EP3487624A1

EP3487624A1 - Portable kit for automated immunoenzymatic assays

Info

Publication number: EP3487624A1
Application number: EP17751483.3A
Authority: EP
Inventors: Niccolò CIPRIANETTI; Laura Boschis; Elena GULI'; Cosimo SPAGNOLO; Erasmo CHESSA
Original assignee: Trustech Srl
Current assignee: Trustech Srl
Priority date: 2016-07-22
Filing date: 2017-07-21
Publication date: 2019-05-29
Also published as: IT201600077085A1; WO2018015931A1

Abstract

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 (Lab-On- Chip), 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).

Description

"Portable kit for automated immunoenzymatic assays"

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) .

Prior art

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.

By way of example, 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 .

Description of the invention 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. A method for the optimal execution of said analysis process is also an integral part of the present invention. In said LOC, 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.

With the kit and the method of the present invention, 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.

Description of the figures

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.

Detailed description of the invention:

The kit object of the present invention comprises a LOC (1) and an instrument (20) for analyzing the same LOC. In a further aspect, 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:

a) Interface region with the instrument: holes (3), (13) that put the microfluidic circuit present on said LOC in communication with the management and analysis instrument. Preferably, 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 ;

b) 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;

c) Region of insertion of the sample to be assayed: one or more sample micro-reservoirs (5) receiving the liquid sample to be assayed. 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;

d) Reaction region: one or more reaction chambers (6) containing immobilized probes, for example through physical adsorption or covalent bond to the surface. In a preferred embodiment, 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;

e) Measurement region: at least one measurement micro- reservoir (7) to accommodate the liquid to be measured;

f) Discharge region: at least one discharge structure (8) that receives the waste reagents after the use thereof in the various analytical operations. Preferably, said discharge region is associated with an absorbing pad which prevents the escape of waste reagents, also protecting the instrument from contamination problems.

Said regions are in fluidic communication with each other by means of connection channels (9) .

In one embodiment, depicted in figure 1, said LOC consists of a single element which comprises the regions described above.

In an alternative embodiment, depicted in figures 6 and 7, 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. For example, 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. Also the arrangement of the various regions in the LOC may be subject to changes that do not affect the functionality thereof.

In a preferred embodiment, said LOC houses 4 analysis lines. In this embodiment, 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.

For measurements with LOC with 4 lines loaded with two standard solutions and the blank, the software subtracts the absorbance value of the blank from the values obtained on the other 3 lines .

If the 4-line LOC is instead used for 4 different assays, the calibration curve is entered to the memory of the software and the value read is a qualitative value, useful where it is necessary to carry out a rapid screening of multiple analytes. In a further embodiment, 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) . When said LOC is inserted in said instrument (20), said structures open, thereby creating suitable pressures-depressions through the activation of the valve and/or pump on board of said instrument (20) .

For the purposes of the present description, the term "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. In a particularly preferred embodiment, 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.

Where magnets are used, said manifold consists of insulating material and, preferably on the surface, bears tracks of conductive material capable of powering the electromagnets of said manifold.

In the embodiment schematically shown in figures 6 and 7, said LOC consists of two superimposed elements which are joined at the time of the assay, a first element (30) and a second element

(31) . In this embodiment, 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

(13) and the functional regions possibly not present in the first element (30) . 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) . In this embodiment, 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. In a preferred embodiment, 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.

In a preferred embodiment, 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.

In a preferred embodiment, 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) .

In one embodiment, 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. In this embodiment, 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.

The reagents are loaded after the closure of the two elements, a closure which is obtained according to a method known in the art, such as with an adhesive or by heat sealing. Through said holes (33) upstream of said reservoirs, it is possible to introduce said reagents, said holes (33) downstream of said reservoirs serve as a vent for air. 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.

In order to prevent losses of reagents, when liquid, a closing system of said holes (33), (3) and (13) is provided.

In one embodiment, schematically shown in figure 8, 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)

(panel b) , or it is rejected, thereby resulting in the closing thereof (panel a) . The hole (33), (3) or (13) and the magnet

(81) have a size such that the magnet (81) cannot completely return within said hole and consequently, while partially narrowing the channel (9), the pneumatic vacuum condition is maintained. In one embodiment, said magnet is spherical in shape and finds application in semi-circular section channels. In a further embodiment, the magnet is cylindrical in shape and finds application in square section channels. Preferably, said 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) . Optionally, said connection channels (9) are made so as to have a hydrophobic surface to eliminate the risk of surface residues or menisci of liquid.

Preferably, 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. In an even more preferred embodiment, 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.

Preferably, 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 .

If the sample is derived from a preparative process, such as from a pressure extraction process, said process will be carried out by a separate accessory device. In a further embodiment, a dosing mechanism of the sample directly on the LOC is provided. Such a 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. Alternatively, such a dosing can take place on the card via capillarity.

A further peculiarity that distinguishes said LOC from other solutions present in the prior art is given by the conformation of said at least one measurement reservoir (7) . Figure 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. The authors of the present invention have shown that, by virtue of said conformation, a series of advantages are obtained: i) the formation of air bubbles which constitute an important limitation of the prior art microfluidic solutions, is prevented; ii) the absence of pronounced corners allows an optimum flow of the reagents; iii) the absence of pronounced corners, by avoiding stagnation areas, facilitates the washing of the surfaces. The solution of the present invention also contemplates that the connection channels (9) have tapers (10), as depicted in figure 3. When a uniform mixing of the two reagents is required, 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. Moreover, 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. The LOC management carried out by a plurality of valves, allowing a fine and punctual control of the circuit, gives the kit of the present invention a flexibility not found in other systems described in the prior art.

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. By means of a mechanical interlocking, 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. Between the instrument and the LOC there is an interface called manifold having an insulating surface that has the functions of:

putting the LOC in pneumatic contact with pumps and valves; supporting the electromagnets, where present;

supporting the contact tracks of the magnet poles;

bearing the optical system consisting of source and photo detector at the reading region, one above and the other below .

In the embodiment exemplified in figure 1, 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.

In a preferred embodiment, 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.

In a further preferred embodiment, 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.

In a further embodiment, said LOC comprises:

a) Interface region with the instrument: holes (3), (13) that put the microfluidic circuit (2) present on said LOC in communication with a management and analysis instrument; b) Reagent storage region that are freeze-dried reagents: one or more reagent micro-reservoirs (4) adapted to contain the reagents necessary for conducting the assay, for example selected from the group comprising:

Reservoir for secondary antibodies, conjugated antibodies, enzymatic conjugates, aptamers according to the analytical protocol for the selected sample. 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;

- Reservoir for chromogenic reagents such as, for example, TMB;

- Reservoir for washing solutions;

- Reservoir for STOP solution, which is preferably an acid;

c) Region of insertion of the sample to be assayed: one or more sample micro-reservoirs (5) receiving the liquid sample to be assayed;

d) 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;

e) Measurement region: at least one micro-reservoir (7) with transparent surfaces to allow the colorimetric measurement which accommodates the liquid to be measured;

f) Discharge region: at least one discharge structure (8) that receives the waste reagents after the use thereof;

wherein said regions are in fluidic communication with each other by means of connection channels (9) .

The advantage resulting from the use of the block in which the freeze-dried reagent is contained is given by the fact that 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.

Preferably, said instrument comprises:

a) 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;

b) 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);

c) A data reading system (26) that is an optical system;

d) hardware and software (27) for controlling the fluidics, management, analysis of the readings and data communication, independently locally and/or remotely;

e) optionally, an electronic system for stabilizing the temperature of said housing (21), for example at 25 °C;

f) optionally, optical sensors for the position control of liquids ;

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.

In one embodiment, said instrument comprises said hardware and software and interfaces with the user via a display.

In an alternative embodiment, 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.

In a further embodiment, 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.

In one embodiment, said at least one pump (25) is positioned downstream of said LOC and is connected in output to said channels (23) . Preferably, 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.

In this embodiment, 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. By way of example, after actuating said pump (25) , opening the valve (24) placed at the hole (3) which is upstream of said sample micro-reservoir (5), the air entering through said hole (3) opened by said valve (24), by the action of the pump (25), makes the fluid contained in said sample micro-reservoir (5) enter in said connection channel (9) to reach said reaction chamber (3) .

In a further embodiment, 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) . Alternatively, 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.

In one embodiment, each of said channels (23) is controlled individually by a valve (24), wherein each hole (3) is thus connected to a valve (24) . In an alternative embodiment, each of said channels (23) is controlled individually by a pump (25) to which each of said holes (3) is connected. In this embodiment, said pump (25) is a thrust pump. In a further embodiment 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.

In a preferred embodiment, 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. In this embodiment, schematically shown in figure 5, 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) .

In a further embodiment, 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. Or, alternately using said suction pump downstream and said suction pump and upstream of 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 .

With the LOC of the present invention, 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 :

a) Inserting the LOC in the microfluidics management and reading instrument, where said instrument is controlled by software, locally or remotely;

b) Loading the sample in the LOC (1), in said sample micro- reservoir (5), for example by pipette, or by capillary action or suction assisted by the instrument;

c) Starting the desired assay protocol via software, with the automatic recognition of the card by the instrument with the code reading system, where present, or through manual control ;

d) Automated transfer of the sample and reagents, preferably mediated by a suction pump connected to said hole (13), in one or more reaction chambers (6) where, optionally, said sample and reagents come into contact with one or more probes of interest immobilized therein, thus generating the chromogenic signal to be measured at the end of the protocol ;

e) Automatically managing the reaction steps alternated with washing steps subsequent to the transfer of said sample and reagents in the discharge region (8);

f) Optionally introducing a solution called STOP, mixing and transferring the same, where present, together with said chromogenic substrate in the measurement region, where said transfer is obtained by suction, preferably mediated by a suction pump connected to said hole (13);

g) Measuring the absorbance/emission of the chromogenic signal via software through the microfluidic management and measurement instrument;

h) Processing the measured data by means of the software and displaying the results.

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.

According to the present invention, 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. Depending on whether a direct or indirect enzymatic immunoassay must be carried out, the probes, specific antigens or specific antibodies, suitably stabilized with blocking and overcoating operations, are in the reaction chamber, immobilized in advance on the surface. 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. In a preferred embodiment, 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. In the reaction region, 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. In order to ensure a perfect alignment of the fluids in the two converging branches of the fluidics, 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.

By way of example, 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 .

EXAMPLES :

Assay of cereal samples

In samples of cereals, with a competitive immunoenzymatic protocol, concentrations of aflatoxin Bl were measured in the range between 0.01 and 1.2 ng/mL.

Assay of wine samples

In wine samples, with a non-competitive immunoenzymatic assay, the concentration of lysozyme was analyzed in concentrations in the range between 1.5 and 12 ng/mL.

To this end, LOC preloaded with standard target solutions, washing solutions, specific secondary antibodies conjugated to horseradish peroxidase HRP (for non-competitive ELISA protocols) or specific antigens conjugated to horseradish peroxidase HRP (for competitive ELISA protocols), chromogenic solutions TMB, stop solution, were made available.

Analysis of IgE in the blood

In blood samples, specific antibodies may be searched for in multi-line LOCs, for example to search for allergies and/or the immune response after vaccination.

Assay of milk samples

In milk samples, aflatoxin Bl was measured up to concentrations of the order of 10 pg/mL.

By way of example, the method for the analysis of aflatoxin Bl is described hereinafter, wherein said method comprises:

a) Providing a 4-line LOC and inserting the same into the instrument ;

b) Loading the sample to be assayed;

c) Starting the protocol by the instrument;

d) Sequentially mixing the solutions at a known concentration of aflatoxin Bl (for the calibration curve), and the sample to be assayed, with enzymatic conjugate solutions (consisting of aflatoxin Bl chemically conjugated to the enzyme HRP) within the LOC and sequentially transferring such solutions in the reaction chambers (6) by switching on and off said at least one pump (25) and said at least one valve (24 ) ;

e) Incubating for about 15 minutes, and subsequently transferring said solutions towards the discharge region; f) Sequentially transferring the washing solutions in the reaction chamber and subsequently transferring the same towards the discharge region;

g) Sequentially transferring the reagents TMB in the 4 reaction chambers where they remain for about 5 minutes; h) Sequentially transferring the STOP solutions up to the tapering of the circuit simultaneously with the sequential transfer of the colored reagents TMB in such a way that they can be aligned for a proper mixing;

i) Sequentially transferring each reagent TMB, developed, simultaneously with the STOP solution thereof to the measurement region, an operation whereby the two solutions undergo a mixing and then the quenching of the chemical development reaction of the TMB;

) Sequentially measuring the absorbance of the solutions and transferring to the discharge region.

The quantitative assay response of this sample is obtainable in less than 30 minutes. Figure 4, solid line, 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.

Claims

A kit for conducting miniaturized immunoenzymatic assays comprising at least one LOC (1) and an instrument (20) for the analysis of said LOC, wherein said LOC comprises at least six 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 communication channels (9) and said instrument (20) comprises a housing (21) for said LOC, a system that activates the microfluidic communication between said functional regions that are on said LOC and a data reading system.

A microfluidic structure (LOC) for conducting miniaturized immunoenzymatic assays comprising at least one microfluidic circuit (2) divided into at least 6 functional regions, respectively :

a) Interface region with the instrument: holes (3), (13) that put the microfluidic circuit

(2) present on said LOC in communication with a management and analysis instrument ;

b) 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, chromogenic reagents, fluorescent reagents, chemiluminescent reagents, aptamers, washing liquids, buffer solutions and acids, optionally freeze- dried;

d) Reaction region: one or more reaction chambers (6) containing immobilized probes;

e ) Measurement region: at least one measurement micro- reservoir (7) to accommodate the liquid to be measured; f) Discharge region: at least one discharge structure (8) that receives the waste reagents after the use thereof; wherein said regions are in fluidic communication with each other by means of connection channels (9) .

3. A LOC according to claim 2, wherein said LOC consists of a first element (30) and a second element (31) which can be mutually superimposed, wherein said first element (30) comprises at least one of said functional regions in which the micro-reservoirs and the chambers comprises holes (33) at the ends, closed with a protective film (34) and said second element (31) accommodates the connection channels (9) and the interface region with the instrument, i.e. the holes (3) and (13) and the functional regions possibly not present in the first element (30) .

4. A LOC according to claim 3, wherein said second element (31) has protuberances (35) adapted to pierce said protective film (34) .

5. A LOC according to claim 3, wherein 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), said holes (33) at the ends of said micro-reservoirs and chambers, and said second element (31) has the sole function of closure.

6 . A LOC according to claims 2, 3 or 5, wherein said holes (33) are closed by elements (81) with magnetic core placed within said LOC and moved by at least one electromagnet (82) .

7. A LOC according to one of claims 2 to 6, wherein said at least one measurement micro-reservoir (7) is transparent and has a size such that the light beam used for reading crosses it entirely.

8. A LOC according to one of claims 2 to 7, wherein said reaction micro-reservoir (7) has a more or less elongated shape, where the two ends end with a narrowing, leading into the inlet and outlet channels and/or in said holes (33) .

9. A LOC according to one of claims 2 to 8, wherein said connection channels (9) have at least one tapering (10) .

10. A LOC according to one of claims 2 to 9, 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.

11. A LOC according to one of claims 2 to 10, characterized in that 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) and said connection channels (9) have a square section and the side of said square measures about 0.5 mm and said reaction chambers (6) are elongated in shape.

12. A LOC according to one of claims 2 to 11, wherein one or more of said reagents are preloaded in the form of freeze- dried particles (93) loaded onto a block (91) inserted into said LOC, said block (91) comprising a number of teeth (92) so as to form a comb structure in which said freeze-dried particles (93) are inserted.

13. A LOC according to one of claims 6 to 12, wherein said elements (81) have a spherical shape and said channel (9) has a semicircular section.

1 . A LOC according to one of claims 6 to 12, wherein said elements (81) have a cylindrical shape and said channel (9) has a square section.

15. A LOC according to one of claims 2 to 14, comprising structures adapted to separate said reagent micro- reservoirs (4) from said connection channels (9), wherein said structures may be in open position, thus allowing the passage of fluid, or in closed position, thus preventing the passage of fluid.

16. A LOC according to claim 15, wherein said structures adapted to separate are valves, wherein said valves are activated by activating the valve and/or pump on board of the instrument (20) in which said LOC is inserted.

17. A LOC according to claim 15, wherein said structures adapted to separate are magnetic closing devices, regulated by an external electromagnet.

18. A LOC according to one of claims 2 to 17, wherein said probes are adhered on the surface of said reaction chambers and are passivated.

19. An instrument (20) for the microfluidic management and the assay of the LOC according to claims 2 to 17, wherein said instrument comprises:

a) A housing (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 with channels (23), wherein said channels are independent of each other and contained in the portion below the housing area of said LOC;

b) A microfluidic communication management system comprising at least one valve (24), such as a solenoid valve, and at least one pump (25) connected at least one in input and one in output to said channels (23);

c) A data reading system that is an optical system (26); hardware and software (27) for controlling the fluidics, management, analysis of the readings and data communication, independently locally and/or remotely and optionally, means for connecting said instrument with said hardware and software remotely.

20. An instrument according to claim 18, wherein said pump (25) is a suction pump connected downstream of said LOC and connected in output to said channels (23), through said hole (13) located downstream of said discharge region (8) and each of said channels (23) that open onto holes (3) upstream of said micro-reservoirs on said LOC is individually controlled by a valve (24), such as a solenoid valve .

21. An instrument according to one of claims 18 or 19, further comprising optical sensors for the position control of liquids and/or sensor for signaling the insertion of the LOC and/or an optical reading device for identifying the LOC inserted and/or a system for stabilizing the temperature of the housing (21) .

22. A kitaccording to claim 1, wherein said LOC (1) is according to one of claims 2 to 17 and said instrument (20) is according to one of claims 18 to 20.

23. A method for conducting miniaturized and automated immunoenzymatic assays comprising the following steps, not necessarily in this order:

a) Providing a kit according to claim 1 or 21;

b) Inserting said LOC (1) in the housing area (21) on said instrument (20);

c) Loading the sample into the LOC (1), in said sample micro-reservoir (5); d) Starting the desired assay protocol;

e ) Automatically transferring the sample and the reagents in one or more reaction chambers (6);

f) Automatically managing the reaction steps alternated with washing steps subsequent to the transfer of said sample and reagents in the discharge region (8);

g) Transferring an optional solution called STOP, mixing and transferring the same, where present, along with said chromogenic substrate to the measurement region;

h) Measuring the absorbance/emission of the chromogenic/chemiluminescent signal via software through the microfluidic management and measurement instrument; i) Processing the measured data by means of the software, displaying and optionally saving the results.

2 . A method according to claim 22, wherein said immunoenzymatic assay is an ELISA assay and said LOC is a 4-line LOC, wherein one of said lines is preloaded with a control reagent, two of said lines are preloaded with solutions at a known concentration of the analyte, the fourth line is available for loading the test sample by the operator .

25. A method according to claim 22, wherein said LOC is a 4- line LOC, wherein each of said lines is preloaded with a different control reagent for the assay of a panel of 4 different targets in a same test sample.

26. A method according to claim 22, wherein said LOC is a 4- line LOC, wherein each of said lines is preloaded with the same control reagent for the assay of a same target on four different test samples.