Submersible light-directing member for material excitation in microfluidic devices

Abstract

A system for material excitation in microfluidic devices is described. Aspects of the invention resemble a submerged periscope when in use. They allow for light to be redirected along a more advantageous trajectory as does the maritime device. A prism with a reflecting surface may be used to direct a laser beam along one or more microfluidic trenches or channels. Alternately, a reflecting surface may be provided in connection with a simple support. Directing a beam along multiple paths is preferably accomplished by scanning a single laser across or around the reflecting surface provided. Provision may be made for at least a portion of the submersible used to function as an electrode to assist in electrokinetically driving fluids and/or ions within the microfluidic device.

Description

TECHNICAL FIELD

[0002] This relates to systems for detection of material in microfluidic devices. Aspects of the invention provide for direct application of light to excite material within a device to enable fluorescent detection without producing background fluorescence in a chip resulting in signal loss or interference.

BACKGROUND

[0003] In performing any one of several useful types of analyses, microfluidic devices may be employed. Whether applied for the purpose of DNA separation or drug screening, the ability to achieve movement of sample, reagents and buffer within a network of fine trenches or channels can result is great time savings over conventional techniques. Most all common laboratory procedures such as mixing, incubation, metering, dilution, purification, capture, concentration, injection, separation, and detection to be performed on a single microfluidic “chip.” What is more, the chip format allows for parallel tasking and concomitant gains in productivity.

[0004] Data from experiments run on microfluidic devices is commonly extracted utilizing optical detection techniques. Laser-induced fluorescence (LIF) techniques are particularly advantageous. LIF techniques employ laser light to excite material for detection by an optical unit such as a photomultiplier tube (PMT) device or charge-coupled device (CCD) camera. One or more PMT devices or CCD cameras may be used or any combination thereof.

[0005] Most often, LIF detection utilizes a laser beam directed normal to the plane of the microfabricated device, exciting molecules at or adjacent to a detection zone. Another detection scheme employs light reflecting structures integrated within a chip to direct a beam across a detection zone. Such an approach may further utilize a second reflector to allow light detection normal to the plane of the microfluidic device.

[0006] Irrespective of what advantages such systems provide, they suffer from serious drawbacks. Background illumination or scattering of light introduced at detection windows and less-than-perfect reflecting surfaces often adversely affect sample detection capabilities. Furthermore, for some configurations, the layout will require passing the laser light through the material forming the body or a cover of the microfluidic device. Some configurations impose difficult demands on chip flatness or dimensional tolerances. This may introduce background fluorescence (i.e., autoflourescence) that also decreases detection signal accuracy.

[0007] Some have sought to address these problems through material choice to reduce background fluorescence and by use of high-quality optical surfaces to avoid light scattering within chips. Issues associated with cost and reproducibility are presented in either case. The present invention offers an elegant alternative in dealing with either fluorescence or optical challenges.

[0008] Further advantages and utility of the present invention may also be apparent to those with skill in the art upon further consideration of the various features of the present invention.

SUMMARY OF THE INVENTION

[0009] The present invention includes light-directing hardware, microfluidic devices adapted for use with the same, and combinations thereof. Most preferably, a light-directing member provided by a prism including a reflecting surface is sized in coordination with a microfluidic chip so that the end of the prism may reside within a sample waste well with its reflecting surface set to send light through various channels in the microfluidic device. The location of the reflecting surface (whether provided in connection with a prism or simply with a supporting member) in relation to a chip may be varied. For instance, the light directing member can be provided at any location within the chip where light introduction is desired. The location may be in fluid communication with channels in the chip such that the light directing member can be submersed within liquid media contained therein. In order to set the reflecting surface(s) properly with respect to channels in a device, location features may be variously provided.

[0010] Further variation possible in connection with the optical system described herein includes provision to scan light across a reflecting surface to illuminate multiple channels. Also, the light-directing member itself or a conductive member or coating associated therewith may be provided to serve as an electrode for electrokinetically driving material within a microfluidic device.

[0011] It is to be understood that the present invention includes the devices as well as the methodology disclosed. Furthermore, it is contemplated that any of the features of the systems disclosed, alone or variously combined, comprise aspects of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Each of the following figures diagrammatically illustrates aspects of the present invention. The illustrations provide examples of the invention described herein. Like elements in the various figures often are represented by identical numbering. For the sake of clarity, some such numbering may be omitted.

[0013] FIG. 1 is a top view of features of a microfluidic device.

[0014] FIG. 2 is a detail of the sample waste well of the device shown in FIG. 1.

[0015] FIG. 3 is a cross-sectional view of the well as shown in FIG. 2 taken along line A-A.

[0016] FIG. 4 is a cross-sectional view of the well as shown in FIG. 2 taken along line B-B.

[0017] FIG. 5 shows the features in FIG. 2 together with light-directing hardware according to the present invention.

[0018] FIG. 6 is a cross-sectional view of FIG. 5 taken along line C-C.

[0019] FIG. 7 is a cross-sectional view of FIG. 5 taken along line D-D.

[0020] FIG. 8 shows an alternate configuration for a portion of a microfluidic device useable in the present invention.

[0021] FIGS. 9A and 9B show prism configurations complimentary to the device shown in FIG. 8.

DETAILED DESCRIPTION

[0022] In connection with the figures, the following text provides examples or variations of the invention. Turning to FIG. 1, a portion of a chip (2) is shown. It includes a plurality of ingress reservoirs (4) in communication with a plurality of channels (6) which exit into a common waste well (8). Samples and reagents added to selected ingress reservoirs are electrokinetically manipulated to carry out various processes as noted above.

[0023] FIG. 2 shows a detail of the chip in the area of the waste well. Multiple channels (6) empty into well (8). FIG. 3 shows the channels end-on as they enter the well. FIG. 4 shows a channel and the well in cross section, with the well partially filled with liquid to form a meniscus (10).

[0024] FIGS. 3 and 4 both illustrate a preferred manner of producing chip (2). A chip body (12) defines a portion of each of the aforementioned features, with a base backing or covering (14) affixed thereto that completes the channels and reservoirs/wells.

[0025] The microfabricated structure shown in FIGS. 1-3 includes eight separation channels (6). Each separation channel is fluidly coupled to three ingress reservoirs (4). This arrangement permits running up to eight separate procedures in parallel in connection with a single waste well (8). Of course, the number of channels in communication with a given waste well (8) may be greater or smaller. For example, only one channel may be so-connected. Alternately, upwards of 20 to 100 channels may empty into a single waste well. Further, it is contemplated that a number of such functional units as shown in FIG. 1 may be provided on a single chip. A chip preferably has a thickness (T), width (W), and length (L) of 0.005 to 0.5 inches, 0.5 to 2.5 inches and 1 to 10 inches, respectively. Additionally, certain films may be used as chips and be as thin as 0.005 inches.

[0026] Each reservoir (4) is preferably sized to receive sufficient material to run a desired test or experiment and accept an electrode for running the procedure electrokinetically (i.e., employing either or both electrophoretic and electroosmotic phenomena). Suitable materials for the electrodes include platinum or other conducting materials, particularly those resistant to corrosion. The electrodes may be connected to a programmable voltage controller for applying desired voltage differentials across the channels. The electrodes are positioned in the reservoirs such that electrical contact is made with a sample or medium therein suitable for carrying out electrokinetic processes. Exemplary media includes but is not limited to fluids (e.g., buffered solutions, samples, etc.) and gels such as polyacrylamide gel and agarose.

[0027] Still, it is noted that aspects of the present invention need not be employed exclusively in connection with an electrokinetic chip. As will be apparent to one with skill in the art, features as described herein may be used in connection with a microfluidic device that is at least partially pressure driven or otherwise motivated.

[0028] Regardless, a complete chip is preferably configured to include 96 or 384 wells to correspond in number to such standard microliter plates as available through Van Waters and Rogers and other plate microliter plate manufacturers, conforming to what is known as the SBS standard. Conventional dispenser/pipetter means such as available from Beckman Coulter, Packard Instruments, Zymark, and other dispenser/pipetter manufacturers, are often used with such plates. An advantageous manner in which to configure a chip with the channel layout observed in connection with FIG. 1 is by arranging such functional units in a head-to-toe or complimentary fashion.

[0029] Typically, channels (6) will have a rectangular, trapezoidal or “D”-shaped cross-section. However, other cross sectional geometry may be employed. Usually, the channels preferably have a substantially constant or uniform cross-section. Preferably, channels (6) have a height and a width between about 0.1 μm and about 100 μm. It is also preferred that channels have a surface finish that does not result in irregular flow effects.

[0030] Chips according to the present invention may be fabricated in any number of manners. Most preferably, a chip body (12) is formed in plastic off of a micromachined/etched positive. Suitable plastics include acrylics, polycarbonates, polyolefins, polystyrenes and other polymers suitable for microfluidic or electrokinetic applications. Backing (14) is preferably made of a nonconducting film attached at the back of the body. One suitable material for backing (14) is a polymethylmethacrylate (PMMA) film. The backing is preferably attached to body by chemical, thermal or mechanical bonding. Ultrasonic welding (an example of the mechanical welding) may also be employed to fuse various parts together. Of course, as is known in the art, the body of microfluidic devices may be produced directly by etching the intended structures in a substrate. In such instances, a cover including wells or reservoirs openings is preferably placed over channels or trenches in the substrate to complete the device. Alternatively, the channel features can be formed in the cover (or film) by e.g., embossing. The film thereafter being attached to a substrate which may feature wells. Further details as to chip construction may be appreciated to those skilled in the art.

[0031] As is known in the art, voltages may be used to drive the chip. For example, U.S. Pat. No. 6,010,607 to Ramsey describes information on the manner in which voltages may be applied in order to run chip (2). Other techniques as presently known and used in the art can, of course, also be used in driving chip (2).

[0032] Turning now to FIGS. 5-7, features of the present invention are shown in connection with a portion of a chip as described so far. FIG. 5 is a top view of aspects of the present invention showing the features in FIG. 2 together with light directing members. FIGS. 6 and 7 show side and end views taken along lines C-C and D-D, in FIG. 5. In each, optional prism (16) is backed by an optional support member (18). A reflecting surface (20) is provided. As shown in FIG. 6, these components may be submersed in media (99). Media (99) is contained in the chip as described above.

[0033] Reflecting surface (20), may be provided in connecting with prism (16) or support member (18). It may be provided in connection with prism (16) by way of a reflective coating deposited on the angled surface of the prism. The coating chosen should be selected so as to reflect a beam of sufficient intensity to carry out the detection methodology described below. Accordingly, it may be preferred to use aluminum or silver coatings over gold since they absorb a lower percentage of the wavelengths of light produced by such lasers as typically used in detection schemes. However, for other reasons discussed below, it may be more important to utilize a less corrosive material such as gold or platinum for reflecting surface (20). In any event, the material coating may be applied by electro-plating, sputter coating or otherwise as would be known to one with skill in the art.

[0034] A reflective coating may be applied to the outside of prism (16) or on support (18). If both a support and a prism is to be used, a transparent seal (such as provided by epoxy) may be preferred between the parts if the reflecting surface is to be provided on support (18). Passing light through a prism offers an advantage in that it avoids passing light through media contained in a well or reservoir. Accordingly, loss of beam light intensity and fluorescence interaction with this material is avoided. Moreover, passing light though meniscus (10) will not occur, thereby avoiding any lens-type effect this has on beam (22) increasing the difficulty in which is may be directed down the length of fine channels. Indeed, it is for reason of beam divergence that a laser is the most preferred source of light for the invention. The coherent beam offered by such a device allows for greater light intensity as a point of interest for a given distance the light most travels. An alternative method for delivering light to a desired location involves inserting a fiber optic within the well or channel in an orientation to achieve the desired illumination or excitation.

[0035] Regardless of whether a prism is used or not, if the reflective surface is to be provided in connection with support (18), it is possible no coating may be required. Instead a polished surface may suffice, so long as absorption effects of the base metal of support (18) is acceptably low.

[0036] When a prism is provided, instead of using a coating on the prism for reflective surface (20), it may be provided by selecting parameters sufficient to result in total internal refraction within prism (16) to redirect a beam of light (22) instead. This phenomena is described by the equation:

sinθc=n2/i /n1(for n1>n2)

[0037] where θc is the minimum angle at which total internal reflection occurs and n1 and n2 are the refractive indices of the material in which total internal reflection is desired and that of the material external to the material within which total internal reflection is desired. Since n2 will approximate the value of water (n=1.33) for most solutions used in well (8), certain design considerations must be taken into account. To utilize a reflection surface angled at 45° relative to the initial beam trajectory, a prism material having a refractive index>1.88 must be used. Accordingly, for such a setup, any one of a number of rare-earth doped glasses may be used. Where a lower refractive index material is desired, such as quartz (n=1.47) or crown glass (n=1.52), the geometry of the prism may be modified, together with mounting structure associated with the light source to accommodate a higher incidence angle. However, a 45° angle of incidence is preferred in each variation of the invention since it turns a beam by 90°, allowing associated hardware to be setup at orthogonal angles.

[0038] However provided, in the variation of the invention in FIGS. 5-7, reflecting surface (20) is oriented to reflect a beam (22) along a channel (6) as shown in FIG. 6. In so doing, sample material within channel (6) at and above a detection window (24) is illuminated. This in turn causes tagged, labeled or marked material to fluoresce producing light that may be picked up by a detection system (26). To increase signal and enhance illumination, the sample detection channel may also incorporate certain surface coatings or claddings, or be composed of specific materials such that the channel walls will serve as a waveguide reflecting beam (22) inward. Various detection systems may be employed. For example, a system utilizing one or more lenses and a PMT device or a CCD camera (30) may be employed.

[0039] A suitable light collection setup is shown in FIG. 6. In this case, light is collected through lenses (28) from the bottom of the card or chip. The light is imaged through a slit (68) and collected by, for example, a PMT. The slit provides a spatial mask thereby setting the size of a detection region (66). The amount of fluorescent light that is collected from the bottom of the card may thus be controlled by the presence and size of the slit (68). In this manner, an optimum amount of fluorescent light may be collected. Alternatively, sets of pinholes and other variations can be utilized for the mask configurations disclosed herein.

[0040] It is also contemplated that slit (68) may be positioned on top of or on the bottom of backing (14). The slit may be, for example, a layer inside the chip or a layer formed on an outside surface. The slit may also be in the form of a coating deposited on the cover film or backing (14).

[0041] While the present invention may be utilized to direct a beam up a single channel or trench, it is preferred that provision be made to allow detection in multiple channels running more-or-less simultaneously. This may be accomplished using multiple beams, each aligned to reflect into a given channel. More preferably, it is accomplished by scanning a single beam into a number of channels (or simply directing it across a number of channels). In the latter embodiment, a single beam may be provided normal or transversely to a region of parallel channels or channel streams similar to the configurations disclosed in U.S. Pat. Nos. 5,833,826 and 5,741,412, and WO 01/20309. In the former embodiment, several ways of beam scanning are contemplated, the first of which is most clearly shown in connection with FIG. 7. Here, a mirror (32) to be attached to structure under control enabling it to traverse the face of reflecting surface (20) as indicated is provided. Of course, it is contemplated that mirror (32) and its source may be oriented otherwise.

[0042] FIGS. 8, 9A and 9B illustrate other manners in which to scan a beam into multiple channels. FIG. 8 shows a channel configuration with a prism (16) located at the center of a waste well (8). In FIG. 8, channels (6) empty into waste well (8) so each has an axis through a center point shared by prism (16). A circular waste well for such a configuration is preferred, but not required. FIGS. 9A and 9B show alternate side views of the prism in FIG. 8

[0043] The prism configuration in FIG. 9A includes a planar reflecting surface (20). The backside (34) of the reflecting surface may be filled in as shown. Alternately, it may be left open. It is advantageously filled in by material such as epoxy to protect any coating on reflecting surface from corrosive interaction with material in well 8. Also, it provides for a cylindrical body. This may be useful since prism (16) in FIG. 9A is preferably placed in well (8) and rotated in order to direct beam (22) into each channel (6) to enable detection when multiple channels are used in parallel. Rotating a cylindrical body rather than one missing a section of material produces less disruption of material within well (8).

[0044] The reflecting surface associated with prism in FIG. 9B is configured differently although backspace (34) may, likewise, be filled in with material. This will similarly insulate reflecting surface (20). However, to effect scanning a beam into multiple channels with the inventive variation in FIG. 9B, the beam is rotated instead of the prism. It may be preferred that the conical reflecting surface (34) be faceted in order to avoid divergence of beam (22).

[0045] For the variation in FIG. 9B, filling backspace (34) may provide another advantage. Namely, it provides a flat surface at the base of prism (16) useful for positioning reflecting surface (34) with respect to the channels. In order to properly locate reflecting surface to direct a beam up a channel, prism placement can be critical.

[0046] FIGS. 6, 7 and 11, show a manner of accurately and precisely placing reflecting surface (20) to direct a beam as desired. This approach may also be used with the prisms shown in FIG. 9B. A base (36) of a prism, support structure or both abuts a portion of chip (2) maintained as a stable location feature. As shown, backing (14) is maintained in a set location by a platen or fixture (38). Backing (14) is shown bowed or flexed into recesses (40) in platen (38). An advantage of such an approach is the ability to lower the position of reflecting surface (20) with respect to chip (2) so as to be able to bounce a beam off an area inboard of the leading edge of reflecting surface. Further, it allows passing beam deep within the interior of a prism, if used. It also eliminates the requirement to accurately control card thickness.

[0047] For chips where the channels are not at the bottom of structure, but rather formed at an intermediate height within a body, a recessed location-function approach may not be most preferred, or even feasible. Instead, it may be desired to simply locate base (36) against the base of a substantially non-deformable portion of the chip. On the other hand, it may be desirable to locate reflecting surface (20) relative to channels in a chip by way of features other than a base (36). For instance, in connection with the prism arrangement shown in FIG. 9A, base (36) is held so it does not contact chip (2). Instead it rotates above backing (14). Accordingly, stop features incorporated in a holding and actuating mechanism can set the height of reflecting surface (20) relative to the chip. Such an approach may also be used in conjunction with prisms or support members that do not move once placed in relation to a chip. Yet another approach is to locate a reflecting surface by reference to any repeatable feature that may be provided in a chip (2) or chip platten (38).

[0048] Especially in connection with the variations of the invention shown in FIGS. 5-8, 9A and 9B, it is preferable to include an electrode feature in connection with whatever body is submerged in well (8). This may be accomplished by utilizing a conductive material for optional support member (18). Stainless steel, or titanium alloy may be desired for corrosion resistance. Alternately, a coating of gold or platinum may be applied to support (18) so it will resist corrosion. Indeed, a suitably electrically conductive coating may itself function as an electrode even if the underlying material of support (18) is not conductive. Similarly, a conductive coating may be applied to prism (16) so at least a portion of the exterior of this member serves as an electrode. Instead, a simple wire or rod electrode may be affixed to whatever structure prism (16) and/or support member (18) is attached to serve as an electrode for driving chip (2).

[0049] Hydrophilic coatings may be applied to prism 16 and/or support member 18. Hydrophilic coatings may be helpful in avoiding bubbles.

[0050] With any of the systems described herein, it is noted that mounting and actuating structure for the prism or a supporting member for reflecting surface may be provided to advance the reflecting surface into a recess within a chip. Alternately, a chip may be moved in order to submerge a reflecting surface that is mounted in a stationary fashion. Provision of such constructional detail in the form of collateral structure and control for that structure is within the ability of those within the level of ordinary skill in the art.

[0051] Additional details as to the use or other aspects of the system described herein may be drawn from the background that is intended to form part of the present invention, including any of the patents and patent applications cited above, each of which being incorporated by reference herein in its entirety for any purpose. It is noted that this invention has been described and specific examples or variations of the invention have been portrayed. The use of those specific examples is not intended to limit the invention in any way. Additionally, to the extent that there are variations of the invention which are within the spirit of the disclosure and are equivalent to features found in the claims, it is the intent that the claims cover those variations as well. All equivalents are considered to be within the scope of the claimed invention, even those which may not have been set forth herein merely for the sake of relative brevity.

[0052] Also, the various aspects of the invention described herein, in any manner or section of the application including the Abstract, Field of the Invention, Background of the Invention, Summary of the Invention, Brief Description of the Drawings, the Drawings themselves and Detailed Description, may be claimed as set forth therein or be modified and/or used in combination with such other aspects also described to be part of the invention either explicitly, implicitly or inherently in order to form additional variations considered to be part of the invention. Furthermore, it is contemplated that any single or any combination of optional features of the inventive variations described herein may be specifically excluded from the invention claimed and be so-described as a negative limitation.

Claims

1. A system for use in detecting material in a microfluidic device comprising: a first light-directing member separate from said microfluidic device, said first member comprising a proximal end, a distal end, and a reflecting surface adjacent said distal end; and a light source, said light source being aimed to reflect light off said reflecting surface towards a target region of said microfluidic device.

2. The system of claim 1, wherein said target region is an opening of a channel in said microfluidic device.

3. The system of claim 1, wherein said first light directing member comprises a prism.

4. The system of claim 3, wherein said prism includes a coated region providing said reflecting surface.

5. The system of claim 3, wherein said prism is adapted to provide said reflecting surface by total internal reflection.

6. The system of claim 3, wherein said first light directing member further comprising a prism support.

7. The system of claim 6, wherein said prism support is adapted to function as an electrode.

8. The system of claim 3, wherein said prism is at least partially coated with a conductive material layer adapted to function as an electrode.

9. The system of claim 1, wherein said reflecting surface is oriented at an angle of 45° with respect to said length.

10. The system of claim 1, wherein said reflecting surface is planar.

11. The system of claim 1, wherein said reflection surface is provided around an axis parallel to said length.

12. The system of claim 1, wherein said light source is a laser.

13. The system of claim 1, further comprising a means for scanning light from said light source across said reflecting surface.

14. The system of claim 1, further comprising a reflective member that moves to direct light at different target regions.

15. The system of claim 1, wherein said first light-directing member moves to direct light at different target areas.

16. The system of claim 1, wherein said first light-directing member rotates to direct light at different target areas.

17. The system of claim 1, wherein said first light-directing is adapted to direct light at different target areas when light is reflected at differing angular orientations.

18. The system of claim 17, wherein said light source rotates about an axis parallel to said length of said first light-directing member.

19. The system of claim 1, further comprising a second light-directing member, said second member comprising a proximal end, a distal end, and a reflecting surface adjacent said distal end, said second light-directing member being positioned relative to said first light-directing member so to reflect light directed by first member outside of said microfluidic device.

20. The system of claim 19, further comprising said microfluidic device.

21. The system of claim 1, further comprising said microfluidic device.

22. The system of claim 20, wherein said microfluidic device is supported by a platten and comprises a body portion and a backing portion.

23. The system of claim 21, wherein said microfluidic device is supported by a platten and comprises a body portion and a backing portion.

24. The system of claim 20, wherein said distal end of said first light-directing member sets the position of said reflecting surface relative to said microfluidic device upon advancement to contact said backing and depressing said backing to said platten.

25. The system of claim 24, wherein said platten is recessed in a region opposite said first light directing member.

26. The system of claim 21, wherein said distal ends of said light-directing members set the position of said reflecting surfaces relative to said microfluidic device upon advancement to contact said backing and depressing said backing to said platten.

27. The system of claim 26, wherein said platten is recessed at a region opposite said light directing members.

28. The system of claim 20, wherein said microfluidic devices further comprises a waste well adapted to receive said first light-directing member.

29. The system of claim 28, wherein said microfluidic device further comprises a plurality of channels oriented radially about said waste well.

30. The system of claim 21, wherein said microfluidic device further comprises a plurality of parallel channel portions and recessed portions adapted to receive said first and second light-directing members across said plurality of channels portions.

31. The system of claim 1, further comprising a light collecting apparatus to collect light fluorescing from a sample material to be detected.

32. The system of claim 31, further comprising a slit for imaging said collected light onto said light collecting apparatus, said slit being positioned in a path of said light to said light collecting apparatus.

33. The system if claim 32 wherein said light collecting apparatus is a PMT.

34. A method for detecting material in a microfluidic device having a reservoir with a media contained therein, said method comprising: directing light at a first light-directing member separate from said microfluidic device, said first light-directing member comprising a distal section submersed in said media wherein said light reflects off a reflecting surface on said distal section towards a target region of said microfluidic device.