FIELD OF THE INVENTION
The present invention relates to a microfluidic device and a method for providing a reagent to a microfluidic device.
The polymerase chain reaction (PCR) is a highly sensitive method for the amplification of segments of genomic DNA (gDNA) or complementary DNA (cDNA). PCR has many applications, for example the detection of trace amounts of nucleic acids to determine the presence of disease causing organisms, gene expression, genotyping, genetic engineering or modification, and forensic science applications. PCR amplification provides outstanding target identification and quantification over a large range of analyte concentrations. However, simultaneous and quantitative analysis of many analytes by PCR has proven to be extremely challenging. Intercalating dye fluorescence-based detection is only capable of determining total dsDNA concentration and therefore concurrent analysis of multiple templates in a single reaction vessel is not possible using this detection method. Fluorescent probe technologies (i.e., Taqman, molecular beacons, or other chemistries) can be used for low-level multiplexing of reactions as each target can be amplified using a different color fluorescence probe as a signaling reporter. Probes are also sequence specific, reducing false positives from primer-dimer formation or nonspecific amplification. A typical method for multiplexing with either conventional microtiter plate or microfluidic real-time-PCR (rt-PCR) is to use a small number of reaction wells, each containing three different color probes. However, it is generally considered challenging to design multiplexed primer and probe sets as they require an additional level of careful design and optimization to insure compatibility with each other. Multiplexing by this method is ultimately limited, by instrumentation and spectral overlap between dyes, to four-color detection, with one color typically reserved for an internal standard dye.
The optimization challenges for multiplexed PCR also create a hurdle to rapid reconfiguration of the assay for emerging diseases or other time-sensitive applications that require analysis of new sequences. Other problems encountered in multiplexed PCR include primer complementarity producing dimers, non-specific interactions between extraneous DNA and amplicons or the primers/probes, and an inherent bias where short sequences are amplified faster than longer ones. Additionally, while using multiplexed reactions divided among a small number of reaction wells can work well for small numbers of targets (∼3-12) in systems such as the GeneXpert (Cepheid) or the JBAIDS (Idaho Technology Inc.), this approach becomes cumbersome for significantly larger numbers of targets. Cartridges used with these technologies must be individually loaded with relatively large amounts of liquid reagents either at the point of care or at the point of manufacture. Technology such as the OpenArray Real-Time PCR System from Life Technologies uses preloaded primer/probe sequences dried in an array of approximately 3000 microwells. This technology does not perform sample cleanup or preparation, and it requires multiple, separate pieces of instrumentation to process, load, and analyze samples. Additionally, the preloading of primer/probe sets must be done using printing-based technology that greatly increases the time and expense required for device production.
 Berti, F., et al.: "Microfluidic-based electrochemical genosensor coupled to magnetic beads for hybridization detection", Talanta, vol. 77, no. 3, 15 January 2009, pages 971-978
, describes the development of a rapid and sensitive enzyme-linked electrochemical genosensor using a microfluidic-based platform. In the experimental work a microfluidic-based platform, produced by DiagnoSwiss S.A., was used. This platform consists of a disposable cartridge called ImmuChip™, an instrument called ImmuSpeed™ and its associated software ImmuSoft™. ImmuChip™ consists of eight parallel microchannels etched in a polyimide substrate at distances compatible with conventional 96-well plate automation. Each microchannel contains a housing with a sample deposition reservoir, and it was etched in a polyimide body with inlet/outlet in contact with microelectrode tracks. The microchannels have a length of 1 cm and a total volume of 65 nL. The upper part of each channel contains an array of 48 gold working microelectrodes.
SUMMARY OF EMBODIMENTS OF THE INVENTION
The present invention provides, according to a first aspect, a microfluidic device as defined in claim 1. The microfluidic device includes a plurality of reaction wells; and a plurality of solid supports. Each of the solid supports has a reagent attached thereto, wherein the reagent is attached to the solid support via a labile reagent/support bond such that the reagent is configured to be cleaved from the support via a cleaving operation.
In some embodiments, the cleaving operation comprises a thermal operation, an addition of a chemical and/or an application of light to the reagent/support bond. The reagent may include a primer for a PCR reaction. The labile reagent/support bond may include a streptavidin-biotin bond and/or avidin-biotin bond. The cleaving operation may include a thermal operation. In some embodiments, the streptavidin-biotin bond is configured to release the reagent from the support when the solid supports are incubated at about 50-99°C. The solid supports may be microbeads, such as magnetic microspheres. In some embodiments, the solid supports comprise a marker. The marker may include a predefined support shape, a predefined support size, a magnetic property and/or optical property of the support. The marker may include a fluorescence marker.
The plurality of wells each include a solid support containment region and a reaction region, and the solid support containment region is configured to receive a single one of the plurality of solid supports. In some embodiments, the plurality of wells and the plurality of solid supports are sized and configured such that only a single one of the plurality of solid supports is received in a corresponding single one of the plurality of wells. The solid support containment region may have a cross-sectional area that generally corresponds to a size and/or shape of the single one of the plurality of solid supports. In some embodiments, the reaction may occur or be initiated in the solid support containment region which may serve as the reaction region in this case. The reaction region may have a cross-sectional area that is larger than the cross-sectional area of the solid support containment region. The microfluidic device includes a sealing member (for example an elastomeric membrane, such as polydimethylsiloxane) or an immiscible fluid to fluidically isolate each of the plurality of wells. In use, reaction solution can be added to the wells and the wells are sealed with the sealing member or immiscible fluid such that the reaction wells are fluidically isolated from one another, and in each fluidically isolated reaction well a cleaving operation can be performed to separate the reagent from the solid support so as to deliver reagent to the reaction region. The microfluidic device may include a membrane in the wells. The membrane may be an extraction membrane. In some embodiments, the membrane includes a monolithic aluminum oxide membrane. In some embodiments, a channel is connected to the membrane that is configured to provide a fluid connection to the plurality of wells via the membrane. In some embodiments, the wells are configured to receive a plurality of supports. In some embodiments, a pattern of microposts are between the membrane and the channel to the support the membrane but allow even fluid flow from the plurality of wells via the membrane. In some embodiments, reagents are configured to be released from multiple beads in one of the plurality of wells to effect a reaction between reagents.
The present invention also provides, according to a second aspect, a method as defined in claim 15. The method is for providing a reagent to a microfluidic device, the device comprising a plurality of reaction wells, the method comprising: providing a plurality of solid supports in the plurality of reaction wells and providing reaction solution to the wells, wherein each of the solid supports has a reagent attached thereto such that the reagent is attached to the solid support via a labile reagent/support bond and the reagent is configured to be cleaved from the support via a cleaving operation. The plurality of wells each include a solid support containment region and a reaction region, with the solid support containment region being configured to receive a single one of the plurality of solid supports. A sealing member or an immiscible fluid is provided to seal the wells, such that the plurality of wells are fluidically isolated from one another. A cleaving operation is performed to release the reagent into the reaction solution in the reaction region in each of the plurality of fluidically isolated reaction wells.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain principles of the invention.
Figure 1A is a diagram of a bead having a primer attached thereto.
Figure 1B is a diagram of four beads having different encoding properties.
Figure 2 is a digital image of a microfluidic device having a plurality of wells for holding the bead/primers of Figures 1A-1B.
Figure 3 is a cross-sectional side view of the microfluidic device of Figure 2.
Figure 4 is a bar graph showing a measured amount of DNA (ng/µL) and indicating an amount of primer released from the beads at room temperature and at 95°C and for a sample without primer beads.
Figures 5A-5D are diagrams illustrating a microfluidic device configured to receive individual beads in respective wells.
Figure 6 is a plot of data showing PCR amplification of three different template sequences using three different bead sets.
Figure 7 is a digital fluorescence microscopy image showing a relatively strong signal from the intercalating dye for the wells of the device of Figures 2-3 containing amplified template molecules and virtually no signal from the blank reaction well in the center of the image.
Figure 8 is a graph of the fluorescence signal from each well in the device of Figures 2-3 plotted against the PCR cycle number for an rt-PCR experiment.
Figure 9 is a schematic drawing of a microfluidic device.
Figure 10 is a perspective view of the device of Figure 9 showing alignment of the substrates.
Figure 11 is a cross-sectional side view of the device of Figure 9.
Figure 12 is a top view of a schematic diagram showing a microfluidic device having photoresist lanes to control fluid flow.
Figure 13 is an optical microscopy image of the device of Figure 12 showing 100 µm by 100 µm pads where a gold layer was removed. Bead wells are located in the center of the wells.
Figure 14 is a fluorescence image of a dye solution isolated on 444 pads in the device of Figure 12. No bridging and few stray droplets are seen.
Figures 15A-15B are fluorescence images of microfluidic devices with 440 hydrophilic reaction region (100 µm x 100 µm wells) that isolate 250 pL using a photoresist spacer 25 µm tall (Figure 15A) and 75 pL using a 7.5 µm spacer (Figure 15B).
Figure 15C is a fluorescence image of a microfluidic device including 1500 reaction regions (50 µm x 50 µm wells) with a 9 µm spacer to isolate 22.5 pL volumes.
Figures 16A-16C are fluorescence images of digital PCR reactions in a microfluidic device demonstrating single copy amplification in 22.5 pL reactions with 1500 reaction wells on each device with a nominal copy number/ well of 0.7 (Figure 16A), 0.2 (Figure 16B), and 0.04 (Figure 16C). Signals from cycles 20-25 were summed to improve the signal to noise for identifying positive wells.
Figure 17A is an SEM image of a loaded primer bead of 3.3 µm diameter loaded into a 4.15 µm diameter, 5.7 µm depth well.
Figure 17B is a fluorescence microscopy image of a device illustrating greater than 90% bead occupancy in the wells.
Figures 18A-18D is a series of fluorescence images showing the magnetic loading of beads. A clump of more than 15,000 beads may be seen as a large mass in each frame, and arrows indicate the direction in which the clump of beads is being drawn by the magnet under the device.
Figures 19A-19C are decoded images of the same twelve reaction wells imaged with different fluorescence filter sets such that beads of interest are highlighted with white squares. Figure 19A identifies Qdot 605 and primers for the mecA gene (365/10nm ex); Figure 19B identifies Qdot 655 and primers for S. mutans; and Figure 19C identifies beads without Qdot labels that carry primers for the nuc gene found in S. aureus gDNA. Both Qdot 605 and Qdot 655 labeled beads fluoresce brightly. An empty bead well is shown in Figure 19C that is highlighted with a white hexagon. Figure 19D is an assay image showing positive results for mecA and nuc genes.
Figure 20 is a graph of the amplification in 5 µL reactions after bulk bead-based capture of target DNA.
Figures 21A-21C are images of sections of an array from a microfluidic device after 30 cycles of PCR amplification showing an increase in SYBR green fluorescence in positive wells for three different primer sets encoded with with color fluorescence.
Figure 22A is a schematic image of a microfluidic device in which different well sizes preferentially retain correspondingly sized beads.
Figures 22B-22C are fluorescence microscopy images illustrating the bead acceptance patterns of the device of Figure 22A.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The scope of the invention is defined by the claims.
Like numbers refer to like elements throughout. In the figures, the thickness of certain lines, layers, components, elements or features may be exaggerated for clarity.
As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y." As used herein, phrases such as "from about X to Y" mean "from about X to about Y."
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and/or clarity.
It will be understood that when an element is referred to as being "on," "attached" to, "connected" to, "coupled" with, "contacting," etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, "directly on," "directly attached" to, "directly connected" to, "directly coupled" with or "directly contacting" another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.
Spatially relative terms, such as "under," "below," "lower," "over," "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of "over" and "under." The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly," "downwardly," "vertical," "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.
It will be understood that, although the terms "first," "second," etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a "first" element discussed below could also be termed a "second" element without departing from the teachings of the present invention. The sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.
The term "oligonucleotide" refers to a nucleic acid sequence of at least about five nucleotides to about 500 nucleotides (e.g. 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 21, 22, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 or 500 nucleotides). In some embodiments, for example, an oligonucleotide can be from about 15 nucleotides to about 30 nucleotides, or about 20 nucleotides to about 25 nucleotides, which can be used, for example, as a primer in a polymerase chain reaction (PCR) amplification assay and/or as a probe in a hybridization assay or in a microarray. Oligonucleotides of this invention can be natural or synthetic, e.g., DNA, RNA, PNA, LNA, modified backbones, etc., or any combination thereof as are well known in the art.
Probes and primers, including those for either amplification and/or detection, are oligonucleotides (including naturally occurring oligonucleotides such as DNA and synthetic and/or modified oligonucleotides) of any suitable length, but are typically from 5, 6, or 8 nucleotides in length up to 40, 50 or 60 nucleotides in length, or more. Such probes and or primers may be immobilized on or coupled to a solid support such as a bead, chip, pin, or microtiter plate well, and/or coupled to or labeled with a detectable group such as a fluorescent compound, a chemiluminescent compound, a radioactive element, or an enzyme.
Polymerase chain reaction (PCR) may be carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195
; and 4,965,188
. In general, PCR involves, first, treating a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) with one oligonucleotide primer for each strand of the specific sequence to be detected under hybridizing conditions so that an extension product of each primer is synthesized which is complementary to each nucleic acid strand, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith so that the extension product synthesized from each primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, and then treating the sample under denaturing conditions to separate the primer extension products from their templates if the sequence or sequences to be detected are present. These steps are cyclically repeated until the desired degree of amplification is obtained. Detection of the amplified sequence may be carried out by adding to the reaction product an oligonucleotide probe capable of hybridizing to the reaction product (e.g., an oligonucleotide probe of the present invention), the probe carrying a detectable label, and then detecting the label in accordance with known techniques, or by direct visualization on a gel. The amplified sequence can also be detected by adding an intercalating dye to the reaction mixture and monitoring the fluorescence signal strength, which will be proportional to the total mass of double stranded DNA. Although embodiments according to the present invention are described with respect to PCR reactions, it should be understood that other nucleic acid amplification methods can be used, such as reverse transcription PCR (RT-PCR) including isothermal amplification techniques such as rolling circle amplification or loop-mediated isothermal amplification (LAMP).
DNA amplification techniques such as the foregoing can involve the use of a probe, a pair of probes, or two pairs of probes which specifically bind to DNA containing a polymorphism or mutation of interest, but do not bind to DNA that does not contain the polymorphism of interest under the same hybridization conditions, and which serve as the primer or primers for the amplification of the DNA or a portion thereof in the amplification reaction. Such probes are sometimes referred to as amplification probes or primers herein.
The term "reagent" refers to any substance or compound, including primers, the nucleic acid template and the amplification enzyme, that is added to a system in order to bring about a chemical reaction, or added to see if a reaction occurs. Amplification reagents or reagent refer to those reagents (deoxyribonucleotide triphosphates, buffer, etc.) generally used for amplification except for primers, nucleic acid template and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.). In the present invention, each of the solid supports has a reagent attached thereto, wherein the reagent is attached to the solid support via a labile reagent/support bond such that the reagent is configured to be cleaved from the support via a cleaving operation; and the plurality of wells each include a solid support containment region and a reaction region, wherein the solid support containment region is configured to receive a single one of the plurality of solid supports.
The term "magnetic" as used herein includes ferromagnetic, paramagnetic and super paramagnetic properties.
In general, an oligonucleotide probe which is used to detect DNA containing a polymorphism or mutation of interest is an oligonucleotide probe which binds to DNA encoding that mutation or polymorphism, but does not bind to DNA that does not contain the mutation or polymorphism under the same hybridization conditions. The oligonucleotide probe is labeled with a suitable detectable group, such as those set forth below. Such probes are sometimes referred to as detection probes or primers herein.
Kits useful for carrying out the methods of the present invention will, in general, comprise a plurality of solid supports, each of the solid supports having reagents attached thereto and other reagents for carrying out the methods as described above, such as restriction enzymes, optionally packaged with suitable instructions for carrying out the methods. The kits may also include containers for housing elements included therein. Such containers include, but are not limited to, vials, ampoules, tubes, capsules, bottles, syringes, bags, microfluidic chips and cartridges, including preloaded bead devices.
As illustrated in Figure 1A,
a solid support (in this case, a bead 10)
may have primers 20A, 20B
attached thereto. The primers 20A, 20B
may be a pair of biotinylated primers, and the bead 10
may be streptavidin-labeled such that binding of the biotinylated primers to the beads occurs. In some embodiments, the beads 10
may include a marker, such as an optical marker, that may be used during analysis to identify the primers 20A, 20B
anchored to the respective beads 10A.
For example, as shown in Figure 1B,
different encoded beads 10A, 10B, 10C, 10D
may be marked for identification of different attached primer sets during analysis. Various pre-encoding methods may be used to provide a marker on the bead 10
or other solid support, including a predefined size, shape, magnetic property and/or fluorescence doping used alone or in combination with other encoding methods. Both custom-sequence biotinylated primers and streptavidin labeled paramagnetic magnetic beads can be readily purchased from commercial vendors or made in a suitable quantity in an appropriately equipped laboratory.
Although embodiments according to the present invention are described herein with respect to beads 10
magnetic microspheres) it should be understood that any suitable solid support may be used, whether porous, superficially porous, or nonporous. Supports may include, but are not limited to magnetic or non-magnetic particles or granules made of materials such as polymer or plastics, glass, silicon dioxide or metal or semimetal oxides (including but not limited to aluminum oxides, titanium oxides, zirconium oxides or other oxides), quantum dots, photolithographically fabricated particles or structures (discrete or nondiscrete), filter membranes or elements, solid surfaces of vials, microtiter plates, microfluidic or macrofluidic reaction wells, silicone or other rubbers, metals and metal particles, naturally occurring supports or absorbents suited for these purposes.
Although embodiments according to the present invention are also described herein with respect to PCR reactions, it should be understood that the microfluidic devices, beads and reaction methods described herein may be used in various other reactions, e.g.,
where reagents are cleaved from a bead into a well to participate in a reaction. For example, the reaction may be any nucleic acid transcription and/or amplification-related reaction, including but not limited to PCR reactions, real-time PCR (rt-PCR), digital PCR (dPCR), reverse transcription of RNA into cDNA (RT), PCR of cDNA from previous RT step (RT-PCR), RT-PCR using real-time or digital quantification, immuno-PCR (iPCR) and its variants, loop-mediated isothermal amplification (LAMP), rolling circle replication, and/or non-enzymatic nucleic acid amplification methods (e.g.,
"DNA circuits"). For example, the reaction may include enzyme-linked immunosorbent assays (ELISA) where the fluorogenic substrate is bound to the support surface to be cleaved at some point for subsequent reaction, single molecule array (SiMoA) or digital ELISAs where the fluorogenic substrate is bound to the support surface to be cleaved at some point for subsequent reaction, reactions in which multiple beads are used to deliver different reagents for combinatorial chemistry, reactions where the beads deliver a catalyst reagent, and/or reactions where "click" chemistry reagents are delivered in stoichiometries determined by stochastic bead loading.
The microfluidic device of the present invention comprises a plurality of solid supports, wherein each of the solid supports having a reagent attached thereto, and wherein the reagent is attached to the solid support via a labile reagent/support bond such that the reagent is configured to be cleaved from the support via a cleaving operation. Any suitable coupling chemistry may be used to bind the primers 20A, 20B
to the beads 10.
In some embodiments, the primers 20A, 20B
may be attached to the beads 10
using labile chemical bonds, such as bonds that may be cleaved using various techniques. For example, thermal energy may be applied to break or cleave a chemical bond or light may be applied to break a photo-cleavable bond. Thermal heating may be accomplished by conductive contact with a hot surface, Joule heating of a solution, resistive heating of a substrate that is adjacent the beads 10,
exposure to electromagnetic radiation and/or electromagnetic induction. In some embodiments, the chemical bonds may be cleaved using ionizing radiation, a chemical agent, an enzyme, an electrochemical process, a change in pH, or other suitable cleaving operations. For example, a change in pH may be caused by electrochemical reactions occurring at the surface of the region holding the beads 10
or a change in conditions may be directly or indirectly caused by an activation of an initiator reagent, which itself may be activated by a physical changes such as heat, light, electrochemical processes, change in pH, etc. The primers 20A, 20B
may be anchored to the bead 10
such that the primers may remain anchored during storage, sample preparation and/or the washing steps before the PCR. A cleaving operation may be performed when the PCR reaction takes place to disengage the primers 20A, 20B
from the beads 10.
Depending on the particular labile chemical bond used, the cleaving operation may include the application of heat, light and/or addition of a chemical to disengage the primers 20A, 20B
from the beads 10.
When the primers 20A, 20B
are disengaged from the beads 10,
the primers may readily participate in the PCR reactions, and when the primers 20A, 20B
are anchored to the beads 10,
the primers 20A, 20B
generally do not react in the PCR reaction unless template DNA is in close proximity. Although embodiments according to the present invention are described with respect to the primers 20A, 20B,
it should be understood that the any suitable reagent may be used as elements 20A, 20B
and bound with any suitable chemical bond. Any reagent, such as a probe or an oligonucleotide, may be used. Suitable cleavable bonds in addition to streptavidin-biotin include, but are not limited to photo- cleavable biotin, avidin, streptavidin, and captavidin, and may be commercially available, for example, from Ambergen (PC Biotin Phosphoramidite, PC Amino-Modifier Phosphoramidite and PC Spacer Phosphoramidite).
 Figures 2-3
illustrate a microfluidic device 50
that includes a plurality of reaction wells 60.
In the microfluidic device of the present invention, the plurality of wells each include a solid support containment region and a reaction region, wherein the solid support containment region is configured to receive a single one of the plurality of solid supports. The beads 10
and primers 20A, 20B
attached thereto may be pre-loaded into the wells 60.
The device 50
may further include a substrate 62
glass) and a microfluidic portion 64
having a channel 66
for fluidically connecting to the wells 60.
As illustrated in Figure
3, the wells 60
may be formed on a membrane 68,
such as a monolithic aluminum oxide membrane (AOM). The membrane 68
may be used to purify DNA from a sample. Other suitable materials for sample cleanup may be employed including polymer membranes or inorganic or polymer surfaces that can perform a solid phase extraction or another method of separation. The membrane can be supported on a patterned grid or array of microposts 70
connected to a channel such that the membrane is fully supported but fluid may still flow through it freely and evenly. Accordingly, the primers 20A, 20B
on the beads 10
may be preloaded into the wells 60 and stored for a period of time before use. The wells 60
may each contain beads 10
having many copies of a single primer pair and be labeled for ease of analysis.
In some embodiments, the primers 20A, 20B
are attached to the beads 10
via a streptavidin-biotin bond. The streptavidin-biotin bond is one of the strongest known non-covalent bonds found in biological systems, but it is heat labile. When biotinylated primers are reacted with streptavidin-coated microspheres before PCR, the primers are significantly captured onto the surface of the beads. Washing or other sample processing steps in a suitable buffer can be conducted without generally releasing the primers. However, upon heating the beads 10
or otherwise denaturing the streptavidin, large amounts of primers are released from the surface and can participate in PCR reactions. Figure 4
is a graph of measurements using a NanoDrop 2000 UV-Vis absorbance detector of the supernatant liquid from a suspension of beads labeled in this manner with primers specific for a region of Staphylococcus aureus
target DNA. As illustrated, at a range of temperatures from room temperature (RT) to <50 °C, there is no detectable release of primers, even after several days' storage at 4°C or when stored dry at room temperature in the wells of the microfluidic device for more than 2 months. When incubated at 95 °C, primers are released and can be readily detected in the supernatant.
High levels of bead functionalization with all biotinylated primer sets tested to date have been observed. The attachment is stable for more than 60 days when stored dry at room temperature, and beads stored in solution at 4 °C for >11 months have been tested and activity in PCR reactions was still observed.
In some embodiments, a bead set may be labeled with various primers for a PCR reaction (e.g
., forward and reverse primers, and/or hybridization probe) as illustrated in Figure 1B
. The beads 10
may be decoded by dyes, size, shape, magnetic properties, or other detectable and/or optical physical properties or a combination of properties such that each bead set with predefined primer pairs may be distinguished from one another. Figure 5A
illustrates a microreaction well array 100
that includes a plurality of wells 110.
Each well 110
includes a support (e.g. bead) containment region 112
and a reaction region 114.
Dimensions of the beads and the wells should be proportional such that the appropriate amount of reagent (primers, probes, etc.) is delivered. The bead containment region 112,
if used, may be similarly sized to the bead diameter. The reaction region 114
can range from submicron to millimeter dimensions with volumes ranging from subfemtoliter to hundreds of microliters. The bead containment regions 112
and the bead 10
can range from submicron to millimeter size as appropriate for the desired reaction. The bead containment region 112
is sized and configured to contain a single bead 10.
Therefore, the beads 10
may be loaded into the individual reaction wells 110
separately. As shown in Figure 5B,
a reaction solution 120
is added to the wells 110
and the wells are sealed with a cover 130
or other sealing member such as an immiscible fluid or oil such that the reaction wells 110
are fluidically isolated from one another. As shown in Figure 5C,
a cleaving operation is performed to separate the primers 20A, 20B
from the beads 10.
For example, the device 100
may be heated to release the primers 20A, 20B
from a streptavidin-biotin bond. However, depending on the type of bond between the primers 20A, 20B
and the bead 10,
other cleaving operations may be used, including the application of light or chemicals. As illustrated in Figure 5D,
the bead encoding and PCR reaction results may be read to determine 1) the type of bead 10 in
the wells 110
(and the corresponding primers in the reaction in the well 110),
and 2) the presence and/or concentration of each respective template molecule being detected. For example, if fluorescence encoding of a particular wavelength is used on the beads 10
to identify the relevant primer 20A, 20B,
then the fluorescence encoding signal is read at that wavelength before, during, or at the end of PCR. The signal from the intercalating dye or probe used to determine the concentration or presence of amplicon can be read at another wavelength. This signal along with the encoding information may then be used to determine the presence and/or concentration of each template molecule.
Accordingly, in this configuration, the beads 10
may be stochastically distributed in the microwells 110
without a complicated deterministic loading or printing technique. The primer set and/or probe of each of the beads 10
in the microwells 110
may be determined after the beads 10
are loaded and/or after the reaction is performed. Therefore, relatively large numbers of very small reaction wells 110
that can be loaded stochastically from a mixture of all the desired bead sets may be used. The device 100
may use a set of microwells 110
patterned into a substrate that mates to a sealing member or cover 130
or an overlay of immiscible fluidic such as oil, liquid polymer, or fluorocarbon liquid that will seal in a manner where each well 110
is fluidically isolated from its neighbors such that each PCR reaction is fully isolated. In some embodiments, the device 100
(having a substrate and wells) and the cover 130
may be made from a combination of one rigid and one deformable elastomeric material so that a sufficient seal can be made by the application of pressure to the external surfaces of the device 100.
The use of pre-encoding methods (size, fluorescence doping, shape, magnetic properties, among others, used alone or in combination) may be implemented for very small reaction volumes (e.g
., between about 1 cL to 1 µL or less than about 1 µL) where only a single bead 10
is needed to provide the necessary primers or other specific reagent(s). In this case, beads 10
may be loaded into microreaction wells 110
such that each well 110
contains only one bead 10
and sufficient volume for the PCR reaction to take place. In some cases, the volume of the bead well that is not displaced by the bead may be sufficient volume for a reaction to occur. In this case, the bead well can serve to both hold the bead as well as to define the reaction volume. The different sets of primer-labeled and/or probe-labeled beads 10
can be mixed together into a single solution. The identity of the primer set (or other reagent) can then be determined from the encoding of the beads 10.
Sampling may be conducted either by simple inclusion within the well 110
(generally at high concentrations of template) or by direct hybridization to the primer sets or other affinity reagents (including but not limited to antibodies or aptamers) bonded to the bead 10
(generally at lower concentrations of template). In some embodiments, hundreds to billions of parallel, singleplex quantitative PCR reactions may be performed within a single, small-footprint device 100.
 Figures 9-11
show a microfluidic device 200
that includes two substrates 210, 220
that are separated by a channel-forming photoresist or other spacer 230.
The height of the spacer 230
defines a gap between the substrates 210, 220
and a channel 232.
The substrates 210, 220
include respective hydrophobic regions 212, 222
and hydrophilic regions 214, 224
that spatially correspond to one another such that when the substrates 210, 220
are in an assembled configuration (Figure 10),
the hydrophobic regions 212, 222
and hydrophilic regions 214, 224
align with one another. One of the substrates 210
includes apertures 240
that fluidically connect to the channel 232.
One of the substrates 220
further includes etched bead wells 260.
In this configuration, beads 10
as described herein may be positioned in the bead wells 260.
A non-aqueous fluid 242
and an aqueous solution 244
may be positioned in the channel 232
to form reaction regions 246
of generally isolated aqueous solution 244
that is in fluid contact with a respective one of the beads 10.
In some embodiments, the beads 10,
the non-aqueous solution 242
and the aqueous solution 244
may be loaded into the device 200
as follows. A plurality of beads 10
may be inserted into the apertures 240
and loaded into the wells 260.
For example, the beads 10
may be magnetic beads, and a magnet applied to an exterior of one of the substrates 210, 220
may be used to drag the beads 10
across the channel 232
such that the beads 10
fall into the wells 260
and remain in place. The wells 260
may be sized and configured to accommodate a single bead 10.
A buffer may be flushed through the channel 232
to remove any excess beads 10
that were not loaded into the wells 260.
An aqueous fluid may then be added to the apertures 240
followed by a non-aqueous fluid. The aqueous fluid may form reaction regions 246
adjacent the hydrophilic regions 214, 224
that are separated by the non-aqueous fluid 242.
Although the microfluidic device 200
is described above with respect to wells 260
and the reaction region 246
in the hydrophilic regions 214, 224,
it should be understood that the reaction region 246
and wells 260
may instead be positioned in a hydrophobic region such that the reaction occurs in a non-aqueous fluid depending on the reaction environment desired. In some embodiments, the aqueous fluid is a fluid including the components of a desired reaction, such as a PCR master mix. The substrates 210, 220
may be formed of any suitable material, such as plastic, glass, or silicon or combinations thereof. Lipophobic patterning can also be used, for example a fluorocarbon-based coating with a fluorocarbon-based oil phase.
The hydrophilic regions 214, 224
and hydrophobic regions 212, 214
may be formed by depositing a hydrophilic or hydrophobic layer on the substrates 210, 220
and may or may not require subsequently removing part of the layer, depending on the hydrophilic or hydrophobic properties of the substrates 210, 220.
For example, gold may be deposited on a hydrophilic substrate, such as silicon or glass, and then etched to form hydrophilic regions of the substrate and hydrophobic regions of gold. Alternatively, patterning may be done by selective modification of the existing surface, such as plasma treatment of regions of native surfaces of plastic substrates.
As described above, the beads 10
may be loaded into the wells 260
randomly, and a subsequent operation may be used to determine which bead type corresponds to specific locations on the device 200.
For example, if the beads 10
include an optical marker that is associated with the particular primer or other reagent attached to the beads 10,
then an optical imager, such as a microscope, may be used to optically identify which primer is associated with a given well location for subsequent analysis.
In some embodiments, the wells 260
may be sized and configured to preferentially retain a particular bead type based on a size and/or shape so that, for example, larger beads would be retained by larger wells and smaller beads would be retained by smaller wells. For example, as illustrated in Figure 22A,
a schematic of a silicone chip design with 2.1 µm bead wells 360A
and 5.6 µm bead wells 360B
to hold 3 and 6 µm diameter beads, respectively, is shown. In this chip design, the smaller silicone wells expand to accept a slightly larger diameter bead, but smaller beads will not be retained in larger diameter bead wells. The outer outlines indicate reaction well geometry around each bead well. Figure 22B
is a fluorescence microscopy image showing chip loaded with only 3 µm beads. Figure 22C
is an image showing the chip loaded with a mixture of 3 and 6 µm beads and illustrates the ability to preferentially load beads into particular wells based on size encoding.
Moreover, in some embodiments, nonspecific, "parasitic" reactions may be substantially reduced by the isolation of the reaction into multiple, smaller volumes using the solid supports in solid reaction wells, e.g.,
to reduce primer dimers. Segmentation of the reaction into multiple smaller reactions using these principles is applicable to many different reactions. By multiplexing using the beads 10
according to some embodiments, the formation of an emulsion (such as used with emulsion PCR) is not generally required to accomplish reaction segmentation. Emulsions may increase the complexity and/or cost of preparation, limit the range of reaction volumes and require chemical species with a propensity to form emulsions).
In addition, it should be understood that some or all of the processes described herein may be automated, e.g., using pumps, valves, optical imaging devices, and the like.
Embodiments according to the present invention will now be described with respect to the following non-limiting examples.
PCR was performed in standard 200 µL PCR-grade polypropylene tubes with a total reaction volume of 10 µL with sets of beads containing biotinylated primer sequences for S. aureus,
the mecA gene found in Methicillin-resistant S. aureus
(MRSA), and Streptococcus mutans
purchased from IDT (Coralville, IA) and bonded to streptavidin-coated beads (Bangs Laboratories, Inc., product# PMS3N/10098). Two different concentrations of beads were used, one containing 2.5 µg of beads (∼60 nM equivalent primer concentration) and one containing 5 µg of beads (∼120 nM primer concentration). Approximately 300-400 copies of purified gDNA from each species were added to the corresponding reaction tubes. The PCR master mix containing all the reagents needed for PCR except primers was added and the tubes were thermocycled for 50 cycles. Figure 6
shows the subsequent analysis performed on the supernatant using an Agilent Bioanalyzer 2100 using the DNA1000 kit. The first lane for each analyte shows the results for PCR reactions that contained 2.5 µg of beads, and the second lane shows result from reactions that contained 5 µg of beads. Blanks were also run for each bead concentration. Primer dimers (typically indicative of high primer concentrations) are evident for the Methicillin-susceptible S. aureus
(MSSA) and MRSA blank reactions. Reducing the amount of beads to 2.5 µg eliminated dimer formation for these reactions. Strong amplicon bands were seen for tubes containing template DNA for MSSA and MRSA at the 2.5 µg bead concentration and S. mutans
at the 5-µg bead concentration. Dimers were not significant at either bead concentration in samples that contained template DNA. These results demonstrate how primers can be attached to beads, delivered to reaction wells, and then participate in specific PCR amplification reactions after a cleaving operation.
The device 50
of Figures 2-3
was used to place beads into specific wells 60
deterministic loading) in a known pattern. In contrast to multiplexed reactions in a single well, each reaction is singleplex, so an intercalating dye can be used for detection, reducing the optimization time greatly as compared to traditional multiplex PCR that requires multiple primer/probe sets with different fluorophores. Reconfiguration or incorporation of new bead sets for different assays may be accomplished simply by replacing the bead sets or adding more reaction wells to the chip or device 50.
Transfer of liquids onto the device 50
was conducted by hand; however, it should be understood that liquid handling could be easily automated. Pre-labeled beads with unique primers may be loaded into individual wells 60
using simple reagent dispensing technologies. The solution is allowed to dry, leaving the beads on the surface of the AOM 68
(or other solid phase extraction surface or material).
The sample can be pretreated by heat, reagent addition, centrifugation, enzymatic digestion, or other methods. The sample is dispensed into each well 60
that already contains preloaded primer beads and a vacuum is applied at the waste reservoir or channel 66
to pull the sample fluid through the AOM 68
to capture the analyte on the AOM. Wash buffer(s) can then be dispensed into the reaction wells 60
and removed by vacuum applied at the channel 66
if desired. As the primers are anchored to the beads, they will not be washed away during these sample clean up steps. After sample cleanup, a PCR master mix containing all reagents needed for PCR except primers is added to all the reaction wells 60
and the device 50
is thermocycled. As the master mix contains no template-specific primers, the same master mix can be used in all reactions. The elevated temperatures used for denaturation of DNA during PCR also denature the streptavidin, causing the subsequent release of the biotinylated primers during the first rt-PCR cycle thereby initiating amplification of target DNA. The released primers freely interact with target DNA in the reaction well whether or not it was directly hybridized to the primers on the beads before PCR was initiated. Coupling chemistries other than streptavidin-biotin can be used in some embodiments, including attachment of primers to beads using chemical bonds that can be cleaved using heat, a chemically induced change, or a photo-induced change of the bonds. Since the primers are released freely into the solution contained within the reaction well 60,
non-hybridized DNA and double stranded DNA (dsDNA) that is trapped by the solid phase extraction membrane 68
(such as an AOM) is easily detected. Therefore, rapid sample clean up can be performed on both single stranded (ssDNA) and dsDNA without a denaturation step that is normally required to convert dsDNA to ssDNA before hybridizing the sample DNA directly oligomers bound to beads. Since the primers are attached to the beads, in some embodiments, DNA can be extracted with the AOM without washing them away, then the release of the primers into solution allows PCR for amplifying/detecting the extracted DNA. The AOM may permit rapid sample cleanup. DNA extraction could also be performed through hybridization of the target ssDNA to the primers coupled to the beads or adsorption of target DNA to the bead surfaces.
Although the above example is discussed with respect to PCR reactions, it should be understood that similar procedures may be used for any suitable reaction, such as any nucleic acid transcription and/or amplification-related reaction, enzyme-linked immunosorbent assays (ELISA) where the fluorogenic substrate is also bound to the support surface, single molecule array (SiMoA) where the fluorogenic substrate is also bound to the support surface, reactions in which multiple beads are used to deliver different reagents for combinatorial chemistry, reactions where the beads deliver a catalyst reagent, and/or reactions where "click" chemistry reagents are delivered in stoichiometries determined by stochastic bead loading.
The system was tested using an rt-PCR chip such as the device 50
of Figures 2-3,
in which the microfluidic portion 66
was constructed of PDMS, and the substrate 62
was constructed of glass. A bead set labeled with the forward PCR primer was mixed with a set of beads labeled with the reverse PCR primer in a 1/1 ratio in reaction wells 1-3 (Figure
7). A 1-µL aliquot (containing ∼10 µg of beads) from a set of beads with both the forward and reverse primer attached was added to each of wells 4-6 (Figure 7).
Genomic DNA was added to wells 1-6 in 10 µL volumes that delivered a total of 300-400 copies of template DNA to each well. Vacuum was applied to the waste until the wells were dry, Then 5 µL of master mix containing 0.28 units of Platinum Taq
DNA polymerase (Invitrogen, Carlsbad, CA), lx Platinum qPCRSupermix-UDG (Invitrogen, Carlsbad, CA), 1% Blocker BSA (Thermo Scientific, Rockford, IL) and lx SYBR Green was added to each well. No additional primers were added. PDMS prepolymer was added to the waste reservoir to seal it. The device 50
was thermocycled from 66 °C to 95 °C for 60 cycles with a fluorescence image taken at the end of each cycle.
 Figure 7
shows a background subtracted fluorescence microscopy image of the rt-PCR chip after the 50th
amplification cycle. A background subtracted set of images was used to calculate the average signal intensity from the SYBR Green intercalating dye. Figure 8
is a graph of the amplification plots from each well. The cycle threshold (CT
) value where amplification is first evident is consistent for all reaction wells. This experiment demonstrates successful, reproducible on-chip rt-PCR can be performed using this technology. There was no discernible difference between a single bead set where a mix of the two primers were attached to the beads in a single reaction and a mix of two bead sets where only forward primers were attached to one bead set and only reverse primers were attached to another bead set. The preloaded biotinylated primers remained firmly attached to the beads during the sample cleanup step but were released into solution during PCR and successful amplification of template sequences was demonstrated.
The device 200
of Figures 9-11
is made from a silicon substrate (substrate 220)
and a glass substrate (substrate 210)
using photolithography. Silicon wafers were coated with 5 nm of titanium (Ti) and 100 nm of gold (Au) using magnetron sputtering. The metalized wafer was coated with positive photoresist and patterned with photolithography so that arrays of either 50 µm x 50 µm or 100 µm x 100 µm square regions were exposed. The Au layer was removed from the exposed regions by treatment with aqua regia,
leaving behind a layer of oxidized titanium that is very hydrophilic. The substrate was treated with 1-octadecanethiol to form a self-assembled monolayer on the Au surface only that is very hydrophobic. (Thiols such as 1-octadecanethiol form strong bonds with the Au, but not the oxidized titanium.)
Initial testing with the wafer side only (i.e. open and without glass) was performed by covering it with aqueous buffer containing dye. Mineral oil was gently flowed over the surface, and the majority of the aqueous phase was pushed off the gold-alkylthiol surface. Small droplets of aqueous buffer remained at the regions where the Au had been removed. The areas on the wafer covered by each droplet were well defined by the pattern, but droplet height (and therefore, volume) varied greatly.
A microfluidic device 200
with a patterned glass cover or substrate 210
was made to better control droplet size (Figures 9-11).
Silicon wafers were fabricated as described above, but a photo-patterned border 1 mm wide and 9-25 µm high was made with an epoxy-based negative photoresist (KMPR1010, Microchem Corp., Newton, MA). A glass substrate was coated with 5 nm of Ti and 20 nm of Au. The Au was removed by photolithography to leave an array of hydrophilic squares (regions 214)
complementary to corresponding squares (regions 224)
on the silicon substrate 220 (Figure 9).
Vias or apertures 240
were drilled through the glass substrate 210
using abrasive powder blasting. The glass substrate 210
was aligned over the silicon substrate using a Finetech die bonder (FINEPLACER, Berlin, Germany) and permanently bonded using epoxy (LOCTITE Hysol E-120HP). Figure 10
shows a schematic of the alignment, and Figure 11
shows a diagram of the cross section of the chip. The metal film on the glass is mostly transparent at these thicknesses, making alignment and optical interrogation of the hydrophobic regions possible. After treatment in ethanol saturated with octadecanethiol for ≥12 hours, the chip was rinsed with pure ethanol and then stored dry.
Alternative methods for patterning the metal films on the substrates 210, 220
may be used to form hydrophilic and hydrophobic regions, including patterning of the substrate with a photoresist and then metal deposition with lift off and/or various sputtering techniques, such as argon ion sputtering. Additional techniques for forming hydrophilic and hydrophobic reasons will be readily apparent to those of skill in the art.
The chip was designed to include barriers or "lanes" 250
of negative photoresist to divide the array into separate regions so that the flow of immiscible phase could be better regulated (Figures 12-13).
Fabrication was similar to that described above, and the lanes and outer border of the chip were patterned during the same step of photolithography. The Au was removed from areas where the photoresist was patterned as the alkylthiol treatment caused resist delamination from the Au after prolonged treatment. A solution of dye was added to fill the chip as previously described. Mineral oil was then added and vacuum was used to pull it through the chip. As can be seen in Figure 14,
droplets were isolated with mineral oil filling the interstitial space (defined by the hydrophobic regions on the substrates). A few small, stray droplets were observed but little to no bridging between wells was observed. The vast majority of droplets display similar and regular morphology.
Devices with 440 reaction regions measuring 100 µm x 100 µm were fabricated using KMPR borders either 25 µm or 7.5 µm tall to form reaction volumes of either 250 pL or 75 pL. Higher density arrays incorporating 1,500 reaction wells (50 µm x 50 µm pads with 9 µm tall KMPR) with volumes of 22.5 pL each within the same overall area were also fabricated and tested. Figures 15A-15C
show the three chips filled with aqueous buffer containing fluorescein dye. Aqueous solutions were added as 2.5-5 µL droplets to one via, and vacuum was applied at the other via to fill the chip. After filling with buffer, mineral oil was added to the via and pulled through the chip. The mineral oil wet the Au/alkylthiol surface, forcing the aqueous solution from the bulk of the chip, leaving isolated droplets at the hydrophilic defined reaction regions. The filling/droplet isolation process can be very fast, typically requiring only ∼2 minutes or less to define the isolated reaction wells.
Use of chips in rt-PCR and digital PCR experiments
Digital PCR using free solution primers at 250 nM was used to verify chip performance at the single-target-molecule level in 22.5 pL reactions (1,500 per chip). Figures 16A-16C
show results from 0.7, 0.2, and 0.04 copies of template DNA/well (nominal concentration). Positive hits indicating that at least one target copy was present totaled ∼1100 (73%), 360 (24%), and 140 (9%), respectively, out of a total of 1,500 wells. Assuming a Poisson distribution between wells, calculated concentrations of 1.3, 0.27, and 0.094 copies/well are reasonably close to the nominal concentrations, allowing for pipetting errors at submicroliter volumes during sample preparation. It should be noted that although the system has not been optimized for rapid thermocycling, detection of single copies of DNA was accomplished in less than 20 minutes. Shorter cycling times may be achieved with increased optimization of various parameters.
Demonstration of bead-loading using a magnet for high sampling efficiency and high bead well occupancy
Some chip designs may use glass and silicon components to partition sample and reagents into isolated reaction wells, but these elements can be duplicated with analogous structures and surface chemistries in plastics, such as high-volume manufacturable, monolithic polymer devices.
Although gravity-based loading has been observed to result in >55% occupancy of 3.28-µm diameter magnetic primer-coated beads for the silicon and glass chip design, sampling efficiency from the total bead population was poor (<1%). To implement sampling of rare target sequences by hybridization to primers on the beads, sampling efficiency, defined here to mean the total number of beads loaded into bead wells divided by the total number of beads in the slurry incubated with the sample, should be as high as possible. Modifications to the bead well geometry and the use of a suitable blocking buffer was shown to significantly improve occupancy and bead sampling efficiency using magnetic loading. Magnetic manipulation of the beads can greatly speed delivery of the beads to the bead wells but can be problematic since the beads align along the magnetic field lines in chains that cause multiple loading in non-optimized geometries as noted in a recent paper in which magnetic loading was unsuccessful. [Kan, C. W. et al. Lab on a Chip, 2012, 12, 5, p. 977-985
DRIE was used to fabricate arrays of bead wells ranging in size from 2.5 to 5.4 µm in diameter with depths of 4.4, 5.7, and 7.0 µm into Si wafers. Wafers were cut into sections of 3-4 arrays each. Starting Block PBS buffer (Thermo Scientific) was used to block the arrays and prevent bead adsorption (currently, 0.5% BSA is used instead of Starting Block due to PCR inhibition problems observed on-chip). An aliquot of beads (∼15,000) was placed at one side of the array, and a magnet was translated along the backside of the silicon to draw the beads over the array of bead wells while observing the beads with a microscope. After pulling the beads across a significant number of wells, the array was gently washed with deionized water, sputter coated with ~5 nm of Au/Pd, and imaged with SEM. Most arrays etched to 4.4 µm deep showed poor to moderate retention of beads after vigorous washing, but retention should be acceptable with more gentle washing. Arrays etched to 7.0 µm depth displayed many instances of double bead loading when examined by SEM. While good retention was seen for 3.5 µm diameter bead wells, multiple passes over the arrays were needed before appreciable loading was achieved. Arrays etched to a depth of 5.7 µm with diameters of 4.1 µm (Figures 17A-17B)
loaded quickly and demonstrated good bead retention. Chips fabricated with bead wells of this geometry can be loaded with <15k beads and typically show 80% to >90% bead occupancy after washing, yielding a sampling efficiency >8-10%. Figures 18A-18D
show a series of images taken during the loading process. Beads are deposited into wells from the bright clump of fluorescent beads as it is moved over the array. Washing with buffer after loading removes most loose beads, leaving most wells loaded with single beads. Rare instances of double loading might be further reduced by decreasing the well depth to a value between 4.5 and 5.7 µm. It was observed that bead populations >10k can be translocated much faster than smaller populations of beads; therefore, sampling efficiency may improve as the number of bead wells is increased. Increased optimization of the geometry of the magnet and automation of magnet movement may further improve occupancy and minimize loading time. In some embodiments, various ratios of beads to the wells or containment regions may be used to improve bead containment. For example, it is currently believed that, where a bead has a diameter d
, and the solid support containment regions have a cylindrical shape having a diameter of 1.05d
and a depth of 1.2d
. This ratio may be particularly beneficial when using magnetic bead loading as described herein. In addition, this ratio may be used in any suitable bead array loading application or device, such as SiMoA.
Reducing dimer formation by reducing reaction volume
Testing of biotinylated primer sets for the three targets was completed and the effect on primer dimer formation when the total volume of the master mix is divided into smaller, isolated reactions was explored. Massively parallel multiplexing-in-space may require a compatible thermocycling program for all primer sets. While significant primer dimer formation for nuc, S. mutans,
primer sets is typically noted when performing PCR at low annealing temperatures in conventional volumes (5-10 µL) in polypropylene PCR tubes, reduced primer dimer formation on-chip in smaller volumes has also been observed. While it is possible that these observations may be due to differences in surface interactions, an alternative explanation may be that although the concentration of primers remains the same, the total number of primer molecules in a given reaction (and therefore the likelihood of amplifying a nonspecific interaction) decreases with reduction of the total primer population. Additionally, trace amounts of malformed primers that form during synthesis that are not removed during purification may also cause nonspecific amplification. Segmentation of the reaction such that the occurrence of these impurities in any given reaction is very low will reduce the instances of these nonspecific reactions. It is anticipated that the greatly reduced volumes used for single bead reactions (millions to a thousands of times less than a standard 10 µL reaction) will allow the use of primer sets that are not currently compatible in conventional volume reactions because of primer dimer formation in some sets at low annealing temperatures. The ability to relax primer design constraints would greatly improve the accessibility of the technique and greatly accelerate reconfiguration to include new target sequences for the detection of emerging diseases. Often, dimer formation is a rare event, but when it occurs it is amplified and spreads throughout the entire reaction volume. When a given volume of PCR master mix is divided among many isolated wells, rare instances of dimer formation (or trace impurities) are effectively quarantined in one of many small reaction volumes, and the majority of the reactions remain unaffected.
Reaction Volume and Dimer Occurrence
|Primer Set||PCR Reaction Volume|
|5 µL (n=9)||300 nL (n=50)|
|S. mutans (16SrRNA)
|S. aureus (nuc)
Preliminary testing was performed to compare the degree of dimer formation in 5 µL reactions in PCR tubes with that observed in 300 nL on-chip reactions. Three stock master mixes were prepared, each containing 500 nM of a different primer set (Table 2), 1% Blocker BSA, 1x Platinum Quantitative PCR SuperMix-UDG, 1x SYBR Green I, and 0.0625 units/µL additional Platinum Taq DNA polymerase. Nine aliquots (5 µL) were then dispensed into propylene PCR tubes and thermocycled for 60 cycles with a 60 °C annealing temperature. After thermocycling, the PCR mixes were analyzed using capillary gel electrophoresis on an Agilent Bioanalyzer 2100 with a DNA 1000 chip. Dimer formation was observed in 11-22% of these reactions. Fifty on-chip reactions (300 nL each) were prepared from master mixes formulated as above and thermocycled on-chip using rt-PCR. Only one of the on-chip reactions showed primer dimers in the SYBR Green I fluorescence signal. Positive controls for S. mutans
16S rRNA and mecA
sequences indicated successful amplification for 11/11 reactions, respectively.
Hybridization with primer-functionalized beads
Preliminary investigations into the ability of the primer-labeled beads to sample DNA from dilute solution using bulk beads and standard PCR have been performed. Target gDNA (∼350 copies/µL) was digested for 15 min at 37 °C with restriction enzymes (such as EcoRV, Bpu10I, and SfaNI) to cut the DNA near the targeted region. The digestion was then heated to 95 °C for 5 min to denature the DNA. Ten µL of this digest (∼3,500 copies) was then incubated for 10-15 minutes with 5 µL of bead slurry (∼25 µg beads). Three, 15 µL wash steps were performed with PCR buffer (w/o polymerase). A 1.5 µL aliquot was then added to a PCR master mix to form a 5 µL reaction that was thermocycled 60 times. Example electropherograms plotted as gels are shown in Figure 20.
If a carryover of 0.5 µL between wash steps is assumed, then only ∼0.013 copies of target DNA was transferred in the liquid phase. Therefore, these preliminary results indicate that the beads are capturing DNA in some manner. The mechanism of DNA attachment to the beads may be specific hybridization or nonspecific adsorption. While hybridization is preferred for efficient sampling and the lowest possible limits of detection, some degree of nonspecific adsorption is not likely to be detrimental to the assay since the identity of the target is determined by PCR amplification.
 Figures 21A-21C
show a representative section from an example encoded bead-based assay for the detection of MRSA by multiple genetic markers (nuc
with S. mutans
as a negative control).
Three aliquots of streptavidin-coated beads (Bangs Laboratories, Inc., product# PMS3N/10098) were placed into separate 200 µL PCR-grade polypropylene tubes. A different set of forward and reverse 5'-biotinylated primers were incubated with each aliquot so that one aliquot contained primers for nuc
gene found in Staphylococcus aureus
gDNA, another contained primers for the mecA
gene found in Methicillin-resistant S. aureus
(MRSA), and the third contained primers for Streptococcus mutans
gDNA. After incubation, the beads were washed with buffer to remove any primers not bonded to the bead surface. Beads with primers for the mecA
gene were then incubated with biotin-labeled Qdot® 605 nanocrystals (Invitrogen, Carlsbad, CA), and beads with primers for S. mutans
gDNA were incubated with biotin-labeled Qdot® 655 nanocrystals (Invitrogen) for encoding purposes. Beads with primers for the nuc
gene found in S
were incubated in buffer only. After incubation, the bead sets were washed with buffer to remove any unbound biotin-labeled Qdot nanocrystals. In this manner, a rudimentary encoding scheme was implemented; however, encoding is not limited to this method and can be done using the various other methods described herein. Aliquots of beads from each set were then mixed together to form a slurry containing all three bead types. Bead type was readily discernible using fluorescence microscopy by monitoring the emission from the Qdot605, Qdot655, or autofluorescence of the unlabeled beads (Figures 19A-19C).
A microfluidic chip such as the device 200
in Figures 9-11
was used to isolate individual beads in separate reaction wells (∼22.5 pL each). Briefly, silicon wafers were coated with 5 nm of titanium and 100 nm of gold using magnetron sputtering. Glass substrates were also coated with 5 nm of titanium and 20 nm of gold. Photolithographic patterning was used on both the silicon wafers and glass substrates to form an array of 1,500 reaction regions, each region defined as a square measuring 50 µm on a side, where the gold was removed leaving a hydrophilic oxide surface. A single bead well measuring ∼ 4.1 µm in diameter and 5.7 µm deep was etched into the center of each reaction region using photolithography and deep reactive ion etching. A layer of negative photoresist (KMPR1010, MicroChem Corp., Newton, MA) was patterned onto the surface of the silicon wafer to form a pattern of channels and walls ∼ 9 µm tall. After fabricating holes in the glass substrate for the addition of reagents, it was aligned over the silicon wafer so that the hydrophilic oxide pattern complemented that on the silicon wafer. The two were then bonded using epoxy so that a gap of ∼9 µm separated the glass substrate from the silicon wafer. The device was filled with a saturated solution of 1-octadecanethiol in pure ethanol and left for >12 hours so that a self-assembled monolayer of the alkylthiol formed on the gold surfaces of the glass and silicon substrates.
The microfluidic device was filled with buffer, and a small amount of the mixture containing the three different bead types was then loaded into the device. A magnet was applied to the outside of the device so that the magnetic beads were pulled over the array of bead wells etched into a silicon substrate. A single bead was observed to load in ∼80% to greater than 90% of the bead wells. The array was washed with buffer to remove loose beads. The array was then imaged by fluorescence microscopy to determine the type and location of each loaded bead.
PCR master mix was prepared with lx Platinum qPCRSupermix-UDG and 0.11 units/µL additional Platinum Taq
DNA polymerase (Invitrogen, Carlsbad, CA), 0.55% Blocker BSA (Thermo Scientific, Rockford, IL), and 1x SYBR Green I (Invitrogen, Carlsbad, CA), and approximately 15 copies of MRSA gDNA (ATCC# 700699D-5) per 25 pL reaction. The master mix was then loaded into the microfluidic device so that it completely filled the gap between the glass and silicon. Mineral oil was then added to the device and transported across the well array by vacuum. The mineral oil preferentially wet the alkylthiol-treated gold surfaces, leaving behind isolated -25 pL aqueous droplets sandwiched between the exposed hydrophilic oxide surfaces.
The chip was placed onto a thermocycling stage where it was heated to 50 °C for 12 seconds, 90 °C for 120 s, and then 30 cycles of 90 °C for 5 s and 60 °C for 30 s. The array was imaged after every cycle to monitor fluorescence emission from the SYBR Green I intercalating dye. Approximately 94% of the reaction wells containing beads identified as those carrying primers for nuc
gene found in S. aureus
gDNA showed evidence of PCR amplification, as did ∼94% of reaction wells containing beads carrying primers for the mecA
gene found in Methicillin-resistant S. aureus
(MRSA). Only ∼6% of wells identified as containing primers for S
gDNA showed a positive result. Approximately 2% of the bead wells were identified as containing both one MRSA primer bead and one mutans
primer bead. It is likely that some double loading of beads carrying primers for the nuc
gene occurred, causing the false positives seen for S. mutans
primer sets, but this could not be readily discerned from the weak autofluorescence signal from the polystyrene shell of the bead (as no encoding dye was used with this set). It can be anticipated that further optimization of bead well geometries and more highly refined encoding methods can be readily implemented to reduce false positive signals.
A small region of a chip is shown in fluorescence microscopy images of 12 reaction wells with ∼25 pL volume each are shown in Figures 19A-19D. Figure 19A
is an encoding image with 365/10 nm ex. and 605/55 nm em. Three beads labeled with Qdot 605 and primers for the mecA
gene found in MRSA gDNA are highlighted with white squares. Figure 19B
shows an encoding image at 445/50 nm ex. and 665/30 nm em. Four beads labeled with Qdot 655 and primers for S. mutans
gDNA are highlighted with white squares. Figure 19C
shows a third encoding image at 556/20 nm ex. and 620/60 nm em. All bead sets autofluoresce weakly at these wavelengths, and both Qdot 605 and Qdot 655 labeled beads fluoresce brightly. Beads without Qdot labels (highlighted with white squares) carry primers for the nuc
gene found in S. aureus
gDNA. An empty bead well is highlighted with a white hexagon. Figure 19D
shows an assay image at 470/40 nm ex. and 525/50 nm em. after thermocycling 30 times. SYBR Green I fluorescence in the presence of significant amounts of double stranded DNA can be readily detected, indicating detection of the analyte. Reaction wells outlined with squares showed large increases in signal, whereas the other reaction wells only showed bead autofluorescence or a weak signal from non-intercalated SYBR Green I, similar to that shown by the well that contained no primers (white hexagon).
Results from whole chip
| ||nuc-MSSA||mecA-MRSA||S. Mutans|
Accordingly, PCR probes (for example Taqman and molecular beacon probes, among others) can improve the specificity of PCR, and these can be easily attached to the beads along with the primers if the probes are biotinylated or otherwise functionalized for attachment to a solid surface. As the beads are deterministically loaded into the reaction wells, a single detection fluorophore may be used for all probe sets. Additional optimization may be performed if probes are incorporated. PCR reactions that do not require probes can be used on the same chip with reactions that use probes if similar thermocycling and master mix conditions are used for both types of reaction. In some cases, universal primers could be added to the master mix in free solution and specific probes could be attached to the beads in place of specific primer sets.
While some embodiments of the invention uses a solid-phase extraction membrane 68 (Figures 2-3)
to purify the DNA before PCR, other implementations of the device can use direct hybridization of target DNA to its sequence-specific primers bonded to the microbeads. When the bead mixture is incubated with analyte DNA or cDNA, the primers attached to the beads will act as hybridization probes, capturing and purifying the sequence specific to that bead. Primers can be designed to optimize these hybridization events by controlling their length and sequence or the addition of novel nucleobases. Interfering components of the sample matrix (extraneous gDNA, RNA, cell membrane components, etc.) can be washed away, leaving purified target DNA hybridized to the primers bonded to the beads.
Some embodiments may also be implemented in a digital PCR format where many reaction wells can contain one or more beads or surfaces of the same set labeled with primers. However, it will be understood that in the microfluidic device according to the present invention the solid support containment region of each reaction well is configured to receive a single one of the plurality of solid supports. Quantification may then be based upon endpoint detection. Quantification of targets using isothermal amplification may also be accomplished in a manner similar to digital PCR approaches.
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. Although a few exemplary embodiments of this invention have been described, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. The scope of the invention is defined by the claims.