ES2405507B1 - DEVICE AND PROCEDURE FOR CLEANING A PLATE OF POCILLOS. - Google Patents

Abstract

Device and method for cleaning a well plate # The invention describes a device for cleaning a well plate that is kept upside down at all times, comprising a base (2) having deposits (3) of fluid from cleaning; and a support (4) provided with needles (6) designed to press air and / or said cleaning fluid against the inverted interior of the wells, exerting a cleaning effect much greater than current devices. The invention also relates to a cleaning procedure using the above device.

Description

Device and procedure for cleaning a well plate

OBJECT OF THE INVENTION

The present invention belongs to the field of biotechnology, and more specifically to the field of tests carried out using well plates.

A first object of the invention is a device designed for cleaning a well plate that is kept upside down at all times.

A second object of the invention is directed to a method for cleaning a well plate using the above device.

BACKGROUND OF THE INVENTION

The ELISA technique (acronym for Enzyme-Linked ImmunoSorbent Assay, or Enzyme-linked Immuno-Absorption Assay) is an immunoassay technique in which an immobilized antigen is detected by an antibody bound to an enzyme capable of generating a detectable product such as color change or some other type; Sometimes, in order to reduce the costs of the assay, we find that there is a primary antibody that recognizes the antigen and that in turn is recognized by a secondary antibody that carries the aforementioned enzyme linked. The appearance of dyes allows the antigen in the sample to be measured indirectly by spectrophotometry.

This technique is used in many laboratories to determine if a particular antibody is present in a patient's blood sample. Although the procedure is routine and simple, it involves a large number of variables, such as reagent selection, temperature, volume and time measurement, which if not adjusted correctly can affect the successive steps and the test result.

One of the steps that is repeated several times when performing the ELISA technique is cleaning the well plate. This is an important operation, since if the cleaning is not done correctly the results could be falsified due to the presence of reagent remains in the wells. In fact, this is not an exclusive problem of the ELISA technique, since in general it is necessary to carry out a thorough cleaning of any plate to be used a second time.

At the moment to clean the wells of a plate a certain volume liquid by gravity is deposited in each one of the wells of one (or several rows). Subsequently said liquid is aspirated. This pour-aspiration process is repeated several times for each row. Then it goes to the next row, and so on. It is therefore about alternating liquid discharge and aspiration. A drawback of this system is that liquid entrainment phenomena frequently occur from well to well. Also, sometimes remains of reagents remain on the upper edges of the well due to the meniscus that forms the liquid with the wall of the well.

DESCRIPTION OF THE INVENTION

Sowing of plates in an inverted position is a novel technique developed by the inventors of the present application and described in application P201130501, which is directed to a device and method for applying a fluid to the wells of a plate while maintaining it in an inverted position, that is, with the wells down. This technique takes advantage of the predominant effect of surface tension over small distances so that a liquid, for example a liquid with a reagent, adheres to the inner surface of a well small well while in an inverted position. A great advantage of this procedure is that it is possible to fill all the wells with liquid in a much faster, clean, uniform and simultaneous way throughout the plate. Another important advantage is that it allows reagent volumes to be used much smaller, up to 1000 times, than those used using traditional techniques.

The inventors of this application have been based on similar principles for, according to a first aspect of the invention, to develop a novel device comprising means for cleaning the wells of a plate by keeping it in an inverted position. This device complements the one described in the previous application to allow the performance of the ELISA technique, or similar ones, keeping the plate in an inverted position both during the sowing or reagent application operations and in the cleaning operations.

Carrying out the cleaning of the wells keeping them in an inverted position has the additional advantage of achieving a more complete cleaning, since the remains adhered to the well, as well as the cleaning liquid, no longer tend to get stuck in the bottom of it as was the case with prior art devices. On the contrary, when the well is in an inverted position, the force of gravity itself facilitates that these remains tend to leave the well

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The cleaning means mentioned may in principle be any, provided that they allow the well to be cleaned keeping it in an inverted position at all times. However, in a preferred embodiment of the invention said means essentially comprise a base and a support, on which the plate will rest in an inverted position during cleaning operations. Both the base and the support are normally made of plastic, methacrylate, polypropylene or other similar material. Each of these parts is described in more detail below.

a) Base

The base constitutes the part on which the support will rest when the device is in operation and comprises at least one suitable tank for housing a washing fluid. The deposit will normally consist of a small blind hole in the upper surface of the base, preferably with a volume of between 100 and 300 microliters.

b) Support

The support is a somewhat more complex piece than the previous one, being essentially formed by a lower sheet and an upper sheet arranged in parallel:

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 The bottom sheet is essentially a flat plate that is configured to rest on the base. In this context, this means that the bottom sheet has a shape somehow complementary to the shape of the base, so that both "fit" into their correct position. For example, the upper part of the base and the lower sheet could comprise complementary flanges that facilitate the correct positioning of the support on the base, thus being both "embedded" one on the other.

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The lower sheet further comprises at least one needle that passes through it substantially perpendicularly. In this context, the term "substantially" refers to the fact that small deviations from normal are possible. Said at least one needle is positioned such that, when the support is placed in its correct position on the base, its lower end is inserted into the at least one base deposit.

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 The upper sheet comprises at least one hole of a size corresponding to a well of the plate, and is located parallel to the first so that the upper end of the at least one needle protrudes through said hole, between them being a adequate space to create the void. In this document, the expression "suitable for creating the vacuum" means that said space between the upper and lower sheets will be closed in some way at the edges of the support and will have at least one mouth or connection to couple a small vacuum pump or similar to create the void.

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 The top sheet is also configured so that the well plate in an inverted position rests on it, for example by means of suitable flanges that facilitate the correct positioning of the well plate in an inverted position on it.

Thus, the operation of this device is as follows. The at least one tank of the base is filled with a cleaning liquid and then the support is placed on the base. In carrying out this operation, the lower end of the at least one needle of the support is inserted into the reservoir, while the upper end of the needle protrudes slightly through the at least one hole in the upper sheet. Next, the plate to be cleaned upside down is placed on top of the support such that said upper end of the needle is within a well. Finally, vacuum is applied between the lower and upper sheets. This has the effect, first, of aspirating remains adhered to the well; then, to cause the ascent of the cleaning liquid through the needle, projecting on the bottom of the well to drag more adhered remains; and finally, when the cleaning liquid has run out, cause a stream of air to flow through the needle and project onto the bottom of the well, drying it.

When the device is mounted and there is a well on the support, the upper end of the needle could in principle be at any height inside the inverted well as long as the pressure of the cleaning liquid and the air is sufficient to reach the walls of the well and exert an effective cleaning function. However, in a preferred embodiment of the invention the distance protruding the upper end of the at least one needle above the upper sheet of the support is slightly less than the depth of a well of a plate. The expression "slightly smaller" here refers to the needle protruding a height such that its end is at a distance from the bottom of the well small enough that a hypothetical drop of liquid coming out of the needle contacts the bottom of the well before of sliding down the needle by the action of gravity, but large enough so that the mouth of the needle is not blocked. Thus, the closer the needle is to the bottom of the well, the greater the drag effect of the materials attached to the well for the same pressure of the cleaning liquid or the air.

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In another particular embodiment of the invention, the upper surface of the upper sheet comprises a coating that facilitates that the support of the inverted plate on said upper sheet is airtight, for example a coating comprising rubber, silicone, rubber, fiberglass, plastic polymers, or combinations thereof. The addition of this coating hinders the entry of air between the edges of the well and the top sheet, thus facilitating the creation of a vacuum in the space between the bottom and top sheets. In addition, the addition of this coating allows to control the distance protruding the needles on the top sheet, since the distance will be greater for thin coatings and smaller for thick coatings. This allows the use of the device with well plates with different depths. Therefore, in a further preferred embodiment of the invention the coating is removable. For example, it could have a suitable adhesive side to be fixed to the upper surface of the topsheet.

The present description has been made with the intention of covering the most basic version of the device, which may comprise only a reservoir, a needle and a hole, in which case the cleaning of several wells would be performed sequentially one after the other. For that reason, the description has mentioned at all times "at least one deposit", "at least one needle" and "at least one hole." However, it is evident that a particularly preferred embodiment of the invention comprises a plurality of tanks / needles / holes configured as a matrix whose positions would correspond to those of the wells of a plate, for example, a device with a matrix of 6, 12, 24, 48, 60, 72, 96 or 384 could be conceived tanks / needles / holes whose positions would correspond to those of the wells of the corresponding plates.This would allow to simultaneously wash all the wells of one of such plates, or in two operations if a device of 48 tanks / needles / holes is used for a 96 well plate, and so on.

A second aspect of the invention is directed, in general, to a method of cleaning a well plate characterized in that it is carried out by holding the plate in an inverted position. Although this procedure could be carried out in different ways, it can preferably be performed using the device described above, in which case it comprises the following operations:

1) Fill the at least one tank with a cleaning liquid.

2) Place the support with the lower blade resting on the base, so that the lower end of the at least one needle is inserted into the at least one reservoir.

3) Place the plate in an inverted position on the upper sheet so that the upper end of the at least one needle is within a well of the inverted plate.

4) Apply the vacuum in the space between the lower plate and the upper plate of the support, which causes:

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first the aspiration of the content adhered to the well,

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then the ascent of the cleaning liquid through the needle, washing the well from top to bottom,

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Finally, the cleaning liquid is exhausted, a stream of air that draws any remaining debris and dries the well

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 represents a perspective view of a conventional 12-well plate.

Fig. 2 represents a view of a support corresponding to an example of a device of the invention.

Fig. 3 represents a view of a base corresponding to an example of the device of the invention.

Fig. 4 represents a cross section of the support and the base shown in the previous figures before mounting the device.

Fig. 5 shows a section of the device of the invention assembled, just before placing a plate.

Fig. 6 shows the device section of the previous figures during the cleaning of a plate.

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PARTICULAR EMBODIMENT OF THE INVENTION

A particular embodiment of the device (1) of the invention and its mode of operation is described below with reference to the attached figures. As mentioned above, the device (1) of the invention can be designed to be coupled to plates (100) of any number of wells (101), for example 6, 12, 24, 48, 60, 72, 96 or 384 wells. This example describes a 12-well plate (100) (101) solely for simplicity.

Fig. 1 shows a conventional 12-well plate (100) (101), which are arranged according to a 3 x 4 matrix. The representation is schematic, showing more details of the shape of the plate (100) in the sections of Figs. 4-6.

The support (4) is shown in Fig. 2, although some internal details that are hidden in that figure can be seen more clearly in the section of any of Figs. 4-6. The support (4) is formed by the lower sheet (5) and the upper sheet (7) arranged in parallel, with a space between them to create the vacuum (a cut has been made in the piece so that these elements can be seen ). It is observed how the needles (6) protrude through the holes (8) of the upper sheet (7). These needles (6) are fixed to the bottom sheet (5), as seen in the section of Figs. 4-6, where you can also see how the needles (6) protrude below the support (4).

Fig. 3 shows the base (2), which in this example consists essentially of a rectangular piece made of polypropylene and equipped with 12 tanks (3) also arranged according to a 3 x 4 matrix in positions corresponding to the wells (101) of Fig. 1.

Thus, once the tanks (3) of the base (2) have been filled with a cleaning liquid, the support is placed

(4) on the base (2) such that the needles (6) enter the tanks (3), as shown in Figs. 4-5. In order to ensure its correct location, notches, projections, steps or the like can be devised that require a correct positioning of both pieces. Next, the plate (100) is supported on the support (4) such that each well (101) rests on the upper sheet (7) around one of the holes (8). In this position, the upper ends of the needles (6) will be inserted into the wells (101), near their bottom, as seen in Fig. 6. In a similar way to that described above, to ensure proper positioning of the plate (100) on the support (4) it is possible to conceive notches, projections, steps or the like that indicate the user the proper position.

The cleaning is then carried out by applying the vacuum in the free space between the lower (5) and upper (7) plates of the support (4). Although it has not been represented in the figures, it is understood that the support (4) has a connection mouth that allows coupling a tube through which to make the vacuum with the help of a compressor or the like. When applying the vacuum, there are three different phases of operation that result in the optimal cleaning of the wells (101). First, the vacuum absorbs a part of the residues or remains that are attached to the inside of the well (101). Secondly, the cleaning liquid of the tanks (3) is absorbed by the vacuum, rises through the needles (6) and is projected strongly towards the inner surface of the wells (101), washing the well (101) and dragging the rest of the waste. Finally, once the liquid in the reservoir (3) has run out, the vacuum forces the air rise through the needles (6) and projects it against the inside of the wells (101), dragging the last remains that could remaining and drying the well (101).

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Claims (14)

one.

Device (1) for cleaning a well plate (100) (101), characterized in that it comprises means for washing the well (101) by keeping said plate (100) in an inverted position.

2.

Device (1) according to claim 1, wherein said means comprise:

-

 a base (2), comprising at least one reservoir (3) suitable for housing a washing fluid; Y

-

a support (4), comprising

a lower sheet (5) configured to rest on said base (2) and comprising at least one hollow needle (6) that passes it substantially perpendicularly so that, when the support (4) is supported on the base (2 ), its lower end is introduced into the at least one deposit (3) of the base (2); Y an upper sheet (7) comprising at least one hole (8) of a size corresponding to a well (101) of the plate (100), and is located parallel to the lower sheet (5) so that the upper end of the at least one needle (6) protrudes through said hole (8), there is a space between the lower sheet (5) and the upper sheet (7) to create the vacuum, and where said upper sheet (7) is configured so that the plate (100) of wells (101) in an inverted position rests on it.

3.

Device (1) according to claim 2, wherein the at least one reservoir (3) has a volume of between 100 and 300 microliters.

Four.

Device (1) according to any of claims 2-3, wherein the distance protruding the upper end of the at least one needle (6) above the upper sheet (7) of the support (4) is slightly less than the depth of a well (101) of a plate (100).

5.

Device (1) according to any of claims 2-4, wherein the upper part of the base

(2) and the lower sheet (5) of the support (4) comprise complementary flanges to facilitate correct positioning of the support (4) on the base (2).

6.

Device (1) according to any one of claims 2-5, wherein the upper sheet (7) of the support (4) comprises suitable flanges to facilitate correct positioning of the well plate (100) (101) in an inverted position on the support (4).

7.

Device (1) according to any of claims 2-6, wherein the upper surface of the upper sheet (7) comprises a coating that facilitates that the support of the inverted plate (100) on said upper sheet (7) is airtight .

8.

Device (1) according to claim 7, wherein the coating comprises rubber, silicone, rubber, fiberglass or plastic polymers, or combinations thereof.

9.

Device (1) according to any of claims 7-8, wherein the coating is removable.

10.

Device (1) according to any of claims 2-9, comprising a plurality of reservoirs (3), holes (8) and needles (6) forming a matrix whose positions are equivalent to those of the wells (101) of a plate (100).

eleven.

Device (1) according to claim 10, wherein the matrix of deposits (3), holes (8) and needles (6) has 6, 12, 24, 48, 60, 72, 96 or 384 positions.

12.

Cleaning procedure of a plate (100) of wells (101), characterized in that it is carried out by keeping the plate (100) in an inverted position.

13.

Cleaning process according to claim 12 which is carried out using the device (1) of any of claims 2-11, which comprises the following operations:

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 refill the at least one tank (3) with a cleaning liquid;

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 place the support (4) on the base (2), so that the lower end of the at least one needle (6) is inserted into the at least one reservoir (3);

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 placing the plate (100) in an inverted position on the upper sheet (7) so that the upper end of the at least one needle (6) is within a well (101) of the inverted plate (100); Y

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 apply the vacuum in the space between the lower plate (5) and the upper plate (7) of the support (4), which causes

ES 2 405 507 Al first the aspiration of the content adhered to the well (101), then the ascent of the cleaning liquid through the needle (6) that washes the well (101) from top to bottom, Finally, the cleaning liquid is exhausted, a stream of air through the needle (6) that draws any remaining debris and dries the well (101). ES 2 405 507 Al ES 2 405 507 Al SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201131702 SPAIN Date of submission of the application: 21.10.2011 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

X

WO 2010134964 A1 (PROTEDYNE CORP et al.) 25.11.2010, the whole document; in particular, page 5, 2nd paragraph and figure 3. 1.12

TO

EP 1637887 A1 (BTI HOLDINGS INC) 22.03.2006, the whole document. 1-13

TO

EP 0903181 A2 (ASTLE THOMAS W) 24.03.1999, the whole document. 1-13

TO

EP 0304765 A2 (HOECHST JAPAN) 01.03.1989, the whole document. 1-13

TO

US 5650122 A (HARRIS PAUL C et al.) 07.27.1997, the whole document. 1-13

TO

CN 2608989 Y (WANG FENGYING) 31.03.2004, (summary) EPODOC DATABASE [online], Recovered from: EPOQUENET, E.P.O., [retrieved 06.06.2012]. 1-13

TO

O & APOS et al. A SIMPLE TECHNIQUE FOR HARVESTING LYMPHOCYTES CULTURED IN TERASAKIPLATES. Journal of Immunological Methods 1979 VOL: 27 No: 3 Pags: 219-224 ISSN 0022-1759. 1-13

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 27.03.2013

Examiner A. Maquedano Herrero Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201131702 CLASSIFICATION OBJECT OF THE APPLICATION G01N37 / 00 (2006.01) G01N35 / 10 (2006.01) G01N35 / 02 (2006.01) B08B3 / 04 (2006.01) Minimum documentation sought (classification system followed by classification symbols) G01N, B08B Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, BIOSIS, XPESP, NPL State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201131702 Date of Completion of Written Opinion: 03.23.2013 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 2-11, 13 1, 12 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 2-11, 13 1, 12 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201131702  1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

WO 2010134964 A1 (PROTEDYNE CORP et al.) 11.25.2010

D02

EP 1637887 A1 (BTI HOLDINGS INC) 03/22/2006

D03

EP 0903181 A2 (ASTLE THOMAS W) 24.03.1999

D04

EP 0304765 A2 (HOECHST JAPAN) 01.03.1989

D05

US 5650122 A (HARRIS PAUL C et al.) 07/22/1997

D06

CN 2608989 Y (WANG FENGYING) 03.31.2004

D07

O & APOS et al. A SIMPLE TECHNIQUE FOR HARVESTING LYMPHOCYTES CULTURED IN TERASAKIPLATES. Journal of Immunological Methods 1979 VOL: 27 No: 3 Pags: 219-224 ISSN 0022-1759. 11/30/1978

 2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The application claims a device for cleaning a well plate that is kept upside down at all times. The device comprises a base with deposits of cleaning liquid and a support with needles for releasing air and / or said cleaning liquid against the inverted inside of the wells. It also claims a cleaning procedure carried out by this device. D01-D07 represent the prior art. Of them, D01 is the closest. Describes a procedure to remove residues that remain in the wells of the plates used in research. The plates are reversed during the washing phase mechanically. In this way, both cleaning and disposal of waste after washing is facilitated. The difference with respect to the request is that in D01 the inversion of the plate is only made to carry out the cleaning. On the other hand, in the application a needle system is used to press the washing liquid under pressure, while no needle is described in D01. Claims 1 and 12 of the application, being drafted so generally, are anticipated by the content of D01. The rest of the claims, being based on the technical characteristics that differentiate one and the other document, do not. Therefore, claims 2-11 and 13 of the application are considered to meet the requirements of novelty within the meaning of article 6.1 of Law 11/1986 and of inventive activity within the meaning of article 8.1 of Law 11/1986 . Claims 1 and 12 do not meet any. State of the Art Report Page 4/4