Abstract:
Rapid and specific detection of biological cells and biomolecules is important to biological assays across diverse fields including genomics, proteomics, diagnoses, and pathological studies. Microarrays and microfluidics increasingly dominate such detection techniques due to the ability to perform significant numbers of tests with limited sample volumes. A snap chip assembly is provided for the transfer of a microarray of reagents within semi-spherical liquid droplets on a transfer chip to a target assay microarray on an assay chip following assembly of the two chips and physical contact of the droplets with the target array. Reagents in nanolitre quantities are spotted on both chips and selectively transferred as liquid droplets between transfer chip and assay chip within the contact areas. Using the snap chip structure the inventors performed immunoassays with colocalization of capture and detection antibodies with 10 targets and bead-in-gel droplet microarrays with 9 targets in the low pg/ml regime.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This patent application claims the benefit of U.S. Provisional Patent Application U.S. 61/528,898 filed Aug. 30, 2011 entitled “Methods and Devices for Multiplexed Microarray Microfluidic Analysis of Biomolecules” and U.S. Provisional Patent Application U.S. 61/528,792 filed Aug. 30, 2011 entitled “Methods and Devices for Multiplexed Microarray Microfluidic Analysis of Biomolecules.” 
     
    
     FIELD OF THE INVENTION 
       [0002]    This invention relates to the field of bio-analysis and more particularly to a method of providing multiplexed microfluidic analysis via microarray-to-microarray transfer. 
       BACKGROUND OF THE INVENTION 
       [0003]    Rapid and specific detection of biological cells and biomolecules, such as red blood cells, white blood cells, platelets, proteins, DNA, and RNA, has become more and more important to biological assays that form a crucial element in diverse fields such as genomics, proteomics, diagnoses, and pathological studies. For example, the rapid and accurate detection of specific antigens and viruses is critical for combating pandemic diseases such as AIDS, flu, and other infectious diseases. Also, due to faster and more specific methods of separating and detecting cells and biomolecules, the molecular-level origins of diseases are being elucidated at a rapid pace, potentially ushering in a new era of personalized medicine in which a specific course of therapy is developed for each patient. To fully exploit this expanding knowledge of disease phenotype, new methods for detecting multiple biomolecules (e.g. viruses, DNA and proteins) simultaneously are required. Such multiplex biomolecule detection methods must be rapid, sensitive, highly parallel, and ideally capable of diagnosing cellular phenotype. 
         [0004]    One specific type of biological assay increasingly used for medical diagnostics, as well as in food and environmental analysis, is the immunoassay. An immunoassay is a biochemical test that measures the level of a substance in a biological liquid, such as serum or urine, using the reaction of an antibody and its antigen. The assay takes advantage of the specific binding of an antibody to its antigen. Monoclonal antibodies are often used as they only usually bind to one site of a particular molecule, and therefore provide a more specific and accurate test, which is less easily confused by the presence of other molecules. The antibodies picked must have a high affinity for the antigen (if there is antigen in the sample, a very high proportion of it must bind to the antibody so that even when only a few antigens are present, they can be detected). In an immunoassay, either the presence of antigen or the patient&#39;s own antibodies (which in some cases are indicative of a disease) can be measured. For instance, when detecting infection the presence of antibody against the pathogen is measured. For measuring hormones such as insulin, the insulin acts as the antigen. Conventionally, for numerical results, the response of the fluid being measured is compared to standards of a known concentration. This is usually done though the plotting of a standard curve on a graph, the position of the curve at a response of the unknown is then examined, and so the quantity of the unknown found. The detection of the quantity of antibody or antigen present can be achieved by either the antigen or antibody. 
         [0005]    An increasing amount of biological assays, such as immunoassays and gene expression analysis, are carried out using microarrays, such as DNA microarrays, protein microarrays or antibody microarrays, for example. A microarray is a collection of microscopic spots such as DNA, proteins or antibodies, attached to a substrate surface, such as a glass, plastic or silicon, and which thereby form a “microscopic” array. Such microarrays can be used to measure the expression levels of large numbers of genes or proteins simultaneously. The biomolecules, such as DNA, proteins or antibodies, on a microarray chip are typically detected through optical readout of fluorescent labels attached to a target molecule that is specifically attached or hybridized to a probe molecule. The labels used may consist of an enzyme, radioisotopes, or a fluorophore. 
         [0006]    A large number of assays use a sandwich assay format for performing the assay. In this format, a capture probe molecule is immobilized on a surface. In the subsequent steps, a sample solution containing target molecules, also called analytes is applied to the surface. The target or analyte binds in a concentration dependent manner to the capture probe molecules immobilized on the surface. In a subsequent step, a solution containing detection probe molecules is applied to the surface, and the detection probe molecules can then bind to the analyte molecule. The analyte is thus “sandwiched” between the capture probe and detection probe molecules. In some assays, a secondary probe molecule is also applied to the assay, which can bind the detection probe molecule. The secondary probe can be conjugated to a fluorophore, in which case the binding result can be detected using a fluorescence scanner or a fluorescence microscope. In some cases, the secondary probe is conjugated to radioactive element, in which case the radioactivity is detected to read out the assay result. In some cases, the secondary probe is conjugated to an enzyme, in which case a solution containing a substrate has to be added to the surface, and the conversion of the substrate by the enzyme can be detected. The intensity of the signal detected is in all cases proportional to the concentration of the analyte in the sample solution. 
         [0007]    Another type of cell and biomolecule separation and detection method uses microfluidic devices to conduct high throughput separation and analysis based on accurate flow controls through the microfluidic channels. By designing patterned fluidic channels, or channels with specific dimensions in the micro or sub-micro scales, often on a small chip, one is able to carry out multiple assays simultaneously. The cells and biomolecules in microfluidic assays are also typically detected by optical readout of fluorescent labels attached to a target cell or molecule that is specifically attached or hybridized to a probe molecule. 
         [0008]    However, for protein analysis it remains very challenging to develop multiplexed assays. A number of recent attempts have been made to develop improved multiplexed antibody microarrays for use in quantitative proteomics, and various researchers have begun to examine the particular issues involved. Some of the general considerations in assembling multiplexed immunoassays have been found to include: requirements for elimination of assay cross-reactivity; configuration of multianalyte sensitivities; achievement of dynamic ranges appropriate for biological relevance when performed in diverse matrices and biological states; and optimization of reagent manufacturing and chip production to achieve acceptable reproducibility. In contrast to traditional monoplex enzyme-linked immunoassays, generally agreed specifications and standards for antibody microarrays have not yet been formulated. 
         [0009]    The challenge of multiplexed immunoassay is further compounded when using complex biological samples, such as blood and its plasma and serum derivatives or other bodily fluids. The dynamic range of concentration of protein in blood has been found to span 11 orders of magnitude. Thus, when identifying low abundance proteins in blood, it has to be made against a background of proteins 11 orders of magnitude more numerous. As an analogy, if we were to identify a single person among the entire world population it would correspond to less than 10 orders of magnitude, as the world population is still less than 10 billion people. 
         [0010]    Immunoassays and other assays exploiting microarrays exploit microfluidics. Microfluidics is concerned with handling and manipulating minute amounts of reagents. A major challenge in microfluidics is the mismatch between conventional liquid handling systems and the small scale of microfluidics, which constitutes a major obstacle to the more widespread adoption of microfluidics in laboratories and clinical settings, and has been described as the “world-to-chip” interface. The difficulty lies in delivering solutions from macroscopic containers such as vials or microplates to the microscopic reservoirs and channels of microfluidics rapidly, and without wastage. The interfacing problem becomes particularly challenging when large numbers of reagents need to be delivered to a microchip. Complex integrated microfluidic circuits have been built using so called multilayer soft lithography, see for example J. M. K. Ng et al in “Components for Integrated Poly(dimethylsiloxane) Microfluidic Systems” (Electrophoresis, Vol. 23, pp 3461-3473), but the delivery of reagents remains cumbersome, and often large external reservoirs with dead volumes are used, multiple tubings need to be manually connected, and reagent loading remains serial, all of which contribute to limit the versatility of these technologies. Many microfluidic chips are still loaded manually using pipettes which is slow, and with a lower limit for the volume of approximately 200 nl, but with little dead volume on the other hand, see for example L. Gervais et al in “Toward One-Step Point-of-Care Immunodiagnostics using Capillary-Driven Microfluidics and PDMS Substrates” (Lab on a Chip, Vol. 9, pp 3330-3337). 
         [0011]    Microarrays although typically considered apart from microfluidics also depend on the transfer of minute amounts of reagents. In microarrays, the macro-to-micro challenge was addressed using large number of pins to transfer minute amount of liquids from microtiter plates to chips by repeatedly printing them onto multiple chips to minimize waste. The upload and transfer are controlled by capillary effects that need to be precisely engineered, see for example R. A. George et al in ““Ceramic Capillaries for use in Microarray Fabrication” (Genome Res., Vol. 11, pp 1780-1783) and R. Safavieh et al in “Straight SU-8 Pins” (J. Micromechanics and Microengineering, Vol. 20, 055001, 2010). Inkjet spotters with front-loading have also been developed and used to produce microarrays, see for example H. Li et al in “Hydrogel Droplet Microarrays with Trapped Antibody-Functionalized Beads for Multiplexed Protein Analysis” (Lab on a Chip, Vol. 11, pp 528-534) and M. Pia-Roca et al in “Addressable Nanowell Arrays Formed Using Reversibly Sealable Hybrid Elastomer-Metal Stencils” (Anal. Chem., Vol. 82, pp 3848-3855). The number of nozzles is typically much lower than that for pin spotters, however the programmability and rapid dispensing of droplets on-the-fly compensates for the limited parallelism. More recently, a novel system named the top spot has been presented which is made of a spotting head that is filled using capillary forces and for which dispensing is effected by compression of air above the nozzles, see for example C. Steinert et al in “TopSpot™ Vario: A Novel Microarrayer System for Highly Flexible and Highly Parallel Picoliter Dispensing” (Biomed. Microdevices, Vol. 11, 755-761). This system is overall simpler than inkjet spotters, but lacks individual addressing of the nozzles and requires larger volumes for loading the head. All these systems however remain reliant on robotics and are quite complex. 
         [0012]    Recently, several groups proposed novel approaches to transfer minute amounts of reagents by using a “storage chip”. In this way, an array can first be formed on one or several chips using high precision inkjet spotters, and subsequently all reagents transferred to another chip, or mixed with a sample, at once. Du, Ismagilov and colleagues have developed an elegant approach called the “SlipChip”. With a “SlipChip”, nanolitre droplets of reagents are first trapped in channels and recesses which serve as reaction chambers, then a sample is loaded in a microchannel running parallel to the recesses, and then both are merged by sliding the two microstructured chips, see W. Du et al in “SlipChip” (Lab on a Chip, Vol. 9, 2286-2292). 
         [0013]    To date, “SlipChips” have been used to deliver a single sample to an array of reagents, such as the delivery of single sample to 48 crystallization wells or to different chambers for sandwich immunoassays, see Du and W. Liu et al in “SlipChip for Immunoassays in Nanolitre Volumes” (Anal. Chem., Vol. 82, pp. 3276-3282), these examples represent a 1-to-N transfer. Alternative chip-to-chip transfer methods based on reagent diffusion from sol-gels and hydrogel spots have recently been proposed in the context of cell-based drug screening. First, the transfer of drugs and drug metabolites from sol-gel spots to cell monolayers on a flat substrate was demonstrated by M. Y. Lee et al in “Metabolizing Enzyme Toxicology Assay Chip (MetaChip) for High-Throughput Microscale Toxicity Analyses” (Proc. Natl. Acad. Sci. U.S.A., Vol. 102, pp. 983-987) and then the transfer from alginate gel droplets to cells encapsulated in collagen by T. G. Fernandes et al in “Three-Dimensional Cell Culture Microarray for High-Throughput Studies of Stem Cell Fate” (Biotechnol. and Bioeng., Vol. 106, pp. 106-118) and M-Y. Lee et al in “Three-dimensional Cellular Microarray for High-Throughput Toxicology Assays” (Proc. 
         [0014]    Natl. Acad. Sci. U.S.A, Vol. 105, pp. 59-63). More recently, Khademhosseini and colleagues adopted a similar approach to transfer drugs from approximately 200 μm wide posts made of either PDMS in “A Sandwiched Microarray Platform for Benchtop Cell-Based High Throughput Screening” (Biomaterials, Vol. 32, pp. 841-848) or a hydrogel in “Drug-Eluting Microarrays for Cell-Based Screening of Chemical-Induced Apoptosis” (Anal. Chem., Vol. 83, pp. 4118-4125) that were coated or loaded, respectively, with a drug library by inkjet spotting. The library was delivered at once to an array of 400 μm wide micro-wells on a microscope slide by clamping the chips and letting the drug diffuse into the buffer contained in each well. The wells were seeded with cells from a single cell line. This approach allowed selective delivery of a single drug per well, however a minor misalignment persisted possibly due to shrinkage of the PDMS. In summary, for the chip transfer methods described to date, manual alignment based on visible structures on the chip was used, and the transfer followed an N-to-1 or a 1-to-N arrangement with N different reagents being reacted or mixed with a single kind of sample. 
         [0015]    In conventional multiplexed sandwich assays in both array and bead formats, the detection antibodies are applied as a mixture, which is much simpler than multi-spotting, but gives rise to interactions among reagents that each constitute a liability for cross-reactivity, which in turn entails lengthy and costly optimization protocols and which severely limits the performance of these assays. Recently, we proposed the antibody colocalization microarray (ACM), see M. Pla-Roca et al in “Antibody Colocalization Microarray: A Scalable Method for Multiplexed and Quantitative Protein Profiling” (submitted to Mol. Cell. Proteomics), which depends on the addressing of each capture antibody spot by a single detection antibody, thus colocalizes each pair and reproducing assay conditions that are found in single-plex ELISA assays, but only requires less than a nanolitre of antibody reagents. The execution of an ACM requires first spotting the capture antibody, removing the slide from the spotter, incubating it with sample, washing and rinsing it as needed, and placing it back for the spotting of the detection antibody followed by binding and incubation. ACM depends on the transfer of N different reagents to N spots each with a different reagent as well, representing an N-to-N transfer. Local addressing was achieved using a custom built microarrayer with precise alignment mechanisms, but unlike approaches with mixing of reagents, spotting needs to be performed as part of the assay, which is cumbersome, and constitutes an obstacle to the adoption of ACM by others. 
         [0016]    Here, we present the snap chip for the collective transfer of a microarray of reagents contained within semi-spherical liquid droplets to a target microarray following assembly of the two chips and physical contact of the droplets with the target array. Nanolitres of reagents are spotted on both slides using an inkjet spotter, and selectively transferred from liquid droplets on a transfer chip to an assay chip within the contact areas. A process with back-side alignment and a hand-held snap apparatus were developed to allow for simple and reliable transfer of reagents of an entire microarray. Using the snap chip, we performed multiplexed sandwich immunoassays with colocalization of capture and detection antibodies with 10 targets simultaneously with detection limits in the low pg/ml in buffer and in 10% serum. Finally, we established a protocol for long term storage, three month in this study, of both the assay and transfer chips. 
       SUMMARY OF THE INVENTION 
       [0017]    It is an object of the present invention to provide microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays. 
         [0018]    In accordance with an embodiment of the invention there is provided a method comprising:
   providing an assay chip comprising a plurality of first locations disposed on a surface of the assay chip, each first location being a spot comprising at least a capture antibody;   providing a transfer chip comprising a plurality of second locations disposed on a surface of the transfer chip, each second location being a spot comprising at least a detection antibody;   orientating the surface of the assay chip with the plurality of first locations opposite and in predetermined orientation with the surface of the transfer chip with the plurality of second locations;   bringing said surfaces of the assay chip and transfer chip together to within a predetermined spacing;   keeping the assay chip and transfer chip together for a predetermined time; and   separating the assay chip and transfer chip.   
 
         [0025]    In accordance with an embodiment of the invention there is provided a device comprising:
   an assay chip comprising a plurality of first locations disposed on a surface of the assay chip, each first location being a spot comprising at least a capture antibody;   a transfer chip comprising a plurality of second locations disposed on a surface of the transfer chip, each second location being a spot comprising at least a detection antibody; wherein   the surface of the assay chip comprising the plurality of first locations is opposite to, in predetermined with, and within a predetermined spacing of the surface of the transfer chip with the plurality of second locations.   
 
         [0029]    In accordance with an embodiment of the invention there is provided a method comprising: 
         [0000]    generating a capture antibody chip;
       providing a first assay chip comprising a plurality of first locations disposed on a surface of the first assay chip, each first location being a spot comprising at least a capture antibody;   providing a transfer chip;   orientating the surface of the first assay chip with the plurality of first locations opposite and in a first predetermined orientation with the surface of the transfer chip;   bringing said surfaces of the first assay chip and transfer chip together to within a first predetermined spacing;   keeping the first assay chip and transfer chip together for a first predetermined time; and   removing the first assay chip thereby generating the capture antibody chip from the transfer slide with the transferred capture antibodies;   exposing the surface of the transfer chip to a sample for analysis under predetermined conditions;
 
generating a detection antibody chip;
   providing a second assay chip comprising a plurality of second locations disposed on a surface of the second assay chip, each second location being a spot comprising at least a detection antibody;
 
executing a detection step;
   orientating the surface of the detection antibody chip with the plurality of second locations opposite and in a second predetermined orientation with the surface of the capture antibody chip with the transferred capture antibodies;   bringing said surfaces of the second assay chip and transfer chip together to within a second predetermined spacing;   keeping the detection antibody chip and capture antibody chip together for a predetermined time; and   separating the detection antibody chip and capture antibody chip.       
 
         [0042]    Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0043]    Embodiments of the present invention will now be described, by way of example only, with reference to the attached Figures, wherein: 
           [0044]      FIG. 1  depicts a “SlipChip” according to the prior art of Du, Ismagilov et al; 
           [0045]      FIG. 2  depicts a microfluidic delivery system for multiplexed analysis according to the prior art of Juncker et al; 
           [0046]      FIG. 3A  depicts a process flow for microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention; 
           [0047]      FIG. 3B  depicts a process flow for microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention; 
           [0048]      FIG. 4  depicts a protocol for minor alignment and schematics for microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention; 
           [0049]      FIG. 5A  depicts a first mechanical structure for snap assembly and microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention; 
           [0050]      FIG. 5B  depicts a second mechanical structure for snap assembly and microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention; 
           [0051]      FIGS. 5C and 5D  depict third and fourth mechanical structures for snap assembly and microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention; 
           [0052]      FIG. 6A  depicts fluorescence images of assay chip after snapping and transfer according to an embodiment of the invention; 
           [0053]      FIG. 6B  depicts measured intensity profiles for the fluorescence from the spots within a row of one array in the assay chip of  FIG. 6A ; 
           [0054]      FIG. 6C  depicts fluorescence images of a high density assay chip after snapping and transfer according to an embodiment of the invention; 
           [0055]      FIG. 7  depicts fluorescent micrographs of a representative slide with 16 replicate arrays incubated together with a close-up of a single array as fabricated according to an embodiment of the invention; 
           [0056]      FIG. 8  depicts assay results and binding curves for antibodies in buffer solution measured using microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention; 
           [0057]      FIG. 9  depicts assay results and binding curves for antibodies in 10% serum measured using microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention; 
           [0058]      FIG. 10  depicts binding curves measured using microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention as a function of storage time; 
           [0059]      FIGS. 11A through 11C  depict assay and transfer structures for microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays and resulting snap chip assembly prior to separation according to an embodiment of the invention; 
           [0060]      FIG. 12  depicts assay and transfer structures for microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays for a snap chip assembly featuring electrodes; 
           [0061]      FIG. 13  depicts assay results and binding curves for 9 different Ab pairs measured using microarray-to-microarray transfer of reagents according to an embodiment of the invention; 
           [0062]      FIG. 14  depicts a process flow for double snap-chip based microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention; 
           [0063]      FIG. 15  depicts a detailed schematic of the double snap-chip based microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention presented in  FIG. 14 ; 
           [0064]      FIG. 16  depicts a scan of an assay slide with 3,136 spots using the double snap chip process described above in respect of  FIG. 14 ; and 
           [0065]      FIGS. 17A and 17B  depict binding curves for 40 proteins measured simultaneously measured using microarray-to-microarray transfer of reagents according to an embodiment of the invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0066]    The present invention is directed to bio-analysis and more particularly to a method of providing multiplexed microfluidic analysis via microarray-to-microarray transfer. 
         [0067]    Within the following description reference may be made below to specific elements, numbered in accordance with the attached figures. The discussion below should be taken to be exemplary in nature, and not as limiting the scope of the present invention. The scope of the present invention is defined in the claims, and should not be considered as limited by the implementation details described below, which as one skilled in the art will appreciate, can be modified by replacing elements with equivalent functional elements or combination of elements. Within these embodiments reference will be made to terms which are intended to simplify the descriptions and relate them to the prior art, however, the embodiments of the invention should not be read as only being associated with prior art embodiments. 
         [0068]    Referring to  FIG. 1  there is depicted a “SlipChip” according to the prior art of Du, Ismagilov et al in “SlipChip” (Lab on a Chip, Vol. 9, 2286-2292). The SlipChip consists of two plates, but in contrast to the previous methods in the prior art, the two plates are designed to be in contact and are not separated during use. As depicted in  FIG. 1  in first assembly  110  the bottom plate contains an array of wells which have been preloaded with reagents. Additionally, the bottom plate contains an array of disconnected ducts that are involved in loading the ‘SlipChip”. The top plate serves as a lid for the wells of the bottom plate as shown in second image  120  that also contains an array of wells that are complementary in pattern to the array of wells in the bottom plate and connect to the ducts of the bottom plate in a continuous fluidic path. The user receives the chip in the assembled form depicted by second image  120 . 
         [0069]    The sample is added through the fluidic path provided by the ducts and wells as shown in third and fourth images  130  and  140  respectively. To expose the sample wells to all of the corresponding reagent wells simultaneously the top plate is slipped relative to the bottom plate as shown in fifth image  150 . Mixing takes place and the results of the experiments are read out as shown in sixth image  160 . Sliding two pieces of a device is common in devices that regulate fluid flow, from a standard high-performance liquid chromatography (HPLC) valve to more sophisticated microfluidic devices, see for example M. Tokeshi et al in “Flow Analysis in Microfluidic Devices” (Chapter 6, oo149-166, published by Wiley) and M. Kuwata et al in “Sliding Micro Valve Injection Device for Quantitative Nano Liter Volume” (8 th  Int. Conf. Miniaturized Systems for Chemistry and Life Sciences, 2004, pp. 342-344). 
         [0070]    In addition, sliding has been used to induce reactions and to induce shear flow in shear-driven chromatography, see for example G. Desmet et al in “The Possibility of Generating High-Speed Shear-Driven Flows and Their Potential Application in Liquid Chromatography” (Anal. Chem., Vol. 72, pp 2160-2165) and Y. Cai et al in “Channel-Free Shear Driven Circular Liquid Chromatography” (Lab on a Chip, Vol. 8, pp. 1784-1786). The “SlipChip” builds on these advances, and the advances in plug-based microfluidics, to provide a platform that delivers controlled volumes of samples to many reaction wells. 
         [0071]    Now referring to  FIG. 2  there is depicted a microfluidic delivery system  2000 A for a single compartment of a microarray and a schematic  2000 B of arrayed microfluidic dispensers for use in multiplexed analysis according to the prior art of Juncker et al in US Patent Application 2010/0,298,163 entitled “Microfluidic Microarray System and Methods for the Multiplexed Analysis of Biomolecules.” Referring to first to third stages  200 A through  200 C respectively, the method and system used to deliver one or more fluid solutions to the micro-compartments of a microarray is shown. As shown in first stage  200 A a reservoir or liquid transfer needle  210  of a microfluidic microarray system includes a reservoir which is filled with a liquid  215 . The reservoir is in fluid flow communication with, and makes up part of, a fluid conduit  220  defined in the tip of the liquid transfer needle  210 . The terms “needle” and “pin” and “capillary” will both be used herein to describe such a liquid transfer needle in a fluid handling and distribution portion of larger microfluidic microarray system of the present invention. The liquid  215  is maintained and thus held back within the fluid conduit  220  by a capillary pressure P 1  generated at the interface of the liquid  215  in the reservoir. The needle  210  is located above a microarray  230  having at least one microfluidic micro-compartment  225  defined therein. 
         [0072]    Although a variety of different sizes and shapes of the microfluidic micro-compartment  225  are possible, such micro-compartments may for example be approximately between 50 and 150 micrometers (μm) in cross-sectional width (i.e. diameter in the case of a circular micro-compartment well and length or width in the case of a square shaped compartment), and the micro-compartments may be spaced apart by distance substantially corresponding to the cross-sectional width of each of the plurality of micro-compartments (the spacing may however be less than or greater than the individual micro-compartment widths). 
         [0073]    Second image  200 B shows the transfer of liquid  215  from the reservoir and the fluid conduit  220  into one of the micro-compartments  225 . The transfer of fluid takes place automatically upon engagement of fluid flow communication of the needle  210  with the micro-compartment  225 , due to a capillary pressure P 2  of the micro-compartment  225  which is more negative than the capillary pressure P 1  of the reservoir and fluid conduit  220 . Although direct contact is not necessary, a defined amount of liquid may be transferred to the micro-compartment upon contact between the liquid transfer needle  210  and the microfluidic micro-compartment  225 . Due to the difference in capillary pressures P 1  and P 2  between the needle  210  and the micro-compartment  225 , the liquid  215  within the needle is “sucked” into the micro-compartment  225  until it is filled. When the micro-compartment is filled, it no longer generates a negative capillary pressure, and thus the flow of fluid from the needle to the micro-compartment is automatically interrupted. Upon disengagement of the pin  210  from the surface of the micro-compartment, as shown in third image  200 C, the dispensed liquid  235  remains separately in the micro-compartment  225 . The same needle  210  can then be used to service multiple such micro-compartments  225  in sequence, until the reservoir is empty. 
         [0074]    However, it would be apparent to one skilled in the art that the approach of a single needle whilst an improvement over the state of the art still represents significant time within a microarray system to move the needle across every micro-compartment to dispense the necessary liquid into each. This would be further compounded if multiple liquids were required in a predetermined pattern across a microarray. Juncker, as shown in schematic  2000 B, considers this issue and addresses it with an array of needles  245 . As shown a plurality of micro-compartment arrays  250  are depicted wherein each comprises a matrix of micro-compartments  240 . The microarray system comprises a matching array of needles  245  which can be positioned once and multiple dispensing operations performed concurrently. In principle each needle  245  may be filled with a different liquid. However, whilst reducing the number of needle placement and dispensing steps the process now places increased fabrication and assembly tolerances on the array of needles  245  to ensure that these all make contact with the micro-compartments  240  as required and that no cross-contamination arises from misalignment of the needles  245  relative to the micro-compartments  240 . 
       Experimental Materials 
       [0075]    Within the descriptions of experiments presented below using microarray-to-microarray transfer equipment according to embodiments of the invention different materials were employed. For multiplexed microfluidic analysis rabbit anti-goat immunoglobulin G (IgG) (H and L chains, referred to as H+L) labeled with the fluorescent dye Alexa Fluor 488 and goat anti-mouse IgG (H+L) labeled with Alexa Fluor 647 were employed. Antibody and antigen pairs used included human epidermal grow factor receptor 2 (HER 2), Endoglin (ENG), Leptin (LEP), fibroblast growth factor (FGF), osteopontin (OPN), tumor necrosis factor receptor-II (TNF RII), granulocyte macrophage colony-stimulating factor (GM-CSF), chemokine (C-C motif) ligand 2 (CCL 2), chemokine (C-C motif) ligand 3 (CCL 3), interleukin-1 beta (IL 1β), and labeled streptavidin Cy 5. Other materials included phosphate buffered saline (PBS), Tween-20 (polysorbate 20 which is a surfactant and spreading agent), bovine serum albumin (BSA), and normal human female serum (single donor), and BSA-free StabilGuard® Choice Microarray Stabilizer. Slides were coated with either nitrocellulose or aminosilane. 
         [0076]    Scanning and Analysis: 
         [0077]    Within the experiments presented below a commercial microarray laser scanner (LS Reloaded™ by Tecan) was used to scan slides. For the one-step assays, a 488 nm and 633 nm laser were used simultaneously to image capture antibody spots and the transferred proteins. For sandwich assays, only the 633 nm laser was used. The fluorescence intensity was computed by subtracting the background signal in the vicinity of each spot. All the experiments were performed in triplicate, and the data was analyzed using analysis software (Array-Pro Analyzer) and graphics were produced with graphical software (SigmaPlot). The lower limit of detection (LOD) of the sandwich assays were calculated from the negative controls without antigen incremented by three times the standard deviation between three independent assays. 
         [0078]    Microarray Fabrication: 
         [0079]    The procedure for the microarray-to-microarray transfer using snap chips for conducting multiplex immunoassays with colocalization of each capture and detection antibody pair is shown in  FIG. 3A  according to an embodiment of the invention. Capture antibodies  350  are spotted onto an assay chip  360  using inkjets  340  to form a first matrix as shown in first step  300 B. Similarly, biotinylated detection antibodies  320  are spotted onto a transfer chip  330  using inkjets  310  to form a second matrix as shown in second step  300 A. For example a glass slide with an approximately 10 μm thick nitrocellulose coating may be used as assay chip  360  as the three-dimensional structure of the nitrocellulose provides a high antibody binding capacity, and will in subsequent steps absorb the solution from the transfer chip  330 . 
         [0080]    For the transfer chip  330  native glass slides whilst possible typically yield liquid droplets that tend to spread out on the hydrophilic glass thereby forming a thin layer that impacts the subsequent transfer step. A glass slide with hydrophobic coating typically yields rounded droplets which help ensure fluidic contact to the assay chip  360  during the transfer operation, but for the diameters used here, required large volumes of liquid. Within initial experiments employing the microarray-to-microarray transfer method of the invention larger diameters were selected for the droplets on the transfer chip  330  as these allowed for relaxed the alignment constraints while ensuring complete overlap between the capture antibody spot and biotinylated detection antibody spot. 
         [0081]    Within the experiments reported in this specification using an embodiment of the invention glass slides with an aminosilane coating with an intermediate contact angle of approximately 65° were used for the transfer chip  330 . These afforded a suitable compromise between a rounded droplet while reducing the volume required, see R. Briard et al in “Crack Bridging Mechanism for Glass Strengthening by Organosilane Water-based Coatings” (J. Non-Cryst. Solids, Vol. 351, pp. 323-330). The assay chip  360  may then be incubated with a protective coating  370 . 
         [0082]    Once fabricated the spotted transfer chip  300 C and spotted incubated assay chip  300 D were transferred to the snapping system as described below in respect of  FIGS. 4 and 5 . Following snapping and separation, droplets were visible on the nitrocellulose coating of the spotted assay chip  300 D, but no or very little residue was visible on the aminosilane coating of the spotted assay chip  300 C, indicating that the transfer was both reliable and efficient. Next the combined slide  300 E is incubated, for example with streptavidin-Cy5  380  before being characterized with laser fluorescence based test system giving emitted fluorescence  390 . 
         [0083]    Accordingly, for experiments reported below in respect of microarray-to-microarray snap fit processes typical processes and parameters for preparing the slide based microarrays were as follows. For the assay chip, such as assay chip  300 B in  FIG. 3A , capture antibody solutions containing 400 μg/ml antibodies and 10% glycerol in PBS were spotted on a nitrocellulose slide at a relative humidity of approximately 60%, each spot containing approximately 1.2 nl. Detection antibody solutions containing 20 μg/ml antibodies, 20% glycerol, and 1% BSA were spotted on an aminosilane slide to form the transfer chip at a relative humidity of 80% to prevent evaporation; each spot contained approximately 8 nl. Spotting was performed using an inkjet spotter (Nanoplotter 2.0 by GeSiM). The center-to-center spacing between spots was 800 μm for a large scale array, and 1 mm for an assay although it would be apparent that other values may be employed. 
         [0084]    After spotting, an assay chip was typically incubated for 1 hour at room temperature with a humidity of 60%. A slide module gasket with 16 modules (Grace Bio-Labs Inc.) was clamped on the slide dividing it into 16 wells for immunoassays. After incubation the assay chip was rinsed twice with PBS containing 0.1% Tween-20 (PBST) for 5 min on a shaker at 450 rpm and once with PBS for 5 min on the shaker at 450 rpm. 
         [0085]    Now referring to  FIG. 3B  there is depicted a procedure for the microarray-to-microarray transfer using snap chips for conducting multiplex immunoassays with colocalization of each capture and detection antibody pair according to an embodiment of the invention. Capture antibodies  3500  are spotted onto a first transfer chip  3600  using inkjets  3400  to form a first matrix as shown in first step  3000 A. These are then transferred to an assay slide  3650  to form a matrix of transferred capture antibodies  3550  in second step  3000 B. For example, first transfer chip  3600  may be a glass slide with an aminosilane coating with an intermediate contact angle of approximately 65°. The assay slide  3650  with transferred capture antibodies  3550  may then be incubated with a protective coating  370  in third step  3000 G. 
         [0086]    Next, detection antibodies  3200  are spotted onto a second transfer chip  3300  using inkjets  3100  to form a second matrix as shown in fourth step  3000 C. For example a glass slide with an approximately 10 μm thick nitrocellulose coating may be used as assay chip  360  as the three-dimensional structure of the nitrocellulose provides a high antibody binding capacity, and will in subsequent steps absorb the solution from the second transfer chip  3300 . For the second transfer chip  3300  a glass slide with hydrophobic coating is typically employed to yield rounded droplets which help ensure fluidic contact to the assay slide  3650  during the transfer operation, but for the diameters used here, required large volumes of liquid. Within initial experiments employing the microarray-to-microarray transfer method of the invention larger diameters were selected for the droplets on the second transfer chip  3300  as these allowed for relaxed the alignment constraints while ensuring complete overlap between the capture antibody spot and biotinylated detection antibody spot. 
         [0087]    Once fabricated the spotted second transfer chip  3300  and spotted incubated assay chip  3650  were transferred to the snapping system as described below in respect of  FIGS. 4 and 5A  to perform fifth step  3000 D. Following snapping and separation, droplets were visible on the nitrocellulose coating of the spotted assay chip  3650 , but no or very little residue was visible on the aminosilane coating of the spotted second transfer chip  3300 , indicating that the transfer was both reliable and efficient. Next the combined slide was is incubated in sixth step  3000 E, for example with streptavidin-Cy5  3800  before being characterized with laser fluorescence based test system in the seventh step giving emitted fluorescence  3900 . 
         [0088]    Accordingly using the procedure described above in respect of  FIG. 3B  the inventors employed a commercial inkjet spotter to spot 0.65% alginate solutions mixed with cAb-coated polystyrene microbeads onto aminosilane slides at precise coordinates, and fluorescently labeled dAbs in a 1% agarose solution on another slide in a mirrored pattern. The alginate cAb droplets were gelated immediately by adding a calcium solution and the agarose dAb droplets by cooling the slide to 4° C. Next, the cAb slide was blocked with bovine serum albumin for 1 h, and incubated with a sample for 1 h, and briefly dried. The two slides were then clamped together using the snapping system presented below in respect of  FIGS. 4 and 5  before the combined slide was incubated for 1 hour and read out using a microarray scanner. 
         [0089]    Microarray-to-Microarray Mirror Alignment: 
         [0090]    In order to ease the microarray-to-microarray transfer minor patterns and alignment markers are provided on the assay chip and transfer chip according to some embodiments of the invention where visual alignment of the assay chip/transfer chip alignment is made. As indicated in  FIG. 4  during the spotting process  4000 A, the bottom right corner of each slide is pressed against a mechanical stop  470  on the slide deck. However, during the transfer process  4000 B the two slides face one another and for the transfer chip, the bottom-right corner becomes the bottom-left corner. Typically, the position of the spots on the slides is not absolute, but relative to the first spot and to the corner to which the slide was aligned, which is suitable for most applications, but not for snap chip applications because minor alignment is performed relative to an opposite corner. The alignment following mirroring is further complicated by the fact that in most inkjet spotting systems the inkjets do not spot perfectly straight, and that the size of the glass slides is not accurate as these are mass produced consumable items, and that it would thus not be possible to align the spots by aligning the assay chip to the bottom-right and the transfer chip to the bottom-left corner. 
         [0091]    Two approaches have been considered for achieving the required overlay accuracy during the transfer process  4000 B. Within the first approach the spots were provided at exact coordinates in a minor pattern on both slides and then each slide aligned relative to the bottom-left edge on each moiety of the snap system. The second approach was to spot an alignment mark on the back-side of the transfer side, having predetermined relationship to the rightmost spot of the top row of the assay chip, while aligning it relative to the bottom right corner, flip it, align it again relative to the bottom right corner and use the image recognition system of the inkjet to align the first spot exactly atop the alignment mark. 
         [0092]    This second approach being shown by first to third schematics  400 A through  400 C respectively in spotting process  4000 A in Figure B. In this manner, both slides will be aligned to the same edge (i.e. bottom right when seen from the top) and the alignment accuracy is independent on the size of the slides. Within the experiments presented within this specification the second approach was employed. Accordingly as shown by first schematic  400 A an assay chip  420  is patterned with capture antibody spots  430  using inkjet(s)  410 . The back of the transfer chip  440  is patterned with the reference spot  450  in second schematic  400 B whilst in third schematic  400 C the front side of the transfer chip  440  is shown with reference spot  450  visible through the transfer chip  440  whilst the detection antibody spots  460  are disposed on the transfer chip  440  using inkjet(s)  410 . Assembly process  4000 B in  FIG. 4  depicts the assay chip  420  and front side of transfer chip  440  ready for assembly with the axis of symmetry between them. 
         [0093]    Snapping of Microarray Slides: 
         [0094]    The assay chip, such as assay chip  400 A in  FIG. 4 , and the transfer chip, such as transfer chip  400 C in  FIG. 4 , according to an embodiment of the invention are placed in a snap apparatus, shown in open state  500 A and closed  500 B in  FIG. 5A  together with optical micrograph  500 C. As shown the snap apparatus comprises comprising first precision milled vacuum chuck  535 A, second precision milled vacuum chuck  535 B, and four steel rods  515 . Each of the first and second precision milled vacuum chucks  535 A and  535 B respectively comprise a recess for inserting and aligning the assay and transfer chips and serve to hold them in place prior to snapping them together. To keep the precise minor symmetric pattern alignment between the two slides, the assay chip  525 A and the transfer chip  525 B are pushed against the bottom right corner and the bottom left corner in the recess of their respective vacuum chucks. The four steel rods  515  are fixed to the first precision milled vacuum chuck  535 A and serve to guide the second precision milled vacuum chuck  535 B which has four holes matching the pattern of the steel rods  515  assembled into the first precision milled vacuum chuck  535 A. A steel plate, shown in optical micrograph  500 C, is used according to an embodiment of the invention during snapping to support the first and second precision milled vacuum chucks  535 A and  535 B respectively whilst they were being manually clamped together with clamps  550 . 
         [0095]    The first and second precision milled vacuum chucks  535 A and  535 B are clamped to at predetermined pressure. Kapton spacers  510  with a thickness of approximately 25 μm were placed between the assay chip  525 A and transfer chip  525 B to provide control of the gap between them during clamping and to avoid excessive “squeezing” of the droplets during snapping. A typically clamping duration being one minute. Approximately 500 μm thick rubber cushions  520  were inserted between each of the assay chip  525 A and transfer chip  525 B and their respective one of the first and second precision milled vacuum chucks  535 A and  535 B accommodate small imperfections and improve pressure distribution the pressure across the slides. Following snapping, a liquid bridge between the assay chip  525 A and transfer chip  525 B is established, and the detection antibody droplets  545  and associated reagents were transferred to the assay chip  525 A from the transfer chip  525 B upon subsequent separation. As shown assay chip  525 A also shows the nitrocellulose pads  540 . 
         [0096]    It would be evident that the snap apparatus as depicted in  FIG. 5A  may be varied without departing from the scope of the invention. For example, the clamping process may be automated, additional alignment verification means incorporated such as providing contacts on the assay chip and transfer chip such that only in correct alignment will all such contacts provide electrical connections, and that the chucks may be machined from optically transparent materials allowing with suitable absorber materials other than rubber the visual alignment of the slides prior to confirming the snap operation. 
         [0097]    Now referring to  FIG. 5B  there is depicted a second mechanical structure for snap assembly and microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention. In contrast to the snap apparatus, shown in open state  500 A and closed  500 B in  FIG. 5A  together with optical micrograph  500 C the second mechanical structure comprises base and cover elements  560  and  570  respectively made from polyoxymethylene (POM). POM offers high mechanical stiffness, good machining characteristics, and excellent mechanical stability under 40° C. Base and cover elements  560  and  570  reduce the overall dimensions to approximately 98 mm×55 mm×30 mm and weight to approximately 232 g. Formed within base element  560  is a first recess  562  and posts  590  are inserted into holes machined within the base  560 . A second recess  572  is formed within cover element  570  together with machined holes  574  to accept the posts  590  when the cover element  570  is flipped and aligned to the base element  560 . 
         [0098]    Subsequently the assay slide  582  with patterned nitrocellulose pads  582  is assembled into the base element  560  and is retained through pressure from a rubber element  565  inserted within the recess  562  of the base element  560 . Similarly the transfer slide  585  is retained through pressure by a rubber element  565 . Accordingly inversion of one or other of assay slide  582  and transfer slide  585  within their respective base or cover elements  560  and  570  respectively and engagement of the base and cover elements  560  and  570  respectively via posts  590  and holes  574  provides the desired flip-chip process as described above in respect of  FIGS. 3A through 5A  respectively. Maintenance of the engagement of the base and cover elements  560  and  570  respectively is achieved through four screws as depicted in assembled unit image  500 F. It would be evident to one skilled in the art that variants of the design described above in respect of  FIG. 5B  may be implemented without departing from the scope of the invention. 
         [0099]    However, in some instances rather than clinical type environments, or even in such environments, it would be beneficial to have a disposable snap-chip design that allows for high volume, low cost manufacturing through injection molding for example. Such an approach is depicted in  FIG. 5C  wherein a third mechanical structure for snap assembly and microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention is presented. Accordingly, a clam shell comprising base  5100  and cover  5150  joined by a hinge  5130  is depicted wherein base  5100  has a first recess for holding the assay chip  5200  and cover  5150  has a second recess for holding the transfer slide  5200 . The assay chip  5200  and transfer slide  5300  being retained and positioned via rubber elements  5500 . Accordingly closure of the cover  5150  over the base  5100  aligns the assay chip  5200  and transfer slide  5300  by virtue of first and second pairs of pillars  5120  and  5110  respectively engaging first and second pairs of holes  5170  and  5160  respectively. 
         [0100]    The base  5100  and cover  5150  being held in position by spring loaded retainer clips  5400  which engage slots within each of the first and second pairs of pillars  5120  and  5110  respectively as depicted by closed and locked configuration schematics  5000 B and  5000 C respectively. It would evident that other closures may be employed without departing from the scope of the invention wherein these provide pressured contact to ensure interfacing of the transfer slide  5300  and the assay chip  5200 . It would also be evident that clear materials compatible with injection molding such as polymethyl methacrylate (PMMA) may be employed to allow alignment and engagement of the transfer slide  5300  and assay chip  5200  to be visualized as they are performed. Further, as presented schematically in  FIG. 5D  a three element assembly may be employed such that a base  52  has hinged connections to first cover  51  and second cover  53 . First cover  51  may for example support the transfer slide whereas the second cover  53  supports a slide with streptavidin-Cy5  3800  for example such that the assay chip  5200  in base  52  may be incubated and then characterized with a laser fluorescence based test system. 
         [0101]    Accuracy of Microarray-to-Microarray Transfer: 
         [0102]    We characterized the alignment accuracy for 256 spots arrayed over a slide, 16 spots on each of the 16 nitrocellulose pads, by spotting and transferring IgGs labeled with two different fluorescent dyes respectively and scanning the nitrocellulose slide immediately after transferring. The average center-to-center distance between the spots following transfer to the assay chip was 147 μm, with the largest distance being 216 μm. We observed a position shift from the left to the right side of the slide during spotting, which doubled following mirrored transfer, indicating that there was an angular misalignment between the slide deck and the motorized inkjet stage. To achieve complete overlap between corresponding spots, each capture spot was serviced with 1.2 nl of solution yielding a 300 μm spot on the nitrocellulose slide, while 8 nl of detection antibody solution were applied and produced a droplet that was 700 μm in diameter on the transfer chip. 
         [0103]    Microarray-to-Microarray Transfer of Antibody Reagents: 
         [0104]    The inventors have evaluated the use of the snap chip for implementing immunoassays. An array of 256 fluorescent IgGs was transferred from a transfer chip to an assay chip patterned with an array of 1024 fluorescent anti-IgGs as shown in optical micrograph  600 A in  FIG. 6 . 20% glycerol was added to the detection buffer to prevent drying of the detection antibodies while the assay chip was dried under a stream of nitrogen prior to the transfer to promote the absorption of the detection antibody droplets in the nitrocellulose while minimizing lateral spreading. Visual inspection reveals a selective and homogeneous transfer of proteins across the entire slide as shown in optical micrograph  600 B for one 64 array of fluorescent anti-IgGs of the 1024 fluorescent anti-IgGs. The fluorescence intensity profile of the two proteins in the one-step assay show excellent overlap in the spot locations as evident in  FIG. 6B . 
         [0105]    The 1024 array of anti-goat IgGs were labeled with Alexa 488 (green) and spotted on centre-to-centre spacing of 800 μm whilst the 256 array of goat IgGs were labeled with a centre-to-centre spacing of 1600 μm. Intermediate spots were loaded with a solution of PBS. In optical micrograph  600 A the square borders represent the edges of the 16 nitrocellulose pads disposed on the glass slide. Fluorescence intensity profiles of the green (Alexa 488) and red (Alexa 647) protein spots in the row marked by the arrow in optical micrograph  600 B are shown in  FIG. 6B . 
         [0106]    Now referring to  FIG. 6C  there is depicted a schematic showing dAb transfer slide  6000 A and assay slide  6000 B. The assay slide  6000 B comprising Alexa 532 labeled goat IgG (Ab 1) antibodies coated to beads (Ab 1 coated beads-in-gel droplets  6100 ). The dAb transfer slide  6000 A comprising agarose solution in buffer (Agarose in buffer  6300 ) and Alexa 633 labeled anti-goat IgG (Ab 2) antibodies dissolved in agarose solution and spotted to every second spot in every second row (Ab2 in agarose  6200 ). It would be evident to one skilled in the art that the performance of multiplexed assays is severely limited owing to cross-reactivity between antibodies and antigens which occurs because detection antibodies are applied as a mixture. 
         [0107]    Accordingly the inventors have developed antibody colocalization microarrays to eliminate cross reactivity by spotting each dAb on the spot of the corresponding cAb on a nitrocellulose slide, see M. Pla-Roca et al in “Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples” (submitted to Nature Methods). Further the inventors have also recently introduced beads-in-gel droplet microarrays which are 3D antibody microarrays made of porous alginate droplets with the entrapment of antibody-coated polystyrene microbeads that allowed for more sensitive multiplexed protein assays in serum, see for example H. Li et al. in “Hydrogel Droplet Microarrays with Trapped Antibody-Functionalized Beads for Multiplexed Protein Analysis” (Lab on a Chip, Vol. 11, pp. 528-534). Accordingly combining the processes described above in respect of  FIGS. 3A and 3B  in the embodiment presented in  FIG. 6C  results in microarray-to-microarray transfer of antibodies with the advantages of antibody colocalization microarray and of beads-in-gel droplet microarrays to produce handheld, highly sensitive and scalable multiplex immunoassay chips. 
         [0108]    Subsequent to transfer the beads-in gel slide was evaluated using fluorescence imagery with 532 nm and 633 nm filters resulting in first optical micrograph  6000 C wherein the spacing of dots on the combined slide, and hence the dAb transfer slide  6000 A and assay slide  6000 B was 1 mm whilst accuracy of combining the dAb transfer slide  6000 A and assay slide  6000 B on the prototype snap apparatus was &lt;150 μm. It would be evident that improvements in the machining tolerances, materials, etc employed within the snap apparatus that improved tolerances may be achieved. Also shown in  FIG. 6C  is an optical micrograph of high density assay array  6000 D comprising 16 196 spot arrays configured as 14×14 assay spots thereby providing an overall 3,136 assay locations upon a standard glass slide. Visual inspection reveals a selective and homogeneous transfer across the entire slide. 
         [0109]    Accordingly it would be evident that by adjusting the design of the snap apparatus to accommodate larger glass slides that microarray-to-microarray transfer and assay of very high counts can be achieved with high selectivity and homogeneity. 
         [0110]    10-Plex Sandwich Microarray-to-Microarray Immunoassays in Buffer and Serum: 
         [0111]    To evaluate the use of microarray-to-microarray transfer for multiplexed sandwich immunoassays, we selected 10 proteins, including one breast cancer biomarker (HER 2), 4 cancer related proteins (ENG, LEP, FGF, OPN), and 5 cytokines (TNF RII, GM-CSF, CCL 2, CCL 3, IL 1β). The experiment flow employed was that shown in  FIG. 3A . To avoid undesired adsorption of antibodies to the transfer chips, the spotting solution containing the detection antibody was supplemented with 1% BSA, which helped increase the transfer efficiency as BSA molecules competitively interact with surface amino groups and therefore minimize the attachment of antibodies. 
         [0112]    Fabrication of the 10-plex sandwich immunoassays varied slightly from the process described above for other microarray assay and transfer chips as follows. After blocking with Stabilguard® for 1 h on a shaker at 320 rpm, the assay chip was incubated with the sample solutions containing the mixture of 10 proteins that were spiked into the buffer or the 10% serum solution for 1 hour on the shaker at 320 rpm. A dilution series was used to establish a binding curve with the protein concentration ranging from 200 ng/ml to 0.0128 ng/ml for the HER 2, ENG, LEP, FGF, and OPN proteins, and from 50 ng/ml to 0.0032 ng/ml for TNF RII, GM-CSF, CCL 2, CCL 3, and IL 1β proteins, with a dilution factor of 5, and a control with 0 ng/ml for all the 10 proteins. 
         [0113]    The slide was then rinsed twice with PBST and once with PBS on the shaker at 450 rpm for 5 minutes, the slide module gasket was removed, and the slide dried under nitrogen. Next, the assay chip and the transfer chip were clamped on the snap apparatus, snapped together for 1 minute, then separated, and the assay chip was incubated in a Petri dish saturated with humidity for 1 hour. Then a slide module gasket was clamped on the assay chip, and the slide was rinsed three times with PBST and once with PBS on the shaker at 450 rpm for 5 minutes and incubated with 2.5 μg/ml of streptavidin conjugated Cy 5 for 20 minutes on the shaker at 320 rpm. The slide was then rinsed twice with PBST, once with PBS and once with DI water on the shaker at 450 rpm for 5 minutes, and dried before scanning. 
         [0114]    Using the microarray-to-microarray assays, the inventors obtained pg/ml sensitivity for all the 10 proteins in PBS buffer solutions, as shown in  FIG. 8 . Referring to  FIG. 7  there is depicted a fluorescent micrograph of a representative slide  700 B with 16 replicate arrays incubated with PBS and 10% serum samples, and a close-up of a single array  700 A identified by the dashed lines within representative slide  700 B. For scale the bar on the close-up of single array  700 A is 1 mm. 
         [0115]    From the measured fluorescent data a four-parameter logistic equation was used for curve fitting, see J. W. Findlay et al (AAPS Journal, Vol. 9, pp.E260-267) wherein 9 out of 10 curves fit the data well.  FIG. 8A  depicts the assay results and binding curves for HER 2, ENG, LEP, FGF, and OPN whilst  FIG. 8B  depicts the assay results and binding curves for TNF RII, GM-CSF, CCL 2, CCL 3, and IL 1β. As the affinity of the antibodies for these five proteins was higher that that of the other 5 proteins the assay range was adjusted. The error bars are standard deviations between triplicate experiments performed using the microarray-to-microarray snap process according to an embodiment of the invention. 
         [0116]    The curve of CCL 3 in  FIG. 8B  does not fit well with the assay data at low concentrations suggesting that more optimization is needed. The LOD values of the assays are presented below in Table 1. As evident from these results for 9 out of 10 antibodies these values were lower than the LOD obtained from pin spotting colocalization immunoassays reported in the prior art, see for example see M. Pla-Roca et al in “Antibody Colocalization Microarray: A Scalable Method for Multiplexed and Quantitative Protein Profiling” (submitted to Mol. Cell. Proteomics), probably due to the better intra-spot homogeneity. Indeed, colocalization arrays are double spotted with pins, whereas for the snap chips according to embodiments of the invention both the assay chip and transfer chip are spotted with inkjet, and the antibodies on the transfer chip remain in solution. These results indicate that high sensitivity may be achieved using snap chips which might rival the one obtained with enzyme-linked immunosorbent assay (ELISA). 
         [0117]    To explore the applicability of snap chips and microarray-to-microarray transfer for immunoassays using blood, the inventors performed a multiplexed assay for the same ten proteins spiked in 10% serum. These results are presented in  FIG. 9  wherein  FIG. 9A  depicts the assay results and binding curves for HER 2, ENG, LEP, FGF, and OPN whilst  FIG. 9B  depicts the assay results and binding curves for TNF RII, GM-CSF, CCL 2, CCL 3, and IL 1β. The LOD of some proteins, such as TNF RII and OPN, is higher in 10% serum than in PBS which may be ascribed to interferences from matrix proteins, see for example C. Pfleger et al (J. Immunol. Methods, Vol. 329, pp. 214-218), or to endogenous patient proteins. For ENG, LEP, OPN, TNF RII, GM-CSF, CCL 2, and CCL 3, the sensitivity after correcting for the 10 fold serum dilution exceeds the physiological range for healthy persons, and for HER 2 and IL 1β it lies within the range. For example, based on the paper by Rutkowski et al. in “Cytokine Serum Levels in Soft Tissue Sarcoma Patients: Correlations with Clinico-Pathological Features and Prognosis” (Int. J. Cancer, Vol. 100, pp. 463-4′71), the level of TNF RII in healthy people is 3180±600 pg/ml24. The LOD for TNF RII obtained is 30 pg/ml, which is a hundred times lower than the average concentration in blood. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 LOD values obtained from 10-plex immunoassays in PBS and in 10% serum (pg/ml). 
               
             
          
           
               
                   
                   
                   
                 LOD (R&amp;D 
                 Average 
                   
               
               
                   
                 LOD 
                 LOD 
                 System 
                 Concentration 
               
               
                 Protein 
                 (3σ) 
                 (2σ) 
                 (2σ) 
                 (Healthy Control) 
                 Reference 
               
               
                   
               
             
          
           
               
                 HER 2 
                 155 
                 81 
                 n/a 
                 ≦15,000 
                 Kong et al, J. Clin. Pathol., 
               
               
                   
                   
                   
                   
                   
                 59, 373-736 
               
               
                 ENG 
                 138 
                 74 
                 30 
                 150,000 
                 Takahashi et al, Clin. Cancer. 
               
               
                   
                   
                   
                   
                   
                 Res., 7, 524-532 
               
               
                 LEP 
                 52 
                 28 
                 8 
                 26,430 ± 19,400 
                 Aliustaoglu et al, Med. 
               
               
                   
                   
                   
                   
                   
                 Oncol., 2010, 27, 388-391 
               
               
                 FGF 
                 85 
                 51 
                 3 
                 n/a 
                 n/a 
               
               
                 OPN 
                 263 
                 171 
                 24 
                 123,000 
                 Bramwell et al, Clin. Cancer. 
               
               
                   
                   
                   
                   
                   
                 Res., 12, 3337-3343 
               
               
                 TNF RII 
                 36 
                 21 
                 2 
                 3180 ± 600  
                 Rutkowski et al, Int. J. 
               
               
                   
                   
                   
                   
                   
                 Cancer, 100, 463-471 
               
               
                 GM-CSF 
                 6 
                 3 
                 3 
                 900 ± 90  
                 Scholl et al, Breast Cancer 
               
               
                   
                   
                   
                   
                   
                 Res. Treat., 39, 275-283 
               
               
                 CCL 2 
                 15 
                 10 
                 5 
                 173 
                 Kim et al, Breast Cancer 
               
               
                   
                   
                   
                   
                   
                 Res., 11, R22 
               
               
                 CCL 3 
                 3 
                 2 
                 10 
                 88.3 
                 Kim et al, Breast Cancer 
               
               
                   
                   
                   
                   
                   
                 Res., 11, R22 
               
               
                 IL 1β 
                 14 
                 8 
                 1 
                 40 
                 Yurkovetsky et al, Clin. 
               
               
                   
                   
                   
                   
                   
                 Cancer. Res., 13, 2422-2428 
               
               
                   
               
             
          
         
       
     
         [0118]    Storage of Snap Chips: 
         [0119]    It would be evident to one skilled in the art that if the snap chip could be stored, it would allow dissociating the production of the slides which requires advanced equipment such as the inkjet spotter from the execution of the assay which can be done at low cost without need for peripheral equipment. Using TNF RII, the inventors evaluated the possibility of storing snap chips in a freezer at −20° C. wherein fluorescence measurements were made on samples after the snap fit process wherein the assay chips had been stored for 1 month and 3 months and plotted against baseline results from an as freshly spotted assay chip. Based on these results presented in  FIG. 10  it appears that the antibodies loose some of their activity over time, yet the LOD obtained for 3 months storage remains well below the average physiological concentration in healthy patients for this marker. These results indicate that it is possible to store snap chips although some optimization of storage conditions rather than the simple method employed in the results presented may be necessary in order to avoid loss of activity of the antibodies, and to develop protocols for slide storage in a refrigerator at 4° C. or at room temperature as well as within freezers. Using the results presented in  FIG. 10  the LOD values obtained for slides that were fresh, 1 month and 3 months old were 4 pg/ml, 3 pg/ml, and 18 pg/ml respectively. The LOD of each curve was calculated as background intensity incremented by 2σ and is indicated in  FIG. 10  by the arrows for each test. 
         [0120]    Within these experiments the inventors spotted both the assay and transfer chips, stored them for either 1 month or 3 months, and then performed the immunoassays before comparing them to freshly spotted slides. The assay chips were blocked with StabilGuard® after incubation with capture antibodies and both assay chips and transfer chips were immediately stored in an air tight bag with desiccant and placed in a −20° C. freezer. Prior to usage, the sealed bag was left at room temperature for approximately 30 minutes before opening to avoid condensation on the surface of the slides. Next, the transfer chips were incubated in a Petri dish saturated with humidity for 30 minutes to hydrate the glycerol before the antibody transfer process. 
         [0121]    It would be evident to one skilled in the art that alternate structures may be implemented in order to provide the required snap chip assembly in order to provide microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays. Referring to  FIGS. 11A through 11C  there are depicted a silicon micro-machined transfer chip  1100 A, silicon micro-machined assay chip  1100 B, and snap chip assembly prior to separation according to an embodiment of the invention. Referring to  FIG. 11A  the silicon micro-machined transfer chip  1100 A is depicted as comprising a silicon substrate  1140  that has been processed according to standard photolithography and semiconductor processes to provide a pattern of posts  1110  and transfer wells  1120 . Patterned into the bottoms of each transfer well  1120  are aminosilane regions  1130 , these being formed for example through chemical vapour deposition (CVD) or liquid phase deposition processes, see for example S. Fiorilli et al in “Vapor-Phase Self-Assembled Monolayers of Aminosilane on Plasma-Activated Silicon Substrates” (J. Colloid and Interface Science, Vol. 321, pp. 235-241) and F. Zhang et al in “Chemical Vapor Deposition of Three Aminosilanes on Silicon Dioxide: Surface Characterization, Stability, Effects of Silane Concentration, and Cyanine Dye Adsorption” (Langmuir, Vol. 26(18), pp 14648-14654). Also shown is cross-section X-X through silicon micro-machined transfer chip  1100 A wherein the height of the posts  1110  of d POST  are defined through the photolithography openings provided during processing prior to etching of the silicon. 
         [0122]    Now referring to  FIG. 11B  the silicon micro-machined assay chip  1100 B is depicted as comprising a silicon substrate  1180  that has been processed according to standard photolithography and semiconductor processes to provide a pattern of recesses  1150  and assay wells  1160 . Patterned into the bottoms of each assay well  1160  are nitrocellulose regions  1170 , these being formed for example through ultrasonic deposition, see for example C-C Chen et al in US Patent Application 2005/0,191,484 entitled “Process for Forming Nitrocellulose Films” or casting as employed by Sartorius Stedim Biotech. Also shown is cross-section X-X through silicon micro-machined assay chip  1100 B wherein the depth of the recesses  1150  of d RECESS  are defined through the photolithography openings provided during processing prior to etching of the silicon. 
         [0123]    Now referring to  FIG. 11C  a cross-section of the assembled snap chip is shown comprising silicon micro-machined transfer chip  1100 A and silicon micro-machined assay chip  1100 B is depicted. Accordingly the post  1110  of the silicon micro-machined transfer chip  1100 A has engaged the recess  1150  of the silicon micro-machined assay chip  1100 B such that the two chips are aligned in the plane parallel to their surfaces and that the spacing d between the silicon micro-machined transfer chip  1100 A and silicon micro-machined assay chip  1100 B is accordingly defined by d=d POST −d RECESS . Accordingly the patterned aminosilane regions  1130  and nitrocellulose regions  1170  are aligned with respect to each other. Within this cross-section the droplets of detection antibodies and any formations of capture antibodies have been omitted for clarity. 
         [0124]    It would be evident that whilst the profiles depicted for post  1110  and recess  1150  are sloped and accordingly typical of wet chemical etching of silicon as defined by its crystal planes that other techniques may be applied as are well known in the prior art for providing vertical walls to the post  1110  for example. In this case with vertical posts a coarse alignment of the silicon micro-machined transfer chip  1100 A and silicon micro-machined assay chip  1100 B is converted to a fine alignment as the posts  1110  move within the recesses  1150  due to the wall geometry as the silicon micro-machined transfer chip  1100 A and silicon micro-machined assay chip  1100 B are brought together. It would also be evident that such a micro-machined assay chip also allows for improved handling in the subsequent characterization/measurement steps. 
         [0125]    Referring to  FIG. 12  there are depicted silicon micro-machined transfer chip  1200 A and silicon micro-machined assay chip  1200 B according to an embodiment of the invention. However, unlike silicon micro-machined transfer chip  1100 A and silicon micro-machined assay chip  1100 B in Figure lithe silicon micro-machined transfer chip  1200 A and silicon micro-machined assay chip  1200 B are each provided with first and second electrical contacts  1210  and  1220  respectively that couple to the transfer wells and assay wells. Within transfer wells the aminosilane regions  1230 , for example, are still provided but adjacent are open regions  1220  of the transfer wells. Within the assay wells the nitrocellulose regions  1250 , for example, are still provided by adjacent to these are gel regions  1240 . Accordingly when assembled capture antibody and detection antibody etc are within a structure allowing application of an electric field along the length of each test cell as first electrical contact  1210  on silicon micro-machined transfer chip  1200 A is at one end of the test cell and second electrical contact  1220  on the silicon machined assay chip  1200 B is at the other end of the test cell. 
         [0126]    Accordingly after assembly of the snap chip an electrical field can be applied, for example to induce electrophoresis, wherein after the electrical field is removed, the snap chip separated the silicon micro-machined assay chip  1200 B can be tested but now due to the well defined structural characteristics of the silicon micro-machined assay chip  1200 B the fluorescent probe, or whatever characterization technique is employed, can be located accurately one or other end of the test cells according to the particular testing being performed. It would be evident that such a technique may also be modified to include the option to provide the capture antibodies at the opposite end of each test cell so that electrophoresis etc is performed such that the transported protein is then captured. Optionally first and second electrical contacts  1210  and  1220  could be provided on one of the silicon micro-machined assay chip  1200 B and silicon micro-machined transfer chip  1200 A. 
         [0127]    Referring to  FIG. 13  the applicability of snap chips and microarray-to-microarray transfer for immunoassays exploiting antibody colocalization microarray and beads-in-gel droplet microarrays as discussed above was demonstrated with 9 different antibody pairs. These results presented in graph  1300  depict the assay results and binding curves for TNF RII, GM-CSF, CCL 2, IL 1 Beta, CCL 4, IL 5, TNF RI, IL 18 and TNF Alpha. The limit of detection achieved in this experiment being in the pg/ml range for all analytes, and specifically 3 pg/ml for TNF RII (Tumor Necrosis Factor Receptor-II). 
         [0128]    Double Snap: 
         [0129]    Within embodiments of the invention described above in respect of microarray-to-microarray transfer of immunoassays and their exploitation in multiplexed sandwich arrays a factor severely limiting the performance of these multiplexed sandwich assays is cross reactivity. However, this may be overcome by exploiting antibody colocalization microarrays (ACMs), see for example Pla-Roca et al in “Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples” (Molecular &amp; Cellular Proteomics, Vol. 11, pp. 1-12). ACM requires spotting with capture antibodies (cAbs) and detection antibodies (dAbs) to the same spot during the assay, which is challenging. To simplify the ACM the microarray-to-microarray transfer method described above in respect of  FIGS. 3A through 13  was developed by the inventors to deliver antibodies from an array of droplets to an array of spots by snapping two slides, an assay slide and transfer slide, together. However, the mirror setup configuration of this approach can create alignment issues due to the imprecision of inkjet spotters. As described supra 10 proteins were measured simultaneously with this microarray-to-microarray transfer of immunoassays, commonly referred to by the inventors as snap chip, but extension to ACM and increased simultaneous protein counts is limited by this inkjet spotter imprecision. Accordingly, the inventors have established Double Snap Chip (DSC) which overcomes the alignment issues and enables higher density, and very high sensitivity multiplex immunoassays. 
         [0130]    Referring to  FIG. 14  there is depicted a process flow for double snap-chip based microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention. Initially cAbs and dAbs are spotted with an inkjet spotter onto aminosilane slide in steps  1400 B and  1400 A respectively. The dAbs slide in the experiments reported below in respect of  FIGS. 16 ,  17 A, and  17 B were performed wherein the dAbs slide was stored at −20° C. after spotting. However, it would be apparent that other protocols may be employed. The cAbs were transferred onto a nitrocellulose slide by snapping for 1 minute as indicated by process step  1400 C. The resulting assay slide was blocked, dried, and stored. For assays, slides were removed from the freezer, and the nitrocellulose slide incubated with a sample overnight, and dried as indicated by process step  1400 D. Next, the processed nitrocellulose slide and dAb slide were snapped together as indicated in step  1400 E and then incubated for 1 hour. Upon rinsing and subsequent incubation with streptavidin-Cy 5 as indicated in process step  1400 F the assay results are obtained using a scanner as indicated in process step  1400 G. 
         [0131]    Referring to  FIG. 15  there is depicted a detailed schematic of the double snap-chip based microarray-to-microarray transfer of reagents for multiplexed sandwich immunoassays according to an embodiment of the invention presented in  FIG. 14 . As depicted cAbs are spotted on a cAb slide ( 1500 A) which was mechanically aligned to the bottom-right corner of an alignment system ( 1500 B) such as described above in respect of  FIGS. 5A through 5D . Next a cAb slide and an assay slide ( 1500 C) are pushed to bottom right and bottom left corner of the snap apparatus chucks and snapped together resulting in first slide  1500 D wherein upon separation the cAb has been transferred to the assay slide. Next the same sequence of printing ( 1500 E), insertion into snap apparatus chuck ( 1500 F), insertion of assay slide into snap apparatus chuck ( 1500 G) and transfer of the dAb to the assay slide upon separation ( 1500 H) is performed. Thus both the spotting and transfer of the cAb and dAb arrays are each done in the same reference frames of the inkjet spotter and snap apparatus the issues of angular misalignment occurring because the inkjet spotter and slides are not perfectly orthogonal or potential non-straight shooting by the inkjet spotter are reduced significantly. 
         [0132]    In the snap chip results presented supra the array density was approximately 130 spots/cm 2  due to the mirroring induced misalignments. With the DSC methodology of sequentially transferring the cAb array and upon sample incubation the dAb array onto a slide, as depicted in  FIGS. 14 and 15  the average center-to-center distance between aligned spots was reduced to 30 μm although other spacing were also employed, the largest being 80 μm. Accordingly the array density is increased to 400 spots/cm 2 . Accordingly as depicted in  FIG. 16  3136 spots were completed with zero failures using an alignment apparatus such as described in respect of  FIG. 5A .  FIG. 16  depicts a scan of an assay slide after transfer using 532 nm and 633 nm laser sources. Alexa 532 labeled goat IgG functioned as cAbs, and Alexa 633 labeled anti-goat IgG were spotted and transferred on every second spot. The center-to-center spacing between the spots in this instance was 450 μm. The variations in spot size between rows are presumed to be due to inkjet spotting which was done two rows at a time. The scale bar is 1 mm. 
         [0133]    Using the DSC technique immunoassays were performed for 40 proteins simultaneously. The results from this are presented in first to fourth graphs  1700 A to  1700 D in  FIGS. 17A and 17B . The results are split based upon fluorescent intensity and antigen concentrations for readability rather than antigen type. For 36 of the proteins the measured LODs were in the pg/ml range with the best being EGF which was measured at 1.1 pg/ml 
         [0134]    DSC as with the single snap chip approach allows high sensitivity, multiplexed immunoassays to be performed with low handling complexity and reduced process complexity. Assay slides with cAbs and dAbs can be prepared ahead of time and stored, thus avoiding the need of a microarrayer during the assay process. This for end users of such techniques is of great practical importance as it allows immunoassays to be performed in a wider range of environments rather than solely well equipment clinical analysis laboratories. As presented above the DSC could be extended to 1,568 targets assuming duplicate spots, and to further higher counts with improved spotting. The DSC approach therefore provides a useful and powerful tool for antibody-based proteomics, notably for biomarker discovery and validation in blood for cancer and other diseases 
         [0135]    It would be evident to one skilled in the art that whereas glass and silicon have been presented for providing the transfer chip and assay chip that combinations thereof may also be employed as well as other materials including but not limited ceramics, plastics, and glasses not usually associated with glass slides as the provide enhanced characteristics such as for example being molded with enhanced dimensional control. It would be evident that in other embodiments of the invention that the manufacturing tolerances of the clam shell as discussed supra in respect of  FIG. 5C  may be sufficient that similarly toleranced substrates for the assay chip  5200  and transfer chip  5300  may be inserted and interfaced without the requirement for the rubber elements  5500 . 
         [0136]    The above-described embodiments of the present invention are intended to be examples only. Alterations, modifications and variations may be effected to the particular embodiments by those of skill in the art without departing from the scope of the invention, which is defined solely by the claims appended hereto.