Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles
  • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C1/00—Magnetic separation
  • B03C1/005—Pretreatment specially adapted for magnetic separation
  • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C1/00—Magnetic separation
  • B03C1/02—Magnetic separation acting directly on the substance being separated
  • B03C1/28—Magnetic plugs and dipsticks
  • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0668—Trapping microscopic beads
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0887—Laminated structure
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C2201/00—Details of magnetic or electrostatic separation
  • B03C2201/20—Magnetic separation of bulk or dry particles in mixtures
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C2201/00—Details of magnetic or electrostatic separation
  • B03C2201/26—Details of magnetic or electrostatic separation for use in medical or biological applications

Definitions

• the present disclosure relates to microfluidics, and more particularly to microfluidic devices for sample processing.
• 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.
• one type of protein such as actin in the cytoplasm or albumin in blood
• 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
• MS Mass spectrometry
• MS is primarily applied in combination with electrophoresis or chromatography as a "post column” separator and detector.
• proteomics 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.
• the proteins are submitted to proteolytic digestion by trypsin, that cut proteins after specific aminoacids.
• trypsin proteolytic digestion by trypsin
• 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.
• 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.
• 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.
• CE Capillary Electrophoresis
• 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.
• FITC fluorescein isothiocyanate
• anti-FITC direct assay
• This group used streptavidin-modified magnetic beads of 1 -2 ⁇ m in diameter, and demonstrated that the assays had physiologically relevant sensitivity ( ⁇ g.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.
• 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.
• the object of the present disclosure is to solve these challenges.
• 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.
• 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.
• an optical detection based for instance on fluorescence, luminescence, electroluminescence, chemiluminescence, light absorption, light diffraction, light refraction
• an impedancemetric detection based for instance on fluorescence, luminescence, electroluminescence, chemiluminescence, light absorption, light diffraction, light refraction
• an impedancemetric detection based for instance on fluorescence, luminescence, electroluminescence, chemiluminescence, light absorption, light diffraction, light refraction
• an impedancemetric detection based for
• 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,
• microchannels and detection areas may be implemented with a multiplicity of microchannels and detection areas.
• 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.
• 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.
• 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.
• a non-activable magnetic field produced by permanent magnets may be implemented (i.e., not switchable).
• 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.
• microparticles or nanoparticles in microfluidic systems of the invention may provide additional advantages.
• 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.
• 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.
• 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.
• 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.
• 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.
• a reporter i.e., a compound or device capable of indicating (e.g., visually, aurally, tactilely, etc.) presence and/or concentrations of the analytes.
• a method combining the advantages of using magnetic particles, with the advantages of using a reporter of analyte binding with improved resolution is disclosed.
• 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.
• 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.
• a method for detecting or quantifying an analyte in a sample 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.
• 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.
• 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.
• 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.
• the modification of the substrate is detected and the secondary species may include an enzyme, and/or a catalyst.
• 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.
• 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.
• 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.
• this optical property may include fluorescence emission, luminescence, and/or light absorption.
• electrochemical detection may be enabled by, for example, secondary species modification of the redox state of a substrate.
• 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.
• 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.
• 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.
• 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.
• 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.
• biological fluids such as, for example, urine or blood
• Such analysis may be facilitated by first developing a proteinase K enzymatic microreactor allowing distinction between healthy protein and pathogenic proteins.
• 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.
• 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.
• 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.
• 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.
• detection may be fluorimetric, and may use a microscope 8.
• field generators for creating a magnetic field may include permanent magnets 9.
• magnets e.g., electromagnets
• 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 sample is then flowed in microchannel 1 , while an analyte 204, e.g., a prion protein, binds to the primary antibody.
• 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.
• secondary antibody 203 is bound to fluorescent moieties, such as Fluorescence Isothiocyanate FITC or Alexa Fluor.
• fluorescent moieties such as Fluorescence Isothiocyanate FITC or Alexa Fluor.
• they can be coupled to enzymes, such as peroxidases, like Horse Radish persoxydase HRP.
• peroxidases like Horse Radish persoxydase HRP.
• 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.
• 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.
• the SAF34 antibody recognizing the " N-terminal domain of PrP may be bounded onto magnetic beads as follows: Reagents (for 1 mg of beads):
• EDC E6383, Sigma-Aldrich:7,5 mg in 200 ⁇ l PBS S-NHS (56485, Sigma-Aldrich): 7,5 mg in 100 ⁇ l PBS
• MOPS sourced from prod. no. M 1254, Sigma-Aldrich
• PrP recombinant PrP
• recPRP recombinant PrP
• the concentration after dilution can be controlled by BCA test, or by UV absorption at 260 and 280 nm, using equation:
• the selected secondary antibody is Sha31, however, one of skill in the art will recognize that other secondary antibodies are suitable.
• 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.
• 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.
• 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).
• 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.
• 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.
• 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
• 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.
• Nanodrop e.g., Nanodrop ND-100
• 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:
• 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.
• the field of view of the microscope constitutes the second zone for analysis, and the epifluorescence microscope is positioned downstream.
• step D b i S is executed.
• D b i s A solution of 4-methylumbelliferylphos ⁇ hate 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).
• Example 5 Integrated system with in situ fluorescence detection and "stop and go.”
• step D b i s the solution of 4-methy]umbelliferylphosphate is flowed into the microchannel in a "stop and go" mode.
• the 4-methylumbelliferyl ⁇ hosphate is flowed for a given time period at 300 ⁇ l/hour, then stopped, then restarted at 300 ⁇ l/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.
• Fig 6A top curve
• 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.
• 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.

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