WO2012120169A1

WO2012120169A1 - Microplate for biological tests

Info

Publication number: WO2012120169A1
Application number: PCT/ES2012/070108
Authority: WO
Inventors: Roberto Hernan Izquierdo; Antonio Cruz Pacheco
Original assignee: Zf Biotox, S.L.
Priority date: 2011-03-09
Filing date: 2012-02-23
Publication date: 2012-09-13
Also published as: ES2358699B1; ES2358699A1

Abstract

The invention relates to a microplate for biological tests, which includes a series of wells formed by a receptacle (2) that is open on the upper side thereof and has a bottom that is flat or closed over the entire surface thereof, the lower part of the wall of the receptacle being provided with an opening (3) adjacent to the flat bottom.

Description

MICROPLACE FOR BIOLOGICAL TESTS

Field of the Invention

The present invention relates to a microplate for biological assays, especially for cellular assays or immunoassays (ELISA type).

More specifically, the microplate of the invention is of the type that comprises a plurality of small receptacles, called wells, which are open on its upper face and have an outlet opening in the lower part that flows into a vacuum chamber.

Background of the invention

There are a large number of laboratory operations that require the handling of multiple small volume samples. For example, both immunological techniques and cell-based assays require the continuous addition and removal of liquids. In order to handle this sample complexity, some types of containers commonly known as well plates or microplates are used, where biological and chemical reactions take place with the immobilized agents to the plate substrate. Such plates are typically made of plastic (such as polypropylene, polystyrene or the like) and have a series of flat-based wells arranged in a geometric pattern that simplifies the organization and conduct of operations. An extended general purpose plate is one that contains 96 wells in an 8 x 12 well rectangular pattern. There are other microplates available with different numbers of wells and lower volumetric capacities, such as 384, 1536 or even 3456 well plates, where access for the handling and evacuation of liquids from the wells becomes increasingly narrow and difficult.

In all experimental and screening techniques carried out in microplates, several steps are used where reagents, nutrients or wash solutions are added to each well and then removed. With the purpose of To obtain greater flability and accuracy of the results, it is often necessary to empty the wells between these steps and rinse them with a wash solution several times to remove the unspecified reagent remains. In the course of a screening test, you can get to work with a large number of microplates, making it necessary to evacuate each well in a large number of times. In immunological assays for example, the reagent is an antibody that specifically binds to an antigen. Commonly, a color reaction is formed in those wells where the antigen was present. Reagents from all wells should be washed in order to prevent color formation in the wells where the antigen was absent. Another example where you have to evacuate fluid from the wells of a microplate is in cell culture based assays. The cells used in experimental processes are seeded in microplates and need several washes between different reactions and treatments. All these washing operations can be performed manually or automatically. In manual operations, washing is carried out by individually aspirating the liquid from each well with a Pasteur pipette connected to a vacuum system or, in the best case, a whole column or row of wells at the same time. This manual method, in addition to being laborious, has other drawbacks such as the risk of contact of the suction pipette with the samples, the probability of liquid contamination between adjacent wells or the lack of homogeneity in the performance of test to test. Therefore, this process has been automated and there are currently several commercial equipment available that automatically add the reagents, evacuate the liquid from the wells, and dilute the samples in series. All these equipment operate in a similar way. Usually, the microplates are in a horizontal position and a series of needle-shaped collectors added by the part a washing solution to each well on top of the plate, followed by a rapid removal of the washing liquid by another set of aspiration needles, using a vacuum suction device. The suction pipettes must remove as much liquid as possible so that the washing is effective and minimizes the residual amount of reagents present in the wells base. This step requires precise and sophisticated technology that often consists of liquid sensor technologies. Technologies that use liquid sensors impose sophistication and extra cost on the equipment. Another fact that contributes again to the increase in the price of washing operations is that sometimes new sets of pipettes must be used for each wash in order to avoid contamination between successive washes, since the washing needles physically touch the liquid content in the wells.

Another of the limitations imposed by current technology is the difficulty of performing overflow washes in wells that have a very small diameter, due to spatial limitations where both the dispensing and suction needles must coexist. The overflow washing technique consists in adding and removing the liquid from the wells simultaneously in order to achieve a more vigorous washing of the wells. The disposal of residual liquids is of vital importance especially when working with very small amounts of liquids. The robots are programmed to go down to an exact fixed location in the space, but very subtle variations in the alignments between wells or the variations in the depth of these between different manufacturers, can lead to inconsistent washing. Finally, very thin needles may increase the risk of clogging during fluid evacuation. Various systems on the market try to avoid this problem by using ultrasound, which again makes current technology more expensive. In view of this background, a washing method that dispenses with the use of needles would contribute to a decrease in equipment maintenance, avoid residual contamination between washes, avoid the problem of clogging the aspiration needles, increase the processing speed and as a whole it would make it possible to reduce the washing operations of conventional microplates. Therefore, a simple, fast and low-cost needleless evacuation method that overcomes all the above-mentioned drawbacks would be required.

In the state of the art, the evacuation of the wells by their lower base through the use of vacuum is already described in numerous liquid evacuation systems. On the one hand, patents such as PCT / US02 / 00594, GB2422795 A, EP1366818 A1, EP1262759 Al, US2002098125 Al, US6133045 A, W09811989 A1, US4927604 A, EP0359249 A or E04021018 teach us the use of a vacuum source for the purpose of extracting a solid phase by means of filters that can retain bacteria or molecules such as nucleic acids or proteins. Another patent, such as WO2004113874 A2, teaches us the evacuation of liquids from the bottom of the rounded or concave base wells while the analytes are retained by a magnetic force to the well's walls. Finally, patents such as WO9102073 A or US4090850 A employ vacuum suction systems in conjunction with permeable membranes that retain the analytes present in immunoassays. Specifically, US4090850 A shows a washing method for radioimmunoassays whereby the flat base of the wells has a central hole through which the liquid is evacuated by suction while the radio ligands under study are trapped in a disk of cellulose paper

To facilitate the exit of liquids by means of a pressure difference caused by the vacuum, all the The patents described above possess some type of opening in the same base of the wells. None of the patents cited can therefore claim their use for substrates fixed to the base of the wells, such as immunoassays and cell assays. This is due to the fact that in all the patents described, the area destined for the biological substrate and the opening destined for the evacuation of liquids coexist in the same plane, being thus compromised the reduced space destined to the biological substrate of the wells of a microplate . That is why none of these patents can respond to the problem of washing microplates from flat wells where biological substrates such as antigens or cells are attached.

Description of the invention

The present invention aims at a microplate for biological assays, constituted so as to solve the exposed problem and allow the washing of plates or microplates used in the performance of cellular assays or immunoassays in which the biological substrates are immobilized or adhered to the bottom. or base of the wells and whose washing so far required the use of a large number of pipette sets, with the consequent economic and time expenditure.

The microplate of the invention is of the initially indicated type, consisting of a plurality of wells in the form of open receptacles on its upper face and provided with an outlet opening in the lower part that leads to a vacuum chamber.

According to the invention, each well is constituted by a receptacle whose bottom is flat and closed on its entire surface, having a hole in the wall that is located adjacent to the flat bottom.

The flat bottom allows biological substrates to be immobilized or adhered to it, not the position of the hole compromising the study area where said substrate is fixed.

The special arrangement of the exit orifice allows the wells to be usable not only for analytes that are in suspension, but also for tests in which biological substrates anchored or adhered to the flat bottom or base of the well are used. The evacuation hole is located in the lower part of the well wall in order to minimize any residual liquid residue during the suction evacuation process.

The vertical arrangement of the holes also offers an advantage over the horizontal position of the known technique. This advantage is given by a greater capacity of liquid retention in the well by surface tension forces, because the hole is in a vertical plane and therefore perpendicular to the horizontal plane where the terrestrial gravitational force is maximum.

The holes will be of a diameter small enough to retain the volume of the microplate wells due to the surface orifice tension, and in turn with a diameter large enough to allow the evacuation of the liquid from the wells when a pressure is applied negative.

The diameter of the holes will be wide enough to avoid obstruction problems in the liquid outlet.

The invention, therefore, solves the problem of washing plates with immobilized biological substrates or adhered to the flat bottom of the wells, such as cell assays and immunoassays, allowing the complete evacuation of liquids without the need to alter or lift the biological substrates by a Aspiration close to them and at the same time avoiding problems such as the obstruction of the needles and the potential contamination of the samples transmitted by the aspiration needles in automated processes. For the rest, the Microwell washing will be done by suction through a vacuum chamber located below the wells, connected to a suction source. The lower surface of the interior of this chamber may be equipped with hydrophobic properties in order to facilitate the maximum evacuation of liquid from the wells.

The present invention contemplates this chamber as an integrated part in the microplate, by means of its lower enclosure with a plastic base, in which case it will respect the standard measures established for the microplates. At the base of this lower chamber is an outlet connector to which a certain vacuum is applied, and the liquid is collected in a waste container.

Alternatively, the microplate may be open at its bottom, as is the case with conventional microplates, in which case the vacuum would be made by positioning the microplate on a platform that tightly closes the microplate base. This vacuum-connected platform is now available in the market.

The liquid evacuation rates of the wells can be controlled by different vacuum suction levels depending on the washing requirements.

The present invention offers a low cost solution that can be applied in both manual microplate washing and automated washing.

Brief description of the drawings

The attached drawings show a microplate for biological tests constituted according to the invention and given as a non-limiting example. In the drawings:

Figure 1 is a perspective of a microplate for cell assays or immunoassays (ELISA type).

Figure 2 is a cross section of the microplate, taken along the line II-II of Figure 1.

Figure 3 is a perspective of one of the wells that become part of the microplate of Figure 1. Detailed description of one embodiment

A microplate constituted according to the invention is shown in Figure 1, which includes a structure defining a support 1 in which the wells 2 are mounted, aligned in rows and columns. The microplate shown in Figure 1 is 96 wells in which the different tests will be carried out, followed by the corresponding washes.

The wells 2, as shown in figure 3, are constituted by a cylindrical configuration receptacle, with a flat bottom and provided at the bottom of its wall with an outlet orifice 3. The wells with the described constitution are mounted, as shown in figure 2, with the hole 3 located inside a vacuum chamber 4 which is connected, through the corresponding nozzle 5, to a suction or vacuum source. The hole 3 is located adjacent to the bottom of the well.

This arrangement allows the entire plate to be evacuated simultaneously by a lower chamber common to all wells. Evacuation could also be carried out selectively by rows or columns, by independent lower chambers, coinciding with said rows or columns.

A microplate design with a closed base can be contemplated, which forms the suction chamber, or alternatively an unsealed microplate, in this case an extra platform is needed on which the microplate would fit tightly to thereby perform the vacuum on said platform.

A hydrophobic base can also be used at the bottom of the lower chamber, in order to facilitate the withdrawal of liquids by suction, as well as various suction speeds that are adapted to gentle or more energetic washes and to adapt the suction force so as to allow a continuous evacuation in the case of overflow washing. The hole 3 of the wells 2 will be small enough to contain the liquid inside the well, due to the surface tension forces imposed by each hole, however allowing the evacuation of the liquid due to the suction produced in the vacuum chamber 4 .

If the hole 3 were located at the base or bottom of the wells 2, the study area where the biological sample is located would be compromised and would invalidate the process. Since the field of the invention is that of microplates with a high number of wells, the area of the evacuation orifice acquires a very relevant character compared to the area under analysis. On the contrary, since the hole 3 is located on the side wall, there is a flat, continuous and closed bottom on its entire surface, which allows any biological test to be carried out without the study area where the biological sample is located Be compromised.

The position of the hole 3 in the wells 2 of the microplate of the invention allows the wells to be washed, even with biological agents adhered to a substrate that forms the base or bottom of the microplate well.

The invention is of special application in microplates with a high number of wells, for example to microplates with 1356 wells and more, where current washing needles have difficulty entering and where the experimentation area is so small that they do not make it possible to place the evacuation hole 3 at the base or bottom of the well.

Another advantage of the invention is that since the hole 3 is in an upright position, the liquid retention capacity by surface tensions is even greater than if the hole was located at the bottom and occupied a horizontal position. This is because in the decomposition of forces, the downward gravitational force is blocked by the well's own base. The invention is directed to microplates with flat-bottomed wells, not only because physically it is where greater evacuation of liquids can be achieved by this microplate design, but also because this is the format that is mostly used for cellular and ELISAS assays.

Claims

1.- Microplate for biological tests, comprising a series of open wells on its upper face and each equipped with at least one liquid outlet orifice that flows into a vacuum chamber, characterized in that each well is constituted by a flat bottom receptacle, closed over its entire surface, and has at the bottom of its wall a hole located adjacent to the flat bottom.