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Title:
DEVICE FOR NUCLEIC ACID HYBRIDIZATIONS AND IMMUNOASSAYS
Document Type and Number:
WIPO Patent Application WO/2013/190062
Kind Code:
A1
Abstract:
The present invention is related to automated and semi-automated strip-based assays, such as line probe assays (LiPA), involving nucleic acid hybridisations, and line immunoassays (LIA), and methods for performing strip-based assays on a device. Specifically, the present invention relates to a device and a method for performing strip-based assays.

Inventors:
D HONDT ROBERT LEON (BE)
MERSCH GUY GASTON MARIE (BE)
VAN POUCKE STEFAN OMER GERMAINE (BE)
ROELS STEVEN HUGO ODILON (BE)
VERDOODT LIA LOUISE MARIA (BE)
Application Number:
PCT/EP2013/062931
Publication Date:
December 27, 2013
Filing Date:
June 20, 2013
Export Citation:
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Assignee:
INNOGENETICS NV (BE)
International Classes:
G01N35/00; B01L3/02; B01L9/00; C12Q1/00; G01N35/10
Foreign References:
EP2193848A12010-06-09
EP1477815A12004-11-17
DE19835833A12000-02-17
US5055263A1991-10-08
Attorney, Agent or Firm:
BIIP CVBA (Diegem, BE)
Download PDF:
Claims:
Claims

1. A device for processing multiple biological samples, comprising a dispensing unit, at least one reagent vessel, and at least one reaction vessel suitable for accommodating one or more reagent strips, said device characterised in that the dispensing unit dispenses fluid based on positive displacement.

2. A device according to claim 1, wherein said dispensing unit comprises a single channel dosing chamber.

3. A device according to claim 1, wherein said dispensing unit comprises a multiple channel dosing chamber. 4. A device according to claim 3, wherein said multiple channels are separately operable.

5. A device according to claims 1-4, wherein said dispensing unit is capable of dispensing fluid in multiple dosing units of volume.

6. A device according to claims 1-5, wherein dispensing unit uptakes reagent fluid from the lowest-lying area in a reagent vessel.

7. A device according to claim 6, wherein said lowest-lying area in a reagent vessel is created by slopes and/or indentation.

8. A device according to claim 6, wherein the lowest-lying area in a reagent vessel is created through tilting of the reagent vessel. 9. A device according to claim 6, wherein the lowest-lying area in a reagent vessel is created through either providing slopes in the reaction vessel, providing an indentation in the reagent vessel, tilting the reagent vessel, or a combination of any of the previous.

10. A device according to claims 1-9, further comprising at least one heating block.

11. A device according to claim 10, wherein at least one heating block provides simultaneous heating for at least one reagent vessel and at least one reaction vessel.

12. A device according to claims 1-11, further comprising at least one pivoting means.

13. A device according to claim 12, wherein at least one pivoting means provides pivoting for at least one reaction vessel and optionally also for at least one heating block or at least one reagent vessel.

14. A device according to claims 1-13, further comprising waste removal means.

15. A device according to claims 1-10, wherein at least one reagent vessel or at least one reaction vessel can be covered by a lid.

Description:
Device for nucleic acid hybridizations and immunoassays

Field of the invention

The present invention is related to automated and semi-automated strip-based assays, such as line probe assays (LiPA), involving nucleic acid hybridisations, and line immunoassays (LIA), and methods for performing strip-based assays on a device. Specifically, the present invention relates to a device and a method for performing strip-based assays.

Background of the invention In clinical testing emphasis is often on identification or quantification of nucleic acids and proteins present in a sample of a patient by probe-target nucleic acid hybridizations and antibody-antigen immuno-assays, respectively. Clinical laboratories increasingly rely upon automated assay equipment in order to handle large numbers of assays efficiently in terms of time and cost, and further, to increase the reliability of such assays by decreasing the amount of human intervention involved in such assays. This reduction in human intervention necessitates a corresponding increase in equipment and devices which ensure the accurate performance of such automated assays.

In the past decades, many high throughput automation devices have been introduced. Typical examples are described in e.g. US patent 5,698,450; US patent applications 2008/0318323, 2011/0262896, 2011/0275058, 2002/0098117; and WO2004/004905. All these devices are technically very complex and expensive. The dispensing, agitating and heating of reagents and other assay solutions, and the controlled pre-incubation, incubation, washing, and detection of the target of interest in the patient sample can, with such automated devices, still be problematic.

It is of vital importance that the patient samples be handled in a manner which prevents their contamination and mishandling of reagents in the assay protocol, respectively. Also, analyses of a limited number of patient samples on a high throughput automated device are cost-ineffective. A common shortcoming of such devices is their increased usage of reaction buffers and other liquids per run, as compared to the amount of buffers required during manual processing. This deficiency is largely related to the need of priming the tubings that supply dispensing units. Because of the increased liquid reagent usage, stemming from significant amount of liquid remaining within and not being retrievable from device surfaces and tubings (so called "dead volume"), said devices often do not allow efficient utilisation of reagents provided in common commercially-available diagnostic kits. The problem becomes even more pronounced if insufficient amount of samples is collected to perform a full run on an automated high- throughput apparatus - which is a common occurrence in diagnosing rarely occurring syndromes or diseases. In such instance, in order to save on reagents that will become lost in the dead volume of an apparatus, laboratory personnel will likely revert to either performing the analysis manually or waiting with performing an automated run until sufficient number of samples is gathered; both of which "solutions" are a common cause of delays in obtaining often urgently-awaited diagnostic results. Oftentimes, the analyses of only a few patient samples, e.g. in the laboratory of a small hospital or a medical home practice, are very time-consuming, often run consecutively, require trained personnel to perform precise measurements, etc. Each step of these analyses typically needs to be repeated several times, each time with inclusion of proper controls, to acquire statistically significant data. The processes are also often wasteful of costly reagents, prone to human mistakes, require numerous man-hours, and are generally slow.

Nucleic acid hybridizations in particular demand precise temperature control. Rates of hybridization and equilibrium concentrations of nucleic acid double strands depend strongly on temperature and therefore accurate comparisons between hybridization experiments require that the experiments be run at the same temperature and in the same well controlled conditions. In addition, precise temperature programming during diagnostic test is critical to ensure sensitive and specific binding.

There is thus a need for a device that can reduce the extent of manual manipulation during nucleic acid hybridisations and immunoassays, is flexible for processing different amounts of samples according to different protocols, allows precise temperature control, and which in addition minimises reagent waste and is

characterised by low dead volume. Summary of the invention

The present invention is a device for preforming strip-based assays in an efficient manner, using a straightforward arrangement of reagent and reaction vessels, which allows for simple automation of dispensing, agitation and heating steps of the assay. In particular, the invention provides a device for processing multiple biological samples in an automated manner, each sample comprising multiple nucleic acids, proteins or other target molecules or species of molecules. In further aspects, a device is provided that is characterised by low dead-volume and comprises a positive displacement dispensing unit for dispensing reagents, which uses only minimal volume of each reagent required per step of the assay for the analysis of each said sample. According to another embodiment, the device further comprises an integrated heating block for simultaneous heating of assay solutions in reagent containers and the reaction trays.

Brief description of the figures Figure 1 is a perspective view of a preferred embodiment of the device according to the present invention. A, B and C show the same embodiment of said device but with differently positioned removable elements (A and B) or without them (C).

Figure 2 is a perspective view of the layout of the assay platform of a possible embodiment of the device according to the present invention. A and B show the same embodiment of the device from different perspectives.

Figure 3 provides a perspective view of an alternative embodiment of the device of the present invention (A), view of the layout of its assay platform (B), and a cross-sectional view (C) along the line A- A of the assay platform from B.

Figure 4 A shows a dispensing and aspirating system of the embodiment of the device of Figure 1 and according to the present invention. B and C schematically show a dispensing and aspirating system, respectively, according to another embodiment of device of the present invention shown in Figure 3.

Figure 5 A and B show a transverse and longitudinal section, respectively, of the temperature controllable tray comprising reaction vessels of the device of Figure 1 and according to one possible embodiment of the present invention. C and D is a transverse and longitudinal section, respectively, of the temperature controllable tray comprising reagent containers and reaction trays of the device of Figure 3 and according to another possible embodiment of the present invention.

Figure 6 is a perspective view of reaction trays of possible of embodiments of the present invention. A and B are the reaction trays of the device from Figure 1 and 2 and according to a possible embodiment of the present invention. C shows the reaction trays of the device of Figure 3 and according to another possible embodiment of the present invention.

Figure 7 A schematically shows a sloped shape of a reagent vessel according to a possible embodiment of the present invention, which facilitates accumulation of reagent liquid in the lowest- situated point (concavical indent shown in B) within the bottom surface of a reagent tray.

Definitions As used herein "automated" refers to applications and methods that are primarily performed on a programmable platform, such as a device or robot, with minimal manual manipulations and human involvement. "Semi-automated" as used herein shall refer to applications and methods that are partially performed on such programmable platform but are also combined with substantial amount of manual handling.

As used herein, a "sample" or "biological sample" shall refer to a biological specimen such as any tissue, body fluid or organic matter-containing material obtained from a human. Biological samples in accordance with the invention include peripheral blood, plasma, serum, bone marrow, urine, bile, mucus, cerebrospinal fluid, stool, biopsy tissue including lymph nodes, respiratory tissue or exudates, gastrointestinal tissue, cervical swab samples, semen or other body or cellular fluids, tissues, secretions, or materials. Often biological samples are diluted or contained within a various diluents, transport media, preservative solution, or other fluids. As such, a biological sample of the present invention is intended to encompass a biological sample contained within a diluent, transport media, and/or preservative or other fluid intended to hold a biological sample.

As used herein, "reaction vessel" refers to any container, tube, vial, or other vessel appropriate for accommodating a reaction strip and holding reaction fluid that can be utilised in an assay. Whereas, as used herein "reagent vessel" will be understood as any container, tube, vial, or other vessel appropriate for holding assay reagent fluid that can be utilised in an assay.

As used herein "positive displacement" refers generally to the idea of moving a liquid volume with an advancing mechanical part, such as a piston head, so that said volume is displaced by said part and pushed forward. A common example of a positive displacement fluid dispenser includes a piston pump. The pressure of the dispensed fluid can be relatively low as compared to pressure pumps, which provides a better flow of material from the dispenser to form a uniform and controlled fluid application.

The term "reagent strip" as used herein shall refer to a commonly used diagnostic tool that is well recognised in the art. As used herein, a reagent strip or, simply, a strip should be understood as relatively thin, narrow, comparatively long piece of material, in molecular biology applications usually being nitrocellulose, nylon, or polyvinylidene fluoride (PVDF), impregnated or conjugated with an at least one reagent to a given substance, used in testing for that substance in a body fluid or other secretion. As used herein, a strip-based assay or a strip-assay shall designate any substance-detection test utilising a strip-immobilised reagent allowing capturing and detection on said strip of the substance of interest, usually in a defined position of a strip.

Two popular types of strip-based assays are line probe assay (LiPA), using nucleic acid hybridisation to detection probes as detection principle; and line immunoassay (LIA) that employs formation of immunocomplexes between peptides or proteins and spec antibodies, for the detection of the presence of any of the latter.

Line probe assay (LiPA) technology usually involves the following steps: First, nucleic acid is extracted from clinical specimens. Next, polymerase chain reaction (PCR) amplification of the DNA target under question is performed using labelled, for example biotinylated, primers. Following PCR amplification, the labelled PCR products are hybridised with specific oligonucleotide probes immobilised on a strip. The captured labelled hybrids are detected by usually colorimetric development, enabling detection of the presence of DNA targets and test controls. The post- hybridisation reaction leads to the development of coloured bands on the strip at the site of probe binding and is observed by eye.

In line immunoassay (LIA) applications, purified, recombinant, or synthetic antigens are fixed as fine lines on the strip, allowing formation of immunocomplexes with a range of antibodies from body fluids, mainly serum or plasma, when applied on the strip. Alternatively, specific antibodies, usually monoclonal or recombinant, can be immobilised in lines on a strip and form immunocomplexes with their target proteins or peptides when the latter are present in a biological sample applied on the strip. In both instances, such formed immunocomplexes are then usually detected by means of labelled, for example biotinylated, secondary antibodies and a colorimetric

development resulting in the appearance of visible bands (or lines) on the strip where target molecule was present.

Description of the invention

The present invention provides a device for preforming reagent strip-based assays in an efficient manner and with minimal buffer usage, using a straightforward arrangement of reagent and reaction vessels, which allows for simple automation of dispensing, agitation and heating steps of the assay.

The invention is a device that can be used to carry out strip-based nucleic acid hybridizations, such as LiPA, and immunoassays, such as LIA, in an automated, accurate, safe, economical, and user friendly manner. The device comprises an assay platform consisting of a dispensing unit with low dead- volume, reaction vessels suitable for accommodating one or more reagent strips, and optionally reagent containers or vessels, a waste container, a heating means; and an agitation means. The present invention further provides a device capable of precise thermal and fluid control, which is user-friendly and easily programmable.

According to one embodiment of the invention, said device comprises a dispensing unit or head that utilises positive displacement, also known as volumetric dispensing mechanism, for the supply of reagent fluid to the dispenser head and to provide uniform and consistent application of dispensing units onto the surface of reagent strips.

Positive displacement generally refers to mechanism of setting fluid into motion through physical action of a mechanical element, such as a piston head or a tooth of a revolving gear, advancing towards fluid's position, pushing on it and thus displacing it or pushing it out from its initial location. Fluid dispensing based on positive

displacement is believed to ensure most consistent deposit sizes independent from environmental factors. Thus, in a preferred embodiment, the present invention provides a device for processing multiple biological samples in an automated or semi-automated manner, comprising at least one reagent vessel, and at least one reaction vessel suitable for accommodating one or more reagent strips, and a dispensing unit that dispenses fluic based on positive displacement.

In particular, reciprocating-type positive displacement dispenser heads comprising a barrel with a piston or a plunger, such as piston pumps, pipettes, or syringes; allow precise controlling of liquid manipulation and are characterised by much lesser dead volume as compared to the commonly used pressure dispensers with tubings.

Therefore, such dispenser heads allow much better usage of buffer and reagent volumes available in commercial assay kit. In a possible preferred embodiment, the device according to the present invention attains dead volume as low as 1 ml or less, which is largely accomplished by the use of an advantageous positive displacement dispensing unit without or minimal amount of tubings, and, additionally, specific shape of reagent vessels. Positive displacement dispensing tools are known in the art, one example is provided in application WO1996008440. They can be electro-mechanically operated, e.g. using a stepper motor or a solenoid to advance and retract a piston inside the syringe barrel.

In a preferred embodiment, the positive displacement dispensing unit of the device according to the invention comprises a single channel dosing chamber, such as a singular syringe barrel with a retractable piston or plunger on the opposite end of the disposing end.

In another possible embodiment, said dispensing unit comprises a multiple channel dosing chamber, whose channels preferably can be separately programmed and operable. Such multichannel head would preferably comprise a singular piston in each physically separated channel or barrel of the chamber, which could possibly be separately programmable from other singular piston heads positioned in the

neighbouring channels.

In another preferred embodiment the dispensing unit of the device according to the invention is capable of dispensing reagent fluid in more than one dosing unit of reagent volume per dispensing head movement. In such instance, as the positive displacement dosing unit is positioned perpendicularly in relation to the plane of the reaction vessf suitable for accomodating strips, and while it moves from one strip compartment in said reaction vessel to another, it consecutively produces consistent reagent fluid deposits in said compartments as a result of an advancing piston's downward movement inside of the dosing unit barrel. In such instance, the piston's head can be stopped at different positions inside of the barrel, each time dispensing a portion of fluid volume or a dosing unit. In a preferred embodiment, volumes of more than one possible dosing unit can be programmed for different dispensing cycle of the dispensing unit.

Dispensing cycle as used herein should be understood as consecutive disposing of reagent liquid of fixed volume by a multiple-dosing dispensing unit that is performed per unidirectional horizontal movement of said multiple-dosing dispensing unit parallel with respect to and above the reaction vessel.

The device according to the present invention largely accomplishes significant reduction in dead volume through implementation of a dispensing unit that extrudes fluid using positive displacement principle, as described in the afore-mentioned embodiments. In additional embodiments of the present invention, a further

improvement in reduction of excessive buffer usage can be achieved by using reagent vessels of a shape that allows collection of reagent fluid in a particular spot or area of the vessel, thus increasing its local availability during the uptake by a dispensing unit. In a general embodiment of the present invention, a device is provided, wherein dispensing unit uptakes reagent fluid from the lowest-lying area in a reagent vessel.

In a particular embodiment, the inside part of a reagent vessels comprises slopes that facilitate accumulation of the fluid in the lowest point within the reagent vessel. In an alternative embodiment, said reagent vessel comprises an indentation such as such as a groove, concavity, or a hollow point. Alternatively, lowest-lying area in a reagent vessel is created through tilting of the reagent vessel caused by the action of pivoting or agitation means. In a particular embodiment, the lowest-lying area in a reagent vessel can be obtained by the combination of any of the above, i.e. slopes, indentation, or tilting. As a result, the present invention provides a straight-forward device for automation < strip-based assays, which is compatible with and allows for optimal use of reagent volumes provided in commercially available kits. According to another embodiment said device further comprises at least one heating block, likely a metal block, wherein said metal is preferably aluminium. In a particular embodiment, said heating block provides simultaneous heating for at least one reagent vessel and at least one reaction vessel. The device may further also comprise an edge pivoting means that allows agitation of reagents that is advantageous during incubation phases according to certain assays. Said pivoting means may provide pivoting to a singular chosen component of said device, such as advantageously the reaction vessel, or, preferably, provide pivoting to multiple components optionally positioned in a tray. In a preferred embodiment pivoting means provides pivoting for at least one reaction vessel and optionally also for at least one reagent vessel or at least one heating block.

In another aspect of the invention, it is advantageous that said device further waste removal means such as a waste aspiration unit or head, preferably mounted next to and moving together with the dosing unit. Such waste aspiration unit can be designed according to any standard implementations known in the art, for example may utilise pressure pump with tubings to aspirate the waste liquid to a removable waste collection vessel. According to another embodiment the device further comprises a tray with reagent vessels and reaction vessels, wherein the reaction vessels can partially be covered with covering means, such as a lid, in order to reduce liquid reagent losses caused by evaporation.

In a preferred embodiment of the present device, the reaction vessel and/or at least one reagent vessel is covered by a lid, either fully or partially. In a further embodiment, said lid can be pressurised or contain locking means such as a clamp, or both.

In another embodiment of the device, at least one reagent vessel and at least one reaction vessel may be positioned in a tray. In a further embodiment, a device according to the present invention may comprise means to read and feed to a computer with appropriate software, a picture of a final result of the assay, most likely herein being a banding pattern on the strip in a reaction vessel. Such means can be exemplified by optical devices various types of cameras with input means and are well known in the art and readily usable is automated assay platforms.

The device according to the invention may be used in an assay for detecting, e.g.

specific nucleic acids or antigens in a sample. Furthermore, the device may be used in an assay for detecting, e.g. aberrations in nucleic acids and proteins and derivatives thereof, and other biochemical compounds present in a bodily sample. Furthermore, the device may be used in multiparameter assays investigating different nucleic acids and/or different immunological properties simultaneously for a given sample. For nucleic acid based applications, typical examples are genotyping assays and drug resistance assays for viral and bacterial pathogens. Multiparameter panels detecting the presence of nucleic acids of disease-causing agents in infectious diseases like e.g. gastro-intestinal panel, sepsis panel, meningitis panel, food-pathogen panel, etc. can be run in the device. Other applications looking at different polymorphisms or mutations in a single or in different genes simultaneously are used often in genetic testing.

Examples are e.g. mutation testing for cystic fibrosis, HLA typing for transplantation or disease association studies, SNP polymorphisms in pharmacogenetics, etc.

For immunological applications, typical examples are confirmatory assays used in infectious disease testing. The device may be used for examining the presence of antibodies against different antigens simultaneously to confirm and discriminate different viruses in a clinical sample, e.g. HTLV I and II or HIV-1 and HIV-2. Other applications which require investigation of antibodies against different antigens simultaneously may be for diagnosis of auto-immune diseases. Yet other assays make use of sub-variants of the same antigen which are being detected by different antibodies.

Although the invention is described herein with reference to particular embodiments of the device, the descriptions are intended to be exemplary in nature and should not be construed in a limiting sense. Those skilled in the art will readily appreciate that various aspects of the invention may be used with differently-designed fluid dispens apparatus operating on the positive-displacement principle, without specific limitation as to the dispensing head shape, design, motor, means of moving the dispensing head and so on.

Figure 1 shows a perspective view of one embodiment of a device (100) that can be used to perform strip-based assays. The device (100) shown in Figure 1 includes a housing (101), a control panel (103), and an assay platform (104). The device (100) can optionally be covered by a lid (not shown). A, B and C show the same embodiment of said device but with differently positioned removable elements (A and B) or without them (C). In C the position of the dispensing head (115) is indicated.

The number of assay platforms (104) can vary to any number, and is preferably 1, preferably 2, or preferably ranging from 1 to 4. Each assay platform can operate independently. In a preferred embodiment the device (100) according to this invention runs stand-alone when performing assays. The device can be programmed in desirable embodiments to perform a variety of dispensing, heating, agitating and incubating steps with each step having a preselected period of time within which to occur. The control logic can be modified to accommodate other assays or other procedures.

Applications on a PC or USB or memory card or the like are required for service software, manufacturing tools, e.g. calibrations and tests, protocol development or protocol download. In an embodiment the control panel (103) is a LCD display and keypad, wherein the LCD display can be standard 7-segment, alpha numeric, or custom designed; the keypad is a membrane style keypad with approximately 6 to 10 keys; and holds indicator and status LEDs, e.g. a power indicator, alarm, and in progress signal. The following approximate physical specifications are used with desirable

embodiments of the device. The overall length is 50 to 80 cm, desirably 62 cm. The overall width is 30 to 80 cm, desirably 32 cm. The overall height is 15 to 60 cm, desirably 48 cm. The total weight is desirably less than 25 kg. These dimensions provide a commercially desirable device that can be readily used in most laboratories for performing automated solid support based nucleic acid hybridizations and immunoassays. Figure 2 shows a perspective view of one embodiment of the assay platform (104) tl can be used to perform strip-based assays according to the present invention. A and B show the same embodiment of the device from slightly different angles. The assay platform (104) includes a dispense head (115), rail for moving the dispense head (133), reagent containers (111), a waste container (112), and a reaction vessel (126).

According to this embodiment, the dispense head (115) comprises a single channel dosing chamber comprising a barrel (146) that terminates with a dispense needle (131).

Figure 3 A shows a perspective view of another embodiment of a device (100) that can be used to perform strip-based assays, comprising a multichannel dispense head (116). The device further includes a housing (101), a cover (102), a control panel (103), and an assay platform (104). As in the device embodiment shown in Figure 1, the number of assay platforms (104) in the present embodiment can also vary to any number, and is preferably 1, preferably 2, or preferably ranging from 1 to 4. Similarly, each assay platform of the present embodiment can operate independently and preferably runs stand-alone when performing assays. The following approximate physical

specifications can be used with the present embodiment of the device: the overall length is 50 to 80 cm, desirably 63 cm. The overall width is 30 to 80 cm, desirably 38 cm. The overall height is 15 to 40 cm, desirably 24 cm. The total weight is desirably less than 20 kg. B shows a perspective view of the possible embodiment of the assay platform (104) of the device embodiment from A. Said assay platform (104) includes a multichannel dispense head (116), reagent containers (111), a waste container (112), and a tray (120) composed of reagent containers (113) and reaction trays (114). C shows a cross-sectional view of the same assay platform embodiment (104) from B, showing a multichannel dispense head (116), reagent containers (111), a waste container (112), and a tray (120) composed of reagent containers (113) and reaction trays (114). The reaction trays (114) can be covered with a cover lid (121) by a handle bar (122). In a preferred embodiment the tray (120) contains a heating means (123) and insulating means (124). In an embodiment according to the present invention agitation of reagents is accomplished with an edge pivoting rocking mechanism (134) and (135). In an embodiment the dispense head (115) is mounted on a dispense head holder (132) and can move along a rail (133) in X direction for parallel transfer of assay reagents to and from the reaction tray (114). According to the most preferred embodiment of the device according to the present invention, the dispensing head (115) or a multichannel dispensing head (116) utilises positive displacement as the mechanism of supplying reagent fluid to reagent vessels. In a preferred embodiment, the dispensing unit is a reciprocating-type positive displacement dispenser comprising at least one barrel and a piston or a plunger that are operate and moved within the barrel electro-mechanically, e.g. using a stepper motor or a solenoid.

In a preferred embodiment, the device according to the present invention is

characterised by a minimised dead volume thanks to the use of positive displacement dispense head (115 or 116), special shape of the regent vessels (111 or 113) and minimised to none involvement of conduits or pumps, e.g. plastic tubing and peristaltic pumps, for assay solution transfer between reagent containers and reaction vessels. In a preferred embodiment the device according to the present invention is free of conduits or tubings for handling assay solutions. Consequently, there is no need for preloading or priming the device according to the present invention with assay solutions as it is required in the state of the art devices. This additionally spares a considerable amount of reagent solution volume and enables full use of tests assays delivered in a diagnostic kit, e.g. a kit for 10 tests contains assay solutions to run 10 test without the need to add extra volume of assay solutions to compensate for waste of said solutions by preloading or priming liquid conduits in the device.

According to one embodiment of the present device, a dispensing head can be positioned and moved together with means for liquid waste aspiration. For example, Figure 4 A shows such dispensing and aspirating system of the embodiment of the device of Figure 1, and according to the present invention. The dispensing part of said system consist of a dispensing head (115) and means for operating it (not shown). The dispensing head according to this embodiment of the present invention comprises a barrel (142) as a single-channel container for reagent fluid, a piston (143) contained inside of the barrel (142), piston arm and piston actuator (144) that can move the piston within the barrel chamber, and the dispense needle (131). The waste aspiration system according to this embodiment comprises a drain feature (140; schematically indicated by an arrow) terminated with a waste aspiration needle (139), which aspirate the liquid waste to a waste collection container such as a waste bottle (not shown) by any of means known in the art, e.g. via a peristaltic pump. According to the present embodiment, both the dispense needle (131) and the waste aspiration needle (139) are moved together by means of a needle height actuator (145) and or needle height solenoid actuator (146). The dispensing and aspirating system of the embodiment shown in Figure 4 A can be advanced by a step motor along a rail (not shown) positioned above the reaction vessel and reagent vessels. Possibly, the step motor moves the dispense head by means of a cord, chain, belt or equivalent flexible device. In a preferred embodiment the dispense head moves in a direction for dispensing, aspirating, and optionally mixing assay solutions. Movements in this direction can be accomplished by the same movement means as for X direction movement of the dispense head. In a preferred embodiment, movement of the dispense head is fully automated and robotic. In another possible embodiment, the dispensing unit of the device of the present invention is a multichannel dispensing head (116) comprising a multiple channel dosing chamber which can be schematically represented in Figure 4 B. Such multichannel dosing head (116) would preferably comprise a singular piston heads in each of the physically separated channels or barrels of the chamber, which could preferably be operable independently from other piston heads contained in the neighbouring channels of the dispensing head. The dispensing head (116) consists of multiple individual dosing chambers terminating in dispense needles (131), and is advantageously advanced along a rail (133) above reaction and reagent vessels by a step motor. The number of dosing chambers is equal to the number of compartments for strip in reaction trays (114). Controlled positive-displacement-based delivery of reagents with the dispense head (116), comprising dosing chambers and actuator, is advantageously compared to the use of conduit and pump reagent delivery. The use of such-designed dosing chambers with dispense needles (131) as the source of the assay solutions eliminates dead volume, thereby reducing the loss of valuable reagents such as antibodies and enzymes that are generally expensive and in a limited supply.

In a preferred embodiment, the dispensing head (116) is controlled in such way that only the exact volume of each assay solution required to carry out the protocol of choice is aspirated from the reagent containers (111 or 113), and the exact same volume of each said assay solution is expelled in the reaction vessel (126) through the disper needles (131) of the dispense head (116) with little to no waste. The exact volume of each assay solution for each step of the protocol is defined herein as one unit. For clarity, according to the user manual of a diagnostic kit, an assay solution can be used in different steps of the protocol at different volumes, each of which represents one unit of the same assay solution. In a preferred embodiment, Figure 4 B and C depict aspirating an assay solution from a reagent vessel (111) into a reaction vessel (126) before the incubation step, and aspirating said assay solution from reaction vessels (126) into the waste container (112) after the incubation step, respectively.

In some embodiments, the waste container (112) includes a drain feature (140), which allows for removal of assay solutions from the assay platform (104). In some embodiments, the drain feature (140) is a plastic conduit and is connected to a waste bottle (141). In some embodiments, the conduit or the waste bottle is coupled to a pump, such as a peristaltic pump, a piston pump, a hydraulic pump, a vacuum pump or the like.

In another preferred embodiment the dispensing unit (115 or 116) is capable of dispensing reagent fluid in more than one dosing unit of reagent volume per full dispensing unit movement. As the dispensing head (115 or 116) moves above different reaction vessels (126) comprising strips, it disposes defined reagent fluid deposits, in a manner that one such fluid deposit or a dosing unit lands in exactly one reaction vessel as a result of movement of a piston or pistons (143) inside of the dosing unit channel (142) or channels, respectively. According to such embodiment, the piston's head (143) can be stopped at different positions inside of the chamber (142), each time resulting in deposition of exactly one dosing unit.

In preferred embodiments, the dispense needles (131) are non-disposable and preferably consist of inert material not interfering with assay solutions and sample material. In some other embodiments, the dispense needles (131) are disposable.

In some embodiments, the dispense head (115 or 116) includes a temperature control feature, which heats the assay solutions as required. In some embodiments, the dispense head (115 or 116) includes a temperature control feature, which cools the assay solutions as required. In some embodiments, a dispense head (115 or 116) includes a temperature control feature, which heats or cools the assay solutions as required. In some embodiments, the dispense head (115 or 116) includes a volume control feature, e.g. optical sensor(s), timer(s), pressure sensor(s) for dispensing and aspirating assay solutions.

In some embodiments, the dispense head (115 or 116) continuously dispenses and aspirates an assay solution or mixture of assay solutions into and from reaction vessels (126) as an agitating means as required during an incubation step.

Figure 5 A and B is a transverse and longitudinal section, respectively, of the reaction vessel (126) positioned on a reaction tray or reaction vessel tray (114), possibly comprising a cavity (125), on a heating block (123), and covered with a lid (121). In a preferred embodiment a reaction vessel (126) is placed in a reaction tray (114). During processing, the cover lid (121) presses the reaction vessels (126) against the reaction trays (114) that are in direct contact with the heating means (123).

C and D is a transverse and longitudinal section, respectively, of tray (120) comprising components as disclosed herein above annotated with the same numerical annotations. In a preferred embodiment the reaction tray has a cavity (125) for cooling air. C and D also provide a perspective view of a reaction vessel (126), that in a preferred embodiment is placed in a reaction tray (114). Also in this embodiment, during processing, the cover lid (121) presses the reaction vessels (126) against the reaction trays (114) that are in direct contact with the heating means (123).

The reaction vessel unit can be a single reaction vessel or the reaction vessel unit can consist of any number of reaction vessels up to the maximum number of reaction vessels that can be hold by the reaction tray (114). In a preferred embodiment the reaction vessel unit contains 1 to 10 reaction vessels, preferably 1, preferably 10 reaction vessels.

Each reaction vessel (126) is designed to hold a reagent strip immobilising at least one reactant, preferably multiple reactants immobilised in parallel lines on the strip. In a preferred line immunoassay (LIA), the reactant will usually be an antigen or antibody. In a preferred nucleic acid hybridization assay (line probe assay or LiPA), the reacta will usually be DNA, RNA, or modifications thereof, such as said nucleic acids having incorporated therein chemical modifications for improvement of nucleic acid duplex formation and stability, e.g. 2-O-methyl groups.

The immobilised reactant may be a component of a sample, or it may be a reagent which acts upon the sample or upon another reagent (e.g., a secondary antibody which binds a primary antibody, or complementary nucleic acids). The reaction component may be reversibly or irreversibly immobilized on the support by any art-recognized technique. It may be bound covalently, or noncovalently, and directly or indirectly.

The strip is sequentially exposed to other reaction components, such as samples, reagents, and washing solutions. At the end of the assay sequence, the assay results are determined.

The samples of the present invention may be biological fluids such as blood, plasma, sera, CSF, saliva, sputum, tears, urine, milk and the like, or tissues which have been solubilized for assay purposes, or standard samples, e.g. consisting of protein materials. The reagents may be antibodies (or other binding proteins), antigens (or other target molecules), nucleic acids, enzymes, enzyme substrates, buffers, chromogens, small molecular inhibitors, fluorophores, and the like.

In some embodiments, there are 10 reaction vessels (126). Optionally, 8 reaction vessels (126) are for analyses of samples and one each for a positive and a negative control. Alternatively, proper controls are already immobilised on reagent strips designed for sample analysis. In this event, preferably, none of the reaction vessels (126) is used for control purposes only.

In a preferred embodiment multiple strips can be processed in parallel on a single assay platform. The means to permit rocking can be a pivoting connection, such as an axle or hinge, an embodiment the axis of rotation is on an end of the tray (120), said end of the tray remains essentially fixed and the other end moves up and down.

The rocking mechanism of this invention is desirably provided by a step motor (stepper motor; 134). The step motor (134) rotates the tray (120) both clockwise and

counterclockwise about an axis of rotation. The axis of rotation of the tray (120) and rail (133) for movement of the dispense head (115) are perpendicular but not necessarily in the same plane.

In some embodiments, a tray (120), a reaction vessel (126) preferably positioned on a reaction tray (114) on a heating block (123), or at least one reagent vessel (111) is pivoted about an axis of rotation, with the reaction trays (114) being furthest from the axis of rotation. The afore-mentioned elements can be gently rocked to enable the reagents, sample or wash solution to spread evenly over the solid support means and mix uniformly with any soluble assay components already in the reaction vessels (126).

In a preferred embodiment an assay solution is removed from the reaction vessel (126) by either rotating the tray (120) to a positive angle of rotation for the tray (120) and aspirating the solution from the reaction vessel (126) by a waste removal means or, in an alternative embodiment, by a dispense head (116). The aspiration position of the tray (120) is desirably held for a time sufficient to aspirate the assay solution completely. In a preferred embodiment, the tray (120) is tilted up to an inclination enabling efficient aspiration of the solutions away from the solid support means.

In a preferred embodiment the number of reagent vessels or containers (111) equals the number of assay solutions to be kept at room temperature and required to run the assay of choice. This number can vary between 1 and 10, preferably between 3 and 7, and is 5 in a preferred embodiment. In some embodiments the reagent containers (111) can be disposable and configured for single use. In some embodiments the reagent containers (111) can be made of reusable material. The assay solutions can be wash solutions, buffers, distilled water, and the like needed to run the assay of choice, and can be supplied as a kit, optionally in prefilled disposable reagent containers. The following operating specifications are used with desirable embodiments of the device and method of this invention. Input voltage for the apparatus can be 110 or 220 volts. Operating temperatures range from about 4 to about 99 degrees Celsius, preferably from ambient temperature to about degrees Celsius. A preferred ambient temperature can be room temperature or even less when the device is placed e.g. in a refrigerated environment.

Figure 6 shows possible embodiments of positioning reagent vessels (126) onto a heating block (123) and covering them with a lid (121).

In one embodiment shown in Figure 6 A and B, a multi-compartment reagent vessel (126) comprising a handle protrusion (152) allowing easier handling, is positioned directly onto the heating block (123). The multi-compartment reagent vessel (123) can be covered by a lid (121) and possibly locked under the lid (121) or stabilised by a clamp (153).

The cover lid (121) prevents evaporation of assay solutions in the reaction vessels (126) and limits contamination of a reaction vessel (126) by assay solutions or patient sample from a neighbouring reaction vessel (126). In a possible embodiment, the lid (121) is transparent, or as shown in another possible embodiment of Figure 6 A and B, the lid (121) comprises a transparent plate positioned within a frame, which allows

observation of the strips possibly present in the reaction vessel. The transparent lid or its elements can be made of glass, or preferably from a transparent thermoplastic such as poly(methyl methacrylate) (PMMA) or polycarbonate (PC), or any other suitable material accepted in the art, while the frame can preferably consist of metal, PMMA, or PC, or another material known in the art. The cover lid (121) can also be constructed from a high temperature plastic to prevent sagging or softening at the higher operating temperatures of the device (100). A suitable plastic may include polysulfone.

Polysulfone possesses the requisite temperature characteristics and is transparent, which allows direct viewing of the solid support means during processing. In a particular embodiment, frame of the lid (121) may also comprise a handle protrusion (151) for easier opening and closing of the reaction vessel compartment. In an alternative embodiment shown in Figure 6 C, the reaction vessels (126) are held in place in the reaction trays (114) by a cover lid (121) that includes a handle bar (122) for opening and closing the reaction trays (114). In this exemplary embodiment, duri processing, the cover lid (121) presses the reaction vessels (126) against the reaction trays (114) that are in direct contact with the heating means (123).

In a possible embodiment the cover lid (121) partially covers the reaction vessels (126) leaving at least one opening to add or remove assay solutions or a patient sample in each reaction vessel (126) according to the assay protocol of choice. Furthermore, there can be two openings, one at each end of the reaction vessel (126), the one closest to the reagent containers (113) for dispensing and aspirating assay solutions and the second one for adding biological sample in the reaction vessel (126).

In some embodiments the reaction vessels (126) can be disposable and configured for single use. In some embodiments the reaction vessels (126) can be made of reusable material. The reaction vessel (126) can be preloaded with strips according to the assay of choice. The reaction vessel (126) may also be of any desired size or form which can be accommodated by the reaction tray (114). A preferred form is cubical or cylindrical. The reaction vessel (126) may be placed in any direction, preferably horizontal or vertical. In a preferred embodiment the reaction vessel (126) is cubical and placed horizontally. In some other embodiments the reaction vessel (126) is cylindrical and placed vertical. The reaction vessel (126) can be supplied as part of a kit.

Assay solutions such as buffers and wash solutions to be used at ambient temperature are desirably placed in reagent containers (111). Other assay solutions to be used at elevated temperatures are desirably placed in reagent containers 113. The volume of assay solutions dispensed to the reaction vessels 126 is less than 0.5 ml, 0.5 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, more than 3 ml. In a preferred embodiment the volume is desirably 1 ml and desirably a multitude of 1 ml for a reaction vessel (126) having a preferred solid support section of approximately 0.32 cm by 10 cm. The aggregate volume of assay solutions dispensed depends on the number of samples being assayed. A desirable reagent container volume is 180 ml for washing buffer and 30 ml for reagent solutions. The approximate volume of reagent solutions required for a complete assay of a full tray (120) is desirably about 180 ml of wash buffer and about 30 ml of each reagent solution. Also provided is a temperature control device configured to control the temperature of the assay solutions in the tray of the assay platform.

In a preferred embodiment the heating means (123) includes a thermal plate that is designed and constructed to maximize heat transfer between Peltier devices and the reagent containers (113) and reaction trays (114), all part of tray (120). The thermal plate is designed to provide rapid temperature response and uniform temperature distribution across the tray (120) in general, and the assay solutions in reagent containers (113) and in the reaction vessels (126) in particular. The thermal plate has minimal thermal mass and a high degree of flatness to maximize thermal contact area. The heating means (123) is typically capable of temperature accuracies of about 0.5° C. A heating means (123) provides temperatures from ambient temperature to 100 degrees Celsius. Preferably, the heating block is manufactured from a solid piece of metallic material of high thermal conductivity and, most preferably, it is casted from an aluminium or copper alloy.

In some embodiments, the heated reagent container (113) can have a lid for insulating said container and avoiding evaporation of assay solutions dispensed herein. In one possible embodiment, each lid can have entry ports, each configured to allow entry of the dosing needles (131) of the dispense head (115).

In some embodiments, the reagent containers (111) and/or (113) contain appropriate shapes, e.g. slopes, or structure, such as groves or indentations, in order to create a lowest positioned area or point within the reaction vessels (111) in which reagent fluid will accumulate. This solution allows for further reducing the losses of assay reagents, minimising dead volume of the device, and fully maximising the use of reagent volumes. A schematic example of such shaped reagent vessel containing a slope (154) is shown in Figure 7. The panel A shows a cross section of a sloped reagent container whereas B shows a possible indentation (155) which serves accumulation of reagent liquid into one spot, form which said liquid can be aspirated with minimal losses through an aspiration needle (131) of a dispensing head (115 or 116). In an alternative embodiment, the lowest point wherein the reagent fluid accumulates in a reagent vessel (111 or 113) is created by either rotating or tilting said vessel by pivoting means. In a preferred embodiment the lowest-lying area in a reagent vessel is created through a combination of any of the above-exemplified solutions including introduction of slopes in the reagent vessel, indentations, or by tilting said vessel. In some embodiments, the tray (120) can also include a cooler, such as a ventilated Peltier junction thermoelectric cooler, a fan, or the like. In some embodiments, passive air flow is used for cooling tray (120). In some embodiments, cooling of reaction vessels (126) and/or reagent containers (113) in tray (120) takes place through cavities for cooling (125).

The herein described device has many significant advantages. Automating and controlling the dispensing of assay solution from a singular doing chamber or multiple dosing chambers, preferably capable of dosing reagents in multiple dosing units, eliminates a significant amount of waste that would occur if the assay solutions were pumped into the reaction vessels with a pump and tubing system. Also, all biological samples are effectively and identically processed within the assay platform.

In some embodiments all reaction trays (114) available in the tray (120) are loaded with a reaction vessel (126) containing a solid support for analysing a biological sample. Each reaction vessel (126) is visited by a dispense needle (131) and receives assay solutions and is processed according to the protocol of choice. In other embodiments, only some reaction trays (114) in tray (120) are loaded with a reaction vessel (126) containing reagent strips for analysing a biological sample. The dispense head (115) is programmed such that all reaction trays (114) are visited by a dispense needle (131) but only those containing a reaction vessel (126) containing a reagent strip for analysing a biological sample receive assay solutions and are processed according to the protocol of choice.

The device according to the invention can be used for processing multiple biological samples, each said sample comprising multiple nucleic acids, proteins or other target molecules or species of molecules. In a preferred embodiment said device is a simple straightforward device for small-scale diagnostic applications, allowing processing of 5, 8, 10, up to 20 reagent strips in said reaction. In an embodiment, an automated method of performing a strip-based nucleic hybridisation assay such as LiPA comprises: providing at least a nucleic acid hybridisation solution in a first reagent container, placing an undeveloped sample into a reaction vessel, contacting the undeveloped sample with the hybridisation solution by automatically activating a dispensing head to transfer the hybridisation solution from within the first reagent container into the reaction vessel, automatically removing the hybridisation solution by the waste aspiration means from the reaction vessel after the incubation step, transferring from a second reagent container a wash solution into the reaction vessel by the dispensing head, and automatically removing the wash solution from the reaction vessel by the waste aspiration means after the incubation step. In a further embodiment the hybridisation solution in the first reagent container and hybridisation solution in the reaction vessel are kept at the same temperature, preferably a temperature optimal for nucleic acid probe - target hybridization, e.g. 49 degrees Celsius. In a further embodiment the first reagent container and reaction vessel are heated by the same heating means.

In an embodiment, an automated method of processing an strip-based immunoassay such as LIA comprises: providing at least an antibody solution in a first reagent container, placing an undeveloped sample into a reaction vessel, contacting the undeveloped sample with the antibody solution by automatically activating a dispensing head to transfer the antibody solution from within the first reagent container into the reaction vessel, automatically removing the antibody solution by the waste aspiration means from the reaction vessel after the incubation step, transferring from a second reagent container a wash solution into the reaction vessel by the dispensing head, and automatically removing the wash solution from the reaction vessel by waste aspiration means after the incubation step. In a further embodiment the antibody solution in the first reagent container and antibody solution in the reaction vessel are kept at the same temperature, preferably a temperature optimal for antibody - antigen interaction, e.g. 37 degrees Celsius. In a further embodiment the first reagent container and reaction vessel are heated by the same heating means.

The embodiments disclosed herein relate to systems and methods for reducing the amount of manual manipulations required for solid strip-based nucleic acid hybridizations and immunoassays used in molecular biology applications, while preserving reagents, ensuring optimal use of available volume of reagents, minimising waste, and controlling temperature and assay solution transport.

The invention may be embodied in other specified forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, and all changes which come within the meaning and range or equivalency of the claims are therefore intended to be embraced therein.

Examples

The two overviews depicted in Figures 8 and 9 represent two standard work flows for use of the device according to the present invention for processing multiple biological samples in a nucleic acid hybridization and immuno-assay, respectively.