading a flow cell, comprising: providing molecules in a fluid between spaced-apart first and second substrates, at least one of the first and second substrates having a nanoscale surface topography including at least one nanoscale groove extending into said substrate; and displacing at least one of the first and second substrates to contact the first substrate against the second substrate, contact between the first substrate and the second substrate causing displacement of the molecules in the fluid into the at least one nanoscale groove and confining the molecules therein.

In a further aspect, there is provided a method of loading a flow cell, comprising: deforming at least part of the flow cell to confine a biological molecule within a nanoscale groove of the flow cell such that a first end of the biological molecule is proximate to a second end of the biological molecule.

DETAILED DESCRIPTION

FIG.1Ashows an instrument10for manipulating a nanofluidic flow cell20. The instrument includes a microfluidic chuck11for receiving the flow cell20. The chuck11is mounted to a sample holder12, which is secured in place with a clamp13. As will be described in greater detail below, various portions and components of the flow cell20can be displaced by the instrument10to analyse molecules within the flow cell20. In the depicted embodiment, the instrument10has a deflection rod14having a rounded or pointed end14A which presses against portions of the flow cell20. Vertical displacement of the deflection rod14is controlled by a Z-axis piezoelectric actuator15, which provides for 100 μm travel of the deflection rod14along the Z-axis. The vertical translation of the pointed end14A of the deflection rod14brings it into contact with the top surface of the flow cell20. The position of the deflection rod14on the X-Y plane of the instrument10is controlled by a horizontal micropositioner16. The micropositioner16is operable to effect minute displacements in the X-Y plane, for example at the level of a micron, of the deflection rod14to align the deflection rod14with the portion of the flow cell20to be displaced.

FIG.1Bshows the flow cell20, which in the depicted embodiment, is used for imaging molecules confined within the flow cell20. The imaging technique in the depicted embodiment is optical microscopy, and more particularly, fluorescence microscopy. Since the flow cell20is used to displace and confine the molecules, as described in more detail below, it can be used for other molecular analysis or manipulation purposes. Non-limiting examples of other purposes include sequencing base pairs, enabling biomarker detection, and identifying and/or characterizing a biomolecule using larger-scale properties rather than single-base measurements. The flow cell20in the depicted embodiment is used to confine and analyse DNA molecules in a fluid solution. It will be appreciated that the flow cell20can be used to analyse or manipulate other molecules and biological molecules in a fluid solution, and is not limited to being used only with DNA molecules.

The flow cell20includes a first substrate21and a second substrate22. One or both of the first and second substrates21,22has a surface upon structures on the scale of nanometers are formed in order to confine the molecules for analysis. The first and second substrates21,22are spaced apart by one or more support members23. In the depicted embodiment, the support members23are the walls of the substrates21,22. In an alternate embodiment, the support members23are spacers or posts.

The first and second substrates21,22and the support members23collectively define a fluidic chamber24for receiving the molecules. The first and second substrates21,22and the support members23define the boundaries of the fluidic chamber24and prevent the solution containing the molecules from leaking out. The fluidic chamber24is therefore a sealed chamber. The expression “fluidic chamber24” refers to a volume or spaced defined by the structure of the flow cell20with typical characteristic dimensions within the range of 1-100 nm when its geometry is confined, as discussed in greater detail below. The fluidic chamber24and/or substrates21,22may include one or more inlets or outlets for admitting or releasing fluid from within the fluidic chamber24.

Still referring toFIG.1B, the second substrate22has a nanoscale surface topography25. The nanoscale surface topography25is an arrangement of nanoscale structures on the second substrate22. More particularly, the second substrate22is patterned with one or more nanoscale structures such that biological or other molecules can be “loaded” into the nanoscale structures and confined therein. A “nanoscale structure” refers to a structure having one or more dimensions at the nanometer level, which is typically between 0.1 nm and 100 nm. Examples of such nanoscale structures include, but are not limited to, textured surfaces having one dimension on the nanoscale, tubes having two dimensions on the nanoscale, and particles having three dimensions on the nanoscale. Examples of nanoscale textured surfaces include, but are not limited to, grooves, channels, pits, and ridges. Examples of nanoscale tubes include, but are not limited to, structures having geometries resembling tubes, solid rods, whiskers, and rhomboids with square, rectangular, circular, elliptical, and other polygonal cross-sections perpendicular to an axis of the tube. Examples of nanoscale particles include, but are not limited to, structures having geometries representing spheres, pyramids, and cubes. The cross-sectional geometry of nanoscale tubes and nanoscale particles may not be constant such that a nanoscale structure may taper in one or two dimensions. The flow cell20therefore has a nanoscale surface-embedded topography25, or “NanoSET”.

Nanoscale structures such as linear groove arrays, concentric circular grooves, rectangular wells, ring-shaped groove arrays, and pit arrays can be fabricated on 100 mm (4″) diameter, 0.17 mm thick D263 borosilicate glass wafers forming the substrates21,22. Examples of such features include 27×27×200,000 nm3and 50×50×200,000 nm3nanoscale channels, 50×600×600 nm3and 50×900×900 nm3nanoscale pits fabricated using electron-beam lithography and reactive ion etching (RIE). 1 μm deep microchannels connecting the nanoscale structures for fluidic coupling to the external microfluidic circuit can also be formed.

In the embodiment ofFIG.1B, the nanoscale surface topography25includes multiple nanoscale grooves25A that extend into the second substrate22from a surface22A thereof. The fluidic chamber24includes the volume of the nanoscale grooves25A. In the embodiment ofFIG.1C, both the first and the second substrate21,22have a nanoscale surface topography25. The nanoscale surface topography25for the second substrate22includes multiple nanoscale grooves25A that extend into the second substrate22from the surface22A. The nanoscale surface topography25′ for the first substrate21also includes multiple nanoscale grooves25A′ that extend into the first substrate21from the surface21A. The fluidic chamber24includes the volume of the nanoscale grooves25A,25A′. The nanoscale grooves25A′ form nanoscale posts25B extending outwardly from a recessed surface of the first substrate21. In the depicted embodiment, each nanoscale post25B has a height of 20 nm. Other heights and widths for the nanoscale posts25B are possible. The nanoscale posts25B help to create a nanoscale gap between the first and second substrates21,22when they are pressed together into stable contact.

In the embodiment ofFIG.1B, the first and second substrates21,22are two 25×25 mm2glass plates separated by a 10 μm vertical support member23around their edges. The first, or “upper”, substrate21has two small inlet holes near the corners for fluid insertion into the fluidic chamber24. The first substrate21forms a deformable lens to vary the fluidic chamber24by deforming the first glass substrate21using the pointed end14A of the deflection rod14. The first substrates21is shown as a convex lens, mounted curved face down. Supports17space the second, or “lower”, substrate22from an illumination/viewing lens18. In the depicted embodiment, the convex lens defined by the first substrate21becomes spherical when deformed, and the curved deformation of the first substrate21occurs along the Z-axis perpendicular to the length of the first substrate21.

It will therefore be appreciated that one or both of the first and second substrates21,22is displaceable toward each other. Still referring toFIG.1B, at least a central part of the first substrate21is displaceable by deformation caused by the displacement rod14to contact the part of the first substrate21against the second substrate22. In an alternate embodiment, the first substrate21is displaced by translation toward the second substrate22. It will be appreciated that the displacement of the first and second substrates21,22is a relative displacement, in that either one of the first and second substrates21,22can be displaced to provide contact between their surfaces21A,22A. The effect of the displacement of the first substrate21is to cause the molecules in the fluid within the fluidic chamber24to be displaced toward the nanoscale grooves25A and confined therein. More particularly, as the volume of the fluidic chamber24is decreased by the displacement of the first substrate21toward the second substrate22, the molecules are increasingly urged towards the nanoscale grooves25A until they remain trapped within the nanoscale grooves25A by contact between the surfaces21A,22A. The molecules when they are within the one or more nanoscale grooves25A are trapped therein by the walls25C of the nanoscale grooves25A, and by the surface21A of the first substrate21. The size, and thus the volume, of the fluidic chamber24varies with the displacement of the first substrate21. The fluidic chamber24before displacement of the first substrate has dimensions on the micron scale, or at least hundreds of nanometers, when the fluid is received therein. When the volume of the fluidic chamber24is decreased by displacement of the first substrate, the fluidic chamber24is squeezed to the nanoscale and made very thin. In such a squeezed configuration, the fluidic chamber24becomes a nanofluidic chamber.

An exemplary use of the flow cell20is now described, with reference toFIG.1B. After the fluid solution containing the DNA molecules is added into the fluidic chamber24, the pointed end14A of the deflection rod14is lowered into contact with the top of the first substrate21. Immersion oil can be used to prevent laser reflections between the lens of the instrument10and the first substrate21. The first substrate21is deformed into contact with the second substrate22and the geometry of the fluidic chamber24is allowed to stabilize. Once the confinement level is high enough, the confinement of the DNA molecules within the nanoscale grooves25A can be visually observed.

Different geometries of the nanoscale grooves25A are shown inFIGS.2A to2D. InFIG.2A, the second substrate22has multiple linear and open nanoscale grooves125A which extend along an entire length of the second substrate22. The nanoscale grooves125A are said to be “open” because the movement of the molecules19therein is constrained only by the walls of the nanoscale grooves125A that are parallel to the longitudinal axis of the nanoscale grooves125A. Stated differently, the molecules19can exit each open nanoscale groove125A at either one of its ends when the first substrate21is contacting the second substrate22. Furthermore, the molecules' conformations can fluctuate in the vertical dimension.

FIGS.2B to2Dshow different possible “closed” geometries for the nanoscale grooves25A. The geometries of the nanoscale grooves25A are said to be “closed” when the movement of the molecules19within the nanoscale grooves25A is constrained. Stated differently, the molecules19are prevented from exiting each closed nanoscale groove25A when the first substrate21is contacting the second substrate22. In these “closed” geometries for the nanoscale groove25A, the molecule19is trapped within the nanoscale groove25A.

The closed geometry of the nanoscale grooves225A inFIG.2Bis circular, forming a closed loop. The molecules19are trapped within the circular nanoscale grooves225A when the first substrate21is contacting the second substrate22. The circular nanoscale grooves225A vary in diameter and are concentric. The closed geometry of the nanoscale grooves325A inFIG.2Cis annular or ring-like. The molecules19are trapped within the ring nanoscale grooves325A when the first substrate21is contacting the second substrate22. The ring nanoscale grooves325A have the same diameter and are spaced apart from each other along the surface22A of the second substrate22. The closed geometry of the nanoscale pits425A inFIG.2Dis rectangular. The molecules19are trapped within the rectangular nanoscale pits425A when the first substrate21is contacting the second substrate22, and the molecules can fold onto themselves within the nanoscale pits425A depending on their size. The rectangular nanoscale pits425A have the same dimensions and are spaced apart from each other along the surface22A of the second substrate22. It will be appreciated that other closed geometries are possible, and include for example, a triangle and any polygon having at least five sides.

Referring toFIGS.2A to2D, after a sample is loaded into the fluidic chamber24, the first substrate21is displaced to be pushed against the second substrate22. This reduces the dimension of the fluidic chamber24, causing the DNA molecules19A to be entropically driven into the nanoscale grooves25A depicted inFIGS.2A to2D. The DNA molecules19A also feel the global confinement potential of the now curved or shrunken chamber. When using a linear or open nanoscale groove125A, such as the ones shown inFIG.2A, the resulting confinement gradient experienced by the linearized DNA molecules19A causes them to drift along the linear nanoscale grooves125A towards regions of lower confinement, and away from the optical imaging centred on the centre of the flow cell20. The DNA molecules19A eventually move out from the field of view over a sufficiently long timescale and from the linear nanoscale grooves125A themselves as the separation between the first and second substrates21,22is increased.

In contrast, the use of the closed geometries of the nanoscale grooves225A,325A,425A ofFIGS.2B to2Dcoupled with the confinement provided by the first substrate21contacting the second substrate22, the DNA molecules19A may not exhibit biased motion along the closed nanoscale grooves225A,325A,425A.FIG.2Ecompares the molecule drift observed in an “open” linear nanoscale groove125A with that of a “closed” ring nanoscale groove325A.FIG.2Edepicts the fraction of the initial number of trapped DNA molecules19A in a single field of view plotted as a function of time for both linear and ring nanoscale groove125A,325A arrays. The closed ring nanoscale grooves325A maintain a higher percentage of the DNA molecules19A in view throughout the measurement period in comparison to the open linear nanoscale grooves125A. The open linear nanoscale grooves125A are essentially empty within about 10 minutes. In the open linear nanoscale grooves125A, the majority of linearized DNA molecules19A escape the linear nanoscale grooves125A or field of view within tens of minutes. In the closed ring nanoscale grooves325A, nearly all of the DNA molecules19A remain within the field of view for the 1-hour observation period. It therefore appears that exploiting nanoscale structures with closed geometries supports extended observation of molecules19within these nanoscale structures. This prolonged control of the molecules19can allow for programmable control of the ambient fluidic environment around the molecules.

Referring toFIG.2C, contact between the first substrate21and the second substrate21causes displacement of the molecules19into the ring nanoscale grooves325A and confines the molecules19therein. The molecules19are therefore top-loaded into ring nanoscale grooves325A. The surface21A or “roof” comes into contact with the surface22A or “floor” so that the ring nanoscale grooves325A become effective sealed. With no place to go, the molecules19become linearized or “straightened-out” within the nanoscale groove325A, and are trapped therein. The molecules19can therefore be observed for long periods of time, which enables extended observations, and increases the chance of observing interactions, especially for weak and slow interactions.

In the depicted embodiment where the molecules19are DNA molecules19A, the circumference of the ring nanoscale groove325A can be proportional to a length of the DNA molecule19A. In the depicted embodiment, the circumference of the ring nanoscale grooves325A is substantially equal to a length of a DNA molecule19A when extended. The circumference of the ring nanoscale groove325A can vary depending on the molecule19to be confined therein. In the depicted embodiment, the circumference is between 13 μm and 18 μm.

The ability to manipulate polymer molecular conformations on the nanoscale and to load them into closed geometry nanoscale structures is believed to improve self-ligation of the molecule. Self-ligation of DNA molecules19A, for example, requires a ligase protein, which catalyzes the formation of phosphodiester bonds, to find one end of the fluctuating polymer. Simultaneously, the ligase must come into contact with the other fluctuating end of the polymer, which eventually leads to formation of a circular polymer. In three-dimensional space, the large number of conformations accessible to the DNA molecule19A makes it unlikely for the polymer ends to find each other, reducing the efficiency of a self-ligation reaction. The closed ring geometry of the nanoscale grooves325A helps to bring the opposed ends of the DNA molecules19A into sufficiently close proximity with each other such that they may interact When the circumference of the ring nanoscale groove325A is similar to a length of the DNA molecule19A extension, within a tolerance determined by the polymer fluctuations, self-ligation may be further facilitated.

FIGS.3A to3Dshow DNA molecules19A trapped within different ring nanoscale grooves325A.FIG.3Ashows a wide field image of μ-DNA molecules19A trapped in an array of ring nanoscale grooves325A.FIG.3Bdepicts an SEM image of a ring nanoscale groove325A array together with an inset of a close-up image showing an approximately 70 nm width. The depth of the ring nanoscale grooves325A is approximately 65 nm. If the ligation enzyme and required reagents are present when the ends of the DNA molecule19A are within close proximity, self-ligation may occur. Several ring circumferences have been fabricated on a single substrate, including circumferences of 13, 14, 15, 15.5, 16, 16.5, 17, and 18 μm, of which examples are depicted inFIG.3Cwith fluorescence images of the single DNA molecule19A in the ring nanoscale grooves325A of different sizes. When the ring circumference is shorter than the extended DNA molecule19A, the ends of the DNA molecule19A overlap (see, e.g. the image with the 13 μm circumference). When the circumference is larger, the ends do not meet (see, e.g., the images with the 16 μm and 18 μm circumferences). InFIG.3C, a circumference of 14 μm was found to be suitable for ligation for the device and solution conditions used.FIG.3Dshows a kymogram of the DNA molecule19A trapped in a 14 μm circumference ring nanoscale groove325A, in the absence of enzyme, in which ligation is not occurring. There is a small gap between the DNA molecules19A polymer ends which occasionally closes due to thermal fluctuations in polymer extension length.FIGS.4A and4Bshow two ligation events. Within each sequence of images inFIGS.4A and4B), a self-ligated DNA molecule19A is trapped within a ring nanoscale groove325A having 14 μm circumference shown in left to right as the first substrate21is slowly raised away from the second substrate22. Accordingly, an embodiment of the nanoscale surface topography25disclosed herein facilitates a gentle, controlled top-loading of DNA molecules19A into circular or ring nanoscale grooves325A, as well as other geometries. This helps to extend observation times of the DNA molecules19A, while establishing observation conditions free of an applied gradient or flow.

FIGS.5A and5Bshow another embodiment of the flow cell120. The first substrate121includes a membrane126. The membrane126in the depicted embodiment has an upper first portion126A and a lower second portion126B. The membrane126is spaced apart from the surface121A of the first substrate121by supporting walls127. The walls127surround an array of microfluidic chambers127A. The microfluidic chambers127A extend between the surface121A and the membrane126. The microfluidic chambers127A are spaced apart so that they are addressable individually, both in terms of fluid exchange and in terms of electrical connection. The microfluidic chambers127A are in communication with outlet vias127B in the surface of the first substrate121. The membrane126in the depicted embodiment enables molecules to be loaded into the individually addressable pores, between the first (top) and second (bottom) substrates121,122. The first portion126A of the membrane126has a thickness between 1-10 nm, and has multiple nanoscale pores128extending through the first portion126A. The second portion126B of the membrane126has a thickness of about 50 nm, and is thus thicker than the first portion126A. The second portion126B of the membrane has a multiple nanoscale grooves25A etched into the second portion126B. The nanoscale grooves25A have a thickness of about 50 nm. The nanoscale grooves25A are vertically aligned with the nanoscale pores128, and in fluid communication therewith.

Referring toFIG.5C, the membrane126partitions the fluidic chamber124, when the first and second substrates121,122are brought into contact, into a first portion124A having the microfluidic chambers127A, and a second portion124B having the nanoscale grooves25A. The nanoscale pores128allow the fluid solution and molecules to be exchanged between the nanoscale grooves25A and the microfluidic chambers127A. In a typical implementation, molecules are confined in the nanoscale grooves25where they are linearized by the displacement of the second substrate122toward the first substrate121, and are drawn up through the nanoscale pores128into the microfluidic chambers127A for electrical sensing. The microfluidic chambers127A are also connected to fluidics which can exchange the solution in the microfluidic chambers127A.

The membrane126therefore creates a “dual-layer” fluidic chamber124. This dual-layer configuration also allows for buffer and reagent exchange between the nanoscale grooves25A and the microfluidic chambers127A, once the first and second substrates121,122are in contact, while also providing sufficient sealing to prevent the escape of molecules trapped in the nanoscale grooves25A. One possible use of the flow cell120is to deliver solution from the microfluidic chambers127A, via the nanoscale pores128in the membrane126, to the molecules which have been confined in the nanoscale grooves25A, without disturbing the molecules which are trapped in the nanoscale grooves25A. For example, trapped linearized molecules can be “immersed” with a solution of small-molecule reagents diffusing through the nanoscale pores128, before they are drawn up through the nanoscale pores128by an applied electrical force. In an alternate embodiment, the second substrate122has nanoscale features as well, such as other grooves or extrusions, to change the confinement geometry.

In the depicted embodiment, the nanoscale surface topography and the nanoscale grooves25A are on only the first substrate121. Stated differently, the nanoscale grooves25A are on the top of the initial liquid layer during loading of the flow cell120. Other features can be fabricated on the second substrate122, and may require alignment with the first substrate121. The nanoscale grooves25A are open-faced and patterned onto a suspended membrane126. In the depicted embodiment, the width of nanoscale grooves25A and nanoscale pores128is different The nanoscale pores128are positioned at one of the ends of the nanoscale grooves25A.

Each microfluidic chamber127A is in contact with one electrical sensor only, for example embedded directly above it There are separate decoupled outlet vias which enable fluid exchange between each microfluidic chamber127A. Each microfluidic chamber127A is isolated when the first and second substrates121,122are in contact, if closed nanoscale grooves25A are used (e.g. ring nanoscale grooves25A). In the depicted embodiment, each of the microfluidic chambers127A is much bigger than the nanoscale grooves25A. This difference in volume or width helps encourage migration of the molecules from the nanoscale grooves25A to the microfluidic chambers127A via the nanoscale pores128, typically with the application of a driving force to drive the molecules through the nanoscale pores128.

Molecules are confined in the nanoscale grooves25A of the flow cell120by deflecting one or both of the first substrate121or the second substrate122. In the depicted embodiment, the second substrate121is displaceable to contact the membrane126through the fluidic chamber124, as shown inFIG.5A. Contact between the part of the membrane126and the second substrate122causes the molecules to displace into the nanoscale grooves25A and confines the molecules within the nanoscale grooves25A. Any reagent or other solution can be exchanged with the confined molecules by admitting the reagent into the nanoscale grooves25A via the nanoscale pores128.

The molecules can also be displaced along the nanoscale grooves25A and through the nanoscale pores128. It is therefore possible to thread the linear or straightened molecule through the nanoscale pore128. In the embodiment where the molecule is a charged DNA molecule, the DNA molecule can be driven along the nanoscale groove25A and through the nanoscale pore128by applying a potential, bias voltage, or electric field. This causes the extended DNA molecule in the nanoscale groove25A to be driven toward the nanoscale pores128, and eventually threaded through them. Since the nanoscale grooves25A confine the DNA molecules in small volumes near the nanoscale pores128and pre-stretch the DNA molecules and eliminate loops and folds in their conformations, only small forces may be required to thread the DNA molecules through the nanoscale pores128. The microfluidic chambers127A may have electrical contacts or other sensors to detect the presence of translocated DNA molecules via the nanoscale pores128.

FIGS.6A and6Bshow another embodiment of the flow cell220. The first substrate221includes a membrane226that is spaced apart from the surface221A by a structure, such as by the walls223of an enclosed microfluidic chamber227A. The membrane226is “suspended” between the fluid in the microfluidic chamber227A and the fluid in the volume defined between the membrane226and the second substrate222. When the first and second substrates221,222are brought into contact, the membrane226is “suspended” between the upper microfluidic chamber227A and the lower nanoscale grooves25A containing molecules and solution. The membrane226in the depicted embodiment is a porous body between the first and second substrates221,222. The membrane226has a typical thickness between 30 and 100 nm. When the first and second substrates221,222are in contact, the membrane226partitions the fluidic chamber224into a first portion224A having the microfluidic chamber227A, and a second portion224A having the nanoscale grooves25A. The membrane226has one or more nanoscale pores228which extend through the membrane226. The nanoscale pores228allow the fluid solution to be exchanged between the microfluidic chamber227A and nanoscale grooves25A of the second substrate222.

The membrane226therefore creates a “dual-layer” fluidic chamber. This dual-layer configuration allows for reagent exchange between the microfluidic chamber227A and the nanoscale grooves25A, while also providing sufficiently sealing to prevent the escape of molecules from the nanoscale grooves25A. One possible use of the flow cell220is to deliver reagents from inlet vias to the microfluidic chamber227A, via the nanoscale pores228in the membrane226, to the molecules which have been confined in the nanoscale grooves25A without disturbing the molecules. Additional features such as nanoscale posts can be added to the surface of the membrane226which is in contact with the fluid, or to the second substrate222, to change the confinement geometry further.

In the depicted embodiment, the nanoscale surface topography25and the nanoscale grooves25A are on the bottom surface of the fluidic chamber224. More particularly, the nanoscale grooves25A extend into the surface222A of the second substrate222. The nanoscale grooves25A form part of the volume of the second portion224B of the fluidic chamber224. Each of the nanoscale pores228are in fluid communication with the nanoscale grooves25A to communicate fluid between the microfluidic chamber227A and the inlets which lead to it, and nanoscale grooves25A. In the depicted embodiment, the width of nanoscale grooves25A and nanoscale pores228is different

The microfluidic chamber227A has an outlet via227B in the surface of the first substrate221. The outlet via227A is in fluid communication with one or more of the nanoscale grooves25A via the nanoscale pores228. For example, multiple nanoscale grooves25A are directly below the suspended membrane226in the depicted embodiment. In the depicted embodiment, the outlet via227B and the microfluidic chamber227A is much bigger than the nanoscale grooves25A.

Molecules are confined in the nanoscale grooves25A of the second substrate222of the flow cell220by deflecting either the first substrate221or the second substrate222. In the depicted embodiment, the second substrate222is displaceable to contact the membrane226through the fluidic chamber224, as shown inFIG.6B. Contact between the part of the membrane226and the second substrate222causes the molecules to displace into the nanoscale grooves25A in the second substrate222and confines the molecules within the nanoscale grooves25A. Any reagent or other solution can be exchanged with the confined molecules by admitting the reagent into the nanoscale grooves25A via the nanoscale pores228. Reagents are admitted into the microfluidic chamber227A above the nanoscale pores228through the outlet via227B, and they diffuse through the nanoscale pores228.

Referring toFIG.1B, at least the second substrate22is made from glass. More particularly, the second substrate22is made from borosilicate glass. D263 glass has a relatively low surface roughness and index of refraction which matches that of the oil-immersion objectives. In contrast, silica substrates are a common aspect of prior art substrates. However, the refractive index of silica is not well matched to high-NA oil immersion objectives. The flow cell20therefore includes a thin-glass nanofluidic slit with nanoscale groove25A arrays. It has been observed to confine molecules into nanoscale features in coverslip-thickness substrates21,22having a thickness in the range of 100-150 μm, in contrast to millimeter-thick fused silica devices used in some conventional devices.

The use of relatively thin D263 borosilicate glass substrates21,22is well-matched to high-NA oil-immersion objectives. In contrast, the refractive index of fused silica is not well-matched to oil immersion objectives resulting in spherical aberrations which reduce image quality and resolution. By replacing 0.5 mm fused silica substrates used in prior work with 0.17 mm D263 substrates21,22, it is believed possible to replace water-immersion objectives characterized by a NA 1.0 with an oil-immersion objective with NA 1.49. Images of λ-DNA molecules19A confined within 50×65 nm2cross-section nanoscale grooves25A in each case are shown inFIG.7A, demonstrating decreased aberrations and typical improvement in signal-to-background ratio.FIGS.7A to7Edepict two examples of a DNA molecule19A labeled with a single fluorophore19B, namely, a λ-DNA molecule with a single Cy5-labeled oligo covalently attached to one end extended in a linear nanoscale groove25A, as well as a mutant pUC19 plasmid labeled with a single Cy5-fluorophore, trapped in an embedded micro-pit (500 nm×2 nm). Accordingly, these depict single-fluorophore19B imaging of biomolecules19extended in nanoscale surface topographies25.

FIG.7Adepicts in the upper panels μ-DNA molecule19A, stained with YOYO-119B, extended in the linear nanoscale groove25A wherein the panel of the upper left is imaged using a 0.5 mm-thick fused silica substrate with a water-immersion objective as in the prior art, while in the panel of the upper right is imaged using a 0.17 mm-thick glass substrate21,22with an oil immersion objective. Also depicted in the lower panels is λ-DNA molecule19A, end-labeled with a single Cy5 fluorophore19B wherein the lower left hand image of a single Cy5 end-label, covalently attached to one end of the DNA molecules19A while the lower right hand image depicts an overlaid two-color image of end-label and YOYO-1 stained DNA molecule19A.FIG.7Bdepicts a histogram of intensity for the single fluorophore image in the bottom left ofFIG.7A.FIG.7Cdepicts visualization of a mutant pUC19 plasmid covalently labeled with a single Cy5 fluorophore19B in an embedded pit wherein the top image is YOYO-1-stained mutant pUC19 plasmids, some of which are labeled with single Cy5 (middle image), while the lower image is an overlaid two-color image of the labeled plasmids trapped in pits.FIGS.7D and7Epresent histograms of intensity for images of labeled plasmids in pits as depicted inFIG.7Cupper and middle panels respectively.

Micro/nanoscale reaction wells may also enhance reactions between single-molecules by increasing the effective cross section of molecules for finding one another. Nanoscale wells may be defined by electron-beam lithography, and micro wells may be defined either with electron-beam lithography or UV photolithography. In some applications, such nanoscale wells may be etched to a depth less than 500 nm so that molecules are confined within the focal plane of the microscope objective for fluorescence visualization.

In some embodiments, the nanoscale surface topography25of the first substrate21is above the surface21A while in other embodiments it is into the surface21A. Similarly, in some embodiments, the nanoscale surface topography25of the second substrate22is above the surface22A while in other embodiments it is into the surface22A. In some embodiments they are both into or both out of their respective surfaces while in other embodiments one may be into and the other out of the surface and vice-versa. For examples, nanoscale posts127may be formed on both surfaces to trap the molecules, while in other embodiments, nanoscale grooves/pits25A may be formed into both surfaces to trap the molecules.

Within the embodiments described above a nanoscale pore128has been described as providing an “outlet” for a material trapped, e.g. biological molecule. However, within other embodiments, the nanoscale pore128may be replaced by one or more other nanoscale sensors. For example, a nanoscale groove or nanoscale groove/nanoscale pore combination may be employed to direct a molecule towards the nanoscale particle wherein the local electric field is enhanced such that the “read-out” is now a Raman spectrum, frequency shift, or other detection means.

Referring toFIG.1B, there is also disclosed a method of loading the flow cell20. The method includes providing molecules in a fluid between spaced-apart first and second substrates21,22. At least one of the first and second substrates21,22has a nanoscale surface topography25including at least one nanoscale groove25A extending into said substrate21,22. The method includes displacing the first substrate21toward the second substrate22to contact at least part of the first substrate21against the second substrate22. Contact between said part of the first substrate21and the second substrate22causes displacement of the molecules in the fluid into at least one nanoscale groove25A and confines the molecules therein.

Referring toFIG.2C, there is also disclosed another method of loading the flow cell20. The method includes confining a biological molecule19,19A within a nanoscale groove25A of the flow cell20such that a first end of the biological molecule19,19A is proximate to a second end of the biological molecule19,19A.

Reference is made now toFIGS.8to13illustrating with more details various embodiments of a flow cell in accordance with the present invention.

FIG.8show a flow cell300for confining molecules in a fluid. This particular flow cell300comprises an upper substrate320, an upper support member313, a center substrate321, a membrane326, a lower support member311and a lower substrate322. The flow cell is provided with sample inlet/outlet323, reagent inlet/outlet324, a center outlet319comprising at least one sensing chamber339and an imaging chamber308.

Referring now toFIG.9, the upper substrate320comprises a pair of diagonally opposed sample inlet323A and sample outlet323B and a pair diagonally opposed reagent inlet324A and reagent outlet324B.

The center substrate321comprises a pair of diagonally opposed sample inlet323E and sample outlet323F vertically aligned and in fluid communication with the sample inlet323A and sample outlet323B of the upper substrate320, respectively.

The upper support member313is positioned below the upper substrate320and above the center substrate321. The upper support member313comprises a pair of diagonally opposed sample inlet323C and sample outlet323D vertically aligned and in fluid communication with the sample inlet323E and sample outlet323F of the center substrate321. The sample inlet323C and sample outlet323D are also vertically aligned and in fluid communication with the sample inlet323A and sample outlet323B of the upper substrate320. The upper support member313further comprises a reagent exchange chamber309in fluid communication with and extending in between the diagonally opposed reagent inlet324A and reagent outlet324B of the upper substrate320. The reagent exchange chamber309is also in fluid communication with the center outlet319of the center substrate321.

The lower support member311is positioned below the membrane326and above the lower substrate322, The lower support member311comprises an imaging chamber308extending in between and in fluid communication with the diagonally opposed sample inlets323G,323E,323C and323A and the sample outlets323H,323F,323D and323B, respectively. In the illustrated embodiment the imaging chamber308occupies most of the surface of the lower support member311and it has a generally circular shape comprising a wide central section with two narrower extremities, symmetrically positioned from the center of the imaging chamber308which are in fluid communication with the diagonally opposed sample inlet323G and sample outlet323H of the membrane326. One of the roles of the imaging chamber308is to receive a sample to be analysed and also to provide an empty space for allowing bending or displacement of the upper layers (i.e.320,313,321,326) of the flow cell300, as shown inFIGS.11B and12B. The imaging chamber also assist in the trapping of molecules in an imaging plane, thereby allowing for long observation times and an optimized signal to noise.

There are different ways to manufacture the flow cell300. In one embodiment, each of the upper substrate320, upper support member313, center substrate321, membrane326, lower support member311and lower substrate322consists of separate individual layers (FIGS.9and10A). In another embodiment, the lower support member311and the lower substrate322consist of an integral bottom component340(FIGS.10B-10C). In this embodiment the imaging chamber is etched in the integral bottom component340, which can be made of any suitable material, including but not limited to glass (e.g. for optimal imaging conditions), such as borosilicate glass.

In another embodiment, the upper support member313and the center substrate321consist of an integral middle component330(FIGS.10B-10C). In this embodiment the center outlet319, the sample inlet323E and the sample outlet323F of the center substrate321are etched in the integral middle component330. Likewise, the reagent exchange chamber309, the sample inlet323C, and sample outlet323D of the center substrate321are also etched in that integral middle component330. The integral middle component330can be made of any suitable material, for instance silicon. In one embodiment the middle component330contains a thin film of silicon (e.g. silicon nitride, silicon oxide or a combination thereof) deposited on a bottom face (i.e. face facing the lower support member311) and that layer of silicon nitride defines the thin membrane326(FIGS.10A-10C). In another embodiment the membrane326consists of a thin film of silicon (e.g. silicon nitride, silicon oxide or a combination thereof) deposited on a bottom face of the individual center substrate321. In embodiments the membrane326has a thickness of about 10 nm to about 100 nm.

Reference is made now toFIGS.11A to11Dshowing a flow cell300, defined herein as a “sensing device” in accordance with one particular embodiment of the invention, wherein the membrane326comprises perforations329each comprised of a nanoscale groove428and a nanopore328. As best shown in the enlarged view ofFIG.11C, each perforation329extends through the membrane326and is vertically aligned and in fluidic communication with the sensing chamber339, and it is also vertically aligned and in fluidic communication with the imaging chamber308.

The nanoscale groove428has a size allowing to trap molecules to be confined therein (e.g. nucleic acid molecules such as DNA and RNA, and other long polymers) whereas the nanopore328has a dimension smaller than such molecules and smaller than the nanoscale groove428. The main purpose of the nanopore328is to translocate trapped long molecules (e.g. polymers) across the membrane, from the imaging chamber to the sensing chamber. This is typically done for sensing applications.

In embodiments the nanoscale groove428has a diameter of about 5 nm to about 100 nm which is greater than the diameter of the nanopore328(about 2 nm to about 50 nm). The nanoscale groove428extends partially (e.g. about 5 nm to about 100 nm) into a lower face327of the membrane326that is facing the imaging chamber308, whereas the nanopore328extends (e.g. about 5 nm to about 100 nm) into an upper face of the membrane326contacting the upper support member313to reach the nanoscale groove428(FIG.11C). Therefore, the combination of the nanoscale groove428and nanopore328provides a fluid communication between the sensing chamber339and the imaging chamber308.

Reference is made now toFIGS.12A-12Dshowing a flow cell300defined herein as a “reagent exchange device” in accordance with another particular embodiment of the invention, wherein the membrane comprises a plurality of nanopores328and the lower substrate322comprises a plurality of nanoscale grooves425. As best shown in the enlarged view ofFIG.12C, the membrane326comprises a plurality of perforations329defined as nanopores328extending through the membrane326. Therefore, in this embodiment, the nanopores328provide a fluidic communication between the sensing chamber339and the imaging chamber308.

The lower substrate322comprises an upper face423in communication with the imaging chamber308, that upper face423comprising at least one (preferably a plurality of) nanoscale grooves425extending partially therein (e.g. about 10 nm to about 1600 nm deep), Like for the sensing device ofFIG.11, the nanoscale grooves425have a dimension allowing to trap molecules whereas the nanopores328have a dimension smaller than the molecules to be confined in the nanoscale groove(s)425. In the reagent exchange device, the the nanopores allow for performing buffer exchange and/or additional of reagents to the trapped molecules confined in nanogrooves. This allows for visualizing reactions in real time.

In embodiments the nanoscale grooves425have a diameter of about 10 nm to about 50000 nm which is greater than the diameter of the nanopore328(about 5 nm to about 50 nm). In embodiments, the flow cell300comprises at least one a nanoscale groove425which is vertically aligned with at least one sensing chamber339. In embodiments, a plurality of nanopores328are in fluid communication with each of the nanoscale groove(s)425.

As best shown in the enlarged views ofFIG.11B and12B, the upper substrate320, the upper support member313, the center substrate321and the membrane326(or the combination thereof including the upper substrate320and middle component330) are displaceable (see concave shape) into the imaging chamber308(see reduce height in the center). Such displacement will cause molecules in the imaging chamber to be confined or trapped into the nanoscale groove(s)425or428for imaging.

For simplicity, the flow cells300ofFIGS.11and12are illustrated with only one sensing chamber339. However, the center outlet319of flow cells300in accordance with the present invention preferably comprises a plurality of sensing chambers339, as well as a plurality of associated components (e.g. corresponding perforations329in the membrane, etc.). For instance,FIG.13, show a flow cell comprising a plurality of sensing chambers339, each chamber339being in fluid communication with the imaging chamber308via a single perforation in the membrane326(e.g. combination of nanopore328and nanoscale groove428).