HK1158134B - Exchangeable carriers pre-loaded with reagent depots for digital microfluidics - Google Patents

Description

Replaceable carrier pre-loaded with reagent reservoirs for digital microfluidics

RELATED APPLICATIONS

This patent application claims priority from U.S. patent application No. 12/285,326 filed on 1/10/2008.

Technical Field

The present invention relates to replaceable carriers pre-loaded with reagents for digital microfluidics, and more particularly, to removable plastic sheets on which reagents are strategically placed at pre-selected locations as replaceable carriers for Digital Microfluidic (DMF) devices.

Background

Microfluidics involves the precise control and manipulation of liquids with only a small geometric volume (typically microliters). Because of the rapid kinetics and level of automation, microfluidics has the potential to convert conventional biological assays into rapid and reliable assays outside the laboratory. A new example of miniaturized biological assays has recently emerged, known as "digital" (or droplet-based) microfluidics. Digital Microfluidics (DMF) relies on manipulating discrete fluid droplets on the surface of a patterned electrode, see for example US7,147,763; US 4,636,785; US 5,486,337; US 6,911,132; US 6,565,727; US7,255,780; JP 10-267801; or Lee et al, 2002, "Electrowetting and removing-on-electric for microscale liquid handling" Sensors & Actuators 95: 259 and 268; pollack et al, 2000, "electric-based action of liquids for microfluidic applications," Applied Physics Letters 77: 1725-1726; and Washizu, M. et al 1998, "Electrical effects of liquid primers for Microbacterium Applications" IEEE Transactions on Industry Applications 34: 732-737. This technique is very suitable for array bioassays, where one can combine or mix fluid droplets to perform various biochemical reactions, similar to processing samples in test tubes. More importantly, the array geometry of digital microfluidics appears to be well suited to large, parallel multiplex assays in nature. In fact, this new technology has found applications in many areas, including cell-based assays, enzymatic assays, protein profiling, and polymerase chain reactions.

Unfortunately, the two major limitations of biofouling and interfacing have limited the range of applications for digital microfluidics. The former limitation, biofouling, is detrimental to all micro-scale analyses-a high surface area to volume ratio has an adverse side effect of increasing the rate at which analytes are adsorbed from solution onto solid surfaces. The present applicant and others have devised various strategies to limit the bio-fouling level of digital microfluidics, but this problem has always been an obstacle, preventing the widespread use of this technology.

A second limitation of digital microfluidics (and all microfluidic systems) is the "world-to-chip" interface-it is most difficult to deliver reagents and samples to these systems without giving up the inherent advantages of rapid analysis and low reagent consumption. One solution to this problem for microchannel-based approaches is to use pre-loaded reagents. These methods generally include two steps:

(1) storing reagents in the microchannels (or in replaceable cartridges), an

(2) Thereafter, the desired test/experiment is performed quickly using the reagent.

The microchannel system employs two strategies-in the first, storage of reagents as droplet solutions, isolation of droplets from each other using air plugs (see "Reagent-loaded cassettes for droplet and fluid delivery in microfluidic Chemistry 77: 64-71" published by Linder et al in 2005) or by immiscible fluids (see "microfluidic-scale testing of reaction conditioning using nanoparticles in microfluidic Chemistry-MS" Journal of the organic Chemical resource 128: 2518-2519 "published by Hatakyama et al in 2006; and" A microfluidic Reagent for droplet and fluid delivery in 2005 "published by Zheng et al in 2005 until the use of droplet and fluid delivery/fluid delivery of droplets of fluid/Reagent of" isolated from each other. In The second strategy, reagents are stored in solid phase in channels and then reconstituted into solutions when performing The assay (The article "The micro-active project" Automatic detection of related molecular activity "Proceedings of SPIE-int. Soc. Opt. Eng. published by Furuber et al, The article" Controlled micro fluidic storage of functional protein from hydrophilic storage "laboratory 4: 78-82" published by Garcia et al in 2004, and The article "Automounted computer system for one-step assays" published by Zimmermann et al in 2008). Preloading of reagents in microfluidic devices is a strategy that has many applications. However, to date, no similar technology has been found in digital microfluidics.

To address the dual challenges of nonspecific adsorption and external and chip interfacing in digital Microfluidics, the applicants developed a new strategy based on a removable polymer cap (see "Low-cost, rapid-prototyping of digital Microfluidics devices" Microfluidics and Nanofluidics 4: 349-355, "published by Abdelwaged and Wheeler in 2008; Chuang and Fan in 2006" Direct writing management of droplets by analysis of parallel selection-aligned micro analysis-electronic program "Proceedings of memories: 19th International Conference logic Systems, Technical software 541; and library et al" published by patent application of simulation of technology and simulation 136: design of technology and simulation of simulation 136 ": see" simulation of molecular analysis and simulation of technology of transport, 358, et al, "Low-cost, et al," read-2007 and simulation of molecular analysis, see "sample and simulation of technology, see" sample, simulation, 76, see "sample, FIGS. The membrane was replaced after each experiment, but the backbone configuration of the device was reused. This effectively prevents cross-contamination between repeated analyses and, perhaps more importantly, introduces reagents into the microfluidic device as a useful medium.

A laser radiation desorption device for manipulating a liquid sample in the form of individual droplets is known from US 2008/0156983a 1. Reagent pre-loaded carriers for use with digital microfluidic devices are disclosed. The pre-loaded carrier has one or more reagent reservoirs located at one or more pre-selected locations and comprises an electrically insulating layer and a hydrophobic surface. The digital microfluidic device includes an array of discrete electrodes and an electrode controller capable of selectively activating and deactivating the discrete electrodes to translate droplets on the hydrophobic surface to the one or more preselected locations on the pre-loaded carrier. Such droplets are further directed onto a specific pad of the digital microfluidic device, on which MALDI (matrix assisted laser desorption/ionization) analysis can be performed.

Disclosure of Invention

To illustrate the principle of using a single electrode plate and a disposable plastic cover, the applicant prepacked the dried enzyme spots on a plastic cover for later use in proteolytic digestion experiments. It has been found that the packaged reagents remain active when stored in a freezer for more than one month. As a precursor to this technology, the present applicant believes that the present invention represents a significant step forward in digital microfluidics, making it an attractive fluid processing platform for a variety of applications. According to the invention, even the use of a two-plate design (with or without two-electrode plates) is suitable for carriers pre-loaded with reagents.

The present invention provides a removable disposable carrier, such as a plastic sheet, pre-loaded with reagents. This new method involves manipulating reagents and samples on a digital microfluidic device to which a pre-loaded carrier is attached. When the assay is complete, the sheet can be removed, if necessary, and the original device can be reused to start another assay after a new pre-loaded sheet is attached.

These removable disposable plastic sheets pre-loaded with reagents facilitate rapid batch testing using digital microfluidic devices without the problem of cross-contamination between tests. In addition, the kit devices and methods disclosed herein facilitate the use of reagent reservoirs. For example, the inventors have made sheets with pre-loaded spots of dry enzymes that are commonly used in proteomics assays, such as trypsin or alpha-chymotrypsin. After digestion of the model substrate ubiquitin, the product-containing slices were evaluated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The present invention is particularly advantageous in improving the compatibility of digital microfluidics with diverse applications, from experimental analysis to point-of-care (point-of-care) diagnostics.

Accordingly, one embodiment of the present invention comprises a carrier (preferably in sheet or film form) pre-loaded with reagents for use with a digital microfluidic device comprising an electrode array comprising discrete electrodes arranged in an array, the digital microfluidic device comprising an electrode controller, the pre-loaded carrier comprising:

-an electrically insulating sheet having a back surface and a hydrophobic front surface, said electrically insulating sheet being removably attached to said electrode array of said digital microfluidic device, wherein said back surface is adhered to a surface of said electrode array, said electrically insulating sheet covering said discrete electrodes such that said discrete electrodes are electrically insulated from each other and from droplets on the hydrophobic front surface,

wherein the electrically insulating sheet has one or more reagent depots located at one or more preselected locations on the hydrophobic front surface of the electrically insulating sheet;

in operation, the electrode controller is capable of selectively activating and deactivating the discrete electrodes to translate a droplet of liquid across the hydrophobic front surface of the electrically insulating sheet; and

the one or more pre-selected locations of the hydrophobic front surface of the electrically insulating sheet are arranged to be accessible to droplets initiated on the hydrophobic front surface of the electrically insulating sheet.

Another embodiment of the invention provides a digital microfluidic device comprising:

-a first substrate having an electrode array mounted on a surface thereof, the electrode array comprising an array of discrete electrodes, the digital microfluidic device comprising an electrode controller capable of selectively activating and deactivating the discrete electrodes,

-an electrically insulating sheet having a rear surface and a hydrophobic front surface, said electrically insulating sheet being removably attached to an electrode array of said digital microfluidic device (preferably said rear surface being adhered to the surface of said array of discrete electrodes), said electrically insulating sheet electrically insulating the discrete electrodes of said electrode array from each other and from droplets on the hydrophobic front surface, said electrically insulating sheet having one or more reagent reservoirs located at one or more pre-selected locations on said hydrophobic front surface of said electrically insulating sheet; (ii) the one or more pre-selected locations on the hydrophobic front surface are arranged to be accessible to droplets initiated on the hydrophobic front surface of the electrically insulating sheet;

wherein selectively activating and deactivating the discrete electrodes under the control of the electrode controller is capable of translating a droplet through the hydrophobic front surface to the one or more reagent depots.

In one embodiment, the device of the present invention may comprise a second substrate having a front surface, said front surface optionally being a hydrophobic surface, wherein said second substrate is spaced apart from said first substrate so as to define a space between said first and second substrates, said space being capable of containing droplets between said front surface of said second substrate and said hydrophobic front surface of said electrically insulating sheet on said electrode array of said first substrate. An embodiment of the inventive device may comprise an array of electrodes covered by a dielectric sheet on said second substrate. In this case, the electrode array on the first substrate is optional and thus may be omitted. The electrically insulating foil may also be pre-loaded with reagent reservoirs on either or both of the first and second substrates.

The present invention also provides a digital microfluidic method comprising the steps of:

-preparing a digital microfluidic device having an electrode array comprising an array of discrete electrodes, said digital microfluidic device comprising an electrode controller connected to said array of discrete electrodes for applying a selected voltage pattern to said discrete electrodes to selectively activate and deactivate said discrete electrodes to cause droplets of liquid sample to pass through said electrode array on said discrete electrodes in a desired path;

-providing a removable and attachable electrically insulating sheet having a rear surface and a front working surface;

-removably attaching said electrically insulating sheet to said electrode array of said digital microfluidic device (preferably with said rear surface adhered thereto), said electrically insulating sheet having a hydrophobic front surface and one or more reagent reservoirs located at one or more preselected locations on said hydrophobic front surface of said electrically insulating sheet; said one or more preselected locations on said hydrophobic front surface of said electrically insulating sheet are disposed to be accessible to droplets initiated on said hydrophobic front surface of said electrically insulating sheet;

-conducting an assay, directing one or more sample droplets through said front working surface to said one or more reagent depots, thereby delivering said one or more sample droplets to said one or more reagent depots reconstituted from said one or more sample droplets and mixing with at least one selected reagent contained in said one or more reagent depots;

-separating out any (or at least one) resulting reaction product formed between the mixed sample droplet and the at least one selected reagent in each (or at least one) of the one or more reagent depots; and optionally

-removing the removable and attachable electrically insulating sheet from the surface of the electrode array of the digital microfluidic device and preparing the digital microfluidic device for a new experiment.

Further features and advantages of the invention will be understood by reference to the following description and drawings. Further features and other preferred embodiments of the invention are defined by the dependent claims.

Drawings

Exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings, which do not limit the scope of the present invention in any way. Wherein:

FIG. 1A shows the adsorption of proteins from a water droplet onto a digital microfluidic device, where the top image of the figure shows the device before the droplet was started, with the corresponding confocal image of the central electrode, and the bottom image of the figure shows the same device after the droplet containing FITC-BSA (7. mu.g/ml) has been cycled 4 times back and forth over the electrode, with the confocal image collected after the droplet was moved. The same treatment was done on both graphs, indicating that confocal microscopy can be used to detect non-specific protein adsorption on the device surface as a result of digital actuation.

FIG. 1B is a mass spectrum of 10 μ M angiotensin I (MW 1296).

Fig. 1C is cross-contamination of digital microfluidic devices: mass Spectrometry of 1. mu.M angiotensin II (MW 1046). The droplets initiated on the same surface of the same device (on which the former droplet had been initiated), cross-contaminating angiotensin I.

Fig. 2 is a schematic illustration of a strategy for the removable pre-load carrier, shown step by step, in which:

(1) fixing a new piece of carrier (in the form of a plastic sheet) with dry reagents on a digital microfluidic device;

(2) activating a droplet of reagent through the top surface of the carrier, exposing the droplet of reagent to the pre-loaded dry reagent, and combining, mixing and incubating them to obtain a chemical reaction product,

(3) discarding the residue after non-specific adsorption of the analyte;

(4) stripping off the carrier with the product droplets or dry product; and

(5) if desired, the product is analyzed.

FIG. 3 is a MALDI-MS analysis using a single digital microfluidic device to process different analytes on different carriers:

a)35 μ M insulin

b)10 μ M bradykinin

c)10 μ M20 mer DNA oligonucleotide

d) 0.01% superscript (ultramarker).

FIG. 4 is a preloaded vector assay. The MALDI peptide mass spectra obtained after the prepended trypsin (upper) and alpha-chymotrypsin (lower) dot digestion of ubiquitin are shown, and the peptide peaks identified by database search in MASCOT are calculated to have a sequence coverage (sequence coverage) of over 50%.

FIG. 5 is a histogram showing the percent activity versus time of a preloaded vector stability assay in which the fluorescence of the protease substrate (fluoroboro-fluorescence-casein) and internal standard is evaluated after 1, 2, 3, 10, 20 and 30 days of storage of the vector as shown in the histogram, stored at-20 ℃ or-80 ℃ and the mean response and standard deviation calculated for 5 replicate vectors per condition.

Fig. 6 is a different embodiment of a digital microfluidic device according to the present invention, wherein:

FIG. 6A is a digital microfluidic device open on one side with a carrier pre-loaded with reagents attached to a first substrate;

FIG. 6B is a digital microfluidic device open on one side with a carrier pre-loaded with reagents and a dielectric layer under the carrier;

FIG. 6C is a digital microfluidic device closed on one side, having a second substrate, thereby defining a space or gap between the first and second substrates;

fig. 6D is a two-sided closed digital microfluidic device having a second substrate, thereby defining a space or gap between the first and second substrates.

Detailed Description

In general, the systems described herein relate to replaceable reagent-preloaded carriers for digital microfluidic devices, more particularly suitable for high-throughput analysis procedures. As required, several embodiments of the present invention are disclosed herein. However, the disclosed embodiments are merely exemplary of the invention, which is to be understood that the invention may be embodied in various and alternative forms. The figures are not drawn to scale and some features may be exaggerated or minimized to show details of particular components while related components may not be shown to prevent obscuring the new features. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention. The illustrated embodiments are directed to alternative reagent pre-loaded carriers for digital microfluidic devices for purposes of teaching and not limitation.

As used herein, the term "about," when used with respect to a size range of particles or with other physical or chemical properties or characteristics, is meant to encompass minor variations that may exist in the upper and lower limits of such size ranges, thereby not excluding such embodiments: on average most sizes are satisfactory, but statistically sizes may fall outside this range. This is done for the purpose of not excluding these embodiments from the invention.

The underlying problem to be solved by the present invention is to provide a device suitable for digital microfluidic devices that allows digital microfluidic devices to be used in high throughput batch processing, while avoiding the biofouling problems of digital microfluidic devices discussed above in the background section. To address how biofouling is problematic, the present inventors have conducted various studies to determine the scope of the problem.

Protein adsorption and cross-contamination analysis for digital microfluidic devices

Confocal microscopy was used to assess protein adsorption on the surface. In general, a droplet containing 7. mu.g/ml FITC-BSA was translated to a digital microfluidic device. The spots were photographed before and after the droplet was initiated, resulting in two images. Residues remain on the surface during droplet start-up due to non-specific protein adsorption, detectable by confocal microscopy. These residues can cause two types of problems with digital microfluidic devices:

(1) the surface may become tacky, impeding droplet movement, an

(2) If multiple experiments are to be performed, cross-contamination problems may arise.

In the invention, the catalyst is used with Ar⁺(488nm) FluoView 300 scanning confocal microscopy (OLYMPUS, Markam, ON) with a laser combined with a 100-fold objective lens (numerical aperture 0.95) to analyze proteins adsorbed ON the surface of the digital microfluidic device (FIG. 1A). The fluorescence of the adsorbed labeled proteins was passed through a 510-525nm band pass filter and each digital image was formed from the average of four frames using FluoView image acquisition software (OLYMPUS).

MALDI-MS was used to assess the amount of cross-contamination of two different peptide samples that were initiated by the same pathway through the same device. Specifically, a 2 μ l 10 μ M angiotensin I droplet was initiated for the first time and a 2 μ l1 μ M angiotensin II droplet was initiated for the second time. As shown in fig. 1B, the profile of angiotensin I produced after the first start-up was fairly clean; however, as shown in figure 1C, the profile produced by angiotensin II is contaminated with the residue from the last start. In these tests, after digital microfluidics initiation, the sample droplets were transported to MALDI targets for crystallization and analysis, implying cross-contamination including: (a) a first initiated adsorption step, and (b) a second initiated desorption step. The intensity of angiotensin I contaminant is estimated to be approximately 10% of the angiotensin II most intense peak (MW 1046). This corresponds approximately to 1% or 0.1 μ M non-specific fouling of angiotensin I in digital microfluidic devices. This result is consistent with the values reported by Luk (less than 8% FITC-BSA adsorbed onto digital microfluidic devices) even though the viscosity of the test peptides is less than that of the proteins (see the article "Pluronic additives: A solution to solutions in digital microfluidics," Langmuir 24: 6382-6389, published by Luk et al 2008). In addition to contamination, the droplets are prevented from moving smoothly due to non-specific adsorption from the last start, especially during the start of angiotensin II samples. Therefore, a higher actuation voltage is required to cause the droplet to move through the next set of electrodes. However, this approach does not work well where the droplets become permanently adhered by adhering firmly to the contaminated surface, and increasing the actuation voltage does not help in this case, let alone if the voltage is too high may result in breakdown of the dielectric and damage to the device.

Replaceable pre-loaded disposable carrier

The present invention provides replaceable, pre-loaded, disposable carriers on which reagents are strategically located at preselected locations on the upper surface. These carriers can be used with the digital microfluidic device as replaceable carriers that are applied to an electrode array of the digital microfluidic device.

Referring to fig. 2, a pre-loaded disposable electrically insulating carrier 10 according to the invention is shown, which carrier 10 has a pre-loaded reagent reservoir 12 mounted on a hydrophobic front surface of an electrically insulating sheet 11. The disposable carrier 10 may be any thin dielectric sheet or film so long as it remains chemically stable to the pre-loaded reagents. For example, any polymer plastic such as saran wrap (saran wrap) may be used. In addition to plastic food packaging films, other carriers including general/office tape and taut paraffin sheets may also be used as alternative digital microfluidic carriers.

The disposable carrier 10 is connected to an electrode array 16 of a digital microfluidic device 14, wherein the rear surface of the carrier 10 is attached to the electrode array 16, and reagent reservoirs 12 placed on the surface of the carrier 10 through which reagent droplets will translate are aligned with pre-selected individual electrodes 18 of the electrode array 16, as shown in steps (1) and (2) of fig. 2. Two drops of reagent 20 and 22 were dropped onto the device prior to testing. The droplets 20 and 22 are preferably spotted onto the device using a pipette tip 36 attached to either the sample vessel 32 or the solvent vessel 34 (see fig. 2). Alternatively, reservoirs 32 and 34 may be connected to or integrally formed with the device such that droplets 20 and 22 may be dispensed upon actuation of the digital microfluidics.

As shown in step (3) of fig. 2, reagent droplets 20 and 22 are initiated through the top surface of the disposable sheet or carrier during the assay to help mix and merge the reagent droplets 20 and 22 with the desired reagent reservoir 12 at the electrodes. After the reaction is complete, the disposable support 10 may be stripped as shown in step (4) and the resulting reaction product 26 may be analyzed as shown in step (5), if desired. A new disposable carrier 10 is attached to the digital microfluidic device 14 for the next analysis round. The product 26 may also be analyzed while the removable carrier is still attached to the digital microfluidic device 14. The above process can be repeated using additional pre-loaded carriers. In addition, droplets containing reaction products may be split, mixed with other droplets, and/or incubated with cell culture medium if they contain cells.

Since the residue 28, 30 from the test on the previous disposable sheet or carrier 10 is removed with the disposable carrier 10, cross-contamination is avoided. The above test is performed using one pre-loaded reagent reservoir 12, but it will be appreciated by those skilled in the art that the pre-loaded carrier 10 may be loaded with multiple reagents for performing serial or parallel tests with multiple droplet reagents 20 and 22.

In one embodiment of the invention, pre-assembled electrically insulating sheet 11 and electrode array 16 may each include alignment marks that align electrically insulating sheet 11 and electrode array 16 when electrically insulating sheet 11 is attached to electrode array 16 such that one or more pre-selected locations 13 on front working surface 11a of electrically insulating sheet 11 are selected to be in registration with one or more pre-selected discrete actuation electrodes 18 of the electrode array. When the reagent reservoirs 12 are in registration with the pre-selected electrodes 18, they may be positioned above the selected electrodes or laterally close to the selected electrodes so that it is over the gap between adjacent electrodes.

Fig. 6A shows a digital microfluidic device with an open side of a carrier 10, wherein the carrier 10 is preloaded with reagent reservoirs 12 for use with the digital microfluidic device 14, the carrier 10 being connected to a first substrate 24. The digital microfluidic device comprises an array 16 of discrete electrodes 17 and an electrode controller 19. The pre-load carrier 10 comprises an electrically insulating sheet 11 having a hydrophobic front surface 11a and a back surface 11 b. The electrically insulating foil 11 is removably attached to a surface 16' of an electrode array 16 of the digital microfluidic device 14. When the electrically insulating sheet 11 is placed on the electrode array 16 of the digital microfluidic device 14, it covers the discrete electrodes 17 and electrically insulates the discrete electrodes 17 from each other and from the droplets 20, 22, 33 on the hydrophobic front surface 11 a. The electrically insulating foil 11 according to the first embodiment of the invention is provided with one or more reagent depots 12, which one or more reagent depots 12 are located at one or more pre-selected locations 13 of the hydrophobic front surface 11a of the electrically insulating foil 11. In operation, electrode controller 19 of digital microfluidic device 14 is capable of selectively activating and deactivating discrete electrodes 17 to translate droplets 20, 22, 33 across hydrophobic front surface 11a of electrically insulating sheet 11, while one or more preselected locations 13 on front working surface 11a of electrically insulating sheet 11 are positioned to be accessible to droplets 20, 22, 33 activated on hydrophobic front surface 11a of electrically insulating sheet 11.

Preferably, the electrically insulating sheet 11 is attachable or connected to the surface 16 ' of the electrode array 16 by means of an adhesive 15, wherein the adhesive 15 contacts the rear surface 11b of the electrically insulating sheet 11 with the surface 16 ' of the electrode array 16 and/or with the surface 24 ' of the first substrate 24. More preferably, the electrically insulating foil 11 comprises an adhesive 15 on the rear surface 11b thereof, the adhesive 15 being capable of contacting the electrode array to adhere the electrically insulating foil to the first substrate 24.

Fig. 6B shows a digital microfluidic device open on one side with a carrier pre-loaded with reagents and a dielectric layer underneath the carrier. The digital microfluidic device 14 (similar to that described in fig. 6A) includes important features such as an electrode controller 19; in addition, the droplets 20, 22, 33 to be translated are also included. However, in the embodiment shown in fig. 6B, the adhesive 15 contacts only the rear surface 11B of the electrically insulating sheet 11 and the surface 24' of the first substrate 24; alternatively, the adhesive 15 may be provided on the entire rear surface 11b of the electrically insulating sheet 11 (not shown). In this embodiment, the digital microfluidic device 14 preferably comprises a dielectric layer 25 applied directly on said surface 16' of said electrode array 16, so that it is sandwiched between said electrode array 16 and said electrically insulating sheet 11.

Fig. 6C shows a closed-sided digital microfluidic device having a second substrate defining a space or gap between the first and second substrates. The digital microfluidic device 14 (similar to that described in fig. 6B) includes important features such as an electrode controller 19; in addition, the droplets 20, 22, 33 to be translated are also included. In this embodiment, the digital microfluidic device 14 preferably further comprises a second substrate 27, said second substrate 27 having a front surface 27', optionally a hydrophobic surface. The second substrate 27 is spaced apart from the first substrate 24 so as to define a space or gap 29 between the first and second substrates 24, 27, said space or gap 29 being capable of containing the droplets 20, 22, 33 between the front surface 27' of the second substrate 27 and the hydrophobic front surface 11a of the electrically insulating foil 11 on said electrode 16 of said first substrate 24. Preferably, the electrode controller 19 also controls the electrostatic charge of the surface 27' of the second substrate. In contrast to fig. 6B, the adhesive 15 of the present embodiment contacts only the rear surface 11B of the electrically insulating sheet 11 and the dielectric layer 25 on the surface 16' of the electrode array 16 of the first substrate 24. Alternatively, the adhesive 15 may be provided on the entire rear surface 11b of the electrically insulating sheet 11.

Fig. 6D shows a two-sided closed digital microfluidic device having a second substrate defining a space or gap between the first and second substrates. The digital microfluidic device 14 (similar to that described in fig. 6A-6C) includes an array 16 of discrete electrodes 17 and an electrode controller 19. The pre-load carrier 10 comprises an electrically insulating sheet 11 having a hydrophobic front surface 11a and a back surface 11 b. The electrically insulating foil 11 is removably attached to a surface 16' of a first electrode array 16 of the digital microfluidic device 14. In this embodiment, the digital microfluidic device 14 preferably further comprises a second substrate 27 having a front surface 27'. According to this embodiment the front surface 27' of the second substrate 27 is not hydrophobic and comprises an additional second electrically insulating sheet 31 having a back surface 31b and a hydrophobic front surface 31 a. Said additional second electrically insulating foil 31 is removably attached to the front surface 27 'of the second substrate 27, wherein the rear surface 31b is adhered to the front surface 27'. The additional second electrically insulating sheet 31 is devoid of or provided with one or more reagent reservoirs 12, the one or more reagent reservoirs 12 being located at one or more preselected locations on the hydrophobic front surface 31a of the additional second electrically insulating sheet 31.

In contrast to fig. 6B, the adhesive 15 of the present embodiment only contacts the rear surface 11B of the electrically insulating sheet 11 and the surface 16' of the electrode array 16 located on the first substrate 24. On the opposite side, the adhesive 15 is provided on the entire rear surface 31b of the additional second electrically insulating sheet 31. Alternatively, the adhesive 15 may be provided on the entire rear surface 11b of the electrically insulating sheet 11 (not shown). Preferably (as shown in fig. 6D), the digital microfluidic device 14 comprises an additional array of electrodes 35 mounted on the front surface 27' of the second substrate 27, said additional array of electrodes 35 being covered by an additional second electrically insulating sheet 31 having a hydrophobic front surface 31 a. As shown in fig. 6B and 6C, the digital microfluidic device 14 of fig. 6D preferably also comprises a dielectric layer 25 applied directly on said surface 27' of said electrode array 35, such that it is sandwiched between said electrode array 35 and said second electrically insulating sheet 31. Another dielectric layer 25 (not shown) may also be placed between the electrically insulating sheet 11 and the surface 16' of the electrode array 16. In an alternative embodiment (not shown) the additional electrode array 35 on the second substrate 27 is coated with a hydrophobic coating and the second electrically insulating layer 31 is not provided.

The disposable carrier 10 may be packaged with a plurality of other carriers and sold with a reagent reservoir containing one or more reagents selected for a particular test type. The carriers 10 in the package may be provided with the same number of pre-loaded reagent reservoirs 12, each comprising the same reagent composition. The reagent reservoir preferably comprises a dry reagent, but may also comprise a viscous gel reagent.

One potential application of the present invention is the culturing and analysis of cells on reagent reservoirs. In such applications, the reagent reservoir may include biological substrates and attachment factors for adherent cells, such as fibronectin, collagen, laminin, polylysine, and the like, as well as combinations thereof. The droplet containing the cells may be directed to a biological substrate reservoir, allowing the cells to attach for adherent cells. After attachment, the cells can be cultured or analyzed within the digital microfluidic device.

Although fig. 2 shows a digital microfluidic device 14 having a single substrate 24 and an electrode array 16 formed thereon, it will be understood by those skilled in the art that the digital microfluidic device may include a second substrate 27 having a front surface 27' that is optionally a hydrophobic surface, wherein the second substrate is spaced apart from the first substrate so as to define a space between the first and second substrates, said space being capable of containing droplets between the front surface of the second substrate and the hydrophobic front surface of the electrically insulating sheet on said electrode array of the first substrate (see fig. 6C). The second substrate may be substantially transparent. In addition to the embodiment depicted in fig. 6C, the pre-load carrier 10 (comprising a first electrically insulating sheet 11 having a hydrophobic front surface 11a and a back surface 11b) may be removably attached to a surface 27' of a second substrate 27 of the digital microfluidic device 14. Also, the electrode array 16 may be coated with a non-removable electrical insulator (not shown).

When the front surface of the second substrate is not hydrophobic, the apparatus may comprise an additional electrically insulating sheet having a back surface and a hydrophobic front surface, said additional electrically insulating sheet being removably attached to the front surface of the second substrate, wherein its back surface is adhered to the front surface of the second substrate. The additional electrically insulating sheet has one or more reagent reservoirs located at one or more preselected locations on the hydrophobic front surface of the electrically insulating sheet.

Furthermore, an additional electrode array 35 may be mounted on the front surface 27' of the second substrate 27 and comprise a layer with a hydrophobic front surface applied on the additional electrode array 35, said layer with a hydrophobic front surface 31a applied on the additional electrode array 35, possibly an additional electrically insulating foil 31, the additional electrically insulating foil 31 having one or more reagent reservoirs 12, said one or more reagent reservoirs 12 being located at one or more pre-selected locations 13 of said hydrophobic front surface. In the two-plate design shown in fig. 6D, the first substrate 24 optionally may be provided without a pre-loaded insulating sheet or carrier 11 on which the reagent reservoirs 12 are mounted.

The invention and the effects of the invention on high throughput testing will be described in connection with the following studies and examples, which are intended to be illustrative only and non-limiting.

Detailed experiments

Reagents and materials

Working solutions of the total matrix (. alpha. -CHCA, DHB, HPA and SA) were prepared in 50% analytical grade acetonitrile/deionized water (v/v) and 0.1% TFA (v/v) at a concentration of 10mg/ml and stored at 4 ℃ in the dark. Stock solutions of angiotensin I, II and bradykinin (10 μ M) were prepared with deionized water, while stock solutions of ubiquitin and myoglobin (100 μ M) were prepared with working buffer (10mM Tris-HCl, 1mM CaCl20.0005% w/v segmented polyether F68, pH 8). All standard stock solutions were stored at 4 ℃. Stock solutions (100 μ M) of digestive enzymes (bovine trypsin and α -chymotrypsin) were prepared with working buffer and stored in aliquots at-80 ℃ until use. The standard solution and enzymes were warmed to room temperature and diluted with deionized water (peptide) and working buffer (protein, enzyme and fluorescent reagents) just prior to the assay. Fluorescence test solutions (3.3. mu.M quenched fluoroboro-fluorescent-casein and 2. mu.M rhodamine B in working buffer) were prepared just prior to use.

Fabrication and operation of devices

Standard microfabrication techniques were used to fabricate digital microfluidic devices in which 200nm thick chromium electrodes were patterned on a glass substrate. Prior to the experiment, the device was loaded with either (a) unmodified support, or (b) reagent-preloaded support. When the unmodified support (a) was used, a few drops of silicone oil were dropped onto the electrode array, which was then covered with a plastic cap. Teflon-AF (1% w/w in fluorinating solution Fluorinert FC-40, 1000RPM, 60s) was then spin coated on the surface and annealed with a hot plate (75 ℃, 30 min). When the pre-loaded carrier (b) is used, the plastic cap is modified prior to application to the device. The modification comprises three steps: a plastic cover was adhered to an unpatterned glass substrate, coated with teflon-AF (supra), and a reagent reservoir was added. The latter step can be implemented as follows: mu.l of enzyme (6.5. mu.M trypsin or 10. mu.M. alpha. -chymotrypsin) was pipetted onto the surface and the drop was allowed to dry. The pre-loaded carriers can be used immediately or sealed in sterilized plastic petri dishes and stored at-20 ℃. Before use, the pre-loaded carrier was heated to room temperature (if necessary), peeled from the unpatterned substrate, applied to a silicone oil-coated electrode array and annealed on a hot plate (75 ℃ C., 2 min). In addition to food packaging films, plastic tape and paraffin can also be used for mounting on the device. The tape was attached to the device by gentle finger pressure, the paraffin was stretched to a thickness of about 10mm, and the device was then wound to form a bubble-free seal.

The 1mm x 1mm electrodes of the device were made in a "Y" shape design with a gap of 10 μm between the electrodes. Applying a driving voltage (400-500V) to the continuous electrode pair_RMS) The 2 μ Ι droplets were allowed to move and merge on the device operating in open plate mode (i.e. without a top cover). The output of a function generator with the working frequency of 18kHz is amplified to generate driving voltage, and then the driving voltage is applied to the exposed contact by hands. The initiation of the droplets was monitored and recorded by a CCD camera.

Analysis by MALDI-MS

Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to evaluate samples initiated on digital microfluidic devices. Two modes were made for matrix/sample spots: conventional and in situ. In the normal mode, the sample is run on the device, collected with a pipette and dispensed onto a stainless steel target. The matrix solution was added and the combined droplets were allowed to dry. In the in situ mode, dispersed droplets containing the sample and matrix are moved, combined, and actively mixed by digital microfluidics, and then dried on the surface. In situ experiments involving pre-loaded carriers, a chip reaction was performed before matrix/crystallization: the droplets containing the sample proteins were driven to dry spots containing digestive enzymes (trypsin and alpha-chymotrypsin). After incubation with the enzyme (room temperature, 15min), a drop of substrate was driven to the spot, the reaction was quenched, and the combined drops were allowed to dry. After co-crystallization, the support was carefully peeled off the apparatus and then fixed to a stainless steel target with double-sided tape. Different analytes use different matrices: the peptide standard and digestive juice are alpha-CHCA, the overproof marker is DHB, the oligonucleotide is HPA, and the protein is SA. At least three replicate spots were evaluated per sample.

Samples were analyzed on a MALDI-TOF Micro-MX MS mass spectrometer (Waters, Milford, Mass.) operating in forward mode (positive mode). Peptide standards and digestion solution evaluations were performed in a reflex mode (reflexon mode) with mass-to-charge (m/z) ranges of 500-2,000. The evaluation of the proteins was performed in a linear mode (linear mode) with mass to charge ratios (m/z) ranging from 5,000 to 30,000. At least 100 spots are collected per pattern and the laser power is adjusted to optimize the signal-to-noise ratio (S/N). The data was then processed by normalizing, baselining, and smoothing with a 15-point running average (15-point running average) for the maximum peak of the analyte. The SwissProt database was searched using Mascot protein identification package to analyze the profile of the enzymatic digests. Database search resulted in 1 allowed missed cleavage site (allowed misstrained cleavage) with mass spectral accuracy of +/-1.2Da, with no other modifications.

MS analysis of peptides/proteins on alternative supports

To elucidate this new strategy, four different types of analytes were processed with a single digital microfluidic device, each with a new removable carrier. As shown in FIG. 3, four analytes included insulin (MW 5733), bradykinin (MW 1060), 20-mer oligonucleotide (MW 6135) and synthetic polymer supersamples 1621(MW 900-2200). Each removable carrier was analyzed in situ using MALDI-MS and no evidence of any cross-contamination was observed. In the applicant's laboratory, conventional devices are typically disposable (disposable); however, in experiments using removable carriers, the devices used typically performed 9-10 trials, but without a drop in performance. Thus, in addition to the problem of no cross-contamination, the removable carrier strategy can also greatly reduce the fabrication and assembly burden required to support digital microfluidics.

In addition to plastic food packaging films, other carriers including office tape and stretch wax film sheets may also be used as alternative carriers. As with food packaging films, it has been found that a carrier formed of adhesive tape and wax film pieces can support droplet movement and facilitate device recycling (number)As not shown). Furthermore, carriers formed from these materials are advantageous in that they do not require an annealing treatment prior to use. However, other aspects make these materials less attractive. The cover formed from the tape is prone to damage the actuation electrode after repeated use (although presumably, low viscosity tapes may not have this problem). In addition, since the tested tape carrier was relatively thick (about 45 μm), a large driving current (about 900V) was required_RMS) To manipulate the droplets. In contrast, the thickness of the taut wax film sheet is about 10 μm, and the driving voltage is not much different from the voltage used for the carrier formed by the food packaging film. However, carriers formed in this manner were found to be non-uniform in thickness and they were less reliable for droplet movement. In short, various carriers are consistent with the concept of removable lids, but because carriers formed from food packaging films perform best at present, experiments were conducted using this material.

Two disadvantages of the removable carrier strategy are trapped bubbles and material incompatibility. In initial experiments, trapped air bubbles were seen between the carrier and the device surface from time to time during use. When a driving voltage is applied to the electrodes near the trapped bubble, arcing is observed, which can damage the device. It has been found that this problem can be solved by wetting the surface of the device with a few drops of silicone oil before the plastic film is used. After annealing, the silicone oil evaporates, leaving a bubble-free seal. The latter issue, material incompatibility, is of greater concern. If aggressive solvents are used, the material in the carrier may leach into solution, interfering with the test. In the experiments of the present invention, no contaminating peaks were found on the MALDI-MS spectra (including the control mass spectrum generated by the bare support surface, not shown), but the possibility of this problem in other settings cannot be excluded. Since a wide variety of materials can be used to form the carrier (see above), the applicant is confident that other alternatives can be used where teflon coated food packaging films are not suitable.

Pre-loaded carrier and stability analysis thereof

In the course of studying alternative carrier strategies to overcome fouling and cross-contamination, it has been recognized that this technology can also form the basis of an exciting innovation in digital microfluidics. Pre-deposition of reagents onto carriers (and provision of several such carriers) this strategy translates digital microfluidics into a convenient new platform for rapid introduction of reagents into the device, and also solves the well-known problems of microfluidics with external to chip interfaces (see Fang et al, "A high-through connected sample interface for microfluidic chip-based chip electrophoresis systems," Analytical Chemistry 74: 1223-.

To elucidate this new strategy, a food packaging film pre-primed with spots of dry digestive enzymes was prepared and droplets containing the simulated substrate ubiquitin were then delivered to these spots using digital microfluidics. After an appropriate period of incubation, droplets containing the MALDI matrix are delivered to these spots, which are then dried and annealed. As shown in fig. 4, MALDI mass spectrometry is consistent with the expected results for peptide mass fingerprints of analytes. In fact, performance was excellent when evaluated with the proteomic search engine MASCOT, with sequence coverage of 50% or more for all experiments.

This approach has been observed to be quite reliable during the pre-loaded vector strategy for optimizing protease assays. First, block polyether F68 was used as a solution additive to help move the analyte droplet (here ubiquitin); this reagent has been found to reduce the ionization efficiency of MALDI-MS (see Boemsen et al, 1997, in the article "abundance of solvents and reagents on matrix-associated chromatography/ionization Mass Spectrometry of proteins and oligonucleotides" Rapid Communications in Mass Spectrometry 11: 603-609). Fortunately, the amount used here (0.0005% w/v) was so low that this effect was not observed. Secondly, autolysis peaks for trypsin and alpha-chymotrypsin are rarely seen, which applicants attribute to low enzyme to substrate ratios and short reaction times. Third, in preliminary experiments, it was determined that the annealing step (75 ℃, 2min) did not affect the activity of the dry enzyme. If reagents sensitive to these conditions are used later, the applicant plans to evaluate carriers made of materials that do not require annealing (for example low-tack tapes). In any case, the reliable performance of these preliminary experiments suggests that this strategy may ultimately find application in a wide range of fields, such as immunoassays or microarray analysis.

As described above, the preloaded vector strategy is similar to the concept of storing preloaded reagents in microchannels (see Linder et al, 2005; Hatakeyama et al, 2006; Zheng et al, 2005; Furuberg et al, 2007; Garcia et al, 2004; Zimmermann et al, 2008; and Chen et al, 2006 "Microfluidic clamped loaded with nanolithiators of reagents: alternative to 96-well plates for sequencing" Current Opinion in Chemical Biology 10: 226-. Unlike prior methods, which typically dispose of the device after use, the pre-loaded carrier strategy of the present invention allows the basic device architecture to be reused in a large number of experiments. In addition, because none of the reagents (and resulting products) are encapsulated within the channels, their form is inherently very convenient for analysis. For example, in this study, this format was convenient for MALDI-MS detection, but it is estimated that a wide range of detector types, such as optical readers and acoustic sensors, may be used in the future. Finally, while this proof-of-principle study utilizes a food packaging film carrier with a single reagent spot, it is estimated that in the future micro array spotter may be used to make pre-loads with many different reagents for multiple analyses.

For practical use, the pre-loaded carrier must be able to remain active during storage. To evaluate the shelf life of these reagent spots, the applicant carried out a quantitative protein digestion assay. The reporter in this assay is quenched fluoroboric fluorescently labeled casein, which has low fluorescence when intact, but high fluorescence after digestion. In this pre-packaged reagent stability assay, droplets containing a reporter are driven to pre-packaged trypsin spots and, after incubation, the fluorescence signal in the droplets is measured using a plate reader (see, as described above, the article "Pluronic assays: A solution to solutions in microorganisms in Digital microfluidics," Langmuir 24: 6382-. In preliminary experiments with freshly prepared pre-loaded carriers, it has been established that the reaction is complete within 30 minutes at the concentrations used. The internal standard rhodamine B corrects and corrects errors, evaporation effects and instrument zero drift which occurs along with the time.

In shelf life experiments, pre-loaded carriers were stored at-20 ℃ or-80 ℃ for various periods of time (1, 2, 3, 10, 20, or 30 days). In each experiment, after thawing the vector it was placed on the device, the droplets were driven to trypsin and incubated for 30 minutes, and the reporter/internal standard signal ratio was recorded. At least 5 different vectors were evaluated in each case. As shown in FIG. 5, the shelf life performance is excellent-the carriers can retain > 75% of their original activity after storage at-80 ℃ for 30 days. The carrier can still keep more than 50% of the original activity when stored at the temperature of minus 20 ℃ for the same time. This difference may be due to the difference in average storage temperature alone or may reflect that the-20 c refrigerator is used in a self-thawing mode (often with temperature fluctuations) while the-80 c refrigerator is at a constant temperature. In any case, these carriers are excellent in preliminary experiments and it is predicted that adjusting the pH or ionic strength of the enzyme suspension buffer, or adding a stabilizer such as trehalose, a disaccharide which has been widely used for the industrial preservation of dry proteins (see "Stability of monomeric antibodies from fresh-dried in the present soft-top" Journal of Immunological Methods 181: 37-43 published by Draber et al 1995) will extend the shelf life in the future.

Briefly, the present inventors have developed a new digital microfluidic strategy that will actually facilitate the unlimited reuse of various devices without fear of fork contamination, and that will enable rapid replacement of pre-loaded reagents. The present invention is capable of converting digital microfluidics into a versatile platform for lab-on-a-chip applications.

The terms "comprises" and "comprising," as used herein, are to be construed as open-ended inclusion and inclusion, not exclusive. In particular, the terms "comprises" and "comprising," when used in this specification including the claims, refer to the inclusion of particular features, steps or components. These terms are not to be interpreted to exclude the presence of other features, steps or components.

The foregoing description of the preferred embodiments of the invention is provided to illustrate the principles of the invention and not to limit the invention to these preferred embodiments. The scope of the invention is defined by all embodiments encompassed by the appended claims and equivalents thereof.

Like features are denoted by like reference numerals even though they appear only in the figures and are not specifically referred to in the description.

Reference numerals:

10 Disposable Pre-filled Carrier

11 electrically insulating foil

11a hydrophobic front surface of an electrically insulating sheet; front working surface

11b rear surface of electrically insulating sheet

12 prefilled reagent reservoir

13 pre-selected position

14 digital microfluidic device

15 adhesive

16, 16' electrode array; surface of electrode array

17 discrete electrode

18 pre-selected individual electrodes

19 electrode controller

20 drops of reagent

21 alignment mark

22 drop of reagent

23 patterned conductive coating

24, 24' a first substrate; surface of the first substrate

25 dielectric layer

26 to obtain a reaction product

27, 27' a second substrate; surface of the second substrate

28 residue of previous test

29 space(s)

30 residues of the previous experiment

31 additional electrically insulating foil

31a, 31b a hydrophobic front surface of an additional electrically insulating sheet; rear surface of additional electrically insulating sheet

32 sample container

33 solvent droplets

34 solvent container

35, 35' additional electrode arrays; additional electrode array surfaces

36 pipette tip

Claims (38)

1. A pre-loaded carrier (10) for use with a digital microfluidic device (14), said pre-loaded carrier (10) having one or more reagent depots (12) at one or more pre-selected locations (13), said pre-loaded carrier comprising an electrically insulating sheet (11) having a hydrophobic front surface (11a) and a back surface (11 b); the digital microfluidic device (14) comprising an array of electrodes (16) arranged by discrete electrodes (17) and an electrode controller (19), the electrode controller (19) being capable of selectively activating and deactivating the discrete electrodes (17) to translate a droplet on the hydrophobic front surface to the one or more pre-selected locations (13) on the hydrophobic front surface (11a) of the electrically insulating sheet (11),

characterized in that said electrically insulating sheet (11):

(a) mounting the reagent reservoir (12) on the hydrophobic front surface (11a) of the electrically insulating sheet (11) prior to connection to a digital microfluidic device (14);

(b) the rear surface (11b) of said electrically insulating sheet (11) is connectable on a surface (16') of said electrode array (16) of a digital microfluidic device (14);

(c) when placed on the electrode array (16), the electrically insulating sheet (11) covers the discrete electrodes (17) such that the discrete electrodes (17) are electrically insulated from each other and from droplets (20, 22, 33) on the hydrophobic front surface (11 a);

(d) peelable from the surface (16') of the electrode array (16) for optional analysis and disposal; and

(e) when peeled off the electrode array (16) of the digital microfluidic device (14), the electrode array (16) can be reused by attaching a new pre-loaded carrier (10).

2. The carrier (10) of claim 1, wherein: the electrically insulating sheet (11) is connectable or connected to the surface (16 ') of the electrode array (16) by an adhesive (15), wherein the adhesive (15) contacts the rear surface (11b) of the electrically insulating sheet (11) with the surface (16 ') of the electrode array (16) and/or a surface (24 ') of a first substrate (24).

3. The carrier (10) according to claim 1 or 2, wherein: the electrically insulating sheet (11) and the electrode array (16) or first substrate (24) each comprise alignment marks (21) for aligning the electrically insulating sheet (11) and the electrode array (16) when connecting the electrically insulating sheet (11) to the electrode array (16) such that the one or more pre-selected locations (13) on the hydrophobic front surface (11a) of the electrically insulating sheet (11) are selected to overlap with one or more pre-selected individual electrodes (18) of the electrode array (16).

4. The carrier (10) according to claim 1 or 2, wherein: the electrically insulating foil (11) comprises a material selected from the group consisting of polymers, plastics and waxes.

5. The carrier (10) according to claim 1 or 2, wherein: the electrically insulating sheet (11) carries a patterned electrically conductive coating (23), the electrically conductive coating (23) being capable of providing a reference or actuation voltage to the electrode array (16).

6. The carrier (10) according to claim 1 or 2, wherein: the one or more reagent depots (12) comprise a single reagent or at least two reagents, in each case selected from the group comprising dry reagents or viscous gel reagents.

7. The carrier (10) of claim 6, wherein: the one or more reagent depots (12) is more than one reagent depot, wherein at least one reagent in each reagent depot (12) is different from the reagent contained in at least one of all other reagent depots.

8. The carrier (10) of any one of claims 1, 2 or 7, wherein: the electrically insulating sheet (11) comprises an adhesive (15) on the rear surface (11b) capable of contacting the electrode array to adhere the electrically insulating sheet (11) to a first substrate (24).

9. A digital microfluidic device (14) comprising:

(a) a first substrate (24), said first substrate (24) having mounted on a surface (24') thereof an array of electrodes (16) arranged as discrete electrodes (17);

(b) an electrode controller (19), the electrode controller (19) being capable of selectively activating and deactivating the discrete electrodes (17) of the electrode array (16); and

(c) the carrier (10) according to claim 1, the carrier (10) comprising an electrically insulating sheet (11) pre-loaded with one or more reagent depots (12) on a hydrophobic front surface (11 a).

10. The digital microfluidic device (14) according to claim 9 wherein: the electrically insulating sheet (11) is connectable or connected to a surface (16 ') of the electrode array (16) by an adhesive (15), wherein the adhesive (15) contacts the rear surface (11b) of the electrically insulating sheet (11) with the surface (16 ') of the electrode array (16) and/or a surface (24 ') of a first substrate (24).

11. The digital microfluidic device (14) according to claim 9 or 10 wherein: the electrically insulating sheet (11) and the electrode array (16) or first substrate (24) each comprise alignment marks (21) for aligning the electrically insulating sheet (11) and the electrode array (16) when connecting the electrically insulating sheet (11) to the electrode array (16) such that the one or more pre-selected locations (13) on the hydrophobic front surface (11a) of the electrically insulating sheet (11) are selected to overlap with one or more pre-selected individual electrodes (18) of the electrode array (16).

12. The digital microfluidic device (14) according to claim 9 or 10 wherein: the electrically insulating foil (11) comprises a material selected from the group consisting of polymers, plastics and waxes.

13. The digital microfluidic device (14) according to claim 9 or 10 wherein: the electrically insulating sheet (11) carries a patterned electrically conductive coating (23), the electrically conductive coating (23) being capable of providing a reference or actuation voltage to the electrode array (16).

14. The digital microfluidic device (14) according to claim 9 or 10 wherein: the one or more reagent depots (12) comprise a single reagent or at least two reagents, in each case selected from the group comprising dry reagents or viscous gel reagents.

15. The digital microfluidic device (14) according to claim 14 wherein: the one or more reagent depots (12) is more than one reagent depot, wherein at least one reagent in each reagent depot (12) is different from the reagent contained in at least one of all other reagent depots.

16. The digital microfluidic device (14) according to any one of claims 9, 10 or 15 wherein: the electrically insulating sheet (11) comprises an adhesive (15) on the rear surface (11b) capable of contacting the electrode array to adhere the electrically insulating sheet (11) to the first substrate (24).

17. The digital microfluidic device (14) according to any one of claims 9, 10 or 15 wherein: the device comprises a dielectric layer (25) applied directly on said surface (16') of said electrode array (16) so that it is sandwiched between said electrode array (16) and said electrically insulating sheet (11).

18. The digital microfluidic device (14) according to any one of claims 9, 10 or 15 wherein: the device further comprises a second substrate (27), said second substrate (27) having a front surface (27 '), optionally a hydrophobic surface, wherein said second substrate (27) is spaced apart from said first substrate (24) so as to define a space (29) between said first substrate (24) and said second substrate (27), said space (29) being capable of containing droplets (20, 22, 33) between said front surface (27') of said second substrate (27) and said hydrophobic front surface (11a) of said electrically insulating sheet (11) on said electrode array (16) of said first substrate (24).

19. The digital microfluidic device (14) according to claim 18 wherein: the second substrate (27) is substantially transparent.

20. The digital microfluidic device (14) according to claim 18 wherein: said front surface (27 ') of said second substrate (27) being non-hydrophobic and comprising an additional electrically insulating sheet (31) having a rear surface (31b) and a hydrophobic front surface (31a), said additional electrically insulating sheet (31) being removably attached to said front surface (27 ') of said second substrate (27), wherein said rear surface (31b) is adhered to said front surface (27 '), said additional electrically insulating sheet (31) having one or more reagent reservoirs (12), said one or more reagent reservoirs (12) being located at one or more preselected locations (13) of said hydrophobic front surface (31a) of said additional electrically insulating sheet (31).

21. The digital microfluidic device (14) according to claim 20 wherein: the device comprises an additional electrode array (35) mounted on said front surface (27') of said second substrate (27), said additional electrode array (35) being covered by said additional electrically insulating sheet (31) having a hydrophobic front surface (31 a).

22. The digital microfluidic device (14) according to claim 21 wherein: the device comprises a dielectric layer (25) sandwiched between the additional electrically insulating foil (31) and the additional electrode array (35) and the front surface (27') of the second substrate (27).

23. A digital microfluidic method, said method comprising the steps of:

(a) preparing a digital microfluidic device (14), the digital microfluidic device (14) comprising an electrode array (16) of discrete electrodes (17) arranged on a first substrate (24); and an electrode controller (19) connected to the electrode array (16) of the array of discrete electrodes (17) for applying a selected voltage pattern to the discrete electrodes (17) to selectively activate and deactivate the discrete electrodes (17) to move liquid sample droplets (20, 22) through the electrode array (16) on the discrete electrodes (17) in a desired path;

(b) providing a pre-assembled carrier (10), the pre-assembled carrier (10) having an electrically insulating sheet (11), the electrically insulating sheet (11) having a hydrophobic front working surface (11a) and a back surface (11b), the electrically insulating sheet (11) having one or more reagent reservoirs (12), the one or more reagent reservoirs (12) being located in one or more pre-selected locations (13) on the front working surface (11a) of the electrically insulating sheet (11);

(c) -attaching the rear surface (11b) of the electrically insulating sheet (11) on a surface (16') of the electrode array (16) of the digital microfluidic device (14), the electrically insulating sheet (11) thereby covering the discrete electrodes (17) when placed on the electrode array (16) and electrically insulating the discrete electrodes (17) from each other and from droplets (20, 22, 33) on the hydrophobic front surface (11a), and setting the one or more pre-selected locations (13) on the front working surface (11a) of the electrically insulating sheet (11) accessible to droplets actuated on the front working surface (11a) of the electrically insulating sheet (11);

(d) conducting an assay by directing one or more sample droplets (20, 22) through said front working surface (11a) to said one or more reagent depots (12), thereby delivering said one or more sample droplets (20, 22) to said one or more reagent depots (12) reconstituted from said one or more sample droplets (20, 22) and mixing with at least one selected reagent contained by said one or more reagent depots (12);

(e) separating out a resulting reaction product (26) formed between the at least one selected reagent in at least one of the one or more reagent depots (12) and the mixed sample droplet (20, 22); and

(f) peeling the attached electrically insulating sheet (11) from the surface (16') of the electrode array (16) of the digital microfluidic device (14) to remove the attached electrically insulating sheet (11) to enable the electrode array (16) to be reused by attaching a new pre-loaded carrier (10).

24. The digital microfluidic method according to claim 23 wherein: the electrically insulating sheet (11) is attached to the surface (16 ') of the electrode array (16) by an adhesive (15), wherein the adhesive (15) contacts the back surface (11b) of the electrically insulating sheet (11) with the surface (16 ') of the electrode array (16) and/or a surface (24 ') of the first substrate (24).

25. The digital microfluidic method according to claim 23 or 24 wherein: the rear surface (11b) is connected to the surface (16') of the electrode array (16).

26. The digital microfluidic method according to claim 23 or 24 wherein: the method comprises a step (g) of analyzing the obtained reaction product (26).

27. The digital microfluidic method according to claim 26 wherein: said step (g) of analyzing said reaction product (26) is performed before or after removing said attached electrically insulating sheet (11) according to step (f).

28. The digital microfluidic method according to any one of claims 23, 24 or 27 wherein: said step (d) of directing one or more sample droplets (20, 22) past said front working surface (11a) comprises dispensing said one or more droplets (20, 22, 33) from one or more sample containers (32), said one or more sample containers (32) being mounted proximate to said front working surface (11a) of said electrically insulating sheet (11) on said array of electrodes (16) arranged by said discrete electrodes (17).

29. The digital microfluidic method according to any one of claims 23, 24 or 27 wherein: the one or more reagent depots (12) include a biological substrate for cell adhesion.

30. The digital microfluidic method according to any one of claims 23, 24 or 27 wherein: after exposing the one or more sample droplets (20, 22) to the at least one selected reagent reservoir (12) in step (d), the mixture of each sample droplet (20, 22) and the at least one selected reagent is translated through the discrete electrodes (17) and combined and mixed with one or more other sample droplets (20, 22).

31. The digital microfluidic method according to any one of claims 23, 24 or 27 wherein: after exposing the one or more sample droplets (20, 22) to the at least one selected reagent reservoir (12) in step (d), a mixture of each sample droplet (20, 22) and the at least one selected reagent is translated through the discrete electrode (17) and exposed to the at least one selected reagent reservoir (12).

32. The digital microfluidic method according to any one of claims 23, 24 or 27 wherein: after exposing the one or more sample droplets (20, 22) to the at least one selected reagent reservoir (12) in step (d), dividing the mixture of each sample droplet (20, 22) and the at least one selected reagent into one or more additional sample droplets, and processing, collecting and analyzing the one or more additional sample droplets.

33. The digital microfluidic method according to any one of claims 23, 24 or 27 wherein: step (d) comprises directing one or more droplets (33) of one or more solvents from one or more solvent reservoirs (34) in flow communication with the front working surface (11a) to the one or more selected discrete electrodes (17) to dissolve the one or more reagents prior to directing the one or more sample droplets (20, 22) to the one or more selected discrete electrodes (17).

34. The digital microfluidic method according to claim 29 wherein: the biological substrate includes any one of fibronectin, collagen, laminin, polylysine, and combinations thereof.

35. A kit for performing the digital microfluidic method according to any one of claims 23 to 34, wherein: the kit comprising a carrier (10) pre-loaded with reagent reservoirs (12) according to any of claims 1 to 8, the carrier (10) having an electrically insulating sheet (11), the electrically insulating sheet (11) having a hydrophobic front surface (11a) and a back surface (11b), the reagent reservoirs (12) being mounted on the hydrophobic front surface (11a) of the electrically insulating sheet (11) before being attached to the digital microfluidic device (14).

36. The kit of claim 35, wherein: the kit comprises a digital microfluidic device (14) according to any one of claims 9 to 22.

37. The kit of claim 35 or 36, wherein: the pre-loaded carrier (10) is packaged with a plurality of other carriers (10) in a single package.

38. The kit of claim 37, wherein: each of the pre-loaded carriers (10) packed in one package has the same number of reagent reservoirs (12), wherein each reservoir (12) comprises the same reagent composition.