Technical Field The present disclosure relates to microfluidics, and more particularly to microfluidic devices for sample processing.

Background of the Present Disclosure

After the success of the human genome project, and sequencing of an increasing number of genomes of various organisms, the focus of methodological research in high throughput analysis is progressively shifting towards the study of proteins known as proteomics, a partner of genomics in systems biology. A first application of proteomics is screening, i.e., the identification of proteins contained in a sample. This task may include separating the proteins with a desired resolution and sensitivity, and identifying each spot. In many applications, it is also desirable to have a quantization of the relative concentration of proteins of interests in a sample. For example, a level of expression of specific "biomarker" proteins may be used for diagnosis, or for determining the effect of drugs on different metabolic paths, for drug discovery. More refined studies of proteins may also involve the characterization of protein post-translational modifications (sometimes useful as biomarkers of diseases), or studies of protein-ligand interactions.

The increasing activity in the field of protein analysis makes the development of new techniques desirable, and various methods are described in Chapter 11 in "Particles for Protein Analysis in MicroFluidic Systems," published in "Colloidal Particles in Micro & Nano-Biotechnologies," A Elaissari, Ed. Wiley, VCH 2009. However various issues make the analysis of proteins more difficult than that of nucleic acids. First, no amplification such as polymerase chain reaction (PCR) exists with respect to proteins, so that one deals with the quantity of a given protein in the initial sample, thereby challenging the sensitivity of the detection method. The challenge is increased by a large dynamic range of concentrations. For example, one type of protein, such as actin in the cytoplasm or albumin in blood, can represent more than 10 or 20% of the total proteinaceous mass, and some others, such as signalling proteins, may be present at sub-picomolar concentrations. These low-abundance proteins often are of substantial interest for biological and medical applications. Another difficulty lies in the hydrophobicity of numerous proteins, in particular the biologically important membrane proteins. These proteins can be difficult to dissolve and to separate, particularly in aqueous media. Overall, low abundance proteins and membrane proteins remain largely unknown. Further, many proteins lie in a relatively limited range of sizes, typically from

15000 Dalton (Da) to 100 000 Da, but may present substantial chemical diversity. Proteins can involve any of 20 amino acids, acidic or basic, and with differing hydrophobicities, in a specific sequence. Such proteins can be folded into complex secondary and tertiary structures, which gives each protein a unique pattern of recognition, signalling, and enzymatic properties. Mass spectrometry (MS) providing mass resolution is one method used in proteomics, but high resolutions are available for a limited range of sizes, typically up to several kilodalton (kDa). Also, resolutions associated with MS are generally insufficient to separate all of the differing proteins and peptides contained in a sample, for example a serum or a cell extract, so that in practice, MS is primarily applied in combination with electrophoresis or chromatography as a "post column" separator and detector.

Another often employed method for proteomics is two-dimensional gel electrophoresis, which involves separation according to isoelectric point in a pH gradient, followed by separation by size using zone electrophoresis in a denaturant SDS buffer. Such a method can be labor-intensive, but it is widely employed due to its relatively high throughput. Another method for proteomics includes a unidimensional or 2D chromatographic separation of the proteins. Such a method can include, for example, fractionation on a cation exchange column followed by a gradient elution on a Cl 8 reverse phase column. After the excision of the subsequent spots (e.g., from 2D gel electrophoresis) or collection of the protein (e.g., after chromatography), the proteins are submitted to proteolytic digestion by trypsin, that cut proteins after specific aminoacids. Such a technique can provide a series of peptides that can often provide a unique "signature" of a protein.

Beside these proteomic applications, in which it is desirable to simultaneously treat a large number of proteins, numerous applications focused on the identification and quantization of a limited number of proteins exist, for example, diagnosis. These applications often rely on immunoassay-type methods, such as agglutination or enzyme-linked immunosorbent assay (ELISA) tests, using specific interaction of proteins with antibodies directed against them. However, even in diagnosis multiparameter analysis involving several to several tens of different biomarkers is desirable, leading to potential for more powerful diagnosis and more accurate treatment design.

For both fundamental research and diagnosis, it is desirable to develop protein analysis methods with increased parallelism, automation and sensitivity. Therefore, it is a goal of the present disclosure to provide microfluidic systems or "lab-on-chips" to enable such analysis methods.

In addition to molecular separation and/or assays, briefly discussed above, the analysis of a biological sample generally involves sample preparation steps that can represent a significant cost and delay in the analysis process. Microfluidics can be beneficial for integrating such steps into a single, fully automated device (e.g., "lab on chip"), particularly through use of micro and nanoparticles.

However, the use of microparticles for protein analysis in microsystems can present difficulties, e.g., preparing the beads, packing the beads in the microchannel, manipulating the fluids in the system, and detecting proteins in microsystems. Such difficulties may also be present, for example, when trying to develop microfluidic systems for the analysis of nucleic acids, and more generally for applications involving the capture or treatment of analytes by a microcolumn inside a microfluidic system.

In articles of Electrophoresis 2002, 23, 3537-3544 and Electrophoresis 2003, 24, 3018-3025, Oleschuk et al described a chamber demarcated by two weirs and applying to Solid Phase Extraction (SPE) and Capillary ElectroChromatography (CEC) on chip. A 200 μm long, 580 μm wide and 10 μm deep cavity was etched into the glass, with its inlet and outlet channels obstructed by 9 μm high weirs to leave a 1 μm gap for sample and reagents introduction. Beads larger than 1 μm in diameter were introduced through a transverse channel (30 μm wide) and trapped between the weirs. Similarly, in Rapid Commun. Mass Spectrom. 2000, 14, 1377-1383, Wang et al. describe an integrated device comprising an enzymatic reactor associated with electrophoresis separation and coupled to electrospray mass spectrometry (ESI-MS). Digestion was obtained faster on chip (5s) than in a classical cuvette (15min) for melittin, based on an increased ratio of enzyme to substrate and decreased diffusion time. Based on the same packing method, Li et al describe, in Molecular & Cellular

Proteomics 2002, 1 :157-168, another example of a partly-integrated device having a channel filled with Cl 8 reverse phase or antibody-coated beads of mixed sizes. The device integrated sequential injection, preconcentration followed by Capillary Electrophoresis (CE) separation and interface to mass spectroscopy analysis (see Fig. 1) leading to detection of fmol of digest peptides and a throughput of 12 samples per hour.

Sato et al describe, in Anal. Chem. 2000, 72, 1144-1147, a method to create a physical barrier in a microchip. In this method, the solid phase, made of polystyrene beads, was retained by a single microfabricated dam, the microdevice being made of glass plates using different technologies for its fabrication (e.g., soft lithography, fast atom beam fabrication, or lamination.) They developed in a sandwich immunoassay using three antibodies (for capture and detection) for the analysis of a colon cancer marker. They obtained reduced time analysis (45 hours to 35 minutes) and detection limits more than one order of magnitude lower than conventional tests.

One disadvantage associated with the above described techniques, which are based on microfabricated weir, is the size of the beads (higher than 40μm in diameter) which can limit the surface to volume ratio. The packed beads can be cumbersome and may involve a high fabrication cost. Hayes et al described in Anal. Chem. 2001, 73, 5896-5902, a heterogeneous immunoassay in a microchannel using magnetic beads. The particles were packed in the microchannel by the application of a strong magnetic field created by a single permanent magnet located directly over the channel. The length of the bed was between 1 and 3 mm. Reagents were passed through the beads reactor and the bed was imaged with an epifluorescence microscope with laser-induced excitation. The bed of beads did not extend across the channel, because the magnetic beads aggregated near the single magnet, which enabled fluorescence detection on the surface of the packed bed and the use of high flow rates. The device was optimized with the analysis of model compounds, fluorescein isothiocyanate (FITC)/anti-FITC (direct assay) and realistic samples such as parathyroid hormone and interleukin-5 with sandwich assay. This group used streptavidin-modified magnetic beads of 1 -2 μm in diameter, and demonstrated that the assays had physiologically relevant sensitivity (μg.mL-1). Particles could be packed, dynamically positioned, flushed and repacked. Moreover, this system consumed low amount of reagents: 100 to 1000 times smaller than conventional assays. This system, however, has other limitations. First, most of the reagent flows in the open space of the capillary, and interaction with the beads may not be optimized. Second, beads absorb some of the light emitted, thereby reducing sensitivity. This latter disadvantage can be avoided by using a non optical detection, e.g. electrochemical detection, but this may limit the range of possible applications.

Other systems, involving the possibility to dynamically manipulate beads, were also proposed e.g in Rida et al, Anal. Chem. 2004, 76, 6239-6246, or US 7,309, 439 to Fenandez. However, they suffer from the same problems as above- In Slovakova et al, (2005). Lab Chip, 5 : 935-42, disclosed a new way to prepare magnetic microreactor inside a microchannel was disclosed, allowing for digestion at a faster rate than in batch mode. This technology, however, still suffers from some drawbacks. First, the plug was opaque, and thus non applicable to optical detection. Therefore, detection was performed off-chip, sunbtantially reducing the interest in using a microfluidic format.

In another implementation, described in Le NeI A, et al., Micro Total Analysis Systems 2007, Paris - JL Viovy, P. Tabeling, S Descroix, L. Malaquin, CBMS Eds, this difficulty in detection was addressed by directly coupling the microfluidic chip to a Mass Spectrometer. However, Mass Spectrometers are large and expensive instruments, and not very sensitive.

There is thus still a challenge, to develop a microfluidic system for analysis, using microcolumns easy to prepare, and allowing for fast, sensitive and low cost detection of analytes.

There is also still a challenge, to develop a microfluidic system for analysis of analytes, in which chromatography or immuno affinity based protocols can be implemented easily. This imposes having, inside microfluidic systems, microcolumns easy to prepare, and allowing for fast, sensitive and low cost detection of analytes. It is another challenge to integrate this microcolumn as user-friendly as possible, with other steps of the global analysis protocol, and particularly with analyte detection. The object of the present disclosure is to solve these challenges.

Summary of the Present Disclosure

According to some embodiments, the present disclosure relates to an integrated microsystem. The integrated microsystem may include a microchannel, a field generator to create a magnetic field in at least one first portion of the microchannel having a direction substantially collinear with the direction of flow in the portion of the microchannel, the magnetic field also presenting a gradient, and wherein the microsystem additionally comprises a detection area in fluid connection with the microchannel. Such a system may enable the creation of a relatively high throughput, low cost device for protein identification and study. Moreover, because the system may be implemented using magnetic elements, recyclability of the device may be relatively high based on the removability of the system components. In some embodiments consistent with the present disclosure, the detection area is configured for application of a detection method to the content of the detection area, the detection method including at least one of an optical detection (based for instance on fluorescence, luminescence, electroluminescence, chemiluminescence, light absorption, light diffraction, light refraction), an impedancemetric detection, an electrochemical detection, or more generally any methods for the detection of analytes, known from those skilled in the art.

In some embodiments consistent with the present disclosure, the microchannel and the detection area may be contained in a single microfabricated microfluidic element. Such a configuration may aid in simplifying fabrication and operation,

One of ordinary skill in the art will understand that the present disclosure, as related to one microchannel and one detection area, may be implemented with a multiplicity of microchannels and detection areas. Also, the microchannels and detection areas can be integrated into one or more complex microfluidic networks, comprising annex channels for loading samples, reagents, washing solutions, or collecting products.

Further, microsystems consistent with embodiments of the present disclosure may be controlled by a flow control, and/or a pressure control system. Such systems may be enabled to regulate and program the flow of different liquids, including the sample containing the analytes, in the microchannel or microfiuidic network. In some embodiments, the flow control may be synchronized with detection.

In some embodiments according to the present disclosure, the magnetic field may be configured to be activable, i.e., switched on and off as desired. Such switching may be performed using electromagnets, and/or permanent magnets in connection with a mechanically mobile magnetic shunt.

In some other embodiments consistent with the present disclosure, a non-activable magnetic field produced by permanent magnets may be implemented (i.e., not switchable). In such embodiments, renewal of the microcolumn of magnetic particles may still be possible by, for example, using a sufficiently strong flow, as described in greater detail herein.

Embodiments of the present disclosure may aid in development of reduced complexity, integrated, and sensitive systems useful in analyte detection methods. More specifically, according to some embodiments of the present disclosure, a method for detecting or quantifying an analyte in a sample is disclosed. The method may include the steps of providing a microsystem similar to those described _. above and hereing, flowing microsystem magnetic microparticles and/or magnetic nanoparticles in the microchannel, the magnetic microparticles and/or magnetic nanoparticles configured to interact with the analyte, flowing the sample into the microchannel, and detecting in the detection area products related to the presence of, or with the quantity of the analyte in the sample.

The use of microparticles or nanoparticles in microfluidic systems of the invention, and more particularly, magnetic particles, may provide additional advantages. For example, such particles may be available within controllable sizes and with inorganic or organic polymer core composition. They can be manipulated using electric fields, pressure driven flow, gravity or simple agitation. They can be coated with biological molecules to make them interact with or bind to a biological entity.

Further, in contrast to a direct coating of the surface of the analytical device, this biofunctionalization can be performed in a batch process, and the particules prepared and characterized in a single batch can be used to prepare many (e.g., thousands) different test elements. For diagnosis applications, in which cost is an issue, this may offer substantial benefits.

Moreover, particles may offer a larger surface to volume ratio than functionalized planar surface (typically increased hundred fold or more, for micron-sized particles), allowing for higher sensitivity and dynamic range. Magnetic beads can also offer additional advantages, for example, because of their magnetic core, they can be magnetically manipulated with an external magnetic field and thus be easily extracted and resuspended in a different solution without centrifugation. Numerous suppliers now propose various colloids for biological assays, demonstrating the practical interest of these materials.

Some advantages of the present disclosure include for example -The manipulation and packing of the beads in the microchannel. The preparation of good and reproducible packed beads columns in microfluidic systems is more difficult than in conventional chromatography microcolumns, due at least in part to the smaller dimensions and weaker mechanical resistance of microchannel, as compared to tubular microcolumns.

-The manipulation of the fluids in the system. This manipulation is in general facilitated by the microfluidic format, in which connections with limited or no dead volume can be easily fabricated. The possibility of integrating complex protocols is one of the powers of microfluidic systems, and one desire of the present disclosure.

Additional problems exist here, in particular with very small volumes of liquid to be manipulated (in the μl or nl range). The conventional tools for fluid manipulation, such as peristaltic, piston or syringe pumps, are poorly adapted to microfluidic systems, and the present disclosure also provides in some embodiments, a way to solve this problem, based on pressure-based pumps.

-Detection. The detection of proteins in microsystems is challenging for several reasons: first, the reduced volume tends to reduce the total quantity of proteins captured. Second, chips are generally fabricated in materials that are not transparent in the UV (quartz chips exist but are extremely expensive), preventing the use of UV absorption, the most common detector in chromatography or capillary electrophoresis of proteins. Finally, staining is not as easy as with gels. This latter disadvantage has to be minored, however, by the fact that staining is an expensive and slow process that the current trend of high throughput and automation tends to avoid anyway.

Binding of analytes onto magnetic microparticles and/or magnetic nanop articles within micro channels consistent with the present disclosure may involve an interaction, such interaction being, for example, an immunoaffmity reaction, an affinity reaction, a nucleic acid hybridization, a hydrogen bonding, a hydrophobic adsorption, and/or an electrostatic adsorption, among others.

Embodiments of the present disclosure may be implemented to bind analytes, and then to elute and detect the analytes, for example, using chromatography methods. Such configurations may yield improved processing speeds, reduced complexity, and additional sensitivity. In some embodiments, the analyte may have some of its physical or chemical properties altered during contact with the magnetic particles (e.g. through digestion, labelling, chemical reaction, denaturation, aggregation), and such an alteration can be measured in the detection area. Where desired, it may be possible to maintain the analytes in the microchannel, and to detect the analytes, for example, indirectly by a reporter, i.e., a compound or device capable of indicating (e.g., visually, aurally, tactilely, etc.) presence and/or concentrations of the analytes.

Previously, some practiced introduction of a secondary fluorescent ligand in the presence of the bound analyte. Such methods have typically been referred to as sandwich immunoassay. However, where magnetic microparticles and/or magnetic nanoparticles are used, the substantial opacity of the magnetic particles makes such techniques difficult.

Therefore, according to some embodiments of the present disclosure, a method combining the advantages of using magnetic particles, with the advantages of using a reporter of analyte binding with improved resolution is disclosed. To accomplish this, the zone where analytes are bound and the zone where they are detected may be physically separated (e.g., located at different portions or segments of a device. According to some embodiments, a differential measure of the content of the sample before and after crossing the first portion of the microchannel. In yet other embodiments, differential measurements of the content of a secondary fluid containing a reporter may be taken before and after crossing of the first portion of the microchannel.

According to yet further embodiments of the present disclosure, a method for detecting or quantifying an analyte in a sample is disclosed. The method may include providing a microsystem, comprising a microchannel, a field generator to create a magnetic field in at least one first portion of the microchannel, the magnetic field having a direction substantially collinear with the direction of flow in the portion of the microchannel, the magnetic field also presenting a gradient, the microsystem optionally comprising a detection area in fluid communication with the microchannel. Further, the method may comprise flowing magnetic microparticles and/or magnetic nanoparticles in the microchannel from the microsystem, the magnetic microparticles and/or magnetic nanoparticles configured to bind the analyte, flowing the sample into the microchannel, flowing in the microchannel at least a fluid different from the sample, and detecting in the fluid a reporter of a binding of the analyte to at least one of the magnetic microparticles and the magnetic nanoparticles in the microchannel. In some embodiments, the fluid may include a secondary species, configured to bind analytes specifically, and yield a signal. The secondary species maybe eluted, and can be detected in the detection area. Alternatively, the secondary species may be detected indirectly, for example, based on action of the secondary species on a substrate flowing through the first portion of the microchannel or other suitable system. In such embodiments the modification of the substrate is detected and the secondary species may include an enzyme, and/or a catalyst.

In some embodiments consistent with the present disclosure, the secondary species may be an enzyme bound to an antibody directed to the analyte, to an aptamer specific to the analyte, and/or to a nucleic acid sequence with specific affinity for the analyte. In such embodiments, the method can be an enzyme-linked assay. Where the enzyme is linked to an antibody, the method can implement a variety of enzymes, antibodies, and substrates, such as those used in, e.g., Enzyme Linked Immuno Assays. In some embodiments the enzyme can include one or more of a peroxidase, a catalase, a reductase, a restriction enzyme, a protease, and a nuclease, among others.

The enzyme maybe enabled to modify optical properties of a substrate. This change of optical properties can be detected in the detection area after flowing a solution containing the substrate in the capture area e.g., the first portion of the microchannel. In some embodiments, this optical property may include fluorescence emission, luminescence, and/or light absorption.

In some embodiments, electrochemical detection may be enabled by, for example, secondary species modification of the redox state of a substrate. In yet other embodiments, the secondary species may modify the charge or a substrate, which may enable impedancemetric detection.

Embodiments of the present disclosure can be implemented with improved sensitivity, particularly when the substrate is not flowed regularly in the microchannel, but instead is introduced as pulses separated by steps in which flow is arrested, and/or diminished. Embodiments utilizing such stepwise flow, or more generally, flow with a non-constant velocity, can lead to improved results.

One of skill in the art will understand that the systems and methods of the present disclosure may be employed in various embodiments, for carrying out microreaction, immunoreaction, enzymatic digestion, ELISA, diagnosis, prognosis, drug delivery, high throughput screening, and the like. Some exemplary embodiments are described below, in which embodiments of the present disclosure are applied to the analysis of prion protein (PrP). It is important to note that while the discussion below focuses primarily on analysis of prion protein, embodiments of the present disclosure are applicable to analysis of any analytes, biomolecules, polypeptides, proteins, metabolites, nucleic acids, cells, organelles, microparticles and nanoparticles, polymers, colloids, infections agents, food components, and environmental samples, among others.

Systems of the present disclosure can be integrated as a technological building block within more complex devices, in particular high throughput screening devices, lab on chips, point of care, laboratory instruments, robots, and the like. Further, methods of the present disclosure can be integrated as part of complex protocols for diagnosis, drug discovery, target discovery, drug evaluation, among others.

Brief Description of the Drawings

Fig. 1 is an exemplary configuration for a micro-fluidic integrated device consistent with some embodiments of the present disclosure;

Fig. 2 is an illustration of an exemplary sandwich capture on magnetic beads according to some embodiments of the present disclosure; Figs. 3 shows exemplary substrates revealing the enzymatic activity of alkaline phosphatase (AP);

Fig. 4 shows the raw results of resulting absorbance for a number of resulting test;

Fig. 5A is an exemplary device for measuring fluorescent intensity consistent with embodiments of the present disclosure; Fig. 5B is an exemplary chart showing the time course of the fluorescence intensity versus percolation flow rate;

Fig. 6A is a plot of exemplary data associated with the type of curve obtained from "stop-and-go" flow procedures, consistent with the present disclosure; and

Fig. 6B is an exemplary graph plotting digestion efficiency maxima versus dwelling time for two different concentrations of recPrP having the same recPrP percolation rates and the same percolated volumes.

Description

It is desirable to detect PrP in biological fluids such as, for example, urine or blood, at pre-symptomatic stages of a disease, and therefore it is desirable to enable analysis of low concentrations of proteins in such substances. Such analysis may be facilitated by first developing a proteinase K enzymatic microreactor allowing distinction between healthy protein and pathogenic proteins. Following such development, an integrated diagnosis system comprising direct and sensitive detection of the protein which withstands enzymatic degradation can be developed. Two different strategies can be considered, the first allowing direct detection of PrP captured by antibodies, while the second involves capture of the protein, followed by subsequent analysis of the eluted protein. Consistent with embodiments of the present disclosure, a method for grafting anti- prion antibodies on magnetic particles was developed, followed by development of a microfluidic system for direct readout of the PrP concentration based on an ELISA test, fluorescent detection, and assembling magnetic particles under an external magnetic field. In so doing, high specificity of the antigen-antibody interaction may be combined with a low detection limit, by means of the concentration of proteins of interest in a small volume, i.e., the volume of a plug of magnetic beads in a micro-fluidic channel (a few hundred nL). PrP was thereby detected on the chip with a sensitivity of the order of about one hundred femtomoles (fM). The immunoassay which is described hereafter is based on the formation of a sandwich on magnetic beads.

Example 1

Fig. 1 is an exemplary configuration for a micro-fluidic integrated device 100 consistent with some embodiments of the present disclosure. Micro-fluidic integrated device 100 comprises a microchannel 1, a microfluidic chip 2, an inlet 3, and an outlet 4.

MicroChannel 1 comprises a first zone 5 configured to capture microp articles or nanoparticles 6, and a second zone 7, configured for detection tasks. In such embodiments, detection may be fluorimetric, and may use a microscope 8. Further, in such embodiments, field generators for creating a magnetic field may include permanent magnets 9. One of ordinary skill in the art will recognize that other types of magnets could be used (e.g., electromagnets) as desired.

Fig. 2 is an illustration of an exemplary sandwich capture on magnetic beads according to some embodiments of the present disclosure. Detection may involve formation of a such a "sandwich" on the magnetic bead. Primary antibodies 202 may be grafted onto magnetic micro or nanoparticles 201, such as microparticles or nanoparticles 6. The functionalized magnetic particles, as well as the flow in microchannel 1, and are immobilized in capture zone 5 of microfluidic channel 1. The sample is then flowed in microchannel 1 , while an analyte 204, e.g., a prion protein, binds to the primary antibody. After washing, a buffer or solution containing a secondary antibody 203, configured to provide additional function for detection, is flowed into microchannel 1, such that secondary antibody 203 may attach to analyte 204.

In some embodiments, secondary antibody 203 is bound to fluorescent moieties, such as Fluorescence Isothiocyanate FITC or Alexa Fluor. In some other embodiments, they can be coupled to enzymes, such as peroxidases, like Horse Radish persoxydase HRP. While conventionally, secondary antibodies are marked with fluorophores, for example, FITC or Alexa Fluor, and/or are coupled to peroxidases, for example, HRP, the presently described immunoassay desires to be integrated into a micro-fluidic chip and therefore is not so marked for reasons described hereafter. Organization of the magnetic particles under a magnetic field may lead to a network of beads organized as a labyrinth or lattice and the magnetic plug formed as a result may occupy a complete section of the channel over a length of about 3 mm. This may, in turn, delimit a substantially or totally opaque area. Therefore, the use of secondary antibody 203 directly marked with fluorophores may not be desirable, possibly resulting in an optical signal that could be substantially or completely hidden by the opacity of the plug formed by the beads. Thus, optical detection may be accomplished at the exit of the plug of magnetic beads.

Example 2

The SAF34 antibody recognizing the ^" N-terminal domain of PrP may be bounded onto magnetic beads as follows: Reagents (for 1 mg of beads):

"MyOne" Dynal COOH magnetic particles lμm, 1 mg

Phosphate buffer PBS 0.1X, pH 7.3

Purified antibodies solutions (100 μg for 1 mg of beads)

EDC (E6383, Sigma-Aldrich):7,5 mg in 200 μl PBS S-NHS (56485, Sigma-Aldrich): 7,5 mg in 100 μl PBS

Tween 20 (P7949, Sigma-Aldrich)

MOPS (sourced from prod. no. M 1254, Sigma-Aldrich)

SAF34 (sourced from SpiBio)

Sha31 (sourced from SpiBio) Streptavi din- alkaline phosphatase (S2890, Sigma-Aldrich) recPrP: kindly donated by H. Rezaei (description in Rezaei et al, Eur. J. Biochem. 2000, 267, 2833-2839).

Method

- Beads functionalization: Clean beads in 10 mM NaOH solution (e.g., 2 times), then in pure water (e.g., 2 times), then in phosphate buffer (e.g., 2 times) using a Dynal beads separator adapted to Eppendorf tubes. Each cleaning step involves ImI of solution.

Eliminate supernatant and add the 100 μl purified antibodies solution. Complete to 0.5 ml with PBS.

Incubate 1 hour in agitator at room temperature (e.g., 20 to 25 degrees C);

Add solution of EDC then S-NHS;

Complete to ImI with phosphate buffer;

Incubate overnight (e.g., 6-10 hours) at 4°C in a rotary agitator; Following incubation, collect beads, eliminate supernatant and clean with PBS added with 0.1% Tween 20,

Repeat 5 times. Prepare PrP solution:

Used for the example herein is recombinant PrP, named "recPRP." It is important to note that some prion proteins are infectious, and should be manipulated in an L2 laboratory.

Dissolve the PrP at 5 mg/m; in MOPS buffer, 20 mM, pH 7,5;

The concentration after dilution can be controlled by BCA test, or by UV absorption at 260 and 280 nm, using equation:

C (mg.mL-1) = 1,55 x A280 - 0,77 x A260 Functionalization of secondary antibody:

For this example, the selected secondary antibody is Sha31, however, one of skill in the art will recognize that other secondary antibodies are suitable. Further, in this example, the Sha31 was coupled to alkaline phosphatise (AP), through biotin-streptavidin interaction. This step for biotinylation of Sha31 anti-prion antibodies, purified beforehand, was carried out with the kit "EZ-Link Sulfo-NHS-Biotinylation Kit" available from Pierce as product no. 21425, however, one of skill in the art will recognize that similar kits, now available or developed in the future, from any suitable provider may be used.

The biotinylated antibodies are then incubated overnight (e.g., 6-10 hours) at 4°C with streptavidin-alkaline phosphatase. The product is referred to as AcII-AP in the following text.

Principle of detection The principle of the detection consists of using AP to transform a substrate into a product having properties that are detectably different. Such properties could include, for example, a change in color, fluorescence, solubility, or redox properties. Figure 3 provides two exemplary substrates that can be processed by AP, in order to provide a UV absorbing substrate (left), or a fluorescent substrate (right). When using AP, all buffers used should be substantially (e.g., to within laboratory tolerances) or completely void of phosphate, to avoid competition between the phosphate and the substrate. In the present example, In the present example Tris Buffered Saline IX pH 7,8, supplemented with 0.1% Tween 20 (TBST) was used, however, one of skill in the art will recognize that other suitable buffers may be implemented.

EXAMPLE 3: Detection of prion protein in microfluidic chip, with off-chip detection.

Reagents: TBST Paranitrophenylphosphate (N7653, Sigma-Aldrich) •

Operation of the chip:

A microfluidic chip can be prepared by soft lithography, as described, for example, in M. Slovakova et al, Lab Chip 2005, 5, 935-942. Magnetic beads are bound with anti-prion primary antibodies, as described in Example 2. A microfluidic channel with dimensions 0.25x1 x20 mm is flanked by two magnets creating a field with a direction and a gradient collinear to the main axis of the channel.

Step A: Magnetic beads prepared with primary antibodies, as in Example 2, are flowed at, for example, 2 ml/hour, into the channel in the presence of the magnets, and immobilized. Step B: The solution containing recombinant PrP is then flowed in the system, at a flow rate of, for example, 100 ml/hour for one hour. Washing is then performed for one hour with TBST buffer.

Step C: The solution containing secondary antibody is flowed into the system under the same conditions, and then rinsed again for 1 hour. Step D: A solution of paranitrophenylphosphate (e.g., commercial solution diluted

5 times) is flowed at, for example, 100 μl/h during 2 hours, and collected. The degradation of the substrate by alkaline phosphatase is measured through the UV absorption at 405 nm using a "Nanodrop" (e.g., Nanodrop ND-100) UV absorption analyzer.

The results are given as a function of time in Fig. 4. The shape is typical of an enzymatic reaction, with linear increase followed by saturation. The speed of reaction (beginning of the curve) allows to determine the limiting reaction speed as a function of the flow rate. This allows to determine the optimal flow rate (good compromise between efficiency and rapidity of the reaction) of perfusing substrate. This flow rate will be used in the following.

Example 4: Integrated system with in situ fluorescence detection. Reagents:

TBST

4-methylumbelliferylphosphate (M8883, Sigma-Aldrich)

Method:

The microfluidic device is prepared as in Example 3, except that an epi fluorescence microscope with a high sensitivity camera and dichroic equipment, for example, as shown at Fig. 5A. Such a device may include a dichroic mirror 164, an excitation filter 168, for example, a DAPI filter (λ _eXC = 358 nm and λ- _m = 461 nm, cooled CCD camera 160, an emission filter 170, a mercury lamp 166, and a computer 162. One of skill in the art will recognize that more or fewer components may be present as desired. In this embodiment, the field of view of the microscope constitutes the second zone for analysis, and the epifluorescence microscope is positioned downstream.

The protocol is substantially the same as in Example 3 up to Step C. Instead of Step D from example 3, step D _b i _S , as defined below is executed.

D _b i _s : A solution of 4-methylumbelliferylphosρhate at 2.5 mg/ml in TBST IX is flowed in the device at different flow rates (reported in Fig 5B). The observed fluorescence, which demostrates the production of 4-methylumbelliferone, is directly measured in the chip in real time from the epifluorescence objective (Fig 5B).

When the flow rate is decreased, the intensity increases, as a consequence of the longer average incubation time for each substrate molecule. However, for a given flow rate, the intensity may not be stable, and for 20 μl/h, the intensity is smaller than that for 50 μl/hour. This is detrimental to sensitivity, thus described below is another embodiment, which substantially or completely alleviates this limitation. Example 5. Integrated system with in situ fluorescence detection and "stop and go."

The example proceeds as in Example 4, except that during step D _b i _s , the solution of 4-methy]umbelliferylphosphate is flowed into the microchannel in a "stop and go" mode. in other words, the 4-methylumbelliferylρhosphate is flowed for a given time period at 300 μl/hour, then stopped, then restarted at 300 μl/h, and so on.

The resulting fluorescence intensity is plotted in Fig 6A (top curve), and the flow rate, with the variable flow time, is represented in the bottom curve. When flow restarts after a period of stoppage, the fluorescence intensity undergoes a strong overshoot, which increases with the duration of flow stoppage. This is due, at least in part, to the accumulation in the formed plug of fluorescent product during flow stoppage. This accumulated product is then flushed during upon flow reinitiation, leading to increased sensitivity. For longer waiting times, the overshoot saturates, because all the substrate has been consumed.

The value of the fluorescence intensity, as a function of the residence time, is plotted in Fig 6B. One can see that, even at a concentration of 100 ng/ml, a significant signal is obtained in about 600 s, i.e., 10 min. This allows reduction in the testing time and in manipulation of test components, as compared to methods of the prior art.

Throughout the present disclosure the term "at least one" is intended to include one and/or "a" single implementation of the referenced object or action. Further, it will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed systems and methods without departing from the scope of the present disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the disclosed systems and methods. It is intended that the specification and examples be considered as exemplary only, with a true scope being indicated by the following claims and their equivalents.