(19)
(11)EP 2 567 213 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
24.01.2018 Bulletin 2018/04

(21)Application number: 11777067.7

(22)Date of filing:  15.04.2011
(51)International Patent Classification (IPC): 
G01N 1/28(2006.01)
G01N 27/00(2006.01)
H01J 49/04(2006.01)
B01L 3/00(2006.01)
(86)International application number:
PCT/CA2011/050205
(87)International publication number:
WO 2011/137533 (10.11.2011 Gazette  2011/45)

(54)

METHOD OF PROCESSING DRIED SAMPLES USING DIGITAL MICROFLUIDIC DEVICE

VERFAHREN ZUR VERARBEITUNG GETROCKNETER PROBEN MITHILFE EINER DIGITALEN MIKROFLUIDISCHEN VORRICHTUNG

PROCÉDÉ DE TRAITEMENT D'ÉCHANTILLONS SÉCHÉS UTILISANT UN DISPOSITIF MICROFLUIDIQUE NUMÉRIQUE


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 05.05.2010 US 331679 P

(43)Date of publication of application:
13.03.2013 Bulletin 2013/11

(73)Proprietor: The Governing Council of the University of Toronto
Toronto, Ontario M5S 1A1 (CA)

(72)Inventors:
  • JEBRAIL, Mais, J.
    Richmond Hill Ontario L4E 3Z3 (CA)
  • YANG, Hao
    Toronto Ontario M4X 1G7 (CA)
  • WHEELER, Aaron, R.
    Toronto Ontario M6H 3M4 (CA)

(74)Representative: Dennemeyer & Associates S.A. 
Postfach 70 04 25
81304 München
81304 München (DE)


(56)References cited: : 
CA-A1- 2 740 113
US-A1- 2007 095 407
US-A1- 2010 081 578
US-A1- 2002 150 683
US-A1- 2008 281 471
US-A1- 2010 087 633
  
      
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description

    BACKGROUND



    [0001] This invention relates to methods of processing of dried samples for subsequent analysis. Blood samples stored as dried blood spots have emerged as a useful sampling and storage vehicle for clinical and pharmaceutical analysis in a wide range of applications. For example, the Newborn Screening Ontario facility at the Children's Hospital of Eastern Ontario evaluates dried blood spot samples from approximately 140,000 babies each year for 28 inherited diseases. In each screening test, a dried blood spot sample is collected and then mailed to facility for analysis by tandem mass spectrometry (MS/MS). Unfortunately, this technique is slowed by an extensive sample preparation regimen (including excision/punching, extraction, evaporation, resolubilization, and derivatization), and in addition, high-throughput screening typically requires robotic sample handling.

    [0002] The success of dried blood spot sampling and MS/MS for newborn screening has led to a surge in popularity for similar techniques for a wide spectrum of applications in clinical labs and the pharmaceutical industry. Dried blood spot sampling methods allow for the collection of small amounts of sample and are convenient for long-term storage and cataloguing. MS/MS methods allow for the unambiguous identification and quantification of many different analytes in a single shot.

    [0003] Unfortunately, the throughput and turn-around-time associated with this technique are problematic as a result of time-consuming sample preparation. In particular, the off-line sample preparation of blood spots on filter paper necessitates the labor intensive and time consuming steps of extraction via centrifugation, in which the analyte is obtained in a supernatant. Furthermore, the maintenance of instruments (sample preparation robots and mass spectrometers) and plumbing (capillary tubes and associated connections) requires many hours of laboratory-time, which reduces the throughput of such analyses. In addition, the costs are magnified by the scale of operation (for example, nearly 150,000 samples are processed a year in Ontario alone).

    [0004] In US 2010/0081578, there is disclosed a known method of sample preparation wherein a support sheet covers the whole array of a digital microfluidic device.

    [0005] It is against the background, and the limitations and problems associated therewith, that the present invention has been developed.

    SUMMARY



    [0006] To achieve this, the method of sample preparation of the invention comprises the features claimed in claim 1.

    [0007] Accordingly, according to the invention, there is provided a method of sample preparation comprising the steps of: providing a solid phase support comprising a dried sample; providing said solid phase support at a first location between an upper plate and a lower plate of a two-plate digital microfluidic device comprising an array of digital microfluidic elements that can be electrically actuated, wherein said first location is dropwise addressable under actuation of said digital microfluidic device; providing an extraction solvent at an additional location that is dropwise addressable under actuation of said digital microfluidic device; actuating said digital microfluidic device to transport a droplet of said extraction solvent to said first location; and incubating said droplet of said extraction solvent and extracting an analyte in said dried sample, wherein a lateral extent of said solid phase support is selected to be sufficiently small to allow actuation of a droplet from a location where said droplet is contacting said solid phase support to another location addressable by said digital microfluidic device.

    [0008] Advantageous embodiments of the invention are claimed in the dependent claims.

    [0009] Methods are provided for the preparation of a sample using a digital microfluidic platform and the optional subsequent mass analysis of an extracted analyte. A sample is dried, on a solid phase support, and contacted with digital microfluidic array. An analyte present within the dried sample is extracted into an extraction solvent by electrically addressing the digital microfluidic array to transport a droplet of extraction solvent to the dried sample spot. The extracted sample may be dried and subsequently processed on the digital microfluidic array for derivatization. The digital microfluidic device may further include an integrated microfluidic channel having an output aperture, and the method may further include contacting a droplet containing extracted analyte with the microfluidic channel and applying a suitable electric field for generating nano- electrospray, thereby enabling the device to be directly interfaced with a mass analysis device.

    [0010] A further understanding of the functional and advantageous aspects of the invention can be realized by reference to the following detailed description and drawings. All embodiments not falling under the claims are presented for information purposes only.

    BRIEF DESCRIPTION OF THE DRAWINGS



    [0011] Embodiments will now be described, by way of example only, with reference to the drawings, in which:

    Figure 1 illustrates steps in sample processing by digital microfluidics. The top image is a schematic of a digital microfluidic device which allows for the processing of 3 blood samples simultaneously. The bottom image is a sequence of frames from a movie (left-to-right) depicting several stages in sample processing including: (1) a dried blood sample; (2) mixing and incubating an extracted droplet with the sample; (3) a droplet containing sample extractate after translation away from the dried sample; (4) a dried extract; (5) mixing and incubating a derivatization reagent droplet with the dried extract; and (6) the dried, derivatized product.

    Figure 2 illustrates three digital microfluidic methods for processing different sample formats, where (a) shows a droplet of blood spotted directly onto the device surface and allowed to dry, (b) In method 2 shows a punch from filter paper bearing dried blood that is positioned on the device surface.

    Figure 3 illustrates the steps in processing blood samples for quantification of amino acid by tandem mass spectrometry. (a) Reaction scheme involving derivatization of the extracted amino acid, followed by derivatization with n-butanol, followed by the formation of a daughter ion by collision induced dissociation in the mass spectrometer. (b) Mass spectrum generated from primary analysis of derivatized phenyalanine (Phe). (c) Mass spectrum generated from the secondary analysis of derivatized Phe showing the loss of 102 amu as a result of collision induced dissociation.

    Figure 4 provides schematics showing the hybrid digital microfluidic device used for in-line mass spectrometry analysis. Figure 3(a) shows the individual layers forming the device, and Figure 3(b) shows the integrated device.

    Figure 5 provides calibration curves generated by digital microfluidic sample preparation for quantification of three amino acids in blood.

    Figure 6 provides a comparison of Met, Phe, and Tyr concentrations in normal (green) and spiked (red) blood samples as biomarkers for homocystinuria, phenylketonuria, and tyrosinemia, respectively. The dashed lines indicate the upper levels for normal concentrations in newborn blood samples. Each data point represents at least four replicate measurements, and error bars represent ±1 S.D.

    Figures 7 (a)-(c) shows a series of frames from a movie (top-to-bottom) demonstrating derivatization and extraction of amino acids, resolubilization in solvent, and analyte solution on a hybrid microfluidic device.

    Figure 8 is an image of sample spraying from the fabricated emitter into a mass spectrometer inlet.

    Figure 9 plots the secondary analysis spectra of (a) Phe and (b) d5-Phe generated from blood samples.

    Figure 10 illustrates the analysis of amino acids in dried blood spots by digital microfluidic methods, where (a) and (b) show frames from a movie depicting sample processing of 3.2 mm diameter punch of a dried blood spot on filter paper by digital microfluidics, and (c) provides a graph of Phe concentrations measured by the digital microfluidic method involving punches from three patients.


    DETAILED DESCRIPTION



    [0012] As required, embodiments of the present invention are disclosed herein. However, the disclosed embodiments are merely exemplary, and it should be understood that the invention may be embodied in many various and alternative forms. The Figures are not to scale and some features may be exaggerated or minimized to show details of particular elements while related elements may have been eliminated to prevent obscuring novel aspects. 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. For purposes of teaching and not limitation, the illustrated embodiments are directed to methods of processing dried samples using a digital microfluidic device..

    [0013] As used herein, the terms, "comprises" and "comprising" are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in this specification including claims, the terms, "comprises" and "comprising" and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

    [0014] As used herein, the terms "about" and "approximately, when used in conjunction with ranges of dimensions of particles, compositions of mixtures or other physical properties or characteristics, is meant to cover slight variations that may exist in the upper and lower limits of the ranges of dimensions so as to not exclude embodiments where on average most of the dimensions are satisfied but where statistically dimensions may exist outside this region. It is not the intention to exclude embodiments such as these from the present invention.

    [0015] As used herein, the term "exemplary" means "serving as an example, instance, or illustration," and should not be construed as preferred or advantageous over other configurations disclosed herein.

    [0016] In a first embodiment, a method of sample preparation is provided in which a digital microfluidic array is employed to extract and prepare an analyte for subsequent analysis. The sample preparation method is especially suitable for use with a subsequent mass analysis step such as a tandem mass spectrometry.

    [0017] Figure 1 illustrates a digital microfluidic device 100 for performing a method according to a first embodiment. The device 100 includes an insulating substrate 105 having formed thereon an array 110 of digital microfluidic elements that can be electrically actuated to transport droplets between array elements. The array elements each include a conductive surface that is coated with a hydrophobic insulator. For example, the device may be formed on a glass substrate, onto which patterned chromium electrodes are formed by photolithography and etching, and coated with Parylene-C and Teflon-AF.

    [0018] Each element is electrically connected to a contact pad (not shown) for electrically addressing the array. The array may further include reagent reservoirs 120 that are suitable for containing a reagent volume. An array element 125 adjacent to a reservoir may be actuated to form reagent droplets and transport the reagent droplets to samples 130 within the array. As shown in Figure 1, the digital microfluidic array 110 may include multiple regions in which various samples may be processed in parallel. Additionally, side walls and a top plate may also be provided for forming a dual-layer device, in which array elements may be actuated based on a voltage difference between the array and an electrode on the top plate electrode. For example, the top plate may be transparent and may comprise an unpatterned indium tin oxide (ITO) coated glass substrate coated with a hydrophobic material such as Teflon-AF™.

    [0019] The bottom portion of Figure 1 provides a series of images from a movie illustrating an example of the present embodiment, in which an amino acid analyte is extracted from a dried blood spot and derivatized for a subsequent mass analysis assay. As shown in the Figure, the images correspond to a single region 140 of the illustrated digital microfluidic device 100.

    [0020] In image 1 in the bottom portion of Figure 1, a droplet of blood is provided to the digital microfluidic array and dried to form a dried blood spot 145. It is to be appreciated that the sample may be dispensed directly onto the array, or may be dispensed onto a sample reservoir and the array may be electrically addressed to extract a droplet from the sample reservoir and transport the droplet to an element of the array.

    [0021] An extraction solvent is then provided to the array, and may be provided by dispensing the extraction solvent to an extraction solvent reservoir 150. As shown in image 2, the array is then electrically addressed to transport one or more droplets 155 of extraction solvent to the dried blood spot. The extraction solvent is incubated (it has been found that approximately 5 to 10 min is sufficient to incubate the extraction solvent) over the dried blood spot and analyte present in the dried blood spot is extracted into the extraction solvent. It will be understood that a suitable incubation time to extract the analyte may depend on the properties of the analyte and extraction solvent. In image 3, the array is again electrically actuated to transport the extraction solvent to a second array element 160, where it is subsequently dried, as shown in image 4.

    [0022] In image 5, the array is electrically addressed to transport one or more droplets 170 of a derivatization reagent from reagent reservoir 165 to the second array element 160. The derivatization reagent is incubated (for example, for approximately 5 to 10 min) to solubilize and derivatize the analyte previously dried onto second element 160. It will be understood that a suitable incubation time to solubilize and derivatize the analyte may depend on the properties of the analyte and the derivatization solvent. In image 6, the derivatization reagent droplet is evaporated to provide a dried derivatized analyte spot 175.

    [0023] Although embodiments disclosed herein illustrate various methods using blood as the sample matrix, it is to be understood that the sample need not be blood, and may be or may include any suitable sample matrix, such as, but not limited to, whole blood, serum, plasma, urine, sputum, and cerebral spinal fluid.

    [0024] For example, as shown in Figure 2(a), a sample 180 could be spotted directly and dried onto a digital microfluidic device 100 at the point of collection, after which the device could be directly processed or transported to a remote testing location for analysis. If the sample is spotted directly onto the device, it is necessary for the device to be provided at the point of collection. This may be achieved by providing only a component of the device, such as the bottom plate of a two-plate digital microfluidic device. After having received the spotted plate at the laboratory, the full device could be assembled and electrically connected to a device controller. It is to be understood that embodiments involving sample spots dried directly onto the surface of a digital microfluidic array device may be performed using a single plate digital microfluidic device, or a two-plate microfluidic device.

    [0025] Alternatively, the sample may be spotted onto a solid phase support, such as an exchangeable carrier or exchangeable sheet, as disclosed in International Patent Application No. PCT/EP2009/062657, titled "EXCHANGEABLE CARRIERS PRELOADED WITH REAGENT DEPOTS FOR DIGITAL MICROFLUIDICS", and filed on September 30th, 2009. The exchangeable carrier is an electrically insulating sheet having a hydrophobic surface, where the insulating sheet may be contacted with a digital microfluidic array device to form an external surface of the device. After contacting the exchangeable carrier with the digital microfluidic array, the digital microfluidic array may be actuated to transport droplets that are provided on or in contact with the exchangeable carrier. This allows for repeated use of the device without requiring device disposal or decontamination of the device surface.

    [0026] Although PCT/EP2009/062657 disclosed the use of a exchangeable carrier for providing a exchangeable device component including dried reagents, the exchangeable sheets may additionally or alternatively employed to remotely obtaining a dried sample and providing the dried sample to another location (such as a laboratory) for testing. The sample is dried and spotted onto the exchangeable carrier prior to device assembly. The sample is dried at a location such that the dried sample spot is accessible to droplets actuated over the front hydrophobic surface of the exchangeable sheet after attachment to the digital microfluidic device. The dispensing of the sample onto an appropriate location of the exchangeable carrier may be achieved using alignment marks or features, or, for example, using an external masking device that places an aperture at the appropriate location to guide the dispensing process. The exchangeable carrier may also include dried reagents at one or more pre-selected positions, where the positions are selected to be accessible to droplets actuated over the front hydrophobic surface of the exchangeable carrier after attachment to the digital microfluidic device. The exchangeable carrier with a dried spot on a surface thereof may be applied to the surface of a single plate digital microfluidic device, or to the channel-facing surface of one or both of the top and bottom plates of a two-plate digital microfluidic device.

    [0027] In an alternative embodiment, the sample may be first dried onto a solid phase support, after which at least a portion of the solid phase support is locally contacted with a location on the digital microfluidic for analyte extraction and optional further sample processing steps. This allows for the remote collection of dried sample in a simple and convenient format for subsequent processing with a digital microfluidic device.

    [0028] An example of this embodiment is shown in Figure 2(b), where a filter paper punch 185 bearing dried blood is contacted with a surface of a microfluidic device at a location that is dropwise accessible under actuation of the digital microfluidic device. In Figure 2(b), the filter paper punch is positioned over a digital microfluidic element and contacted with the surface of the first plate as shown. Prior to processing the sample, the top plate of the digital microfluidic device is installed to complete the assembly of the two-plate device (as described above, a gap or channel is formed by a spacer layer provided between the upper and lower plates of the digital microfluidic device). This method allows for the convenient placement of the solid phase support at any internal addressable location of the digital microfluidic array. Alternatively, a two-plate digital microfluidic device may be pre-assembled, and the filter paper punch may be inserted into the gap between the two plates of the digital microfluidic device and positioned at a pre-determined location that is dropwise addressable by the digital microfluidic array. Any of the surfaces of the digital microfluidic array may be initially contacted with a exchangeable carrier, and the solid phase support may contact the digital microfluidic device indirectly through contact with the exchangeable carrier.

    [0029] After having provided the solid phase support at a suitable location between the upper and lower plates of the digital microfluidic device, where the suitable location is accessible to droplets actuated by the digital microfluidic device, the digital microfluidic array may be actuated to transport one or more extraction solvent droplets to the location of the solid phase support for incubation and extraction of the dried analyte. The extraction and other sample processing steps, such as derivatization of the extracted analyte, may further be performed as described above.

    [0030] The solid phase support has a thickness that is selected to be compatible with droplet actuation in a digital microfluidic device. For example, a suitable thickness range for a two-plate digital microfluidic device is approximately 90 to 450 microns. As noted above, a suitable solid phase is filter paper, which has a thickness that is compatible with digital microfluidic devices. Other suitable solid phase supports include other porous materials, such as, but not limited to paper, cellulose filters, nitrocellulose, polymer based monoliths such as porous polymer monoliths, and hydrogel forming materials. It has been shown (see below) that when a porous solid phase support such as filter paper is sandwiched between two plates of a digital microfluidic device, the support need not be adhered or otherwise fixed to the device, and is held in place after being contacted with a droplet by inherent forces such as capillary forces. In an alternative embodiment, a non-porous solid phase may be employed, for example, as described above in the context of removable carriers. In another embodiment, a punched non-porous solid phase may be adhered or secured to a surface of the digital microfluidic array using one of many suitable techniques, including bonding methods such as thermal bonding and/or gluing. The solid phase support may be selected to have a hydrophobic surface in order to support the transport of a droplet from a location where the droplet is contacting the solid phase support to a location elsewhere on the digital microfluidic array.

    [0031] The lateral extent (e.g. surface area and/or diameter) of the solid phase support is sufficiently small to support the actuation of droplets to and from the solid phase support, In some cases, it may be beneficial to limit the lateral extent of the solid phase support to less than one array element. For example, this can allow the transport of droplets along neighbouring channels without contacting the solid phase support. However, it is to be understood that the lateral extent of the solid phase support need not be smaller than an array element, and the lateral extent of the solid phase support may overlap with neighbouring array elements, as demonstrated in Figure 10.

    [0032] For example, a larger lateral extent of the solid phase support will provide a larger radial extent of dried solvent, and a correspondingly higher amount of analyte for extraction. In some applications, it may be desirable to provide a greater number of analyte molecules, particularly in applications where the analyte concentration range of interest is near to the limit of detection of an analytical method employed for subsequent detection.

    [0033] It may also be useful to provide, on a single digital microfluidic array, multiple solid phase supports, where each solid phase support contains a common dried sample. The multiple solid phase supports may be subjected to extraction steps as described above, where the extraction steps are performed in parallel or in serial format. The extracted analyte from the multiple solid phases may be dried onto a common array element in order to concentrate the extracted analyte.

    [0034] It another embodiment, multiple solid phase supports may be provided on a single digital microfluidic array, where the solid phase supports contain analyte or analytes from different samples for parallel processing on a single digital microfluidic array. This allows for multiplexed extraction from multiple samples, and is beneficial in further reducing labor costs and turn around time. For example, such an embodiment may be useful for the multiplexed extraction and processing of analyte in applications involving high-throughput screening.

    [0035] After utilizing the digital microfluidic array for the extraction and derivatization of the analyte as described above (either using direct sample deposition and drying or indirect dried sample processing), an assay for the analyte may be subsequently performed using a mass analyzer. To prepare the derivatized analyte for mass analysis, the derivatized analyte is resuspended in a solvent compatible with the subsequent mass analysis step.

    [0036] The extraction solvent may additionally contain an internal standard for use with the subsequent mass analysis step. The standard may include a concentration of isotopically labeled analyte. The mass analysis may involve analysis by collision-induced dissociation for tandem mass analysis. In another embodiment, the resuspended analyte may be first provided to a chromatographic separation system (such as a high performance liquid chromatography system) prior to subsequent analysis of the separation system eluent with a mass analysis device.

    [0037] In one embodiment, the analyte is one or more amino acids, fatty acids (acylcarnitines), organic acids, and a combination thereof. The extraction solvent in this case is preferably methanol. A suitable derivatization step is illustrated in Figure 3(a) for the non-limiting case of amino acid (amino acid) analyte, in which a derivatization reagent comprising HCl-butanol transforms each amino acid to its corresponding butyl ester (derivatized amino acid), and subsequent formation of daughter ions via collision induced dissociation makes each analyte convenient to quantify by multiple reaction monitoring. The capacity to carry out similar processes in parallel for many different amino acids simultaneously with minimal steps makes the present digital microfluidic and MS/MS method a useful tool for a wide range of applications, including the screening of metabolic disorders on a large scale.

    [0038] Figures 3(b) and 3(c) provide illustrative primary (MS1) and secondary (MS2) mass spectra for the amino acid, phenylalanine, with peaks at m/z 222 and 120. This technique, using collision induced dissociation and MS/MS, is useful for selectively analysing such species because they exhibit a characteristic loss of butylformate (HCOOC4H9, 102 D), making them easy to identify.

    [0039] In one embodiment, more than one extraction step may be performed using the digital microfluidic array for the purpose of extracting multiple analytes that are beneficially processed using different extraction and/or derivatization solvents. In a one embodiment, the multiple extraction methods are performed on a common digital microfluidic platform. The multiple extraction methods may be performed serially using a common dried sample spot or in parallel using two separate dried sample spots, where the dried sample spots may be dried directly onto the digital microfluidic device, or dried onto an intermediate matrix as described above. The final droplets containing the extracted analyte, in which the droplets are provided in a fluid that is compatible with a subsequent mass analysis step, may be combined and provided to the mass analyzer in a single aliquot for multiplexed mass analysis. In a non-limiting example, the marker succinulacetone may be extracted in order to perform an assay for tyrosinemia type I. The extraction method is similar to that discussed above, in which an acidic extraction solvent containing hydrazine is provided to the digital microfluidic array. Preferably, this extraction solvent comprises an acetonitrile/water/formic acid solution having relative concentrations of 80:20:0.1 % by volume, respectively, and further containing 0.1% hydrazine monohydrate and isotopically labeled succinylacetone (13C5- succinylacetone), as discussed in Turgeon et al. (C. Turegeon et al., Clin. Chem. 54, 657, 2008). The array is then electrically actuated to transfer a droplet of the extraction solvent to a dried sample (e.g. a dried blood spot), which may have already been processed according to the above protocol using a different solvent (e.g. methanol). The extraction solvent droplet containing the extract succinulacetone is then transported to a different element of the digital microfluidic array under electrical actuation of the array, where it may be dried and resolubilized in another buffer prior to analysis.

    [0040] In one embodiment, the digital microfluidic device is interfaced a the mass spectrometer to support a method that may be performed without the need for intermediate manual or robotic liquid handling steps. This overcomes many of the difficulties associated with convention nanoflow electrospray ionization (nESI), which is known to be a complex technique requiring operator expertise and vigilance to achieve reproducible results. For example, this limitation is part of the reason why newborn blood samples are often mailed to a single remote screening facility for processing.

    [0041] Accordingly, in one embodiment, a nESI device is integrated into the microfluidic device to provide a hybrid digital microfluidic and nESI device that may be formed by standard batch-processing. Sample processing is performed as described above, where an analyte is extracted and processed from a dried sample (directly dried onto the digital microfluidic array, or indirectly dried onto a suitable matrix which is contacted with the array), and mass analysis is realized by positioning the hybrid device in front of the mass spectrometer inlet and applying an electrical potential to achieve nESI. This process requires only a few minutes to accomplish and can be implemented by non-experts.

    [0042] Such an integrated system is illustrated in Figure 4, which shows the various layers that form the device in Figure 4(a) and the overall integrated device in Figure 4(b). As shown in Figure 4(a), the device comprises a top plate 200, a digital microfluidic layer 205, and a microfluidic channel layer 210. Top plate 205 comprises a non-conductive and substrate, having formed on its lower surface an electrode that is further coated with a hydrophobic material. Preferably, top plate is transparent and comprises an unpatterned indium tin oxide (ITO) coated glass substrate coated with a hydrophobic material such as Teflon-AF™.

    [0043] Digital microfluidic layer 205 is similar to the digital microfluidic device described above, and comprises an insulating substrate having formed thereon an array 215 of digital microfluidic elements that can be electrically actuated to transport droplets between array elements. Preferably, the array elements each comprise a conductive surface that is coated with a hydrophobic insulator. For example, the device may be formed on a glass substrate, onto which patterned chromium electrodes are formed by photolithography and etching, and coated with Parylene-C and Teflon-AF. As described above, each element is electrically connected to a contact pad (not shown) for electrically addressing the array. The array preferably further comprises reagent reservoirs 220 that are suitable for containing a reagent volume. Vertical spacing elements 225 are provided between top plate 200 and digital microfluidic layer 205 for forming a planar channel within which fluidic droplets may be transported by electrically addressing the digital microfluidic array.

    [0044] Digital microfluidic layer 205 further includes a vertical hole 230 located adjacent to array element 235, enabling droplets residing on the digital microfluidic array to be transported to, and contacted with, the aperture of vertical hole 230, whereby hole 230 may be filled under capillary action.

    [0045] Microfluidic channel layer 210 comprises a microfluidic channel 240 formed in an upper surface of a substrate, and extending to the edge of the substrate. By positioning microfluidic channel layer 210 in contact with the underside of digital microfluidic layer 205 so that an end portion of microchannel 240 is contacted with vertical hole 230, an inlet is formed in microchannel 240 that may be filled by fluid captured by vertical hole 230 under further capillary forces. Accordingly, microfluidic channel 240 may be filled with liquid from a droplet by electrically addressing the digital microfluidic array to transport the droplet to contact vertical hole 230, which leads to the subsequent filling of channel 240 under via capillary forces. The contacting of microfluidic channel layer 210 with digital microfluidic layer 205 also forms an external outlet 245 of microfluidic channel 240. In a preferred embodiment, microfluidic channel outlet 245 is located in the corner of the device. Figure 4(b) illustrates an integrated device 250 containing a sample droplet 260 and a reagent 270 loaded onto a reagent reservoir.

    [0046] In another embodiment, the digital microfluidic array is employed to perform a sample preparation method as disclosed above, and the derivatized analyte is solubilized in a fluidic droplet that is compatible with a subsequent mass analysis step. The droplet is transported to contact and fill the microfluidic channel, and the device is positioned in close proximity to the inlet of a mass analysis device. An electrical conductor is then made to contact the liquid in the microchannel, for example, by removing the top plate and placing a wire into the vertical hole 230. Alternatively, a conductor may be made to contact the contents of the microfluidic channel by forming a secondary access hole and inserting the electrical conductor into the secondary hole in such a way as to provide a suitable fluidic seal, thereby preventing leakage from the microchannel. Electrospray may be subsequently generated in a cone emerging from outlet 245 by applying a voltage between the conductor and the inlet of the mass analysis device. In yet another embodiment, the electrical contact point may be made at the top plate (ITO slide) with a suitable contact means such as a soldered wire or an alligator clip. This contact is connected to the MS power supply for applying a voltage between the microfluidic channel outlet and the inlet to the mass spectrometer.

    [0047] While the present embodiment illustrates the interfacing of a digital microfluidic array with a microfluidic channel located beneath the digital microfluidic array, it is to be understood that the digital microfluidic array may be interfaced with the microfluidic channel in a variety of different geometries. For example, the digital microfluidic array may be interfaced with a microfluidic channel having an inlet that is laterally adjacent to an element of the digital microfluidic array, as disclosed in PCT Application No. PCT/CA2009/001439, filed Oct. 13, 2009 and titled "Hybrid Digital and Channel Microfluidic Devices and Methods of Use Thereof". The methods provided herein are generally automated and are thus typically faster and less prone to operator error when compared to the conventional techniques in terms of sample preparation and reagent use. Specifically, the methods disclosed herein avoid the need to manually process samples dried on filter paper, namely centrifuging the filter paper in an extraction solvent, which involves laborious and time consuming steps.

    [0048] The aforementioned embodiments may be applied for a wide range of sample types, analytes, and processing applications. Although embodiments disclosed above have focused on three specific metabolic diseases (homocystinuria, phenylketonuria, and tyrosinemia), it is to be understood that the scope of the various embodiments includes, but is not limited to a wide range of analytes that are compatible with digital microfluidic array based extraction and processing.

    [0049] Example assays include, but are not limited to, amino acid assays, fatty acid disorders (acylcarnitines), organic acid disorders, and markers for metabolic disorders. Tables 1, 2 and 3 below provide a non-limiting list of various known amino acid disorders, fatty acid disorders, and organic acid disorders, respectively, and their markers. The analytes below merely provide an illustrative list and are not intended to limit the scope of the embodiments provided in the present disclosure.
    Table 1: List of Amino Acid Metabolic Disorders and their Markers
    Amino Acid Disorders Marker(s)
    Argininemia or Arginase deficiency Arginine
    Citrullinemia-I or Argininosuccinate synthase deficiency Citrulline
    Argininosuccinic aciduria or Argininosuccinate lyase deficiency Citrulline, argininosuccinic acid
    Ornithine transcarbamylase deficiency Alanine, Citrulline
    Carbamoylphosphate synthetase deficiency Alanine, Citrulline
    Citrullinemia-II or Citrin-mitochondrial aspartate-glutamate transporterdeficiency Citrulline
    Hyperammonemia-Hyperornithine mia-Homocitrullinuria Syndrome Ornithine
    Phenylketonuria or Phenylalanine hydroxylase deficiency Phenylalanine
    Maple Syrup Urine Disease or Branched chain ketoacid dehydrogenase deficiency Leucine, Valine
    Homocystinuria or Cystathioninebeta-synthase deficiency Methionine
    Non ketotic hyperglycinemia Glycine
    Tyrosinemia I or Fumarylacetoacetase deficiency Tyrosine
    Tyrosinemia II or Tyrosine aminotransferase deficiency Tyrosine
    Tyrosinemia III or 4-hydroxyphenylpyruvic acid dioxygenase deficiency Tyrosine
    5-Oxoprolinuria or Glutathione synthetase deficiency 5-oxoproline
    Biopterin defects Phenylalanine
    Table 2: List of Fatty Acid Metabolic Disorders and their Markers
    Fatty Acid Oxidation Disorders Marker(s)
    Very long-chain acyl-CoA dehydrogenase deficiency Tetradecenenoylcarnitine, Tetradecanoylcarnitine
    Long-chain hydroxyacyl-CoA dehydrogenase deficiency Hydroxyhexadecanoylcarnitine, Octadecenenoylcarnitine, Hydroxyoctadecenenoylcamitine
    Medium-chain acyl-CoA dehydrogenase deficiency Octanoylcarnitine, Hexanoylcarnitine, Decanoylcarnitine
    Short-chain acyl-CoA dehydrogenase deficiency Butyrylcarnitine & Isobutyrylcarnitine
    Tri-functional protein deficiency Hydroxyhexadecanoylcarnitine, Hydroxyoctadecenenoylcarnitine, Hydroxyoctadecanoylcarnitine
    Glutaric aciduria-II or Multiple acyl-CoA dehydrogenase deficiency Butyrylcarnitine & Isobutyrylcarnitine, Isovalerylcarnitine & 2-methylbutyrylcarnitine, Glutarylcarnitine, Octanoylcarnitine, Tetradecanoylcarnitine
    Carnitine palmitoyl transferase deficiency- I Free carnitine
    Carnitine palmitoyl transferase deficiency-II Octadecenenoylcarnitine, Hexadecanoylcarnitine
    Carnitine/acylcarnitine translocase deficiency Octadecenenoylcarnitine, Hexadecanoylcarnitine
    Carnitine uptake defect or 2,4-Dienoyl-CoA reductase deficiency Free carnitine, Decadienoylcarnitine
    Hydroxyacyl-CoA dehydrogenase deficiency or Short/Medium-chain hydroxyacyl-CoA dehydrogenase deficiency Hydroxybutyrylcarnitine
    Propionic acidemia or Propionyl-CoA carboxylase deficiency Propionylcarnitine
    Table 3: List of Organic Acid Metabolic Disorders and their Markers
    Organic Acid Disorders Marker(s)
    Methylmalonic aciduria or Methylmalonyl-CoA mutase deficiency Propionylcarnitine
    Cobalamin defects (A, B) Propionylcarnitine
    Cobalamin defects (C, D) Propionylcarnitine, Methionine
    Multiple carboxylase deficiency Propionylcarnitine, Hydroxyisovalerylcarnitine
    3-Hydroxyisobutyric aciduria Hydroxybutyrylcarnitine
    Isovaleric acidemia or Isovaleryl-CoA dehydrogenase deficiency Isovalerylcarnitine & 2-methylbutyrylcarnitine
    2-Methylbutyrylglycinuria or 2-Methylbutryl-CoA dehydrogenase deficiency Isovalerylcarnitine & 2-methylbutyrylcarnitine
    3-Methylcrotonyl-CoA carboxylase deficiency Hydroxyisovalerylcarnitine
    3-Hydroxy-3-methylglutaryl-CoA lyase deficiency Hydroxyisovalerylcarnitine,
    Mitochondrial acetoacetyl-CoA thiolase deficiency or Beta-ketothiolase deficiency Tiglylcarnitine plus 3-methylcrotonylcarnitine
    Methylglutaconic aciduria or 3-Methylglutaconyl-CoA hydratase deficiency Tiglylcarnitine plus 3-methylcrotonylcarnitine
    2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency Hydroxyisovalerylcarnitine
    Malonic aciduria or Malonyl-CoA decarboxylase deficiency Malonylcarnitine
    Glutaric aciduria-I or Glutaryl-CoA dehydrogenase deficiency Glutarylcarnitine


    [0050] It is to be understood that while the preceding embodiments have included a derivatization step for processing the extracted analyte using the digital microfluidic array, this step may not be required for extraction protocols that are suitable for other analytes. As such, the derivatization step, or any further on-chip processing steps, may be optionally performed, as appropriate for a given analyte or application.

    [0051] Example applications include, but are not limited to, neonatal screening of metabolic disorders (i.e. amino acids and organic acids), other disorders (for example, congenital adrenal hyperplasia, congenital hypothyroidism, biotinidase deficiency and galactosemia), and genetic disorders (e.g. cystic fibrosis and sickle cell diseases).

    [0052] For example, in each newborn blood spot analysis, a sample is obtained by pricking the subject's heel (or by venipuncture) and allowing a spot of blood to dry on filter paper. The dried blood spot is typically couriered to a lab, where 3.2 mm diameter circular discs are punched, and the analytes are extracted, mixed with isotope-labeled internal standards, derivatized, and then reconstituted for analysis by tandem mass spectrometry (MS/MS). As shown in Figure 3(a), the derivatization step transforms each amino acid to its corresponding butyl ester (derivatized amino acid) that allows for a characteristic fragmentation pattern (neutral loss of 102) via collision induced dissociation . Figures 3(b-c) contains representative primary (MS1) and secondary (MS2) mass spectra for the amino acid, phenylalanine, with peaks at m/z 222 and 120. In addition to amino acids, the same derivatization step butylates acylcarnitines (AC), which serve as markers of inborn errors of fatty acid and organic acid metabolism.

    [0053] An additional and clinically relevant advantage of the present digital microfluidic methods is the reduction in sample size that may be achieved relative to conventional processing methods. In some cases, the reduction in volume may be approximately 15-20x. This reduction has the potential to be beneficial for applications in which it is desirable to employ small sample volumes, such as in the testing of newborn patients, from which five spots of blood are typically collected for analysis, and in high throughput screening applications.

    [0054] In the examples provided herein, the small volume required (5 x 5 µL = 25 µL) for certain clinical in-vitro diagnostic assays can be collected as capillary blood with a single needle-prick, but in conventional sample processing methods, the volume (5 x 75-100 µL = 375-500 µL) often requires multiple pricks and tissue-squeezing, which can contaminate the sample with interstitial fluids, invalidating it for testing. Other sample size-related advantages of the present embodiments are a reduction in reagent use (fpr example, 20 µL vs. 170-450 µL), and a reduction in analysis time (~1 h vs. >3.5 h). This reduction in reagents and analysis time, combined with the potential elimination of sample preparation robotic liquid handling systems, makes the present methods an attractive option for diagnostic testing in a time of increasing costs for health care.

    [0055] The following examples are presented to enable those skilled in the art to understand and to practice the present invention. They should not be considered as a limitation on the scope of the invention, but merely as being illustrative and representative thereof.

    EXAMPLE 1


    Device Fabrication and On-Chip Processing of Amino Acids



    [0056] Digital microfluidic devices were fabricated in the University of Toronto Emerging Communications Technology Institute (ECTI) cleanroom facility, using a transparent photomask printed at Norwood Graphics (Toronto, ON). Glass devices bearing patterned chromium electrodes were formed by photolithography and etching and were coated with 2.5 µm of Parylene-C and 50 nm of Teflon-AF. Parylene-C was applied using a vapor deposition instrument (Specialty Coating Systems), and Teflon-AF was spin-coated (1% wt/wt in Fluorinert FC-40, 2000 rpm, 60 s) followed by post-baking on a hot-plate (160 °C, 10 min). The polymer coatings were removed from contact pads by gentle scraping with a scalpel to facilitate electrical contact for droplet actuation.

    [0057] A prototype similar to the device shown in Figure 1 was fabricated to analyze 5-µL blood samples. As shown in the top image, an array of 88 driving electrodes connects a series of 10 reservoirs dedicated to microliter volumes of sample and reagents. As depicted in the bottom image, blood samples are spotted onto the device and dried, after which the sample is extracted into methanol and the solvent is allowed to evaporate. The extract is then derivatized, and the product is isolated by allowing the solvent to evaporate. The entire process requires 50 min to complete (compared with >3.5 h for clinical laboratories, not including mailing time).

    [0058] The device featured an array of eighty-eight actuation electrodes (2.2 × 2.2 mm ea.) connected to ten reservoir electrodes (5 × 5 mm ea.), with inter-electrode gaps of 40 µm. Devices were assembled with an unpatterned ITO-glass top plate and a patterned bottom plate separated by a spacer formed from four pieces of double-sided tape (total spacer thickness 360 µm). Unit droplets (covering a single driving electrode) were -1.8 µL. To actuate droplets, driving potentials (70-100 VRMS) were generated by amplifying the output of a function generator (Agilent Technologies, Santa Clara, CA) operating at 18 kHz. As described elsewhere, droplets were sandwiched between the two plates and actuated by applying driving potentials between the top electrode (ground) and sequential electrodes on the bottom plate via the exposed contact pads. Droplet actuation was monitored and recorded by a CCD camera mounted on a lens.

    [0059] Blood samples were collected from a healthy adult male volunteer after a 10 h fasting period and were kept at -20°C until analysis. Immediately prior to use, samples were thawed and evaluated as described.

    [0060] Working solutions of all amino acids (amino acids) (25, 50, 100 and 500 µM ea.) were prepared in DI water. For derivatization of extracted amino acids, a 3 N HCl-butanol solution was prepared from a mixture of 12 N HCl/neat butanol (1:3 v/v). For analysis of amino acids in blood samples, the extracting solvent (MeOH) contained 25 µM of the appropriate deuterated amino acid (d3-Met, d5-Phe or d4-Tyr). For quantitative analysis of amino acid recovery from blood and for experiments mimicking diseased/healthy infant blood, samples were spiked with 200 µM of the appropriate amino acid (Met, Phe or Tyr). In all experiments, organic solvents were HPLC grade and deionized (DI) water had a resistivity of 18 MΩ·cm at 25 °C.

    [0061] 5-µL droplets containing the sample (i.e., amino acid standards, whole blood or spiked whole blood) were pipetted onto the bottom plate of a device and dried. The top plate was then affixed and two solvents were loaded into the appropriate reservoirs, including MeOH containing 25 µM of deuterated amino acid (extraction solvent), and 3 N HCl-butanol (derivatization solvent). A reservoir volume (10 µL) of extraction solvent was dispensed and driven by digital microfluidic to the dried sample and allowed to incubate (5 min). The extraction solvent was then actuated away from the sample and dried (∼15 min, room temperature) at a second site, after which a reservoir volume (10 µL) of derivatization solvent was dispensed to the dried extract and incubated for 15 min at 75°C. Following the reaction, the top plate was removed and the solvent was allowed to evaporate (∼15 min, room temperature).

    [0062] Calibration curves were generated by digital microfluidic sample preparation for quantification of (a) methionine (Met), (b) phenylalanine (Phe), and (c) tyrosine (Tyr) in blood. As shown in Figure 5, data was generated by plotting the intensity ratios of the daughter ions of each amino acid relative to their deuterated internal standard (i.e., d3-Met, d5-Phe, d4-Tyr, respectively) as a function of amino acid concentration. Each data point represents at least four replicate measurements, and error bars represent ±1 S.D. Regression lines were linear with R2 > 0.996 for each analyte.

    [0063] For comparison, amino acids were also extracted and derivatized on the macroscale using known methods. Amino acid samples (20 µL) were pipetted and dried in a microcentrifuge tube and extracted in methanol (500 µL, 30 min) containing isotope-labeled internal standards. The solution was centrifuged (13,000 rpm, 15 min), and the supernatant transferred to a second tube and evaporated to dryness using nitrogen. The extractate was then resuspended in 3 N HCl-butanol solution (250 µL) to derivatize the amino acids at 65°C for 20 min, followed by evaporation of the derivatized mixture.

    [0064] Most samples were processed by digital microfluidic and then were analyzed offline by nanoelectrospray tandem mass spectrometry (nESI-MS/MS). Such samples (stored dry on device or in centrifuge tube until analysis) were reconstituted in 70 µl of acetonitrile/water (4:1 v/v); samples originating from blood were, in addition, passed through PVDF membrane centrifuge-filters with 0.1 µm pore diameter (Millipore, ON). Samples were injected into an LTQ Mass Spectrometer (Thermo Scientific) via a fused silica capillary transfer line (100 µm i.d.) mated to a New Objective Inc. (Woburn, MA) nanoelectrospray emitter (100 µm i.d. tapering to 50 µm i.d.) at a flow rate of 0.8 µL min, with an applied voltage of 1.7-1.9 kV and capillary temperature of 200°C. Tandem MS/MS analysis was carried out by introducing 30% collision energy to the parent ions and then the fragments over the m/z range of 100-300 were scanned. For amino acid samples, the daughter ions detected in the second mass selection, which exhibit a loss of butylformate (HCOOC4H9, 102 m/z), were observed and used for quantification. Spectra were collected as an average of 50 acquisitions, and replicate spectra were obtained for digital microfluidic-derivatized samples of both control and blood.

    [0065] Some samples were analyzed by nESI-MS/MS in-line on hybrid digital microfluidic-microchannel devices bearing an integrated nESI emitter. In these experiments, hybrid devices were mounted on a 3-axis micromanipulator (Edmund Optics, NJ) positioned near the inlet of the LTQ MS. After sample processing, a spray was generated by applying 2.5-3.0 kV to a platinum wire inserted in the access hole (see Figure 6).

    [0066] Tandem mass spectrometry was used to (i) quantify amino acids in blood samples and (ii) evaluate the recovery efficiency of the digital microfluidic method. For the former (quantification of amino acid in blood samples), calibration plots were generated by plotting the intensity ratio of daughter ions from the extracted amino acids relative to the those of the internal standards (i.e., Met m/z 104:107, Phe m/z 120:125, and Tyr m/z 136:140) as a function of amino acid concentration in standard solutions (25-500 µM in DI water). Data points included in the calibration plots represent an average of at least 4 replicate measurements, and the data in each plot were fit with a linear regression. Blood samples were then evaluated (with on-chip derivatization and extraction, and measurement by MS/MS relative to internal standards, as above), and the values were compared to the calibration plots to determine the amino acid concentrations. For the latter (evaluation of % recovery), blood samples of known amino acid concentrations were spiked with 200 µM of amino acid standards and extracted (as above). Knowing the total concentration of amino acids in blood spots (e.g. native methionine concentration plus spiked methionine), % recovery was obtained by comparing the concentration values (obtained from calibration curves) vs. the known values.

    [0067] The % recovery of amino acids was evaluated quantitatively using a fluorescence-based assay. Control samples (Met, Phe or Tyr; 50 µM of each) were processed by digital microfluidic (as above), excluding the derivatization step. The dried extracts were diluted into 95 µL aliquots of borate buffer (20 mM, pH 8.5) in wells in a 96-well microplate. Upon addition of 5 µL of fluorescamine (5 mg/mL in acetone) the microplate was inserted into a fluorescence microplate reader (Pherastar, BMG Labtech, Durham, NC) equipped with a module for 390 nm excitation and 510 nm emission. The plate was shaken (5 s) and the fluorescence was measured. As a control, identical samples that had not been extracted were evaluated using the same fluorescent assay. To ensure that controls were processed in identical manner relative to extracted samples, each control sample was spotted on a device, dried and then reconstituted in buffer for analyses. Four replicate measurements were made for each sample and control.

    [0068] Nanoelectrospray ionization tandem mass spectrometry (nESI-MS/MS) is used to quantify amino acids in samples of blood processed by the digital microfluidic method. Calibration curves with R2 greater than 0.996 (Figure 4) were generated by analyzing standards processed by digital microfluidic at known concentrations from the abundance ratio of each amino acid to its deuterated standard peak in the secondary (MS2) spectra. The calibration curves facilitated measurement of amino acid concentrations in blood samples from a healthy male volunteer. As listed in Table 4, the values obtained were in the expected physiological range and the precision in the method was high with coefficients of variation (CVs) ranging from 5 to 11%.
    Table 4: Measured (± 1 S.D.) and normal adult concentrations of amino acids in blood.
    Amino AcidMeasured Blood Concentration (µM) in an Adult Male VolunteerNormal Blood Concentration (µM)
    Methionine 25 ± 2 16-33
    Phenylalanine 38 ± 2 41-68
    Tyrosine 46 ± 5 45-74


    [0069] Fluorescence and MS/MS were used as an orthogonal test to evaluate the extraction efficiency of the new digital microfluidic technique. The former method relies on fluorescamine, a fluorogenic reagent that exhibits no fluorescence until it reacts with primary amines. Recovery was determined by comparing the fluorescence intensity of multiple samples before and after extraction. In the latter (MS/MS) method, blood samples were spiked with amino acid and recovery was determined by comparing the amino acid concentration (native amino acid plus spiked amino acid) vs. known concentration. As listed in Table 5, the two orthogonal methods (fluorescence and MS/MS) agree and reveal the new technique to be very efficient and recovery was ≥80% for each standard and blood sample evaluated. As above, the precision of these measurements was high, with CVs ranging from 1 to 10%.
    Table 5: % Recovery of the digital microfluidic method measured by fluorescence and MS/MS (± 1 S.D.)
    Amino Acid% Recovery by Fluorescence in Standards% Recovery by MS/MS in Blood
    Methionine 98 ± 10 100 ± 1
    Phenylalanine 86 ± 9 85 ± 5
    Tyrosine 82 ± 10 84 ± 7


    [0070] To validate the new digital microfluidic method as a platform for analyzing amino acid disorders in blood, spiked blood samples (mimicking diseased states) and non-spiked blood samples (mimicking healthy state) were analyzed by mass spectrometry. Figure 6 shows a comparison of measured concentration of amino acids in normal and spiked blood samples. The dashed line indicates the threshold value for diagnosis of homocysteinuria (67 µM Met), phenylketonuria (120 µM Phe), and tyrosinemia (150 µM Tyr). As shown, the method is useful for distinguishing between these states.

    [0071] As shown in Figure 4, a hybrid digital microfluidic system was fabricated (using process similar to those disclosed above) in which an nESI device was directly integrated with the microfluidic device. The hybrid digital microfluidic-microchannel devices were fabricated in four steps.

    [0072] First, a DMF bottom substrate (layer 205 in Figure 4(a)) bearing an array of DMF driving electrodes) was fabricated as described above, but without a Teflon-AF™ coating). The design was similar to that of the DMF-only devices described above, but with fewer electrodes - 2 rows of 9 actuation electrodes 215 (2.2 ×x 2.2 mm) and 3 reservoir electrodes 220 (5 ×x 5 mm). Moreover, the substrates were first modified by drilling an access hole 230 (∼2 mm diameter) through the substrate using a micro drill-press before the photolithographic processes, where the hole was drilled adjacent to the planned location of an electrode 235. After patterning the electrodes, the opposite side was first coated with 7 µm of Parylene-C for bonding with substrate 210.

    [0073] Secondly, a glass substrate 210 bearing a microchannel 245 nanoelectrospray tip 240 formed in Parylene was fabricated. 37 grams of Parylene-C were deposited on piranha cleaned, silanized glass slide (25 x 55 mm) via vapor deposition. After Cr deposition, a microchannel (25 µm wide x 5 mm long) was photolithographically patterned on the substrate by UV radiation (365nm, 35mW/cm2, 50s) using a Karl-Suss MA6 mask aligner (Garching, Germany).

    [0074] Third, the channel side of substrate 210 was mated to the non-electrode side of substrate 205, placed under pressure in a precision vise (∼20 MPa), and thermally bonded in a vacuum oven (200°C, 24 h). After cooling, the top of substrate 205 was first coated with 2 µm of parylene followed by spin-coating 50 nm of Teflon-AF with a small piece of dicing tape covering the drilled hole. The tape was removed before post-baking on a hot plate (160°C, 10min).

    [0075] Fourth, the top plate 200 was assembled with spacers formed from four pieces of double-sided tape as described above for droplet actuation.

    [0076] Using this hybrid device, droplets are manipulated on the top surface, and are subsequently transferred to microchannels on the bottom of the device through the hole. The principle of operating the hybrid device for on-chip sample analysis is shown in Figure 7, which shows several frames from a movie demonstrating derivatization and extraction of amino acids, resolubilization in solvent, and analyte solution on a hybrid microfluidic device. A blood sample 310 was first spotted on the device 300 and the amino acids were extracted and derivatized as described above. The dried, derivatized sample 320 was then resuspended in acetonitrile and the droplet 330 was actuated towards the access hole 340 such that it filled the underlying channel 350. The filled channel can be seen at 360 in Figure 7(c).

    [0077] As shown in Figure 8, the fluid in the channel was delivered to a corner 400 of the device and nanoelectropsray 410 was generated by applying a high voltage to the counter electrode 420. Representative mass spectra generated from samples processed and analyzed on-chip are shown in Figure 9. The entire process requires ∼1 h from sampling to analysis, and requires only the hybrid digital microfluidic device and a mass spectrometer (i.e., no complex nanoflow pumps, robots, samplers, or control software).

    EXAMPLE 2


    Direct Digital Microfluidic Processing of Sample Dried on Solid Phase



    [0078] In the present example, a digital microfluidic method is demonstrated using the direct processing, on a digital microfluidic array, of samples dried onto filter paper. A digital microfluidic array device was fabricated as described in Example 1, except that in the present case, the two plates were separated by six pieces of double-sided tape (total spacer thickness 540 µm). This increased spacing was provided to accommodate the thickness of the filter paper. A blood sample was collected and dried onto filter paper, and a 3.2 mm diameter filter paper disc was punched for subsequent analysis. The punch was placed onto an element of a digital microfluidic array.

    [0079] A portion of an experiment is depicted in Figure 10(a). As shown, a droplet of extraction solvent 510 was dispensed on the digital microfluidic array 500 and driven to the filter paper punch 520, and the extract was then moved away and dried 530 for further processing (i.e., derivatization and solvent exchange, similar to Figure 1). As can be seen in Figure 10(b), after wetting, the filter paper punch 520 remains adhered to the surface through capillary forces. This process requires ~ 50 min to complete.

    [0080] To evaluate this digital microfluidic method relative to gold standard practices, a series of punches from blood samples containing various concentrations of Phe were processed by this digital microfluidic method, and punches from the same samples were evaluated using the conventional newborn screening technique. As listed in Table 6 below, a paired t-test revealed no significant difference between the two data sets at a 95% confidence level. To validate the new technique for application to clinical samples, dried blood spot punches from three newborn patients of NSO were evaluated by the digital microfluidic method. As shown in Figure 10(b), the new technique correctly identified patients 1 and 3 as suffering from phenylketonuria, and patient 2 as being unaffected.
    Table 6: Measured phenylalanine (Phe) concentration in 3.2 mm dia. punches from filter paper bearing dried blood using digital microfluidic method 2 (left) and standard techniques at NSO (right). A paired t-test (P = 0.05, t = 0.69) revealed no significant difference between the two data sets.
    SampleMeasured Phe Concentration (µM) Using DMF Method 2Measured Phe Concentration (µM) Using NSO Technique
    1 70 70
    2 550 548
    3 93 88
    4 93 92
    5 368 302
    6 534 539
    7 735 871


    [0081] The foregoing description of the embodiments of the invention has been presented to illustrate the principles of the invention and not to limit the invention to the particular embodiment illustrated. It is intended that the scope of the invention be defined by all of the embodiments encompassed within the following claims.


    Claims

    1. A method of sample preparation comprising the steps of:

    providing a solid phase support comprising a dried sample;

    providing said solid phase support at a first location between an upper plate and a lower plate of a two-plate digital microfluidic device comprising an array of digital microfluidic elements that can be electrically actuated, wherein said first location is dropwise addressable under actuation of said digital microfluidic device;

    providing an extraction solvent at an additional location that is dropwise addressable under actuation of said digital microfluidic device;

    actuating said digital microfluidic device to transport a droplet of said extraction solvent to said first location; and

    incubating said droplet of said extraction solvent and extracting an analyte in said dried sample,

    wherein a lateral extent of said solid phase support is selected to be sufficiently small to allow actuation of a droplet from a location where said droplet is contacting said solid phase support to another location addressable by said digital microfluidic device.


     
    2. The method according to claim 1 wherein said solid phase support is selected to be a porous solid phase.
     
    3. The method according to claim 1 wherein said solid phase support is selected to be filter paper.
     
    4. The method according to claim 3 further comprising the step of punching said filter paper to a pre-selected size prior to providing said solid phase support at said first location.
     
    5. The method according to claim 1 wherein said solid phase support is selected from the group consisting of cellulose, nitrocellulose, and a porous polymer monolith.
     
    6. The method according to any one of claims 1 to 5, wherein said step of providing said solid phase support comprising said dried sample comprises the step of receiving, from a remote collection location, said solid phase support comprising said dried sample.
     
    7. The method according to any one of claims 1 to 6 wherein said solvent is a polar solvent.
     
    8. The method according to any one of claims 1 to 7 wherein said analyte is selected from the group consisting of an amino acid, fatty acid, acylcarnitine, and organic acid.
     
    9. The method according to any one of claims 1 to 7 wherein said analyte is a marker of a metabolic disorder.
     
    10. The method according to any one of claims 1 to 9 wherein said sample is selected from the group consisting of whole blood, serum, plasma, urine, sputum, and cerebral spinal fluid.
     
    11. The method according to any one of claims 1 to 10 further comprising the steps of:

    transporting said droplet of extraction solvent to a second location, wherein said second location is addressable by said digital microfluidic device;

    drying said droplet of extraction solvent;

    providing a derivatization solvent at an additional location that is dropwise addressable under actuation of said digital microfluidic device;

    actuating said digital microfluidic device to transport a droplet of said derivatization solvent to said second location; and

    incubating said droplet of said derivatization solvent to obtain a derivatized analyte.


     
    12. The method according to claim 11 wherein said microfluidic device further comprises a microfluidic channel and an electrical contact for electrically contacting a fluid in said microfluidic channel, wherein an inlet of said microfluidic channel is interfaced with said digital microfluidic device such that said digital microfluidic device may be actuated to contact a droplet with said microfluidic channel and fill said microfluidic channel, and wherein an outlet of said microfluidic channel provides an opening in an external surface of said microfluidic device,
    said method further comprising the steps of:

    electrically addressing said digital microfluidic device to transport said droplet of comprising said derivatized analyte and contact said droplet with said inlet of said microfluidic channel to fill said microfluidic channel;

    positioning said outlet of said microfluidic channel adjacent to an inlet of a mass analysis device; and

    generating an electrospray cone, said cone emerging from said microfluidic channel and into said inlet of said mass analysis device, by applying a voltage between said electrical contact and said inlet of said mass analysis device.


     
    13. The method according to claim 12 further comprising the step of performing a mass analysis assay for said analyte.
     
    14. The method according to claim 12 or 13 wherein said electrical contact is located within an opening in said microfluidic channel.
     
    15. The method according to any one of claims 12 to 14 wherein said outlet is located at a corner of said microfluidic device.
     


    Ansprüche

    1. Probenaufbereitungsverfahren, das die Schritte umfasst:

    Bereitstellen eines Festphasenträgers, der eine getrocknete Probe umfasst;

    Bereitstellen des Festphasenträgers an einer ersten Stelle zwischen einer oberen Platte und einer unteren Platte einer digitalen mikrofluidischen Vorrichtung mit zwei Platten, die eine Anordnung von digitalen mikrofluidischen Elementen umfasst, die elektrisch betätigt werden können, wobei die erste Stelle unter Betätigung der digitalen mikrofluidischen Vorrichtung tropfenweise adressierbar ist;

    Bereitstellen eines Extraktionslösungsmittels an einer weiteren Stelle, die unter Betätigung der digitalen mikrofluidischen Vorrichtung tropfenweise adressierbar ist;

    Betätigen der digitalen mikrofluidischen Vorrichtung, um einen Tropfen des Extraktionslösungsmittels an die erste Stelle zu übertragen; und

    Inkubieren des Tropfens des Extraktionslösungsmittels und Extrahieren eines Analyten in der getrockneten Probe,

    wobei eine laterale Erstreckung des Festphasenträgers so ausgewählt ist, dass sie ausreichend klein ist, um eine Betätigung eines Tropfens von einer Stelle, an der der Tropfen mit dem Festphasenträger in Kontakt steht, auf eine andere Stelle, die für die digitale mikrofluidische Vorrichtung adressierbar ist, zu ermöglichen.


     
    2. Verfahren nach Anspruch 1, wobei der Festphasenträger so ausgewählt ist, dass er eine poröse Festphase ist.
     
    3. Verfahren nach Anspruch 1, wobei der Festphasenträger so ausgewählt ist, dass er Filterpapier ist.
     
    4. Verfahren nach Anspruch 3, das ferner den Schritt des Stanzens des Filterpapiers auf eine vorab ausgewählte Größe umfasst, bevor der Festphasenträger an der ersten Stelle bereitgestellt wird.
     
    5. Verfahren nach Anspruch 1, wobei der Festphasenträger aus der Gruppe ausgewählt ist, bestehend aus Cellulose, Nitrocellulose und einem porösen Polymermonolith.
     
    6. Verfahren nach einem der Ansprüche 1 bis 5, wobei der Schritt des Bereitstellens des Festphasenträgers, der die getrocknete Probe umfasst, den Schritt des Empfangens des Festphasenträgers, der die getrocknete Probe umfasst, von einer entfernt gelegenen Sammelstelle umfasst.
     
    7. Verfahren nach einem der Ansprüche 1 bis 6, wobei das Lösungsmittel ein polares Lösungsmittel ist.
     
    8. Verfahren nach einem der Ansprüche 1 bis 7, wobei der Analyt aus der Gruppe ausgewählt ist, bestehend aus einer Aminosäure, Fettsäure, Acylcarnitin und organischer Säure.
     
    9. Verfahren nach einem der Ansprüche 1 bis 7, wobei der Analyt ein Marker für eine Stoffwechselstörung ist.
     
    10. Verfahren nach einem der Ansprüche 1 bis 9, wobei die Probe aus der Gruppe ausgewählt ist, bestehend aus Vollblut, Serum, Plasma, Urin, Auswurf und Liquor cerebrospinalis.
     
    11. Verfahren nach einem der Ansprüche 1 bis 10, das ferner die Schritte umfasst:

    Übertragen des Extraktionslösungsmitteltropfens an eine zweite Stelle, wobei die zweite Stelle durch die digitale mikrofluidische Vorrichtung adressierbar ist;

    Trocknen des Extraktionslösungsmitteltropfens;

    Bereitstellen eines Derivatisierungslösungsmittels an einer weiteren Stelle, die unter Betätigung der digitalen mikrofluidischen Vorrichtung tropfenweise adressierbar ist;

    Betätigen der digitalen mikrofluidischen Vorrichtung, um einen Tropfen des Derivatisierungslösungsmittels zur zweiten Stelle zu übertragen; und

    Inkubieren des Tropfens des Derivatisierungslösungsmittels, um einen derivatisierten Analyt zu erhalten.


     
    12. Verfahren nach Anspruch 11, wobei die mikrofluidische Vorrichtung ferner einen mikrofluidischen Kanal und einen elektrischen Kontakt zum elektrischen Kontaktieren eines Fluids im mikrofluidischen Kanal umfasst, wobei ein Einlass des mikrofluidischen Kanals eine Schnittstelle mit der digitalen mikrofluidischen Vorrichtung aufweist, so dass die digitale mikrofluidische Vorrichtung betätigt werden kann, um einen Tropfen mit dem mikrofluidischen Kanal in Kontakt zu bringen und den mikrofluidischen Kanal zu füllen, und wobei ein Auslass des mikrofluidischen Kanals eine Öffnung in einer Außenfläche der mikrofluidischen Vorrichtung bereitstellt,
    wobei das Verfahren ferner die Schritte umfasst:

    elektrisches Adressieren der digitalen mikrofluidischen Vorrichtung, um den Tropfen von, umfassend den derivatisierten Analyt, zu übertragen und den Tropfen mit dem Einlass des mikrofluidischen Kanals in Kontakt zu bringen, so dass der mikrofluidische Kanal gefüllt wird;

    Positionieren des Auslasses des mikrofluidischen Kanals benachbart eines Einlasses einer Massenanalysevorrichtung; und

    Erzeugen eines Elektrospraykegels, wobei der Kegel aus dem mikrofluidischen Kanal entspringt und sich in den Einlass der Massenanalysevorrichtung bewegt, indem eine Spannung zwischen dem elektrischen Kontakt und dem Einlass der Massenanalysevorrichtung angelegt wird.


     
    13. Verfahren nach Anspruch 12, das ferner den Schritt des Durchführens eines Massenanalyseassays für den Analyten umfasst.
     
    14. Verfahren nach Anspruch 12 oder 13, wobei der elektrische Kontakt innerhalb einer Öffnung im mikrofluidischen Kanal angeordnet ist.
     
    15. Verfahren nach einem der Ansprüche 12 bis 14, wobei der Auslass in einer Ecke der mikrofluidischen Vorrichtung angeordnet ist.
     


    Revendications

    1. Procédé de préparation d'un échantillon comprenant les étapes de :

    fourniture d'un support en phase solide comprenant un échantillon séché ;

    fourniture dudit support en phase solide à un premier endroit entre une plaque supérieure et une plaque inférieure d'un dispositif microfluidique numérique à deux plaques comprenant un réseau d'éléments microfluidiques numériques qui peut être actionné électriquement, dans lequel ledit premier endroit est adressable goutte à goutte par actionnement dudit dispositif microfluidique numérique ;

    fourniture d'un solvant d'extraction à un endroit additionnel qui est adressable goutte à goutte par actionnement dudit dispositif microfluidique numérique ;

    actionnement dudit dispositif microfluidique numérique pour transporter une gouttelette dudit solvant d'extraction vers ledit premier endroit ; et

    incubation de ladite gouttelette dudit solvant d'extraction et extraction d'un analyte dans ledit échantillon séché,

    dans lequel une extension latérale dudit support en phase solide est choisie de manière à être suffisamment petite pour permettre l'actionnement d'une gouttelette à partir d'un endroit où ladite gouttelette est en contact avec ledit support en phase solide vers un autre endroit adressable par ledit dispositif microfluidique numérique.


     
    2. Procédé selon la revendication 1 dans lequel ledit support en phase solide est choisi de manière à être une phase solide poreuse.
     
    3. Procédé selon la revendication 1 dans lequel ledit support en phase solide est choisi de manière à être un papier filtre.
     
    4. Procédé selon la revendication 3 comprenant en outre l'étape de perforation dudit papier filtre à une taille présélectionnée avant la fourniture dudit support en phase solide au niveau dudit premier endroit.
     
    5. Procédé selon la revendication 1 dans lequel ledit support en phase solide est choisi dans le groupe constitué de la cellulose, de la nitrocellulose et d'un polymère monolithique poreux.
     
    6. Procédé selon l'une quelconque des revendications 1 à 5, dans lequel ladite étape de fourniture dudit support en phase solide comprenant ledit échantillon séché comprend l'étape de réception, à partir d'un endroit de prélèvement distant, dudit support en phase solide comprenant ledit échantillon séché.
     
    7. Procédé selon l'une quelconque des revendications 1 à 6 dans lequel ledit solvant est un solvant polaire.
     
    8. Procédé selon l'une quelconque des revendications 1 à 7 dans lequel ledit analyte est choisi dans le groupe constitué d'un acide aminé, d'un acide gras, d'une acylcarnitine et d'un acide organique.
     
    9. Procédé selon l'une quelconque des revendications 1 à 7 dans lequel ledit analyte est un marqueur d'un trouble métabolique.
     
    10. Procédé selon l'une quelconque des revendications 1 à 9 dans lequel ledit échantillon est choisi dans le groupe constitué du sang total, du sérum, du plasma, de l'urine, du crachat et du liquide céphalo-rachidien.
     
    11. Procédé selon l'une quelconque des revendications 1 à 10 comprenant en outre les étapes de :

    transport de ladite gouttelette de solvant d'extraction vers un second endroit, dans lequel ledit second endroit est adressable par ledit dispositif microfluidique numérique ;

    séchage de ladite gouttelette de solvant d'extraction ;

    fourniture d'un solvant de dérivation à un endroit additionnel qui est adressable goutte à goutte par actionnement dudit dispositif microfluidique numérique ;

    actionnement dudit dispositif microfluidique numérique pour transporter une gouttelette dudit solvant de dérivation vers ledit second endroit ; et

    incubation de ladite gouttelette dudit solvant de dérivation pour obtenir un analyte dérivé.


     
    12. Procédé selon la revendication 11 dans lequel ledit dispositif microfluidique comprend en outre un canal microfluidique et un contact électrique pour établir un contact électrique avec un fluide dans ledit canal microfluidique, dans lequel une entrée dudit canal microfluidique est mise en interface avec ledit dispositif microfluidique numérique de manière à ce que ledit dispositif microfluidique numérique puisse être actionné pour mettre en contact une gouttelette avec ledit canal microfluidique et remplir ledit canal microfluidique, et dans lequel une sortie dudit canal microfluidique fournit une ouverture dans une surface externe dudit dispositif microfluidique,
    ledit procédé comprenant en outre les étapes de :

    adressage électrique dudit dispositif microfluidique numérique pour transporter ladite gouttelette de comprenant ledit analyte dérivé et mettre en contact ladite gouttelette avec ladite entrée dudit canal microfluidique pour remplir ledit canal microfluidique ;

    positionnement de ladite sortie dudit canal microfluidique de façon adjacente à une entrée d'un dispositif d'analyse de masse ; et

    génération d'un cône d'électronébulisation, ledit cône émergeant dudit canal microfluidique et dans ladite entrée dudit dispositif d'analyse de masse, en appliquant une tension entre ledit contact électrique et ladite entrée dudit dispositif d'analyse de masse.


     
    13. Procédé selon la revendication 12 comprenant en outre l'étape de réalisation d'un essai d'analyse de masse pour ledit analyte.
     
    14. Procédé selon la revendication 12 ou 13 dans lequel ledit contact électrique est situé à l'intérieur d'une ouverture dans ledit canal microfluidique.
     
    15. Procédé selon l'une quelconque des revendications 12 à 14 dans lequel ladite sortie est située au niveau d'un coin dudit dispositif microfluidique.
     




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    Cited references

    REFERENCES CITED IN THE DESCRIPTION



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    Patent documents cited in the description




    Non-patent literature cited in the description