WO2016062788A1

WO2016062788A1 - Microfluidic chip for biological analysis

Info

Publication number: WO2016062788A1
Application number: PCT/EP2015/074426
Authority: WO
Inventors: Helene ZIRATH; Johannes PEHAM; Heinz Redl; Michael Vellekoop; Lukas BRANDHOFF
Original assignee: Ait Austrian Institute Of Technology Gmbh
Priority date: 2014-10-24
Filing date: 2015-10-22
Publication date: 2016-04-28

Abstract

The present invention provides a microfluidic chip for biological analysis comprising a cylinder configured to accommodate a piston, whereby the cylinder acts as integrated reservoir for a reaction liquid when the piston is partially inserted. The partially inserted piston is further accessible from outside of the chip. By actuating said partially inserted piston, the liquid is pumped from the reservoir into other parts of the chip, for said biological analysis. In particular, the inventive chip is for performing a photometry-based immunoassay such as an ELISA. In further aspects of the invention the following is provided: a set comprising chip and piston, an apparatus for processing the chip, and methods for using the chip and the apparatus.

Description

Microfluidic chip for biological analysis

The present invention relates to the field of microfluidic chips for biological analysis, especially for point-of-care testing (POCT) .

Biological analysis methods such as immunoassays are standard methods in biochemistry for detecting and quantifying analytes (e.g. biomarkers) in solutions. They are also used for diagnosis of patients at hospitals and clinics by analysing human body fluid. A commonly used immunoassay for detection of biomarkers for diagnosis is the Enzyme-Linked Immunosorbent Assay (ELISA) .

ELISAs are commonly performed in microtiterplates , where the analytes are quantified by measuring fluorescence, chemilumines- cence, or colorimetric reactions. Independent of the quantifica^¬ tion method used, the conventional ELISA has several incubation and washing steps, is time demanding, and requires trained per^¬ sonnel as well as large and expensive laboratory instruments. The same applies to many other biological analysis methods.

It is beneficial to bring such biological analysis methods into a format known as POCT (evidently, biological analysis methods also comprise medical analysis methods and in-vitro di^¬ agnostics in medicine) . POCT, also known as bed-side testing, is defined as medical testing at or near the site of patient care, often conducted directly by the physician or caretaker. POCT offers many advantages, chiefly among them a simpler work flow. POCT increases the likelihood that the physician or caretaker will receive the results faster, which allows for immediate clinical management decisions to be made.

POCT devices or methods are known e.g. from US 6,267,722 Bl, WO 2004/097419 Al, WO 2009/137645 Al, US 8,703,439 Bl, CN 103399161 A and WO 2012/021239 A2.

Warsinke (Analytical and Bioanalytical Chemistry 393.5 (2009) : 1393-1405) gives an overview about POCT of proteins. Ac^¬ cording to the document, although many different POCT technolo^¬ gies have been developed, no single technique or system is a clear leader.

Overall, POCT can decrease occupancy rates of hospital beds and other facilitates, because accurate treatment decisions can be made faster. In conclusion, it is an object of the present invention to provide a device that allows for faster, more convenient analy^¬ sis of biological samples such as blood samples from a patient, especially for POC .

In the course of the present invention it turned out that a microfluidic chip adapted to comprise a specialised cylinder- piston arrangement that allows sample storage in the chip is ex^¬ ceptionally suitable to fulfil said object.

Therefore, the present invention provides a microfluidic chip for biological analysis (1),

comprising an inlet (2) for a liquid (7), a channel (3), and a cylinder ( 4 ) ,

the cylinder (4) being connected to the inlet (2) and the channel (3) and having a cross-section larger than the cross- section of the channel (3) ;

characterised in that:

-the cylinder (4) is configured to accommodate at least a first part of a piston (5) , and

-the cylinder (4) is configured such that the piston (5) has an axis of movement inside the cylinder (4) essentially parallel to the plane of the chip, and

-the chip is configured such that at least a second part of the piston (5) is accessible from outside of the chip, and

-the inlet (2) is closable by the piston (5), whereby the piston also acts as a valve, and

-the chip is configured such that the cylinder (4) and the piston (5) define a reservoir (6) for the liquid (7) .

The term "microfluidic" in the context of "microfluidic chip" means that the chip is configured to be used with small volumes of liquid, e.g. in the micro-liter-, nano-liter- or femto-liter- range . Typically, the microfluidic chip is made from a single substrate (e.g. plastics or metal, preferably plastics for re^¬ duced cost), with channels etc. executed as bores, grooves (or recesses or depressions) or notches in the substrate. Typically, it is close to the shape of a flat cuboid (the flatness defining the main plane of the chip or simply "the plane of the chip") with two sides of larger area four narrow sides (of smaller ar- ea) . The term "chip" refers to the characteristic typical flat appearance of the microfluidic chip. However, the inventive chip can also contain electronics, e.g. to integrate parts that would typically be contained in the apparatus of the invention, such as a detector.

The cylinder forms an integral part of the chip, e.g. it is molded, milled, embossed or etched into the chip. The term cyl^¬ inder is used herein as a functional term, meaning that it pro^¬ vides the counterpart to the piston, to be used in a cylinder- piston arrangement. The term cylinder hence shall not be con^¬ strued as to bear a certain geometrical limitation. For instance, it can be a cylinder in strict geometrical terms, but it can also be a cuboid or have any other profile as long as it can function as a cylinder in a cylinder-piston arrangement. The same applies, mutatis mutandis, also for the piston: It is used herein as a functional term and can be executed e.g. in cylin^¬ drical, half-cyndrical or cuboid form. Preferably, the piston is made of metal or plastics.

Beneficially, the cross-section of the channel is very small (e.g. less than 1mm in diameter, or less than 1mm in width and less 1mm in height) to reduce the amount of dead volume. By con^¬ trast, the cylinder has a cross-section that is larger than the cross-section of the channel because it can act as a reservoir for the liquid. The term "cross-section" refers to the mean cross-section as calculated by averaging all cross-sections along the longitudinal axis of the cylinder or the channel, re^¬ spectively. Typically, the cross-section of cylinder and/or channel remains constant along their respective longitudinal ax^¬ is, evidently whereby any cross-section of cylinder or channel, respectively, is equal to the mean cross-section of cylinder or channel, respectively.

The cylinder's cross-section can also have a diameter that varies along the cylinder's longitudinal axis. This is for in^¬ stance possible if the piston is executed as a plunger that con^¬ sists of a larger-diameter part that fits snugly into the large- diameter part of the cylinder (as to avoid leakage) and another part that has a smaller diameter that fits snugly into the small-diameter part of the cylinder (as to be stabilised in its axis of movement) . Care must be taken, however, that the plunger can still act as a valve in respect to the inlet (e.g. by providing a larger-diameter part of the piston that is sufficiently long in respect to the large-diameter part of the cylin^¬ der) , in order for the chip to fulfil the inventive reservoir function .

It is necessary that the cylinder-piston arrangement can be actuated from outside of the chip. Preferably, therefore, the cylinder ends into a narrow side of the chip (cf. Fig IB or 1C) or ends into a groove (cf. Fig. 2C) or, alternatively, the cyl^¬ inder comprises an opening such as a slot to make the piston ac^¬ cessible by a second part of the piston from outside of the chip when at least a first part of the piston is accommodated in the cylinder. Preferably, the chip is configured in a way such that the piston cannot protrude from any side of the chip when par^¬ tially accommodated in the cylinder. This simplifies packaging of the chip.

The terms "first part of the piston" (the part of the piston accomodated in the cylinder) and "second part of the piston" are defined by the relative position of the piston in the cylinder. As such, they hence do not refer to structurally constant parts of the piston (as the piston is movable) but are solely func^¬ tional terms to simplify the present disclosure.

Typically, the cylinder and/or the channel are executed to be open along their longitudinal axis towards a side of the chip (typically not a narrow side) -e.g. as notches or grooves in the chip (the cylinder can be for instance a geometric half- cylinder, cf . Fig. 7) . Said side is, by definition, the "front side" of the chip. The chip can then be covered (e.g. by lamina^¬ tion) by a, preferably transparent, film or foil for sealing such that the liquid does not leak from the front of the chip. In this instance, the inlets are typically executed as notches or grooves in the chip, to be covered by the film or foil which is to be punctured by a needle (e.g. of a syringe) for filling. This embodiment of the cylinder is called "half-cylinder" here^¬ in, regardless of its exact geometry.

However, the chip can also be configured such that covering with a film or foil is not necessary (except possibly for the inlets) . In this instance, channels inlets and cylinders are ex^¬ ecuted as bores in the chip. Also, mixed forms are possible, such that some inlets, channels and/or cylinders are executed as bores in the chip and some as notches or grooves in the chip (in Figs. IB and 6, the cylinders are executed as a bore whereas the channels are executed as notches) . This embodiment of the cylin^¬ der is called "full cylinder" herein, regardless of its exact geometry. To use an inlet (or outlet) that is covered by the film or foil, it can be punctured by a hollow needle or similar (e.g. the inlet can be injected into with liquid by a syringe with a hollow needle attached) .

In an exceptionally preferred embodiment of the present in^¬ vention, the cylinder is executed as a bore in the chip (i.e. a "full cylinder") , as this is beneficial for sealing by the pis^¬ ton. Preferably, the channel is executed as a notch or groove in the chip.

Typically, the inventive chip is a cartridge (for one-time use) to be read out (or processed) in an apparatus which is also disclosed herein.

Beneficially, the inventive chip can be stored packaged, with filled reservoirs for longer period of time (e.g. 6-12 months), e.g. in a cold room and can be unpacked just before use.

The inventive reservoir function is typically achieved by (1) the piston acting as a valve in respect to the inlet (e.g. where the film or foil above the inlet has been punctured by a needle to fill the reservoir with the liquid; cf. Fig 3) and (2) the liquid being held in place in the reservoir. Since the reservoir is sealed on one side by the piston acting as a valve in respect to the inlet, the liquid is captured in the cylinder (capillary forces cannot drive the liquid into the channel) .

Preferably, said chip is "ready-to-use" such that, if at all, only the biological sample is to be inserted manually into the chip (bulk reagents such as washing buffers are preferably in^¬ jected by the apparatus for processing the chip (e.g. through inlet 28), when the chip is already inserted into the appa^¬ ratus) . Beneficially, said apparatus is a POCT apparatus. In a high-workload working environment such as a hospital, even cut^¬ ting down one manual handling step usually to be performed for each patient, possibly each day, (e.g. injecting the detection or capturing antibody into the chip) can have significant bene^¬ ficial economic impact. Microfluidic chips for biological analysis, in general, are known in the art:

The US No. 5,955,028 A describes a microfluidic system com^¬ prising sample wells, microfluidic channels and reactions cham^¬ bers which may be used for optical detection.

The WO 2005/070533 Al describes a microfluidic device for an^¬ alysing component (s) in a fluid, comprising introducing the fluid into the microfluidic device, moving the fluid to a reaction chamber, reacting the fluid with the moiety that binds; moving a washing fluid to the reaction chamber and washing the reaction chamber; illuminating the reaction chamber by a light source emitting radiation; and detecting the radiation emitted.

The US 2004/0018611 Al describes a microfluidic device com^¬ prising a microchannel .

The WO 2013/090106 Al describes a microfluidic device com^¬ prising optical detection means and a holder to perform fluorescence detection of analytes using a detection chamber wherein a binding partner for the analyte is immobilised.

The US 2008/160630 Al describes a microfluidic device com^¬ prising a fluidic network and an integrated circuitry component. The fluidic network comprises a sample zone, a cleaning zone and a detection zone. The fluidic network contains a magnetic parti^¬ cle and/or a signal particle. A sample containing an analyte is introduced, and the analyte interacts with the magnetic particle and/or the signal particle through affinity agents. A microcoil array or a mechanically movable permanent magnet is functionally coupled to the fluidic network, which are activatable to gener^¬ ate a magnetic field within a portion of the fluidic network, and move the magnetic particle from the sample zone to the de^¬ tection zone. A detection element is present which detects opti^¬ cal or electrical signals from the signal particle indicating the presence of the analyte.

The WO 2011/151250 Al describes a microfluidic photolumines- cence detection system comprising a sample inlet, a detection chamber and a mixing chamber.

The WO 2011/156836 Al describes a microfluidic device for spotting oligonucleotide probes.

The US 2001/0038450 Al describes a hand-held assay device measuring chemiluminescence .

The WO 2003/076937 A2 describes an apparatus for conducting a variety of assays for the determination of analytes in liquid samples .

The US 2012/0322683 Al describes a device comprising a fluid- ic network and an integrated circuitry component.

The WO 2012/019109 Al describes an apparatus and methods for the rapid determination of analytes in liquid samples by immuno^¬ assays incorporating magnetic capture of beads on a sensor capa^¬ ble of being used in the point-of-care diagnostic field.

The WO 2000/050172 Al describes a microfluidic device, com^¬ prising a body structure having a microscale cavity disposed therein and an ordered array of a plurality of sets of particles disposed within the microscale cavity.

The WO 2010/080115 A2 relates to reagent storage in microflu^¬ idic systems and related articles and methods.

However, all of these documents fail to disclose or suggest the inventive cylinder-piston arrangement.

A few documents in the prior art disclose a piston or similar element to be used in combination with a microfluidic chip or device :

The US 2003/0057391 Al discloses a microfluidic system having at least one microchannel , the improvement comprising: a pres^¬ sure generation device, having a fluid driven member (i.e. similar to a piston) , and operatively connected to said at least one microchannel, said device having functions selected from the groups consisting of pumping and valving.

Yet there are essential differences between the teaching of said document (and the prior art in general) and the present in^¬ vention: The fluid driven members of the document are not acces^¬ sible for actuation from the outside. Furthermore, the document is silent on the pistons functioning as valves in regard to the inlet. The nature of actuation in the document makes holding of the piston in a "centre position" - such that the inlet is closed but the cylinder still forms a reservoir - impossible without energy input. Long-term storage of liquids in the system is mentioned (paragraph [0036] of the document) yet the reser^¬ voir of the document appears to be screwed onto or plugged into the respective inlet. The document is silent in respect to the exact nature of the reservoir (cf. paragraph [0025] of the docu^¬ ment) .

Consequently, the present invention offers at least the fol^¬ lowing advantages over said document:

The filling of the reservoir is simpler (less parts, no plug^¬ ging/screwing necessary) . The inventive chip (being adapted to comprise a reservoir) is essentially plane (not easily achieva^¬ ble with an external reservoir attached instead) and hence e.g. very suitable for e.g. packaging and handling. The inventive cylinder-piston arrangement leads to very little dead volume, which is advantageous, especially with expensive reagents such as antibodies.

The WO 2012/041479 Al discloses a parallel arrangement of cylinders/pistons for microfluidics . The cylinders are cast into the substrate (i.e. the chip) and the pistons can be actuated in parallel. The liquids are inserted by a common inlet (reference sign 14 in Fig. 1 of the document), such that the apparatus acts similar to a multi-pipette. By pulling the pistons (reference sign 22, Fig. 1 of the document) out of the cylinders by means of a common actuating element (reference sign 30 in Fig. 1 of the document) , the liquid is sucked into parallel channels, thereby achieving parallel filling of the reaction chambers (18 in Fig. 2 of the document) . The mechanism differs markedly from the present invention (e.g. no independent inlets, no valve function for the piston, pulling instead of pushing for actuation) . The teaching of the document does also not suggest the inventive reservoir.

The WO 2008/036045 Al describes a pre-filled reservoir cov^¬ ered by a stretchable foil, in a microfluidic chip. This reser^¬ voir can be opened by a pin valve (cf. Figs. 2A-2D of the docu^¬ ment) . The mechanism differs greatly from the present invention, moreover, the dead volume is higher as well (cf. Fig. 2D of the document) .

In the US 2012/0234393 Al a piston (reference sign 54 in Figs. 13 and 14 of the document) is used to push on an air- filled chamber (reference sign 55 of the document) , in order to cause liquid flow in the chip. The WO 2005/002729 Al and the US 2006/0159564 Al each dis^¬ close injection mechanisms to be attached onto the chip (cf. ab^¬ stract figure of WO 2005/002729 Al and paragraph 4 of claim 1 in US 2006/0159564 Al) . The US 2010/0291588 Al relates to systems and methods including self-contained cartridges with detection systems and fluid delivery systems. Syringes are disclosed (e.g. paragraph [0217] and Fig. 26A of the document) . These mechanisms are very similar to conventional syringes - the cylinder is not part of the chip.

The DE 103 11 731 Al relates to an apparatus for conducting tests on a liquid. Pistons that can be drawn from cylinders for inducing liquid flow in the apparatus are disclosed (e.g. para^¬ graph [0006], Figs. 1 and 2 of the document). The US 4,585,623 relates to a device for performing quantitative chemical and im^¬ munochemical assays. Pistons are disclosed (e.g. reference sign 43, Fig. 1 of the document) . However, neither of these documents discloses or suggests the present invention, in particular not the feature directed to the inlet being closable by the piston, whereby the piston also acts as a valve, or the feature directed to the chip being configured such that the cylinder and the pis^¬ ton define a reservoir for the liquid.

The US 6,645,758 Bl relates to a containment cuvette for PCR and a method of use. A piston (reference sign 113D of the docu^¬ ment) within the cuvette is disclosed. Yet the document does not disclose or suggest the present invention, in particular not the feature wherein a part of the piston is accessible from outside of the chip, the feature directed to the inlet being closable by the piston, whereby the piston also acts as a valve, or the fea^¬ ture directed to the chip being configured such that the cylin^¬ der and the piston define a reservoir for the liquid.

As a further improvement over the prior art the inventive chip can be configured such that only single-use is possible. This is preferably achieved by the piston being non-retractable from the cylinder once it has been fully inserted, e.g. by being not accessible for actuation from outside of the chip any more once fully inserted or by the cylinder comprising irreversible locking means for the piston at the fully-inserted position. Providing the chip such that single-use is mandatory can enforce a certain quality standard to be followed (especially in envi- ronments with unsophisticated healthcare facilities where strong economic pressures exist to save costs even at a detriment to the patient) . Thereby, false-negative or false-positive results can be avoided that may prove detrimental to the patient's health. This is not possible by using microfluidic chips of the art in which the cylinder-piston arrangement (e.g. a conventional syringe) is often external to the chip.

Another improvement over the prior art is that the inventive cylinder-piston arrangement (as it is effectively a "micro- syringe") can be used for fine control of the injected volume of the liquid. If the specific biological analysis protocol re^¬ quires, instead of the entire volume of liquid in the reservoir, only a certain portion of said liquid (e.g. O.l-ΐμΐ) can be in^¬ jected by the cylinder-piston arrangement into the rest of the chip by moving the piston appropriately. Thereby, the liquid in the reservoir can also be used in multiple increments (e.g. lOx Ιμΐ) instead of all at once (e.g. lx ΙΟμΙ) .

Taken together, the prior art does not disclose or suggest the inventive cylinder-piston arrangement.

In a preferred embodiment of the present invention, the chip further comprises a groove (8) (or a recess or a depression) in the chip, adjacent to the cylinder, configured to accommodate the second part of the piston (5) in its axis of movement. The groove's mean cross-section can have a larger diameter than that of the cylinder, the same diameter or a smaller diameter. The latter is for instance possible if the piston is executed as a plunger that consists of a larger-diameter part that fits snugly into the cylinder (as to avoid leakage) and another part that has a smaller mean diameter than the cylinder and preferably fits snugly into the groove of smaller mean diameter (as to be stabilised in its axis of movement) . Care must be taken, howev^¬ er, that the plunger can still act as a valve in respect to the inlet (e.g. by providing a larger-diameter part of the piston that is sufficiently long in respect to the cylinder) , in order for the chip to fulfil the inventive reservoir function.

Preferably, the groove is at the front side (cf. Fig 2C) or back side of the chip. If it is at the front side of the chip and a foil or film is placed onto the front side of the chip, said foil or film can be adapted to (e.g. by being cut) make the part of the piston accommodated in the groove accessible for ac^¬ tuation. Preferably, the film or foil is pre-cut such that the groove in the front side of the chip is left open upon covering the chip with the film or foil.

Beneficially, the chip is characterised in that the groove is configured to accommodate the piston entirely in the axis per^¬ pendicular to the plane of the chip. Thereby no part of the pis^¬ ton will protrude from the side of the chip the groove is formed into .

Preferably, the inventive chip is further characterised in that the groove is stopped at its end (9) alongside the axis of movement and opposite to the cylinder (4), such that the piston, regardless of its relative position in the cylinder, is contain^¬ able within the lateral boundaries of the chip. (I.e. the groove is a stopped groove as opposed to a through-groove.)

In another preferred embodiment of the present invention, the chip is further characterised in that the cylinder (i.e. the chip substrate forming the wall of the cylinder) and/or the groove (i.e. the chip substrate forming the wall of the groove) comprise an opening (10), preferably a slot. In case of the opening being in the cylinder, the opening is positioned such that the piston is accessible from outside of the chip through the opening when at least a first part of the piston is accommo^¬ dated in the cylinder. In case of the opening being in the groove, the opening is positioned such that the piston is acces^¬ sible from outside of the chip through the opening when at least a second part of the piston is accommodated in the groove. For instance, if the groove is in the front side of the chip and the opening in the groove is in the back side of the chip, the film or foil will not have to be cut in order to make the cylinder- piston arrangement accessible for actuation.

In another preferred embodiment of the present invention, the chip is further characterised in that the inlet is directly con^¬ nected to the cylinder, meaning that no channel is necessary for connection between inlet and cylinder (i.e. the cylinder has an opening) .

In another preferred embodiment of the present invention, the inlet is an inlet into or for the reservoir. The inlet is pref^¬ erably an inlet from the outside of the chip. In another preferred embodiment of the present invention, the chip is configured to accommodate the piston entirely in the ax^¬ is perpendicular to the plane of the chip (the piston surface does not protrude from the surface of the chip essentially par^¬ allel to the main plane of the chip, i.e. from the front and back sides) . This offers the advantage that the inventive chip can be stacked more conveniently, i.e. for packaging.

In a further embodiment of the present invention, the chip is characterised in that the cylinder and/or the groove provide en^¬ gaging means for the piston, preferably for one, more preferably for two, especially for all of the following positions (thereby stabilising the piston in said positions) :

-position PI: inlet (2) open, such that the cylinder is configured to provide a reservoir for the liquid (6)

-position P2 : inlet (2) closed, such that the cylinder is configured to hold the liquid in the reservoir (6) .

-position P3 : inlet (2) closed, such that the reservoir (6) is configured to hold less of the liquid than in the position P2. See also Fig. 3.

Preferably, the cylinder, the channel and/or the groove are molded, milled, embossed or etched into the chip.

In a further preferred embodiment of the present invention, the inventive chip is for detecting or quantifying an analyte in a liquid biological sample, preferably characterized in that the analyte is a protein, a peptide, a carbohydrate, a lipid or com^¬ binations thereof, preferably a protein.

In particular, said protein is a marker selected from one of the group of markers for inflammation (such as C-reactive pro^¬ tein, serum amyloid A, interleukin 6, interleukin 8, interleukin 10 or tumor necrosis factor-alpha) , liver function (such as glu^¬ tathione s-transferase, ornithine carbamoyl transferase or L- arginase) , brain injury (such as glial fibrillary acidic pro^¬ tein, S100 calcium binding protein B, neuron specific enolase or brain fibrillary acidic protein) , sepsis (such as procalcitonin or neopterin) , kidney function (such as cytostatin C or neutrophil gelatinase-associated lipocalin) , complement activation ( such cL S C3cL _r C5cL or terminal complement complex) , coagula- tion/fibrinolysis (such as fibrinogen, prothrombin fragment, plasminogen activator inhibitor, thrombin-antithrombin complex or plasmin-alpha (2 ) -antiplasmin) and endothelial activation

(such as soluble thrombomodulin, soluble intercellular adhesion molecule or sE-selectin) .

Preferably, the sample is a body fluid, more preferably the sample is blood and the chip is configured to be connected to a blood cell/plasma separation device. Suitable devices are for instance disclosed in US 4,753,776 A, US 4,816,224 A, EP 04/57183 Al, US 4, 933, 092 A, or in the references given in the directly following paragraph.

Alternatively, and also preferably, the chip can also com^¬ prise a blood cell/plasma separation device. A suitable device is for instance disclosed in WO 2004/061413 A2 or in Yang et al . (Yang, Sung, Akif Undar, and Jeffrey D. Zahn. "A microfluidic device for continuous, real time blood plasma separation." Lab on a Chip 6.7 (2006): 871-880.) or VanDelinder & Groisman (Van- Delinder, Virginia, and Alex Groisman. "Separation of plasma from whole human blood in a continuous cross-flow in a molded microfluidic device." Analytical chemistry 78.11 (2006): 3765- 3771.), or in Haller et al . (Anna Haller, Wolfgang Buchegger, Michael. J. Vellekoop. "Towards an optimized blood plasma sepa^¬ ration chip: Finite element analysis of a novel corner structure in a backward-facing step" Procedia Engineering 25 (2011) 439 - 442) .

In another preferred embodiment of the present invention, the chip is for performing a photometry-based immunoassay comprising a detecting antibody, conjugated to an enzyme, preferably horse^¬ radish peroxidase or alkaline phosphatase, or conjugated to a fluorophore, the detecting antibody being in solution, and a magnetic bead, conjugated to a capturing antibody, the magnetic bead being in suspension. In another preference, the detecting antibody is biotinylated and thereby adapted to be bound to a streptavidin-conj ugated enzyme or streptavidin-fluorophore com^¬ plex. It is evident that, in a typical setup, there is a plural^¬ ity of a magnetic bead, as a magnetic bead typically has a small diameter such as lym. A known brand of magnetic beads is e.g. Dynabeads® Magnetic Beads (Life Technologies) .

Preferably, the photometry-based immunoassay is a photometry- based sandwich immunoassay, with the detecting antibody and the capturing antibody binding the same analyte, preferably on dif^¬ ferent epitopes of the analyte.

Utilizing magnetic beads as surface for immunoassays increas^¬ es the active surface area of the assay reactions compared to planar surfaces (e.g. microtiter plates). Consequently, the vol^¬ ume of the reaction chamber (i.e. where the assay reactions can take place) can be reduced, which accelerates the reaction time due to decreased diffusion time of the biomolecules . In addi^¬ tion, the cost of the assay is reduced, since a lower volume of the necessary reagents is required. The combination of magnetic beads and small reaction chambers according to the present in^¬ vention allows to obtain rapid immunoassay results without the loss of sensitivity. The reaction chamber can serve as a com^¬ bined zone for reaction, washing and detection. This can minimize the risk of losing magnetic beads inside of the microfluid- ic structure since there is preferably no transfer of beads in^¬ side the chip during the assay. A magnet, which can be posi^¬ tioned above or underneath the reaction chamber during e.g. washing, attracts the magnetic beads and avoids flushing the beads out of the reaction chamber.

Therefore, beneficially, the chip further comprises:

a reaction chamber (20) configured to be accessible for photo^¬ metric signal detection (e.g. by one wall executed as a trans^¬ parent plate or foil or film) , preferably characterised in that one of its walls is thinned (cf. reference sign 40), thereby configured for close proximity of an ultrasound sonotrode, a vi^¬ brating element and/or a magnet to the liquid, and/or preferably characterised in that it is adapted to be deeper than the chan^¬ nel when the chip is being used, thereby being configured to re^¬ tain the magnetic bead in the reaction chamber. Being deeper than the channel facilitates retaining the magnetic bead in the reaction chamber. A further advantage of being deeper than the channel is that the reaction chamber has a volume large enough to be sufficient for reactions requiring a larger volume in the reaction chamber.

Beneficially, the channel connected to the reaction chamber leads into a meander-shaped recess (41) in the reaction chamber, see also Fig. 6 and Fig. 7. This facilitates air-bubble-free filling of the reaction chamber with the liquid, if the reaction chamber was filled with air prior to the filling. When the liquid enters the reaction chamber it is guided along the meander- shaped recess until the chamber is filled, thereby reducing en^¬ capsulation of air bubbles. Air bubbles can e.g. hamper the pho^¬ tometric detection or the reaction (e.g. binding of the detec^¬ tion antibody to the analyte) . Alternatively, in another prefer^¬ ence, the reaction chamber comprises a phase guide as disclosed in Vulto et al . (Vulto, Paul, et al . "Phaseguides : a paradigm shift in microfluidic priming and emptying." Lab on a Chip 11.9 (2011) : 1596-1602.) . However, the meander-shaped recess of the present invention is exceptionally well-suited for the purposes of the present invention, compared to said phase guide.

The immunoassay can be performed on the surface of magnetic beads. The spherical beads can consist of a super-paramagnetic core with a polymer shell, especially activated for efficient covalent bonding of the capture antibodies (e.g. Dynabeads® M- 280 Tosylactivated, Carlsbad, California) . For instance, Kou- rilov & Steinitz (Kourilov, Vitaly, and Michael Steinitz. "Magnetic-bead enzyme-linked immunosorbent assay verifies adsorption of ligand and epitope accessibility." Analytical biochemistry 311.2 (2002): 166-170.) describe an immunoassay with magnetic beads, specifically an ELISA.

Beneficially, the chip comprises at least two, preferably at least three, more preferably at least four, especially at least five of said reaction chamber, configured to be used in paral^¬ lel. For instance, this can be used for measuring replicates of the same analyte or different analytes in parallel.

In another preferred embodiment, the chip is characterised in that the cylinder is configured to hold the detecting antibody, thereby acting as the reservoir.

In another preferred embodiment of the present invention, the chip further comprises a further inlet and a further cylinder acting as a further reservoir, as set forth in any of the embodiments disclosed herein, characterised in that the further res^¬ ervoir is configured to hold the magnetic bead in suspension.

In another preferred embodiment of the present invention, the chip further comprises a further inlet and a further cylinder acting as a further reservoir, as set forth in any of the embodiments disclosed herein, characterised in that the further res- ervoir is configured to hold an enzyme or a fluorophore (e.g. a streptavidin-conj ugated enzyme or streptavidin-fluorophore com^¬ plex) to be bound to the detecting antibody (e.g. a biotinylated detecting antibody) in solution. One of the advantages of this embodiment is that, if the enzyme or fluorophore is most stable under conditions (e.g. pH, salt concentration) that differ from conditions the detecting antibody is most stable, they can be stored separately on the same chip ready for use.

In yet another preferred embodiment of the present invention, the chip further comprises a further inlet and a further cylinder acting as a further reservoir, as set forth in any of the embodiments disclosed herein, characterised in that the further reservoir is configured to hold the sample. Using the inventive cylinder-piston arrangement for sample injection has the advantage that the cylinder (possibly in conjunction with a well- defined piston movement) defines the injected volume very pre^¬ cisely, leading to high reproducibility. The chip can also com^¬ prise another inlet (26) configured to be used to fill the cyl^¬ inder via the processing device, which can be used to connect upstream devices such as blood/plasma separation systems.

In another preferred embodiment of the present invention, the chip is for performing a chemiluminescence-based ELISA, charac^¬ terised in that the chip further comprises a meandering channel (21) configured to be used for mixing of a two-component chemi- luminescence reaction substrate. Suitable substrates are for in^¬ stance luminol-based substrates, dioxetane-based substrates or SuperSignal® ELISA substrates (Thermo Scientific) .

Beneficially, the chip of the present invention is of an opaque material, preferably with low intrinsic phosphorescence, more preferably selected from: acrylonitrile butadiene styrene

(ABS) , acrylonitrile butadiene styrene-polycarbonate (ABS-PC) , ABS-polymethyl methacrylate (ABS-PMMA) , polystyrene, polymethyl methacrylate (PMMA) , polypropylene (PP) , and polyoxymethylene

(POM) .

Preferably, the reaction chamber has a high reflectivity to enhance a light signal to be detected, such as a light signal caused by chemiluminescense .

In another preferred embodiment of the present invention, the chip comprises the piston as an integral part. Preferably, the piston is further characterised in that it has a length such that the cylinder is configured to accommodate at most 99%, preferably at most 95%, especially at most 90% of the piston's length, thereby providing that one end of the piston is accessi^¬ ble from the outside of the chip, regardless of the piston's po^¬ sition in the cylinder. This is further enabled by providing the groove and/or the opening or by the chip being configured such the piston can protrude from the narrow side of the chip.

In a preference, the piston has means of actuation, prefera^¬ bly a groove, located at or near the end of the piston and con^¬ figured to be accessible from the outside of the chip regardless of the piston's position in the cylinder. In another preference, the piston has an O-ring (30) or an elastic disc at one of its ends for sealing the liquid in the reservoir.

In another preferred embodiment of the present invention, the chip is characterised in that the piston is partially inserted into the cylinder, thereby defining the reservoir (piston position P2, cf . Fig. 3) . Preferably said reservoir is pre-filled with liquid.

Preferably, the piston is further characterised in that it has a length such that the chip's groove is configured to accom^¬ modate at most 99%, preferably at most 95%, especially at most 90% of the piston's length, thereby providing that one end of the cylinder always remains closed regardless of the piston's relative position between the cylinder and the groove.

In another preferred embodiment of the present invention, the chip holds the magnetic bead conjugated to the capturing anti^¬ body in suspension and/or the detecting antibody;

each in the reservoir of the cylinder configured thereto or, in case of the magnetic bead conjugated to the capturing anti^¬ body, in the reaction chamber.

Beneficially, the chip of the present invention (with or without the reservoir filled with liquid, preferably with the reservoir filled with liquid) is provided sterile in packaging. Sterile, as used herein, has preferably the meaning of being germ-free. This can be achieved for instance by sterilisation by heat or radiation. However, in some embodiments, it is suffi^¬ cient to provide the chip with a reduced load of germs, thus "sterile" can also mean having less than 10000000 colony-forming units (CFU) of mesophilic aerobic germs, preferably less than 1000000 CFU, more preferably less than 100000 CFU, even more preferably less than 10000 CFU, in particular less than 1000 CFU.

Preferably the chip is provided ready-to-use. In a further preference, it is provided with at least further two of said chip, preferably further four of said chip, more preferably fur^¬ ther nine of said chip.

In another aspect of the invention, a chip-piston set is provided, comprising the chip and piston as disclosed herein. The piston of the set is preferably further characterised in that it has a length such that the cylinder is configured to accommodate at most 99%, preferably at most 95%, especially at most 90% of the piston's length, thereby providing that one end of the pis^¬ ton is accessible from the outside of the chip regardless of the piston's position in the cylinder.

In a preference, the piston of the set has means of actua^¬ tion, preferably a groove, located at or near the end of the piston and configured to be accessible from the outside of the chip regardless of the piston's position in the cylinder. In another preference, the piston has an O-ring (30) or an elastic disc at one of its ends for sealing the liquid in the reservoir. This is further enabled by providing the groove and/or the open^¬ ing or by the chip being configured such the piston can protrude from the narrow side of the chip. The O-ring can be made of rub^¬ ber or a rubber-like material, for instance.

In another preferred embodiment of the present invention, the chip of the set is characterised in that the piston is partially inserted into the cylinder, thereby defining the reservoir (pis^¬ ton position P2, cf . Fig. 3) . Preferably said reservoir is pre- filled with liquid.

Preferably, the piston of the set is further characterised in that it has a length such that the chip's groove is configured to accommodate at most 99%, preferably at most 95%, especially at most 90% of the piston's length, thereby providing that one end of the cylinder always remains closed regardless of the pis^¬ ton's relative position. In another preferred embodiment of the present invention, the chip of the set holds the magnetic bead conjugated to the cap^¬ turing antibody in suspension and/or the detecting antibody;

Beneficially, the chip of the set of the present invention is provided sterile in packaging. Preferably the chip is provided ready-to-use. In a further preference, it is provided with at least further two of said chip, preferably further four of said chip, more preferably further nine of said chip.

In a further aspect of the present invention, an apparatus for biological analysis by processing the inventive chip is pro^¬ vided. The apparatus is configured to use the chip as a car^¬ tridge, e.g. for single use. The chip is preferably character^¬ ised in that it has the piston as set forth herein partially in^¬ serted. The apparatus comprises a cartridge holder for the chip as the cartridge, a means to actuate the piston and a detector. The apparatus is beneficially configured to process several chips in parallel.

In a preferable embodiment, the apparatus is for performing a photometry-based immunoassay by processing the chip for perform^¬ ing a photometry-based immunoassay, and characterised in that the detector is a photometric signal detector, preferably a sil^¬ icon photodiode.

Preferably, the apparatus further comprises magnet for cap^¬ turing the magnetic bead in the chip, a vibrating element for dispersion of the magnetic bead in the chip and/or a ultrasound sonotrode for dispersion of the magnetic bead in the chip.

In another aspect of the present invention, the use of the inventive chip is provided. Further, the use of the inventive set is provided. Finally, the use of the inventive apparatus is provided. Said uses are preferably for POCT .

In another aspect of the present invention, a method for performing biological analysis is provided. This method comprises:

A) inserting the chip, as a cartridge, into the cartridge holder of the apparatus, the chip characterised in that it holds the liquid and has the piston partially inserted; and B) actuating the piston with the means to actuate the piston, thereby causing flow of the liquid; and

C) detecting a signal emerging from the liquid with the detector .

If the chip comprises more than one cylinder-piston arrange^¬ ments, the pistons of the cylinder-piston arrangements are pref^¬ erably actuated in the order necessary to conduct the biological analysis (i.e. the specific analysis protocol).

In another aspect of the present invention, a method for performing a photometry-based immunoassay on a liquid sample is provided. The immunoassay comprises a magnetic bead, conjugated to a capturing antibody, in suspension, and a washing solution, and a detecting antibody, conjugated to an enzyme, preferably horseradish peroxidase, or conjugated to a fluorophore, in solu^¬ tion. Said method comprises:

A) inserting the chip, as a cartridge, into the cartridge holder of the apparatus,

the chip characterised in that it holds the magnetic bead in suspension and the detecting antibody in solution before the inserting, each in the reservoir of the cylinder configured thereto, or, in case of the magnetic bead, in the reaction chamber; each of said cylinder with the piston partially inserted

B) preferably positioning the magnet close to the reaction chamber

C) if the magnetic bead is held in the reservoir configured thereto, actuating the piston inserted into the cylinder provid^¬ ing the reservoir holding the magnetic bead, thereby causing the magnetic bead to flow into the reaction chamber, preferably be^¬ ing held therein by the magnet.

D) pumping the sample into the reaction chamber, such that the analyte is captured by the capturing antibody conjugated to said magnetic bead, preferably held therein by the magnet

E) pumping the washing solution, preferably through another inlet, into the reaction chamber, such that the rest of the sam^¬ ple is separated from the captured analyte

F) actuating the piston inserted into the cylinder providing the reservoir holding the detecting antibody, thereby causing the detecting antibody to flow into the reaction chamber and binding to the capturing antibody

G) detecting a signal emerging from the detecting antibody with the detector.

In a preferred embodiment the method further comprises, be^¬ fore the detecting of step G) , pumping a chemiluminescence reac^¬ tion substrate, preferably through another inlet, into the reac^¬ tion chamber, such that an enzyme conjugated to the detecting antibody produces a signal by luminescence.

Beneficially, the method is characterised in that the chemi^¬ luminescence reaction substrate is a two-component chemilumines^¬ cence reaction substrate, pumped into the chip through another two inlets (22, 23), and in that the chip comprises the meander^¬ ing channel (21), and the method further comprises pumping each component of the two-component substrate, for each independent^¬ ly, through another inlet, and then together through the meandering channel such that the two components are mixed, into the reaction chamber (20) .

In a further preferred embodiment of the method, step D) is characterised in that the sample is pumped into the reaction chamber through another inlet (24 or 25) of the chip, preferably through a blood cell / plasma separation device.

In another preferred embodiment of the method, the chip fur^¬ ther holds the sample in the reservoir of the cylinder configured thereto, before the inserting or by filling of the reser^¬ voir (e.g. through inlet 26 or 2) after the chip was inserted into the cartridge holder, with the piston partially inserted into said cylinder configured thereto,

wherein step D) is characterised in that the piston partially inserted is actuated, thereby causing the sample to flow into the reaction chamber

In another preferred embodiment, the method further comprises positioning the sonotrode close to the reaction chamber and sonicating the reaction chamber, thereby causing dispersion of the magnetic bead within the reaction chamber. Preferably the posi^¬ tioning is achieved by intermittently exchanging the magnet with the sonotrode.

In another preferred embodiment, the method further comprises positioning the vibrating element close to the reaction chamber and causing the reaction chamber to vibrate, thereby causing dispersion of the magnetic bead within the reaction chamber. Preferably the positioning is achieved by intermittently ex^¬ changing the magnet with the vibrating element.

Preferably, the method of the invention further comprises un^¬ packing the chip before further use, the chip characterised in that it is sterile in packaging before further use.

Also preferably, the method of the invention further compris^¬ es discarding the chip after at most five uses, preferably at most two uses, more preferably at most one uses. Discarding af^¬ ter one use offers the most advantages in regard to the quality of results and is especially preferred.

Preferably, the detecting of the detecting step is a multi^¬ plex-detecting, by using more than one chip at the same time or by using a chip with more than one of the reaction chamber configured to be used in parallel.

As used herein, the expression of an axis being "essentially parallel" to a plane preferably means that the angle between the axis and the plane is not more than 20 degrees, preferably not more than 15 degrees, more preferably not more than 10 degrees, especially not more than 5 degrees.

Most typically, the chip, apparatus and method of the present invention are for in-vitro use only.

The invention is further described by the following examples and the drawing figures, yet without being limited thereto.

List of reference signs:

(1) chip

(2) inlet (connected to the cylinder)

(3) channel

(4) cylinder

(5) piston

(6) reservoir

(7) liquid (8) groove

(9) the stopped end of the groove (8)

(10) opening

(20) reaction chamber

(21) meandering channel

(22) inlet

(23) inlet

(24) outlet from the cylinder / inlet into reaction

(25) inlet

(26) inlet

(28) inlet

(29) outlet

(30) O-ring of the piston

(40) thinned wall close to reaction chamber

(41) meander-shaped recess in the reaction chamber

(50) syringe

(51) film or foil

(52) puncture in film or foil (e.g. by a needle)

(53) inlet

Figure 1: 1A) Front view of the chip (1) . The chip comprises two inlets (2) each directly connected to a cylinder (4, see Fig. 1B/1C) , and the channel (3).

The chip also comprises an alternative inlet (26) into the cylinder. This inlet can be connected to the apparatus of the invention, which allows filling the cylinder when the chip is inserted into the apparatus as a cartridge. This can be used to connect upstream devices such as a plasma separation device to the chip and/or allows filling of the cylinder with a defined sample volume which can then be injected into the chip by actua^¬ tion of the piston.

The chip also comprises a reaction chamber (20) where the de^¬ tecting of the analyte occurs. The chip comprises two further inlets (22, 23), one for each component of the two-component chemiluminescent substrate. The components are mixed in the me^¬ andering channel (21) . The further inlet 28 is for injection of the washing buffer. Excess liquid runs from the outlet (29) .

IB) In the same view as in 1A) the two cylinders (4; executed as full cylinders) are shown, a part of each can act as the a reservoir when the respective piston is inserted, each reservoir to be filled through the respective inlet 2. Actuation of the respective piston, when inserted into the respective cylinder, will cause the liquid to flow into the reaction chamber.

1C) In the same view as in 1A) the two cylinders (4) are shown executed as half cylinders. Each half-cylinder comprises a slot (10) such that the piston can be actuated when it is in^¬ serted in the cylinder and the cylinder is covered by a film or foil.

Figure 2: 2A) Front and back views of the chip with two cyl^¬ inders executed as full cylinders, each with a piston (5) in^¬ serted. In the top row, the pistons (5) are only partially in^¬ serted, thereby each cylinder acts as a reservoir and the reser^¬ voir has been emptied. In the bottom row, the two pistons are fully inserted. Naturally, the pistons can be actuated inde^¬ pendently of each other. The wall is thinned (40) close to the reaction chamber, e.g. for the magnet or the vibrating element to be more effective.

2B) Front and back views of the chip with two cylinders exe^¬ cuted as half cylinders, each cylinder further comprising the slot (10) in the back, each cylinder with a piston (5) inserted. Not visible is the transparent foil that covers the entire front of the chip. In the top row, the pistons (5) are only partially inserted, thereby each cylinder acts as a reservoir. In the bot^¬ tom row, the two pistons are fully inserted and the reservoir has been emptied. Naturally, the pistons can be actuated inde^¬ pendently of each other. In the back views, it becomes apparent that respective slot (10) can be used to actuate the respective piston from outside of the chip. The wall is thinned (40) close to the reaction chamber, e.g. for the magnet or the vibrating element to be more effective.

2C) Front and back views of the chip with two cylinders exe^¬ cuted as half cylinders, each cylinder adjacent to a groove (8) wherein the groove is stopped (9), each cylinder with a piston (5) inserted. Not visible is the transparent foil that covers the entire front of the chip with exception of the two grooves. In the top row, the pistons (5) are only partially inserted, thereby each cylinder acts as a reservoir. In the bottom row, the two pistons are fully inserted and the reservoir has been emptied. Naturally, the pistons can be actuated independently of each other. In the front views, it becomes apparent that the re^¬ spective groove (8) can be used to actuate the respective piston from outside of the chip. The wall is thinned (40) close to the reaction chamber, e.g. for the magnet or the vibrating element to be more effective.

Figure 3: In this cross-section through a part of the chip the optical axis is parallel to the plane of the chip. The chip, with the inlet (2), is covered by the thin foil (51) . The piston (5) has the O-ring (30) for sealing and is inserted into the cylinder (8) which is a full cylinder in this figure (as opposed to a half-cylinder) . This entails that the thin foil covers the inlets but not the cylinder itself.

Piston position PI: The syringe (50; not drawn to scale) is used to puncture the foil covering the inlet and to fill the reservoir (6) to be filled with the liquid (7) . After filling, the piston is moved to piston position P2.

Piston position P2 : The piston closes the inlet which may otherwise leak due to the puncture (52) in the foil, thereby acting as a valve in respect to the inlet. The liquid (7) is in the reservoir (6) . To cause the liquid to flow into the rest of the chip (the channel leading away from the cylinder is not vis^¬ ible in this cross-section) , the piston is moved to piston position P3.

Piston position P3 : The reservoir is emptied.

Figure 4: Microfluidic chip / apparatus system for chemilumi- nescent ELISA (the inventive cylinder/piston arrangement is not shown) . a) Microfluidic chip for single measurements (i.e. as a cartridge) filled with magnetic beads after magnetic capture (cluster is located in the centre of the chamber) . The ellipti^¬ cal shape of the reaction chamber reduces dead volumes and thus, provides efficient washing of the beads between incubation steps. A meander-shaped mixing channel is integrated in the chip for an efficient mixing of the two-component chemiluminescent substrate, b) After exposing the magnetic beads to ultrasound or to a vibrating element, the beads are dispersed and distributed over the whole reaction chamber, c) Apparatus with integrated silicon photodiode, permanent magnet, ultrasonic sonotrode and fluidic connection ports. When the apparatus is closed, the chip is shielded from ambient light. Figure 5: Microfluidic chip / apparatus system for chemilumi- nescent ELISA performs rapid and highly sensitive immunoassays. Standard curves from IL-8 magnetic beads ELISA. The red curve

(square tokens) represents the signal when measured in the 20 μΐ reaction chamber of the chip with a silicon photodiode. The reaction volume was 20 μΐ and the duration time of plasma incuba^¬ tion, washing, detection antibody incubation and enzyme incubation was two minutes each. The blue curve (diamond tokens) rep^¬ resents the signal when measuring with a commercial available luminometer using a photomultiplier tube as optical detector

(POLARstar Omega, BMG LABTECH GmbH, Ortenberg, Germany) . In this assay the reaction volume was 100 μΐ and each incubation step lasted 10 minutes. Both systems have a limit of detection of 10 pg/ml of IL-8 protein.

Figure 6: Microfluidic chip with piston. The cylinder is exe^¬ cuted as a full cylinder, with the piston accessible for actua^¬ tion at its part protruding from a narrow side of the chip. A) Chip and pistons are shown. Chip size: 29x25x3mm, piston size: diameter 2mm, length 25mm, reaction chamber volume: 20 μΐ . B) Front and back of the chip is shown, with cylinders partially inserted (upper row) and fully inserted (lower row) . The chip can be covered (e.g. by lamination) by a, preferably transpar^¬ ent, film or foil for sealing such that the liquid does not leak from the front of the chip.

Figure 7: Microfluidic chip with piston. The cylinder is exe^¬ cuted as a half cylinder extending through to a narrow side of the chip, with the piston accessible for actuation through the slot comprised in the cylinder wall. Front (left image, without piston) and back of the chip (right image, with piston) is shown. The chip can be covered (e.g. by lamination) by a, pref^¬ erably transparent, film or foil for sealing such that the liq^¬ uid does not leak from the front of the chip.

Figure 8: Apparatus with the microfluidic chip inserted (as a cartridge) .

A-I) Cutaway-view of the open apparatus with the chip inserted. A-II) A downward movement towards the chip brings the upper part of the apparatus, especially the magnet, in close proximity to the chip. Direction of movement is shown by the arrow. A-III) Enlarged cross section of the microfluidic chip inside the closed apparatus, with the magnet being close to the reaction chamber (as also shown in A-II) . The reaction chamber is in close proximity to the magnet and sonotrode/vibrating element inside the closed apparatus, and the O-rings are sealing the fluidic connections.

B-I) Before signal detection, the apparatus is opened and a lateral and downward movement of the upper part of the device places the photodiode (as opposed to the magnet as shown in A-I) in especially close proximity to the reaction chamber. When the apparatus is closed, a large O-ring is shielding the chip system from ambient light. Direction of movement is shown by the ar^¬ rows. B-II) Enlarged cross section of the microfluidic chip in^¬ side the closed apparatus, with the photodiode being close to the reaction chamber (as also shown in B-I) . Typically, a trans^¬ parent or foil covering the chip is between the photodiode and the reaction chamber.

Legend for both A) and B) C: Microfluidic chip; PI: Pistons in cross section; FC : Fluidic connections; M: Permanent magnet; PD: Silicon photodiode; SO: Ultrasonic sonotrode; OR: O-rings, VM: Vibration element.

Figure 9: Apparatus with the microfluidic chip inserted (as a cartridge) . C: Chip, Al : Pneumatic means to actuate pistons in^¬ serted into the chip, A2, A3: Pneumatic means to position upper part of the apparatus that makes contact with the chip (compris^¬ ing the photodiode as detector), A4 : Part of the apparatus that comprises the magnet and pneumatic means to position the magnet.

Figure 10: Front views of a microfluidic chip with four reac^¬ tion chambers (20) . Each reaction chamber can be filled through a different cylinder/piston arrangement (4/5). The pistons (5) are shown in A), the cylinders (4) are shown in B) . Each cylinder is directly connected to its own inlet (2) and its own out^¬ let (24) being at the same time the inlet into the respective reaction chamber. Each reaction chamber is connected to an inlet for magnetic beads (53) , which are inserted and stored in the reaction chamber prior to usage. The magnetic beads are coated with the specific capture antibody before inserting into the chip. The chip further comprises two inlets for the two- component chemiluminescence substrate (22) and one for the wash^¬ ing buffer (28) . The chip also comprises a meandric channel (21) . All reaction chambers have the same outlet (29) . The pis^¬ tons can be actuated independently of each other, washing and injection of chemiluminescence substrate occurs in parallel for all reaction chambers. Each reaction chamber can be pre-filled with a detection agent for a different biological marker (e.g. via the inlet 53) .

Figure 11: Calibration curve of the biomarker IL-8 spiked in 10% plasma. ELISAs on the inventive microfluidic chip were per^¬ formed with an incubation time of 3 minutes each and a volume of 20 yL each, with varying concentrations of IL-8 spiked in. For each concentration of IL-8, the ELISA was repeated three times (n=3) . RLU: Relative Luminescence Units. The limit of detection (LOD) was determined to be 1 pg/mL, with the LOD calculated as 3 times the standard deviation above the negative control with no IL-8 added. The reproducibility between the IL-8 measurements is marked as error bars in the figure (coefficient of variance) .

Examples

Example 1 - Microfluidic cartridge / apparatus system for chemiluminescent ELISA

The present example represents a working embodiment of the pre^¬ sent invention.

The present exemplary embodiments of the inventive chip (as a disposable cartridge, i.e. for one-time use) and the inventive apparatus, in combination, are a system to detect and quantify chemiluminescent signals from automated sandwich-ELISAs per^¬ formed on magnetic beads inside the microfluidic cartridge. It is used for a rapid and quantitative detection of protein- biomarkers from body fluids such as serum, saliva, and urine. The system was tested for detection of the biomarkers IL-6, IL- 8, IL-10, TNF-alpha, and S100B. Biomarker quantification at concentrations between 5 pg/ml and 2000 pg/ml was achieved. This range represents the clinically relevant values in blood plasma. Each incubation step requires 2 minutes, which is a 30-fold ac^¬ celeration compared to conventional ELISA. As a consequence, the total analysis time of the immunoassay is less than 30 minutes. Cost effective photodiodes and a lens-free setup are used as an optical detection unit of the apparatus, which decreases the to- tal cost of the system. Due to the small size of a photodiode, it also favours for a compact and portable system.

The high sensitivity of the system is reached by several as^¬ pects; i) enhancement of signal by using conjugates with multi^¬ ple enzymes (horseradish peroxidase) in a combination with magnetic beads in the immunoassay, ii) the choice of material of the reaction chamber to avoid light adsorption and auto- luminescence, iii) the blue enhanced photodiode mounted with a minimal distance to the reaction chamber, and iv) high-gain amplification of the photodiode signal. By using cost efficient and lens less silicon photodiodes as photo detectors, the system becomes inexpensive and compact. With integrated reagents and waste management, the chip as disposable cartridge is ready to use with no cleaning required after usage.

The chemiluminescent ELISA is performed on the surface of magnetic beads. The spherical beads consist of a super^¬ paramagnetic core with a polymer shell, especially activated for efficient covalent bonding of the capture antibodies (Dynabeads® M-280 Tosylactivated, Carlsbad, California) . During the ELISA, the magnetic beads with immobilized capture antibodies undergo three incubations steps; protein analyte of unknown concentra^¬ tion, biotin conjugated detection-antibody and streptavidin, conjugated with multiple HRP (horseradish peroxidase) . Between the incubation steps, the beads are washed with a continuous flow of PBST (phosphate buffered saline with Triton X-100) . The signal is measured after adding chemiluminescent substrate to the magnetic beads. The substrate consists of two components, which are mixed through a meander-shaped channel before entering the reaction chamber. During the immunoassay, the chip (as a disposable cartridge) is encased in a lightproof container in which a silicon photodiode is integrated for chemiluminescent signal detection. With an established calibration curve, the concentration of protein analytes is calculated according to the measured signal (see Figure 5) . Figure 11 shows that the in^¬ ventive microfluidic chip achieves measurements with high sensi^¬ tivity and reproducibility.

The disposable cartridge consists of white, opaque polymer with a microfluidic channel network and 20 μΐ reaction chambers for each biomarker being analyzed (see Figure 4) . Polymers used for the cartridge have to show very low intrinsic phosphores^¬ cence to guarantee low background signals. Tested materials in^¬ clude acrylonitrile butadiene styrene- polycarbonate (ABS-PC) , ABS-polymethyl methacrylate (ABS-PMMA) polystyrene, and polyox- ymethylene (POM) . The microfluidic chip comprises three inlets, which join in a T-junction and lead to a meander-shaped mixing channel. After this mixing structure, the channel leads to a re^¬ action chamber, in which the magnetic beads are stored. The re^¬ action chamber is ellipse-shaped for an efficient washing, and has a structured bottom to facilitate air-bubble-free filling. Two inventive cylinder-piston arrangements are connected to the reaction chamber, in which one is filled with detection antibody and the second is filled with body fluid for protein quantifica^¬ tion (see Figure 3 and 4) . The detection antibody is stored in the reservoir of one of the two cylinder-piston arrangements, while the body fluid is pumped into the reservoir of the other of the two cylinder-piston arrangements just before the measure^¬ ment. The fluid is transferred from the reservoir by actuating a piston and thereby pushing the fluid into the reaction chamber. The front side of the chip is thermally bonded to a 100 ym thick polycarbonate film for sealing such that the liquid does not leak from the front of the chip. Inlet ports are connected with a chip-holder with O-rings to obtain a tight connection and to load the reagents into the cartridge. Biomarker specific rea^¬ gents (antibody-coated magnetic beads and detection-antibodies) are stored in the chip, while reagents shared by all biomarkers (wash buffer, enzyme, substrate) are pumped into to the chip from a separate container. All reagents and buffer are exiting the chip through a outlet, or are stored in an on-chip waste container. For the ability of multiplex measurement of the dif^¬ ferent biomarkers, the chip design can be extended with multiple parallel reaction chambers, one for each biomarker to be quanti^¬ fied .

The present invention is further exemplified by the follow^¬ ing embodiments:

1. A microfluidic chip for biological analysis (1),

comprising an inlet (2) for a liquid (7), a channel (3), and a cylinder ( 4 ) , - the cylinder (4) being connected to the inlet (2) and the channel (3) and having a cross-section larger than the cross- section of the channel (3) ;

characterised in that:

- the cylinder (4) is configured to accommodate at least a first part of a piston (5) , and

- the cylinder (4) is configured such that the piston (5) has an axis of movement inside the cylinder (4) essentially par^¬ allel to the plane of the chip, and

- the chip is configured such that at least a second part of the piston (5) is accessible from outside of the chip, and

- the inlet (2) is closable by the piston (5), whereby the piston also acts as a valve, and

- the chip is configured such that the cylinder (4) and the piston (5) define a reservoir (6) for the liquid (7) .

2. The chip of embodiment 1,

further characterised in that a groove (8) in the chip (1), ad^¬ jacent to the cylinder (4), is configured to accommodate the second part of the piston (5) in its axis of movement.

3. The chip of embodiment 2, further characterised in that the groove (8) is configured to accommodate the piston (5) entirely in the axis perpendicular to the plane of the chip.

4. The chip of embodiments 2 or 3,

further characterised in that the groove (8) is stopped at its end (9) opposite to the cylinder (4), such that the piston is containable within the lateral boundaries of the chip .

5. The chip of any one of embodiments 1 to 4, characterised in that the cylinder (4) and/or the groove (8) if present comprise an opening (10), preferably a slot, adapted to making the second part of the piston (5) accessible from outside of the chip if at least the first part of the piston (5) is accommodated in the cylinder ( 4 ) .

6. The chip of any one of embodiments 1 to 5, characterised in that the inlet (2) is directly connected to the cylinder (4) . 7. The chip of any one of embodiments 1 to 6, characterised in that the cylinder (4) and/or the groove (8) if present provide engaging means for the piston (5) , preferably for one, more preferably for two, especially for all of the following posi^¬ tions :

- position PI: inlet (2) open, such that the cylinder is configured to provide a reservoir for the liquid (6)

- position P2 : inlet (2) closed, such that the cylinder is configured to hold the liquid in the reservoir (6) .

- position P3 : inlet (2) closed, such that the reservoir (6) is configured to hold less of the liquid than in the position

P2.

8. The chip of any one of embodiments 1 to 7, characterised in that the cylinder (4), the channel (3) and/or the groove if pre^¬ sent (6) are molded, milled, embossed or etched into the chip.

9. The chip of any one of embodiments 1 to 8, for detecting or quantifying an analyte in a liquid biological sample, preferably characterized in that the analyte is a protein, a peptide, a carbohydrate, a lipid or combinations thereof, preferably a pro^¬ tein .

10. The chip of embodiment 9, characterised in that the sample is a body fluid,

preferably characterised in that the sample is blood, and the chip comprises, or is configured to be connected to, a blood cell/plasma separation device.

11. The chip of embodiments 9 or 10,

for performing a photometry-based immunoassay comprising a detecting antibody, conjugated to an enzyme, preferably horse^¬ radish peroxidase, or conjugated to a fluorophore, or biotinyl- ated, the detecting antibody being in solution, and a magnetic bead, conjugated to a capturing antibody, the magnetic bead be^¬ ing in suspension, the chip further comprising:

a reaction chamber (20) configured to be accessible for pho^¬ tometric signal detection, preferably characterised in that one of its walls is thinned (40), thereby configured for close prox^¬ imity of an ultrasound sonotrode, a vibrating element and/or a magnet to the liquid, and/or preferably characterised in that it is adapted to be deeper than the channel (3) when the chip is being used, thereby being configured to retain the magnetic bead in the reaction chamber;

preferably characterised in that the channel leads into a meander-shaped recess (41) in the reaction chamber, and/or

preferably characterised in that the chip comprises at least two, preferably at least three, more preferably at least four, especially at least five of said reaction chamber (20), config^¬ ured to be used in parallel.

12. The chip of embodiment 11, characterised in that the cylin^¬ der (4) is configured to hold the detecting antibody, thereby acting as the reservoir (6) .

13. The chip of embodiment 12, further comprising a further inlet (2) and a further cylinder (4) acting as a further reservoir (6) as set forth in any one of the previous embodiments, characterised in that the further reservoir (6) is configured to hold an enzyme or a fluorophore to be bound to the detecting antibody in suspension.

14. The chip of embodiment 11 or 12, further comprising a further inlet (2) and a further cylinder (4) acting as a further reservoir (6), as set forth in any one of the previous embodi^¬ ments, characterised in that the further reservoir (6) is con^¬ figured to hold the sample.

15. The chip of any one of embodiments 11 to 14, for performing a chemiluminescence-based ELISA, characterised in that the chip further comprises a meandering channel (21) configured to be used for mixing of a two-component chemiluminescence reaction substrate .

16. The chip of any one of embodiments 11 to 15, characterised in that the chip (1) is of an opaque material, preferably with low intrinsic phosphorescence, more preferably selected from: acrylonitrile butadiene styrene (ABS) , acrylonitrile butadiene styrene-polycarbonate (ABS-PC) , ABS-polymethyl methacrylate

(ABS-PMMA) , polystyrene, polymethyl methacrylate (PMMA) , poly- propylene (PP) , and polyoxymethylene (POM) .

17. The chip of any one of embodiments 11 to 16, characterised in that the reaction chamber (20) has a high reflectivity to en^¬ hance a light signal.

18. The chip of any one of embodiments 1 to 17, further compris^¬ ing the piston (5) as an integral part,

the piston (5) further characterised in that it has a length such that the cylinder is configured to accommodate at most 99%, preferably at most 95%, especially at most 90% of the piston's length, thereby providing that one end of the piston is accessi^¬ ble from the outside of the chip regardless of the piston's po^¬ sition in the cylinder (4),

preferably characterised in that said piston (5) has means of actuation, preferably a groove, located at or near one end of the piston and configured to be accessible from the outside of the chip regardless of the piston's position in the cylinder ( 4 ) , and/or

preferably characterised in that the piston has an O-ring (30) or an elastic disc at one of its ends for sealing.

19. The chip of embodiment 18, characterised that the piston is partially inserted into the cylinder (4), thereby defining the reservoir (6), preferably pre-filled with the liquid (7),

the piston (5) preferably further characterised in that it has a length such that the chip's groove (8) if present is con^¬ figured to accommodate at most 99%, preferably at most 95%, es^¬ pecially at most 90% of the piston's length, thereby providing that one end of the cylinder (4) always remains closed regard^¬ less of the piston's relative position between the cylinder and the groove.

20. The chip of embodiment 19, characterised in that the chip holds the magnetic bead conjugated to the capturing antibody in suspension, the detecting antibody, and/or an enzyme or a fluor- ophore to be bound thereto;

each in the reservoir (6) of the cylinder (4) configured thereto or, in case of the magnetic bead conjugated to the capturing an^¬ tibody, in the reaction chamber (20) . 21. The chip of embodiment 19 or 20, characterised in that it is provided sterile in packaging, preferably ready-to-use,

especially together with at least further two of said chip, preferably further four of said chip, more preferably further nine of said chip.

22. A chip-piston set, comprising the chip of any one of embodiments 1 to 21 and the piston (5) ,

the piston (5) further characterised in that it has a length such that the cylinder is configured to accommodate at most 99%, preferably at most 95%, especially at most 90% of the piston's length, thereby providing that one end of the piston is accessi^¬ ble from the outside of the chip regardless of the piston's po^¬ sition in the cylinder (4) .

23. The set of embodiment 22, characterised in that said piston (5) has means of actuation, preferably a groove, located at or near the end of the piston and accessible from the outside of the chip regardless of the piston's position in the cylinder

(4) .

24. The set of embodiments 22 or 23, characterised in that the piston has an O-ring (30) or an elastic disc at one of its ends for sealing.

25. The set of any one of embodiments 22 to 24, characterised that the piston (5) is partially inserted into the cylinder (4), thereby defining the reservoir (6), preferably pre-filled with the liquid ( 7 ) ,

the piston preferably further characterised in that it has a length such that the chip's groove (8) if present is configured to accommodate at most 99%, preferably at most 95%, especially at most 90% of the piston's length, thereby providing that one end of the cylinder (4) always remains closed regardless of the piston's relative position.

26. The set of embodiment 25, characterised in that the chip holds the magnetic bead in suspension and/or the detecting anti^¬ body in solution, each in the reservoir (6) of the cylinder (4) configured thereto or, in case of the magnetic bead conjugated to the capturing antibody, in the reaction chamber (20) .

27. The set of embodiment 25 or 26, characterised in that it is provided sterile in packaging, preferably ready-to-use, more preferably comprising at least two each, preferably at least three each, more preferably at least five each, especially at least ten each of said chip (1) and piston (5) .

28. An apparatus for biological analysis by processing the chip as set forth in any one of embodiments 1 to 21,

the apparatus configured to use the chip (1) as a cartridge, the chip characterised in that it has the piston (5) as set forth in any of embodiments 1 to 21 partially inserted,

the apparatus comprising a cartridge holder for the chip (1) as the cartridge, a means to actuate the piston (5) and a detec^¬ tor .

29. The apparatus of embodiment 28, for performing a photometry- based immunoassay by processing the chip (1) for performing a photometry-based immunoassay,

characterised in that the detector is a photometric signal detector, preferably a silicon photodiode.

30. The apparatus of embodiment 29, further comprising a magnet, a vibrating element and/or a ultrasound sonotrode.

31. Use of one or more selected from: the chip of any one of em^¬ bodiments 1 to 21, the set of any one of embodiments 22 to 27 and the apparatus of any one of embodiments 28 to 30, preferably in point-of-care testing.

32. A method for performing biological analysis,

comprising :

A) inserting the chip (1) as set forth in any of embodiments 1 to 21, as a cartridge, into the cartridge holder of the appa^¬ ratus of any one of embodiments 28 to 30, the chip characterised in that it holds the liquid and has the piston (5) as set forth in any of embodiments 1 to 21 partially inserted; and

B) actuating the piston (5) with the means to actuate the piston, thereby causing flow of the liquid; and

C) detecting a signal emerging from the liquid with the detector .

33. A method

for performing a photometry-based immunoassay on a liquid sample, the immunoassay comprising a magnetic bead, conjugated to a capturing antibody, in suspension, and a washing solution, and a detecting antibody, conjugated to an enzyme, preferably horseradish peroxidase, or conjugated to a fluorophore, in solu^¬ tion,

the method comprising:

A) inserting the chip (1) as set forth in any of previous embodiments, as a cartridge, into the cartridge holder of the apparatus of embodiment 29 or 30,

the chip characterised in that it holds the magnetic bead in suspension and the detecting antibody in solution before the inserting, each in the reservoir (6) of the cylinder (4) configured thereto, or, in case of the magnetic bead, in the reaction chamber (20) ;

each of said cylinder (4) with the piston (5) as set forth in any one of embodiments 1 to 21 partially inserted

B) preferably positioning the magnet close to the reaction chamber (20), preferably on the thinned wall (40)

C) if the magnetic bead is held in the reservoir configured thereto, actuating the piston (5) inserted into the cylinder (4) providing the reservoir holding the magnetic bead, thereby caus^¬ ing the magnetic bead to flow into the reaction chamber (20), preferably being held therein by the magnet.

D) pumping the sample into the reaction chamber (20), such that the analyte is captured by the capturing antibody conjugat^¬ ed to said magnetic bead, preferably held therein by the magnet

E) pumping the washing solution, preferably through another inlet (28), into the reaction chamber (20), such that the rest of the sample is separated from the captured analyte

F) actuating the piston (5) inserted into the cylinder (4) providing the reservoir (6) holding the detecting antibody, thereby causing the detecting antibody to flow into the reaction chamber (20) and binding to the capturing antibody

G) detecting a signal emerging from the detecting antibody with the detector.

34. The method of embodiment 33, for performing a chemilumines- cence-based ELISA, further comprising, before the detecting of step G) , pumping a chemiluminescence reaction substrate, prefer^¬ ably through another inlet, into the reaction chamber, such that an enzyme conjugated to the detecting antibody produces a signal by luminescence.

35. The method of embodiment 34,

characterised in that the chemiluminescence reaction sub^¬ strate is a two-component chemiluminescence reaction substrate, pumped into the chip through another two inlets (22, 23) of the chip, and the chip comprises the meandering channel (21) of em^¬ bodiment 15,

further comprising pumping each component of the two- component substrate, for each independently, through another in^¬ let, and then together through the meandering channel such that the two components are mixed, into the reaction chamber (20) .

36. The method of any one of embodiments 33 to 35, wherein step D) is characterised in that the sample is pumped into the reac^¬ tion chamber (20) through another inlet of the chip, preferably through a blood cell / plasma separation device.

37. The method of any one of embodiments 33 to 36,

characterised in that the chip further holds the sample in the reservoir (6) of the cylinder (4) configured thereto, before the inserting or by filling of the reservoir after the chip was inserted into the cartridge holder, with the piston (5) partial^¬ ly inserted into said cylinder configured thereto,

wherein step D) is characterised in that the piston (5) par^¬ tially inserted is actuated, thereby causing the sample to flow into the reaction chamber (20) .

38. The method of any one of embodiments 33 to 37, further com^¬ prising positioning the sonotrode close to the reaction chamber (20), preferably by intermittently exchanging the magnet with the sonotrode, and sonicating the reaction chamber, thereby causing dispersion of the magnetic bead within the reaction chamber (20)

39. The method of any one of embodiments 33 to 37, further com^¬ prising positioning the vibrating element close to the reaction chamber, preferably by intermittently exchanging the magnet with the vibrating element, and causing the reaction chamber to vibrate, thereby causing dispersion of the magnetic bead within the reaction chamber.

40. The method of any one embodiments 32 to 39, further compris^¬ ing unpacking the chip before further use, the chip characterised in that it is sterile in packaging before further use.

41. The method of any one of embodiments 32 to 40, further com^¬ prising discarding the chip after at most five uses, preferably at most two uses, more preferably at most one uses.

42. The method of any one of embodiments 32 to 41, characterised in that the detecting of the detecting step is a multiplex- detecting, by using more than one chip at the same time or by using a chip with more than one of the reaction chamber (20) configured to be used in parallel.

Claims

Claims :

1. A microfluidic chip for biological analysis (1),

comprising an inlet (2) for a liquid (7), a channel (3), and a cylinder ( 4 ) ,

characterised in that:

2. The chip of claim 1,

further characterised in that a groove (8) in the chip (1), adjacent to the cylinder (4), is configured to accommodate the second part of the piston (5) at least partially in its axis of movement .

3. The chip of claim 2, further characterised in that the groove (8) is configured to accommodate the piston (5) entirely in the axis perpendicular to the plane of the chip.

4. The chip of claims 2 or 3,

5. The chip of any one of claims 1 to 4, characterised in that the cylinder (4) and/or the groove (8) if present comprise an opening (10), preferably a slot, adapted to making the second part of the piston (5) accessible from outside of the chip if at least the first part of the piston (5) is accommodated in the cylinder ( 4 ) .

6. The chip of any one of claims 1 to 5, characterised in that the inlet (2) is directly connected to the cylinder (4) .

7. The chip of any one of claims 1 to 6, characterised in that the cylinder (4) and/or the groove (8) if present provide engag^¬ ing means for the piston (5) , preferably for one, more prefera^¬ bly for two, especially for all of the following positions:

P2.

8. The chip of any one of claims 1 to 7, further comprising

a reaction chamber (20) configured to be accessible for pho^¬ tometric signal detection;

preferably characterised in that the channel leads into a meander-shaped recess (41) in the reaction chamber.

9. The chip of any one of claims 1 to 8, further characterised in that it comprises the piston (5) as set forth in any one of claims 1 to 8 as an integral part.

10. A chip-piston set, comprising the chip (1) of any one of claims 1 to 8 and the piston (5) as set forth in any one of claims 1 to 8.

11. The chip of claim 9 or the set of claim 10, characterised in that the piston (5) is partially inserted into the cylinder (4), thereby defining the reservoir (6), the reservoir being pre- filled with the liquid (7) .

12. The chip or set of claim 11, characterised in that it is provided sterile in packaging and/or ready-to-use.

13. An apparatus for biological analysis by processing the chip or the set of claim 11 or 12,

the apparatus configured to use the chip (1) as a cartridge, the apparatus comprising a cartridge holder for the chip (1) as the cartridge, a means to actuate the piston (5) and a detec^¬ tor .

14. A method for performing biological analysis,

comprising :

A) inserting the chip or the set of claim 11 or 12, as a cartridge, into the cartridge holder of the apparatus of claim 13; and

C) detecting a signal emerging from the liquid with the detector .

15. Use of the chip, set or apparatus as defined in any one of the previous claims in point-of-care testing.