WO2000057153A1

WO2000057153A1 - Improvements in apparatus and method for removing samples

Info

Publication number: WO2000057153A1
Application number: PCT/AU2000/000200
Authority: WO
Inventors: Hong Bing Cheng
Original assignee: Campbell Corporation Pty. Ltd.
Priority date: 1999-03-19
Filing date: 2000-03-17
Publication date: 2000-09-28
Also published as: AUPP930099A0

Abstract

An apparatus for excising at least one sample from an array of samples. The apparatus includes a platform (106) which supports a digital camera (110) which records an electronic image of the sample array that is placed on the platform (106). The recorded image is sent to a computer (120) which enables a user to select various samples for excising. Excision is performed by a cutting tool assembly. The cutting tool assembly includes a rotating cutting head (192) to excise the sample and an ejector pin (174a) to eject the sample into a collection plate.

Description

IMPROVEMENTS IN APPARATUS AND METHOD FOR REMOVING SAMPLES

Technical Field

The present invention relates to a method and apparatus for removing one or more samples from an array of samples and to a cutting tool for use with that apparatus. In particular, the invention relates to an apparatus for excising, and ejecting, biomolecules from an array of biomolecule samples in a gel or solid support.

Background Art

Improvements in laboratory techniques and practices have led to the discovery of an ever increasing number of new biomolecules. New protein purification and detection methods, for example, have allowed the detection of many possibly new proteins.

Due to the large number of known biomolecules, it is now necessary to carry out molecular comparisons of newly discovered molecules to determine to what extent they are similar to, or different from, known molecules. For example, to carry out definitive analysis for proteins it is necessary to obtain amino acid sequence information or determine the masses of peptides after protein digestion. Often, the biomolecules are separated by electrophoresis in polymer based media. Thus, the biomolecules for analysis are usually present as "concentrated" spots on media such as dry polymer membranes or wet gels.

It is then necessary to excise the biomolecule from the media and transfer them separately to a vessel such as a microtitre plate to carry out analysis of the biomolecules. The spots are usually cut out by a laboratory worker or researcher using a scalpel and are placed in a test tube for analysis by application of a reagent or succession of reagents to the sample. Typically, a single membrane may have many hundreds or thousands of spots of biomolecules. In this respect, multiple sample handling using laboratory techniques can be labor intensive, and prone to error and contamination. At the same time, the improvements in laboratory techniques and practices, and the discovery of an ever increasing number of new biomolecules, have increased the need for analysis of sample biomolecules.

The present invention seeks to provide a method and apparatus that ameliorates the problems associated with the manual task described above, or at least to provide an alternative.

Summary of Invention

The present invention provides an apparatus for excising at least one sample from an array of samples, the apparatus including :

(a) means for recording an electronic image of the position of at least one sample relative to the other samples in the array;

(b) means for utilising the recorded electronic image to position a cutting tool assembly above the at least one sample in the array, the cutting tool assembly including at least one cutting head;

(c) means for rotating the cutting head;

(d) means for advancing the cutting head into contact with the at least one sample so as to effect excision of the at least one sample and means for withdrawing the cutting head therefrom; (e) means for ejecting the at least one excised sample at a selected location; and

(f) control means for controlling means (a), (b) (c) and (d), the control means being such that means (b) excises the at least one sample according to the position of the sample relative to the other samples in the array as determined by means (a). Preferably, the cutting head is rotating when it is advanced into contact with the at least one sample.

The apparatus may further include: (g) a table means for supporting the array of samples;

(h) display means for displaying the electronic image of the array on a visual display unit; (i) means for selecting a sample on the array for excising by the cutting tool assembly; and (j) means for moving the table means relative to the cutting tool assembly in the plane of the array.

It will be appreciated by those skilled in the art that the apparatus may be used to excise samples from varying sources. Typically, the array of samples will be present as a non-ordered array of spots on dry polymer membranes or wet gels. The sample will generally be of a biological nature and may include proteins, peptides, polysaccharides, lipids and nucleic acid molecules or complex molecules like glycoproteins, for example. The sample may also comprise a sample of meat, tobacco, membrane etc.

The means for recording an electronic image of at least a part of the array of samples may be a digital camera or scanner that records a digital image of the samples. However, in order to generate an electronic image of the samples in the array, it is necessary to make them visible to the recording means in some manner. Thus, the electronic image may be generated from a scan of the samples which are stained or illuminated or otherwise marked with a visible or fluorescent marker to cause them to be visible to the recording means. Once the electronic image has been recorded there is no requirement to maintain the visibility of the samples on the array as the image may be maintained electronically. Preferably, co-ordinates of the samples are recorded which may then be transformed into a format suitable for interpretation by a computing means. In a preferred embodiment, the computing means controls a transport mechanism suitable for positioning the cutting tool assembly at desired locations in accordance with the recorded co-ordinates and hence the sample locations. Accordingly, excision is then possible regardless of whether or not the samples remain visible to the recording means.

The cutting head of the cutting tool assembly is rotating when it is brought into contact with the sample. This greatly increases the likelihood of successfully removing the sample from the array and subsequently ejecting the sample at a selected coordinate of a receptacle or a microtitre plate or the like

The means for advancing and withdrawal of the cutting head into contact with the array of samples may be a stepping motor arrangement. Preferably, a suspension means is provided to the cutting head to reduce any damage to the array or the table upon which it rests caused by the motion of the stepping motor arrangement.

In a preferred embodiment, an image file relating to a number of arrays of samples is stored on the computer and visible spots present in the array are selected to be excised using a visual display unit and a mouse or keyboard. Upon selecting a particular spot, the cutting tool assembly automatically moves to that co-ordinate position, cuts the spot from the array, collects the spot and transports it to a selected receptacle or the like.

Preferably the array of samples is in a plane, the x-y plane, and in one form of the invention, the cutting tool assembly is movable in both the x and y directions so that the spot to be sampled is located underneath the cutting head.

In a preferred form of the present invention, a cutting tool assembly for use in the apparatus and method of the present invention includes: a cutting head defining a central bore, the cutting head adapted to rotate and sever a sample of material by a cutting motion; an ejector pin disposed in the bore defining a rod which is disposed in and movable along the bore, the ejector pin being capable of extending through and beyond the end of the cutting head; and a means to transport the ejector pin along the bore of the cutting head.

The cutting head may be circular and the ejector pin may be a rod of circular cross section.

In this preferred form, the cutting head may be rotated by means of a belt and pulley arrangement, and is preferably continuously rotated in the same direction during the excising operation. However, the cutting head may be rotated in either direction or in a combination of directions to effect the best possible cutting motion for the particular material from which a sample is to be excised. For certain types of material, it is preferred to effect rotation of the cutting head during the ejection phase to assist with the removal of the sample material.

The cutting tool assembly may further include: (a) a magnet or a piece of ferromagnetic material attached to the ejector pin distal from the end where the ejector pin may extend through the cutting head;

(b) a solenoid disposed around the ferro magnetic material for causing the ejector pin to move in the bore in a direction which expels material from the cutting tip when the solenoid is energised; and

(c) return means for causing the pin to move in the opposite direction when the solenoid is not energised.

If item (b) is a magnet, the return means may also be a magnet. The return means may also be a spring. In the preferred form, the cutting head is removable and disposable. Additionally, the ejector pin may also include a central bore through which pressurised fluid may be passed. The central bore provides for the injecting of a required liquid into the receptacle containing the sample or for the passing of pressurised air to assist with the removal of the sample from the cutting tool assembly.

For particular types of material from which samples will be excised, a static potential difference may develop between the material and the cutting head during the excision process. In this instance, upon attempting to remove the sample from inside the cutting tool assembly by means of extending the ejector pin through the cutting head, the sample may adhere to the outside of the cutting tool assembly by electrostatic attraction. Preferably, the cutting tool assembly includes an electrical connection to a source of high voltage in the event that a static charge develops between the cutting head and the sample, in which case the potential difference may be neutralised thereby enabling the sample to drop from cutting head.

Rotation of the cutting head and severing material from the sample array by a cutting motion combined with the controlled advancement and withdrawal of the cutting head assembly in relation to the sample array enables the use of different types of material for the table upon which the sample array is placed.

For example, if the cutting head were to remove samples from the array by way of punching the material, it would not be possible to use a glass table, as the punching of the cutting head would likely damage the glass. Rotation of the cutting head enables the use of a glass table without resultant damage to the glass. This further enables the use of an illumination means to render the spots in the sample material visible from beneath the table as glass is relatively transparent. This particular arrangement embodies the advantage that the means to render the spots visible may reside beneath the table whilst the means to remove samples may reside above the table without any interference therebetween. The present invention also provides a method for excising at least one sample in an array of samples including:

(a) recording an electronic image of the position of at least one sample relative to the other samples in the array;

(b) utilising the recorded image to position the cutting tool assembly above the at least one sample;

(c) initiating the rotation of the cutting head;

(d) advancing the rotating cutting head so as to bring the cutting head into contact with the at least one sample so as to cut the at least one sample from the array;

(e) moving an ejector pin such that it draws the severed at least one sample into the cutting tool assembly.;

(f) withdrawing the entire cutting head assembly from the sample array; (g) conveying the cutting tool assembly to a selected location;

(h) advancing the ejector pin beyond the reach of the cutting head such that the at least one sample is ejected from the cutting tool assembly at the selected location; and

(i) positioning the cutting head above the next sample in the array to be excised and resuming the process at step (c) until all the samples required have been excised from the array.

The present invention also provides a method for excising at least one sample in an array of samples including: (a) recording an electronic image of the position of at least one sample relative to the other samples in the array;

(c) extending an ejector pin such that it extends beyond the reach of the cutting head;

(d) placing the ejector pin into contact with the at least one sample to secure the at least one sample; (e) initiating rotation of the cutting head;

(f) advancing the rotating cutting head relative to the ejector pin to bring the cutting head into contact with the at least one sample so as to cut the at least one sample from the array; (g) withdrawing the ejector pin such that it draws the severed at least one sample into the cutting tool assembly; (h) withdrawing the entire cutting head assembly from the sample array; (i) conveying the cutting tool assembly to a selected location; (j) advancing the ejector pin beyond the reach of the cutting head such that the at least one sample is ejected from the cutting tool assembly at the selected location; and (k) positioning the cutting head above the next sample in the array to be excised and resuming the process at step (c) until all the samples required have been excised from the array.

In one form, the ejector pin may include a central bore therethrough enabling the supply of fluid therethrough from a reservoir external to the cutting tool assembly. Accordingly, the method may include a step whereby pressurised fluid is passed through the ejector pin bore to assist in removing the sample from the cutting tool assembly. Additionally, the method may include a step whereby liquid is passed through the ejector pin bore to assist in the removal of the sample from the cutting tool assembly or for the purpose of supplying necessary liquid to a receptacle.

In another form of the invention, the method may include effecting rotation of the cutting head during the ejection phase to assist in the removal of sample material.

Preferably, where the use of materials results in the development of substantial electrostatic charges between the cutting tool assembly and the sample material, the method may include a step whereby a source of high voltage is temporarily connected to the cutting tool assembly to neutralise any static charge.

The samples may be separated into the array by known means such as electrophoresis in a polymer matrix which is then placed on a solid support or membrane support. For example, two dimensional electrophoresis separations in polyacrylamide are transferred to supports like PTFE, gortex, PVDF, nylon, nitrocellulose or polypropylene which are particularly suitable for supporting an array of samples for excision using the methods and apparatus of the present invention. Samples may also be excised without having to transfer them to a membrane.

Brief Description of Drawings

A preferred embodiment of the invention will now be described with reference to the accompanying drawings in which:

Figure 1 is a front view of a sampling apparatus detailing the main components;

Figures 2a to 2g detail the sequence of operation of the cutting tool assembly showing in sequence the cutting of a sample from an array and the depositing of that cut sample in a test tube; and

Figure 3 shows the cutting tool assembly with attached stepping motor and spring mechanism.

Detailed Description of the Preferred Embodiments

Figure 1 details the main components of a sampling apparatus in accordance with the present invention wherein samples contained within an array are supported upon a cutting panel which rests on a platform 106. The platform also supports a transport 102 along which a cutting tool assembly 104 is conveyed. The transport mechanism 102 is also capable of movement in the direction perpendicular to the plane of the page thereby providing the cutting tool assembly 104 the freedom to access all relevant parts of the platform 106.

In the particular embodiment of Figure 1 , the platform 106 also supports a structure that houses a digital camera 110 and illumination devices. The illumination devices enable the digital camera 110 to record an electronic image of the sample array that is placed upon the platform 106. The recorded image is then transferred to the computer 120 which in combination with the monitor 125 and suitable software (not detailed herein) enables a user to select from the recorded image various sections of the array for excising and effecting excision of those sections by control of the transport 102 and the cutting tool assembly 104.

Figure 2a is a front sectional view of a cutting tool assembly which forms part of the sampling device of Figure 1 and details the ejector pin retainer 150, a solenoid 152, a support bracket 155, a pulley 167, a first bearing 170, a connecting shaft 172, a ejector pin 174, a second bearing 176, a male luer 190, and a cutting head 192.

The cutting head 192 can be made of various materials including glass, metal or a plastic, such as polypropylene. However, it is preferable that the cutting head 192 is translucent as this makes it possible to determine if acrylamide residue is caught inside the head. The cutting head may be removable and disposable.

In the particular embodiment of Figure 2a, the ejector pin 174 includes a central bore that extends the entire length of the ejector pin. This enables the user of the apparatus to connect a tube or hose to the top section 200 of the ejector pin 174 to deliver fluid through the ejector pin to the end of the ejector pin 174 proximate the cutting head 192. As detailed in Figure 2a, the cutting tool assembly 104 is initially suspended above an array of samples 210 with the ejector pin 174 projecting beyond the tip of the cutting head 192. The cutting tool assembly 104 is directed to the precise location of a sample to be excised as previously described.

As detailed in Figure 2b upon being located correctly, the cutting tool assembly 104 is lowered to a position wherein the ejector pin 174 rests upon the surface of the array of samples 210 and retains the array in position in preparation for the excision of a sample. Contact between the ejection pin 174 and the surface of the array also helps to overcome adhesive forces between the sample and the platform 106 during subsequent removal of the sample after excise. It will be appreciated by those skilled in the art, that it may not always be necessary or desired to rest the ejector pin 174 on the surface of the array of samples 210 prior to cutting with the cutting head 192. Accordingly, in such circumstances, the ejector pin 174 is retained in a retracted position within the bore of the cutting head 192 while excision of the sample occurs. The ejector pin 174 preferably is located approximately 2 mm within the lowermost end of the bore of the cutting head 192. Alternatively, the end of the ejector pin 174 may sit flush with the end of the cutting head 192 when cutting of the sample commences.

Figure 2c details the next step of the process wherein while rotating the cutting head 192 is lowered to contact the array 210. The rotation of the cutting head 192 results in the cutting of a portion of the array 210 around the ejector pin 174. In the preferred embodiment, the rotation of the cutting head 192 is effected by the pulley 167 and bearing arrangement 170 and 190. This arrangement enables the cutting head 192 to rotate whilst the ejector pin 174 remains stationery.

With reference to Figure 2, upon sufficient cutting of the sample from the array 210, the rotation of the cutting head 192 is halted and the ejector pin 174 is withdrawn from the surface of the array. Withdrawal of the ejector pin 174 effectively creates a partial vacuum which draws the excised sample 215 from the array 210 up into the recess formed between the cutting head 192 and the withdrawn ejector pin 174. Extra vacuum can be supplied through the central bore of the ejector pin 174 if required.

It has been found that by applying brief pressure to the sample with the ejector pin 174 and subsequently applying a rotating cutting head 192 to the sample, the excised sample is less likely to be deformed and thus fall out of the cutting tool assembly. It has also been found that the combination of brief pressure and a rotating cutting head reduces damage to the platform 106 and also reduces the likelihood of the sample picking up liquid from the array and thereby causing the array to dehydrate.

The cutting head 192 preferably rotates at a speed in the order of 4000 to 5000 revolutions per minute. The speed of the cutting head 192 must be controlled so as to prevent the cutting head 192 from heating up and thereby causing damage and/or contamination of the sample 210. Furthermore, the time the cutting head 192 is in contact with the array of samples 210 is dependent on the exact nature of sample and it thickness.

At this stage of the process, the cutting tool assembly 104 is raised from the array 210 as detailed in Figure 2e.

The cutting tool assembly 104 is then conveyed to a desired location where the sample is to be ejected. In the instance of Figure 2f, the cutting tool assembly has been conveyed to a location and is suspended above the open mouth of a test tube 218.

Figure 2g details the next step of the process being the ejection of the sample 215 into the test tube 218 by extending the ejector pin 174 beyond the tip of the cutting head 192. If traversing the ejector pin 174 through the central bore of the cutting head 192 is insufficient to eject the sample from the cutting tool assembly, then pressurised fluid may be provided through the central bore of the ejector pin 174 to assist with the ejection of the sample. With a particular type of array material it may be desirable to use pressurised air. Alternatively, where liquid is required to be inserted into the test tube 218, this liquid may be supplied through the central bore of the ejector pin 174 and assist with the removal of the sample from the cutting tool assembly. In the event that a static charge may result between the sample material and the cutting head 192, a source of high voltage may be applied to the cutting head to neutralise the charge and reduce any attractive force preventing the sample from dropping into the test tube.

Having effectively ejected the sample, the cutting tool assembly is then conveyed to the location of the next sample to be excised. The process is then resumed repeating the steps outlined in Figures 2a to 2g until all the desired samples have been removed from the array and placed into test tubes.

It will be appreciated that by repeatedly extending and retracting the ejector pin 174 it is possible to remove small burrs formed on the inside of the cutting head 192.

Figure 3 illustrates a stepping motor 300 which is arranged to advance the cutting head 192 towards and away from the array of samples 210. A suspension mechanism in the form of a spring peg 310 is connected at one end 310a to the gear 312 of the stepping motor 300 and at the other end 310b to the cutting head 192. During cutting of a sample from the array of samples 210 the resultant forces applied by the stepping motor 300 to the cutting head 192 are counteracted by the opening of the spring peg 310 as it hinges about the spring peg 310a.

The cutting head 192 is preferably spring loaded so as to reduce the vertical load applied to the sample being excised and the platform 106. In this manner the likelihood of damage to the cutting head 192, the cutting panel and the platform 106 is reduced. It is particularly important to avoid damage to the cutting panel because they are expensive to replace and because damage thereto may cause contamination of the sample.

Thus the present invention allows the very laborious job of a researcher individually cutting spots from a gel or support using a scalpel to be carried out automatically. As will also be appreciated by those skilled in the art, the cutting motion of the cutting head provides for the use of support tables made from materials that could otherwise be damaged. In particular, the use of a highly transparent material such as glass for the support table enables the sample array to be imaged from beneath the sample array. In this instance, a significant advantage is gained as the imaging and recording apparatus can be mounted and located such that it does not interfere with the excision apparatus (ie the imaging and recording apparatus may be located beneath the support table with the excision apparatus above the table). It will also be appreciated by those skilled in the art that the present invention increases the probability of the sample being successfully cut from the array and delivered to a selected receptacle.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. An apparatus for excising at least one sample from an array of samples, the apparatus including: (a) means for recording an electronic image of the position of at least one sample relative to the other samples in the array; (b) means for utilising the recorded electronic image to position a cutting tool assembly above the at least one sample in the array, the cutting tool assembly including at least one cutting head; (c) means for rotating the cutting head;

(d) means for advancing the cutting head into contact with the at least one sample so as to effect excision of the at least one sample and means for withdrawing the cutting head therefrom;

(e) means for ejecting the at least one excised sample at a selected location; and

(f) control means for controlling means (a), (b), (c) and (d), the control means being such that means (b) excises the at least one sample according to the position of the sample relative to the other.

2. An apparatus according to claim 1 wherein the cutting head is arranged to rotate when it is advanced into contact with the at least one sample.

3. An apparatus for excising at least one sample from an array of samples, the apparatus including:

(c) means for rotating the cutting head; (d) means for advancing the cutting head while it is rotating into contact with the at least one sample so as to effect excision of the at least one sample and means for withdrawing the cutting head therefrom; (e) means for ejecting the at least one excised sample at a selected location; and

(f) control means for controlling means (a), (b) (c) and (d), the control means being such that means (b) excises the at least one sample according to the position of the sample relative to the other samples in the array as determined by means (a).

4. An apparatus according to claim 3 wherein the ejector means is arranged to be brought into contact with the sample to be excised prior to advancing the rotating cutting head.

5. An apparatus according to claim 3 further including:

(g) a table means for supporting the array of samples; and

(h) display means for displaying the electronic image of the array on a visual display unit, (i) means for selecting a sample on the array for excising by the cutting tool assembly; and

(j) means for moving the table means relative to the cutting tool assembly in the plane of the array.

6. An apparatus according to claim 3 wherein the means for recording an electronic image of at least a part of the array of samples is a digital camera.

7. An apparatus according to claim 3 further including suspension means for reducing forces transmitted from the cutting head to the array of samples.

8. An apparatus according to claim 3 wherein the means for advancing the cutting head comprises a stepping motor arrangement.

9. A method for excising at least one sample in the array of samples including:

(b) utilising the recorded image to position the cutting tool assembly above the at least one sample; (c) initiating rotation of the cutting head; (d) advancing the rotating cutting head so as to bring the rotating cutting head into contact with the at least one sample so as to cut the at least one sample from the array;

(e) moving an ejector pin such that it draws the severed sample into the cutting tool assembly;

(f) withdrawing the entire cutting head assembly from the sample array;

(g) conveying the cutting tool assembly to a selected location;

(h) advancing the ejector pin beyond the reach of the cutting head such that the at least one sample is ejected from the cutting tool assembly at the selected location; and (i) positioning the cutting head above the next sample in the array to be excised and resuming the process at step (c) until all the samples required have been excised from the array.

10. A method according to claim 9 including the step of rotating the cutting head during the ejection step (h) to assist in the ejection of the sample.

11. A method according to claim 9 or claim 10 wherein the ejector pin includes a central bore and fluid is passed through the central bore to assist in removing the sample from the cutting tool assembly or to supply fluid to a receptacle.

12. A method for excising at least one sample in the array of samples including:

(d) placing the ejector pin into contact with the at least one sample to secure the at least one sample; (e) initiating rotation of the cutting head; (f) advancing the rotating cutting head relative to the ejector pin to bring the rotating cutting head into contact with the at least one sample so as to cut the at least one sample from the array;

(g) withdrawing the ejector pin such that it draws the severed sample into the cutting tool assembly;

(h) withdrawing the entire cutting head assembly from the sample array; (i) conveying the cutting tool assembly to a selected location; (j) advancing the ejector pin beyond the reach of the cutting head such that the at least one sample is ejected from the cutting tool assembly at the selected location; and (k) positioning the cutting head above the next sample in the array to be excised and resuming the process at step (c) until all the samples required have been excised from the array.

13. A method according to claim 12 including the step of rotating the cutting head during the ejection step (j) to assist in the ejection of the sample.

14. A method according to claim 12 wherein the ejector pin includes a central bore and fluid is passed through the central bore to assist in removing the sample from the cutting tool assembly or to supply fluid to a receptacle.

15. A cutting tool assembly for excising at least one sample from an array of samples, said assembly including: a cutting head defining a central bore, the cutting head adapted to rotate prior to contact with a sample of the array so that upon contact with the sample the sample is severed from the array; an ejector pin disposed in the bore defining a rod which is disposed in and movable along the bore; and a means to transport the ejector pin along the bore of the cutting head.

16. A cutting tool assembly according to claim 15 wherein the ejector pin is capable of extending through and beyond the end of the cutting head.

17. A cutting tool assembly according to claim 16 wherein the cutting head is circular and the ejector pin is a rod of circular cross section.

18. A cutting tool assembly according to claim 16 wherein the cutting head is removable.