Classifications

• • 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
  • B01L3/502715—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 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F16—ENGINEERING ELEMENTS AND UNITS; GENERAL MEASURES FOR PRODUCING AND MAINTAINING EFFECTIVE FUNCTIONING OF MACHINES OR INSTALLATIONS; THERMAL INSULATION IN GENERAL
  • F16K—VALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING
  • F16K99/00—Subject matter not provided for in other groups of this subclass
  • F16K99/0001—Microvalves
  • F16K99/0034—Operating means specially adapted for microvalves
  • F16K99/0042—Electric operating means therefor
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F16—ENGINEERING ELEMENTS AND UNITS; GENERAL MEASURES FOR PRODUCING AND MAINTAINING EFFECTIVE FUNCTIONING OF MACHINES OR INSTALLATIONS; THERMAL INSULATION IN GENERAL
  • F16K—VALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING
  • F16K99/00—Subject matter not provided for in other groups of this subclass
  • F16K99/0001—Microvalves
  • F16K99/0034—Operating means specially adapted for microvalves
  • F16K99/0042—Electric operating means therefor
  • F16K99/0046—Electric operating means therefor using magnets
• • 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
  • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• • 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
  • B01L2300/04—Closures and closing means
  • B01L2300/041—Connecting closures to device or container
  • B01L2300/044—Connecting closures to device or container pierceable, e.g. films, membranes
• • 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
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0627—Sensor or part of a sensor is integrated
  • B01L2300/0636—Integrated biosensor, microarrays
• • 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
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0672—Integrated piercing tool
• • 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
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0819—Microarrays; Biochips
• • 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
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
• • 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
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/087—Multiple sequential chambers
• • 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
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
  • B01L2400/049—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics vacuum
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
  • G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus

Definitions

• the present invention relates to a kit for conducting miniaturized immunoenzymatic assays comprising at least one microfluidic circuit (2) supported on a card called LOC, and an instrument for the analysis of said LOC, wherein said microfluidic circuit (2) is divided into at least 6 functional regions consisting of: a) Interface region with the instrument; b) Reagent storage region; c) Region of insertion of the sample to be assayed; d) Reaction region; e) Measurement region; f) Discharge region; wherein said regions are in fluidic communication with each other by means of connection channels (9) .
• Immunoenzymatic assays are widely used in research, in clinical practice, in quality control. Their flexibility of use and the consequent wide penetration have stimulated the development of methods whose execution is rapid, as operator- independent as possible, reliable and carried out with the aid of the accessible, cost-effective and space-saving instruments.
• US 8.765.062, US 20140271361, US 7.419.821 describe sample analyzers capable of reading analytes previously loaded onto a cartridge.
• the present invention is intended to provide an alternative to the solutions currently available that offers advantages of use, performance and economy .
• the present invention relates to a kit for conducting miniaturized and automated immunoenzymatic assays for Points of Care (POC) .
• Said kit comprises a card, which is a microfluidic structure, defined as LOC (Lab-On-Chip) and an instrument, preferably portable, for the automatic management of reagents and for the automatic reading of the analytical results of said LOC.
• LOC Lab-On-Chip
• a method for the optimal execution of said analysis process is also an integral part of the present invention.
• the reagents are preloaded in special micro-reservoirs and through channels, they reach functional regions present on the same structure, as described in the following paragraphs.
• the immunoenzymatic assays are performed in an automated manner, using a suitable portable instrument that manages the microfluidics of the reagents and carries out the final measurement.
• Said LOC is inserted pneumatically sealed in said management and measurement instrument with a manifold and, after suitable automatic handling of the fluids, the analytical result is read automatically by absorbance measurement.
• Figure 1 exemplary embodiment of the microfluidic structure according to the present invention, with 4 reaction lines.
• Figure 2 shape of the measurement micro-reservoir (7) of the microfluidic structure according to the present invention.
• Figure 3 schematic representation of the taper of the channels which allows the alignment of fluids in the microfluidic structure according to the present invention.
• Figure 4 standard curve for aflatoxin Bl obtained with the method of the present invention (solid line) and on standard ELISA plates (dashed line) .
• Figure 5 schematic representation of the LOC microfluidic management and reading instrument according to the present invention .
• Figure 6 schematic representation of the first element of the LOC, in an alternative embodiment.
• Figure 7 schematic representation of the second element of the LOC, in an alternative embodiment.
• Figure 8 schematic representation of an embodiment of the closure of the holes present on the LOC according to the present invention, a) closed position; b) open position.
• Figure 9 embodiment of a LOC where the reagent is loaded freeze-dried in a block.
• the kit object of the present invention comprises a LOC (1) and an instrument (20) for analyzing the same LOC.
• said LOC (1) and said instrument (20) are independently claimed herein.
• Said LOC (1) which is a single-use device, depicted in an exemplary embodiment in figure 1, and, in a further embodiment, in figures 6 and 7, comprises at least one microfluidic circuit (2) divided into at least 6 functional regions, respectively:
• holes (3), (13) that put the microfluidic circuit present on said LOC in communication with the management and analysis instrument.
• said holes are associated with absorbing structures in such a way as to prevent the escape of liquids and prevent contamination, and a protective film for closing the holes themselves when the LOC is not used, allowing a preloaded LOC to be provided while preventing the evaporation of liquids, alternatively, said closure is obtained with elements with magnetic core placed inside the holes themselves ;
• Reagent storage region one or more reagent micro- reservoirs (4) adapted to contain the reagents necessary for conducting the assay, for example selected from the group comprising antibodies, conjugated antibodies, enzymatic conjugates, aptamers, chromogenic reagents, fluorescent reagents, chemiluminescent reagents, washing solutions, buffer solutions and acids, suitably preloaded and ready to use, optionally freeze-dried;
• the sample is a fluid of analytical interest such as, for example, wine, milk, water or any physiological liquid selected for example from urine, saliva, blood, plasma, serum or is the result of an extraction process previously performed, such as an extraction from cereals, meat, textiles, dried fruits;
• a fluid of analytical interest such as, for example, wine, milk, water or any physiological liquid selected for example from urine, saliva, blood, plasma, serum or is the result of an extraction process previously performed, such as an extraction from cereals, meat, textiles, dried fruits;
• reaction region one or more reaction chambers (6) containing immobilized probes, for example through physical adsorption or covalent bond to the surface.
• said reaction chamber (6) is coated, for example with metals, such as gold, or films of molecules rich in hydroxyl or amine bonds, such as to bind the various probes, preferably said probes are adhered on the surface of said reaction chambers and are passivated to reduce the background noise;
• Measurement region at least one measurement micro- reservoir (7) to accommodate the liquid to be measured;
• Discharge region at least one discharge structure (8) that receives the waste reagents after the use thereof in the various analytical operations.
• said discharge region is associated with an absorbing pad which prevents the escape of waste reagents, also protecting the instrument from contamination problems.
• connection channels (9) are in fluidic communication with each other by means of connection channels (9) .
• said LOC consists of a single element which comprises the regions described above.
• said LOC comprises a first element (30) and a second element (31), where said first and second element are described in detail hereinafter and the superimposition of said two elements results in the LOC (1) with the regions described above.
• Said LOC comprises regions a) to f) described, where the geometry represented in figures 1, 6 and 7 is to be regarded as purely illustrative and not exhaustive. Therefore, also embodiments whose geometry does not reflect that exemplified in the figures are to be considered part of the present invention.
• the LOCs depicted have 4 analysis lines, embodiments with 1, 2, 3, 5, 6 or more analysis lines are possible and are within the scope of the present invention.
• the arrangement of the various regions in the LOC may be subject to changes that do not affect the functionality thereof.
• said LOC houses 4 analysis lines.
• said LOC proved itself surprisingly adapted to carry out screening assays of 4 different samples or 4 different analytes; moreover, this embodiment is particularly useful for obtaining, in parallel to the test, a calibration curve, where a line is used for the analyte, a second line is used as blank and the remaining 2 accommodate assays of solutions of known concentration. In this way, a calibration curve is obtained for performing a quantitative assay in the field.
• the software subtracts the absorbance value of the blank from the values obtained on the other 3 lines .
• said LOC also comprises structures such as, by way of non-limiting example, frangible seals, frangible gaskets, rubber duckbill valves, flexible rubber valves or magnets that separate said reagent micro-reservoirs (4) from said connection channels (9) .
• frangible seals frangible gaskets
• rubber duckbill valves flexible rubber valves or magnets that separate said reagent micro-reservoirs (4) from said connection channels (9) .
• valve means a device that regulates the flow of fluids in the microcircuit .
• Prior art valves applicable to microfluidic devices find application in the present solution.
• said valves are replaced by a magnetic closing device within the holes of the LOC itself.
• Said magnetic closing device is regulated by an external electromagnet, preferably positioned on the manifold which, by attracting or retracting it, causes the displacement of said magnet and the consequent opening/closing of the conduit occupied by the same.
• said manifold consists of insulating material and, preferably on the surface, bears tracks of conductive material capable of powering the electromagnets of said manifold.
• said LOC consists of two superimposed elements which are joined at the time of the assay, a first element (30) and a second element
• the card contains, in said first element (30), at least one of the functional regions: reagent micro-reservoirs (4), sample micro-reservoirs (5), reaction chambers (6), at least one measurement micro-reservoir (7), at least one discharge structure (8) .
• Said micro-reservoirs and chambers present in said first element (30) comprise, at the ends thereof, holes (33) closed by a protective film (34) .
• Said second element (31) accommodates the connection channels (9) and the interface region with the instrument, i.e. the holes (3) and
• Said second element (31) has some protuberances (35), for example spine-like or needle-like. Said protuberances (35), when said first element is superimposed on said second element, are located at said holes (33) on said first element. Said protuberances (35) pierce said protective film (34), putting said regions (4, 5, 6, 7, 8) on said first element (30) in communication with each other through said communication channels (9) on said second element (31) .
• said microfluidic circuit (2) is thus obtained by the superposition of said first element (30) and said second element (31) .
• the advantage of this solution consists in keeping the different regions isolated, thereby eliminating residual risks of cross-contamination during the storage of the card itself.
• said first and second element have holes closed by magnets placed inside the LOC, in such a way as to maintain a pneumatic seal and allow the storage of liquids.
• the above holes are opened when said first and second element are joined, by starting electromagnets placed externally, preferably on the manifold, such as to move the magnets placed inside the LOC, thereby pneumatically opening or closing the holes.
• said LOC (1) with two elements is depicted in figures 6 and 7, and said first element (30) comprises reagent micro-reservoirs (4), sample micro-reservoirs (5), reaction chambers (6), measurement micro-reservoirs (7), discharge structure (8) .
• Said second element (31) accommodates the connection channels (9) and the interface region with the instrument, i.e. the holes (3) and (13) .
• Said at least one measurement micro-reservoir (7) is transparent and with size dependent on the type of light beam and on the sensitive area for the detection, having to fit completely inside of both. An undersized measurement region with respect to the light beam, in fact, would not allow a correct reading of the signal.
• said at least one measurement micro- reservoir (7) is introduced through said connection channel (9) into said discharge structure (8) in a diametrically opposite point with respect to the point where said hole (13) is positioned in output from said discharge structure (8) .
• said first element (30) comprises all the fluidic elements such as communication channels (9) and the different regions such as the reagent micro-reservoirs (4), the sample micro-reservoirs, the one or more reaction chambers (6), the at least one measurement reservoir (7), the at least one discharge structure (8) and a second element (31) that has the sole function of closure.
• fluidics and regions are aligned with each other on the same plane, there are no interruptions or fittings and they are put in contact with the data reading system (26), through a connection plate, manifold, through said holes (3), (13), also on said first element (30) .
• Said first element (30) further comprises said holes (33) at the ends of said micro-reservoirs and chambers. This makes the LOC simple and cost-effective.
• a LOC according to said embodiment is a ready to use LOC, where at the time of the assay the only operation that the operator has to carry out is the introduction of the sample. The introduction of the sample is done through the hole upstream of the reservoir (5) thereof.
• the closure is obtained with elements with magnetic core inside the holes themselves .
• Magnets (81) are inserted before closing said first element (30) and second element (31) of the LOC, and they remain trapped at the level of said holes (33), (3), (13) .
• An electromagnet (82) approached with a mechanical or electromechanical system from the outside of the LOC to a hole allows moving said magnets (81) along the vertical axis of the hole itself. Alternately reversing the polarity of the electric field (83) attracts the magnet upwards, resulting in the opening of the channel (9)
• connection channels (9) have a square section. Even more preferably, the side of said square has a size equal to 1 mm or less, even more preferably said side measures about 0.5 mm. By virtue of these reduced dimensions, the formation of menisci within the connection channel itself is minimized, thus minimizing the risk of fluid residues within the connection channel (9) .
• said connection channels (9) are made so as to have a hydrophobic surface to eliminate the risk of surface residues or menisci of liquid.
• said reaction chambers (6) have an elongated shape, namely a shape such as to maximize the surface/volume ratio. Said maximization promotes the contact of the molecules contained in the liquid within the reaction chamber with the surface of the chamber itself. Since said probes are immobilized on said surface, the execution of the reaction is thus promoted.
• one or more of the reagent micro-reservoirs (4) is connected with a further micro-reservoir via a mixing channel.
• Said LOC is made of any polymeric material capable of adsorbing the probes with or without appropriate surface modifications ( functionalization) .
• the material must be transparent in the range of wavelength of the selected measure, such as monochromatic light at 450 or 620nm.
• said LOC (1) is preloaded, i.e. probes specific for the assay of interest are previously immobilized in said one or more reaction chambers (6) .
• One or more reaction lines are present on each LOC so as to be able to perform even multiple different assays simultaneously or, where particular accuracy is required, so as to obtain a calibration curve, if standard reference solutions are inserted in one or more of said reaction lines .
• a dosing mechanism of the sample directly on the LOC is provided.
• a dosing mechanism can work starting from an unknown amount or a drop of the sample to be assayed, which is sucked into the LOC by the automatic instrument up to a precise point of the microfluidic channel, so as to be able to quantify the volume thereof, such as 10 or 20 microliters.
• a dosing can take place on the card via capillarity.
• FIG. 2 shows the shape of said at least one measurement reservoir (7) in the LOC.
• Said at least one measurement reservoir (7) has a more or less elongated shape, where the two ends end with a narrowing leading into the inlet and outlet channels.
• connection channels (9) have tapers (10), as depicted in figure 3.
• said taper (10) allows obtaining a realignment of the same reagents since the surface tension of the meniscus of the front of an incoming liquid partially blocks the flow thereof, allowing the second liquid, if late, to realign itself.
• said tapers allow a correct flow of the fluid, at the intersection, in the desired downstream direction due to the surface tension that is created and prevents the liquid from flowing into the undesired channel .
• Said LOC may have one to four reaction lines simultaneously present and in the case of LOC with less than 4 lines, adapters are used on the instrument which ensure the optical alignment and the alignment with the holes in the manifold which are connected to the valves and pumps to be used, present on the reading instrument, thus allowing the use of a single reading instrument per LOC comprising one to 4 reaction lines.
• Said LOC is accommodated within the data reading system (26) by means of a suitable mechanical system that introduces it inside the instrument for the assay.
• said LOC is placed on a tray, also removable, inside the instrument, so as to ensure the alignment between the LOC holes and those of the manifold.
• manifold having an insulating surface that has the functions of:
• optical system consisting of source and photo detector at the reading region, one above and the other below .
• said LOC comprises 4 mutually independent reaction lines. This feature allows immobilizing a different probe in each of said regions, so as to allow the parallel execution of 4 independent assays. In addition, it is possible to obtain a calibration curve, using three of the four reaction lines for the calibration and one for the sample.
• said LOC is preloaded with standard solutions of the target Aflatoxin Bl (or Ml), washing solutions, enzymatic conjugates consisting of specific aflatoxin, conjugated with horseradish peroxidase r chromogenic solutions TMB, stop solution.
• said LOC is preloaded with standard solutions of the target lysozyme, washing solutions, specific secondary antibodies conjugated with horseradish peroxidase r chromogenic solutions TMB, stop solution.
• said LOC comprises:
• the freeze-dried reagents may, in one embodiment shown in figure 9, be fixed into a block (91) which is inserted in said LOC (1) through a hole positioned onto the same;
• chromogenic reagents such as, for example, TMB
• Region of insertion of the sample to be assayed one or more sample micro-reservoirs (5) receiving the liquid sample to be assayed;
• Reaction region 4 reaction chambers (6) containing immobilized probes, such as antibodies, aptamers where said probes are adhered on the surface of said reaction chambers and are passivated to reduce the background noise;
• immobilized probes such as antibodies, aptamers
• Measurement region at least one micro-reservoir (7) with transparent surfaces to allow the colorimetric measurement which accommodates the liquid to be measured;
• Discharge region at least one discharge structure (8) that receives the waste reagents after the use thereof;
• connection channels (9) wherein said regions are in fluidic communication with each other by means of connection channels (9) .
• said block (91) comprises a series of teeth (92) which form a comb-like structure.
• the freeze-dried reagent particles (93) are positioned inside said comb-like structure. This means that, upon use, when the buffer to reconstitute said freeze-dried reagent into solution is added to the LOC, said buffer finds said freeze-dried reagent well distributed. Therefore, said freeze-dried reagent is brought into solution in a gradual manner, thus obviating the problem of a sudden dissolution of the freeze-dried reagent that does not allow a homogeneous distribution of the same in the buffer.
• a further aspect of the present invention is said instrument for the microfluidic management and assay of said LOC.
• Said instrument (20) comprises a housing (21) for said LOC, a system which activates the microfluidic communication between the functional regions that are on said LOC, a data reading system, where said instrument functions are managed, at least partially, in an automated manner.
• said instrument comprises:
• a housing, manifold (21) for said LOC wherein said housing comprises, at holes (3), (13) present on said LOC, a number of seals (22) which put said holes (3), (13) in pneumatic sealed connection, for example through needle-like or other structures, with channels (23) provided in the housing itself, wherein said channels are independent of each other and contained in the portion below the housing area of said LOC;
• a microfluidic communication management system comprising at least one valve (24), such as a solenoid valve, and at least one pump (25) connected in input and/or output to said channels (23);
• e) optionally, an electronic system for stabilizing the temperature of said housing (21), for example at 25 °C;
• g) optionally, sensor for signaling the insertion of the LOC; h) optionally, an optical reading device for the identification of the inserted LOC.
• Said software for the control of the fluidics, through the management of said valves and pumps, acquisition and analysis of data, and/or for the control of the position of liquids along said microfluidic paths and/or for the management of the data obtained from said sensors, is installed on hardware (27) contained in the instrument itself, or one or more of said software is external to the instrument, installed on a PC or other device or, alternatively, on a cloud to which said instrument is connected, for example by the Internet through Wi- Fi connection or connection through a SIM card.
• said instrument comprises said hardware and software and interfaces with the user via a display.
• said software and/or hardware are remote and said instrument comprises a means of connection to said software and/or hardware.
• This embodiment is particularly advantageous as it provides a flexible, cost-effective and easy to use instrument. For example, the same operator in charge of collecting the sample can have said kit on site, load the sample into said LOC and insert said LOC into the specific housing in said instrument. The software will proceed with the analysis, the reading and the interpretation of the data, communicating and saving the same, if necessary, remotely.
• said instrument incorporates both functions, by comprising hardware and software and also means for the remote connection to software and hardware.
• Said holes (3), (13) on said LOC allow the connection of said microfluidic circuit with said instrument the for microfluidic management and the reading of said LOC.
• Said holes (3), (13) by means of gaskets come into contact with the channels (23) contained in said instrument that put the same in communication with the at least one valve and the at least one pump on board of the instrument, where said connection is a pneumatic seal connection that allows the use of said valves and/or pumps for moving the liquids contained in said LOC, typically by suction or thrust.
• said at least one pump (25) is positioned downstream of said LOC and is connected in output to said channels (23) .
• said at least one pump (25) positioned downstream of said LOC is in connection with the hole (13) positioned downstream of said discharge region (8) and said pump (25) is a suction pump.
• a suction upon the switching on of said pump (25) , a suction generates at the hole (13) downstream of said LOC (1) . Opening one or more of said holes (3), each controlled independently by said valves (24), determines whether and from which micro-reservoir the contents has to flow towards the micro-reservoir or chamber located downstream of the same.
• said at least one pump (25) which is a thrust pump, is positioned upstream of said LOC and is connected in input to said channels (23) while said at least one valve (24) is downstream of said LOC in connection with the hole (13) positioned downstream of said discharge region (8) .
• said system comprises at least one pump (25) and at least one valve (24) downstream and at least one pump (25) and at least one valve upstream of said LOC.
• Said valves (24), managed by said software, in a preferred embodiment control the opening and closing of the holes (3) and/or (13) on said LOC.
• each of said channels (23) is controlled individually by a valve (24), wherein each hole (3) is thus connected to a valve (24) .
• each of said channels (23) is controlled individually by a pump (25) to which each of said holes (3) is connected.
• said pump (25) is a thrust pump.
• each of said channels (23) is controlled individually by a pump (25) or a valve (24) to which each of said holes (3) is connected.
• said system comprises a pump (25) which is positioned downstream with respect to said LOC, preferably connected through one of said channels (23) to one of the seals (22) to which the hole (13) on said LOC is connected.
• said pump (25) is a suction pump and its activation allows the movement of fluids in said LOC, where the selection of the fluidic circuit to be activated is possible due to the opening of at least one valve (24), wherein each hole (3) is controlled by a valve (24) through said channel (23) .
• the system further comprises a pump (25) upstream of said LOC.
• Said pump (25) upstream of said LOC by working in thrust, allows a movement of fluids within the LOC, for example, by activating said pump (25) upstream, it is possible to move by thrust a sample within the LOC.
• an upward and downward movement of the fluid is carried out which leads to a mixing of the fluid itself .
• Said optical system consists of: at least one monochromatic, LED or halogen light source, emitting at a wavelength of absorption of the chromogenic reagent, typically at 450 nm or 620 nm and stabilized, or emitting at the wavelength of emission of the fluorophores used for the assay and at least one detector or photodiode, or other measurement system, sensitive to the above wavelengths.
• the light beam is positioned perpendicularly with respect to the detector and maintained at a constant distance, such as to remain centered within the area of the LOC measurement region and at the same time centered on the detector .
• the operator's manual intervention is limited to the loading of the sample to be assayed, thus minimizing the operator-dependent variability, as well as the risk of error.
• Said LOCs are made available empty or, more preferably, preloaded.
• the operator has a number of preloaded assay-specific LOCs so as to select the LOC suitable for the specific assay to be performed.
• the kit according to the present invention is adapted to conduct multiple ELISA protocols, such as ELISA Competitive, Non-competitive, Direct, Indirect.
• Said kit is designed to be used also by non-qualified personnel as the method of execution only contemplates the simple operations listed herein, not necessarily in the order indicated :
• the method according to the present invention involves the use of a LOC already loaded with all reagents, including the specific probes.
• the software manages the protocol according to the LOC introduced.
• said measurement is a measurement exclusively of the colorimetric type. For this reason, it is essential that said measurement region is highly transparent, so as not to interfere with the measurement itself.
• Said step h) of processing and displaying the results is performed locally, where said instrument is designed for this functionality, or remotely through hardware to which said instrument is connected, or via the Internet and a cloud system, through the connection ensured, for example, by the SIM card the previously inserted into the instrument. Said results may be saved on any memory, either locally or remotely.
• Quantitative results can be obtained by executing a calibration curve on the LOC at the same time as the analysis of the sample or by preloading said calibration curve via software.
• a specific protocol is applied for each target to be analyzed.
• the probes, specific antigens or specific antibodies are in the reaction chamber, immobilized in advance on the surface.
• the analytes Prior to proceeding with the assay, the analytes are introduced into dedicated inlets into the LOC.
• Each assay protocol involves the transfer of the reagents from the storage regions to the reaction region and then up to the discharge region along the microfluidics channels.
• the sample is introduced into the storage region by means of a short channel connected with the outside through a hole which is preferably hopper-like to facilitate the insertion of a micropipette therein.
• the calibrated introduction of volumes of the order of 20 microliters is thus possible.
• the reagents remain for a predetermined time. After each reaction, washing solutions are made to transit to eliminate any residues of the reaction regions and stop the reactions themselves.
• the last step in the reaction region consists in the introduction of the chromogenic solution, such as TMB, which is allowed to interact for fixed times and then, moved to the measurement region. During this transfer, the chromogen developed can be mixed with a stop solution, such as acid, to quench the reaction. If the quenching with the stop solution is carried out, it is essential to obtain a perfect mixing of the chromogen therewith.
• tapers are provided as described in the description of the card.
• the color of the chromogen when mixed with the stop solution, changes.
• the final result is made to flow in the measurement region, and then the control unit makes the absorbance measurement proceed.
• the kit and the method according to the present invention find application in the agricultural and food industry, for example for the measurement of aflatoxins in milk and cereals, of antibiotics in milk, and of allergens. Further applications are possible in diagnostics, in humans and in animals .
• concentrations of aflatoxin Bl were measured in the range between 0.01 and 1.2 ng/mL.
• the concentration of lysozyme was analyzed in concentrations in the range between 1.5 and 12 ng/mL.
• specific antibodies may be searched for in multi-line LOCs, for example to search for allergies and/or the immune response after vaccination.
• FIG. 4 shows the standard curve obtained using 4 different known concentrations of aflatoxin Bl with the method of the present invention.
• the dotted line on the same figure 4 shows the data obtained by performing a comparative experiment on traditional ELISA plates on the same solutions (comparison between the techniques) . To confirm the validity of the system proposed herein, the results obtained can be superimposed.

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• 2016
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