WO2009125221A2

WO2009125221A2 - Buffers for non-contact microarray spotting

Info

Publication number: WO2009125221A2
Application number: PCT/GB2009/050342
Authority: WO
Inventors: Howard John Manning; Duncan John Hall
Original assignee: Arrayjet Limited
Priority date: 2008-03-09
Filing date: 2009-04-08
Publication date: 2009-10-15
Also published as: WO2009125221A3; GB0806473D0

Abstract

The present invention relates to a method of microarray spotting which utilises improved buffers. In particular it relates to buffers being used in a non-contact microarray system and toamethod of printing samples wherein samples are prepared in a sample buffer, nozzles are then purged in a forwards direction with a system buffer; samples are then aspirated into the nozzles displacing at least a portion of a system buffer without mixing with it; and samples are then printed through the nozzles.

Description

Buffers for non-contact microarray spotting

The present invention relates to a method of microarray spotting which utilises improved buffers. In particular it relates to buffers being used in a non-contact microarray system.

Spotted microarrays, and other patterns of spots of liquid samples, are made by loading liquid samples from a source such as microtitre plates into a printhead and spotting them onto substrates such as coated microscope slides. The samples are typically biological and take the form of aqueous solutions or suspensions. The printhead may consist of solid or split pins, or other contact devices. More desirable, however, are non- contact devices; the printhead may consist of one or more pipettes, each equipped with piezoelectric or other means for projecting drops from a nozzle at its tip, or the printhead may be a multi-nozzle inkjet printhead. The samples are often available only in small quantities, so dead volume is minimised by introducing the samples at the tips or nozzles from which they are subsequently printed.

UK Patent GB1307292 describes a means of introducing or loading multiple different samples into a multi-nozzle inkjet printhead, keeping them separate, printing them without cross-contamination, and purging so that further sets of samples can be handled without carryover. This involves the temporary attachment, to the nozzle plate of the printhead, of a connection block device which has at its lower end multiple capillaries and at its upper end seals, one associated with each capillary, which make contact with the nozzle plate. Samples are aspirated from the source plate via the capillaries and the seals into the printhead, flowing backwards through the nozzles into the chambers associated with each nozzle. Subsequently the connection block is detached and the samples can be spotted by the printhead, forwards through the nozzles, onto the substrates, without contact between the printhead and the substrates.

In the case of contact printheads it is necessary, prior to loading of samples, to wash the tips with a 'system buffer' to remove the previous samples. With non-contact printheads, system buffer is purged from the rear of the printhead, forwards through the nozzles, to remove traces of previous samples prior to the aspiration of samples via the nozzles. If a connection block is being used with a multi-nozzle inkjet printhead, then it is purged at the same time, from the seals towards the capillaries. Some printheads are sensitive to the presence of air, and the purging removes it from the printhead (and the connection block if used), preventing air from entering the printhead during the subsequent aspiration of the next set of samples.

The obvious way to operate any of the systems described above is to employ a system buffer which is identical to the buffer in which the samples are dissolved. In this case when the samples are aspirated, any mixing of the samples with the system buffer merely results in a degree of dilution of the samples rather than contamination of the samples by another buffer. However, the identity of the two buffers imposes a constraint on the system, as the buffer has to be suited to the processes of purging, aspiration and printing, but must also be suitable for the final application on the substrate.

Another possibility is that the system buffer is different from but compatible with the sample buffer. The advantage of this approach is that the two buffers can be optimised, to some extent separately, for different functions within the overall operation of the system. The sample buffer can be optimised primarily for printing of the spots and for the final application on the substrate, while the system buffer is optimised primarily for the processes of purging. For example, the system buffer may contain surfactant to encourage good wetting of the interior of the phnthead in order to exclude air, but the sample buffer may contain less or no surfactant, in order to produce small printed spots on the substrate. The two buffers cannot be optimised entirely separately, because typically mixing during aspiration leads to a degree of contamination of the samples by components of the system buffer, and the printed spots must meet the requirements of the final application despite this contamination. Furthermore in some systems, the system buffer has to be capable of printing despite the fact that the overall purpose is the printing of samples.

The Applicant's original technology was developed using a system buffer consisting of water, glycerol and Triton X100 (octyl phenol ethoxylate). The glycerol increases the viscosity of the water to a level suitable for inkjet printheads; for example, 50% glycerol by weight raises the viscosity of water from 1 mPa.s to 6 mPa.s, just adequate for the Xaarjet® XJ126 printhead that is used by the Applicant. The Triton is a surfactant which lowers the high surface tension of water to a level suitable for the printhead; for example 0.05% Triton by weight reduces the surface tension of water from 70 mN/m to around 30 mN/m.

This system buffer has the considerable advantage that all the components are liquids, miscible in all proportions. It is therefore unlikely that the system buffer will block nozzles, or the capillaries of the connection block. Furthermore, glycerol is a good humectant, preventing evaporation of water from the system buffer in the nozzles which might lead to the appearance of air in the printhead. The result is that the system can be left unattended for many weeks and then re-started with a simple purge of the printhead and connection block. This system buffer is suitable for many applications where a biological sample such as DNA, protein or other water-soluble substance of interest can be dissolved in buffer based on glycerol, or free from glycerol but compatible with it, and printed in the context of glycerol-based system buffer. However, some applications are sensitive to the presence of glycerol on the substrate, and even if the samples are dissolved in buffer free from glycerol, the printed spots are contaminated by a certain amount of glycerol originating in the system buffer.

The present invention is predicated on the inventor's identification of the fact that it would be advantageous if the samples being aspirated did not mix with the buffer into which they were being aspirated, but instead displaced it. In this way the aspirated sample would not be subject to dilution and, further, contamination of the aspirated samples by components of the displaced buffer would be reduced or even eliminated. As a result, a higher proportion of the sample aspirated could be printed usefully than would otherwise be the case.

Thus, it is an object of the present invention to provide a method whereby the proportion of aspirated sample which can be usefully printed is enhanced over known systems.

It is a further object of the present invention to obviate or mitigate some of the problems associated with the prior art.

The present invention provides a method of printing samples utilising a printhead with one or more nozzles capable of spotting the samples onto the substrates, comprising the steps of; preparing one or more samples in a sample buffer; purging the one or more nozzles in a forwards direction with a system buffer; aspirating one or more samples into the printhead, flowing in a backwards direction through the nozzles and displacing at least a portion of the system buffer in the printhead without mixing with it; and printing the samples through the nozzles in a forwards direction, onto the substrates, without contact between the printhead and the substrates.

In embodiments of the present invention, the method may further comprise the step: repeating the purging, aspiration and printing steps with further samples.

Optionally the method may further comprise the step of; purging the system buffer from the one or more nozzles of the printhead (and the connection block, if present) using a shutdown buffer prior to finishing printing.

A first aspect of the present invention is characterized in that the system buffer is substantially immiscible with the sample buffer.

Preferably the sample buffer is aqueous.

Preferably the system buffer is substantially immiscible with water.

In embodiments of the invention wherein the sample buffer is aqueous and the system buffer is substantially immiscible in water, the system buffer may be used to purge the one or more nozzles of the printhead (and connection block, if present), and, when the aqueous sample is subsequently aspirated into the printhead, the system buffer and the aqueous sample buffer will not mix, but instead the sample buffer will displace at least a portion of the system buffer remaining in the one or more nozzles of the printhead following the purging step.

In embodiments of the first aspect of the present invention, the sample buffer and/or the system buffer may contain components to combat the formation of an emulsion. Thus, an anti-emulsion agent in either the sample buffer and/or the system buffer may be utilised to enhance the immiscibility of the sample buffer and the system buffer. In this way, much of each sample can be printed without dilution or contamination by components from the system buffer.

It is much by preference that the samples and the system buffer are substantially immiscible with one another.

In embodiments of the first aspect of the present invention, it is much by preference that the sample buffer and the system buffer are the only buffers utilised in the method.

In embodiments of the first aspect of the invention wherein only two buffers are used, the embodiments preferably use a water-based sample buffer and a system buffer which is substantially immiscible with water

(less than 0.1 % solubility in water). Thus, system buffer is used to purge the printhead (and connection block, if present), and when water-based sample is aspirated, they will not mix with the system buffer but instead will displace it.

Given the immiscibility, and, for example,when the solubility of the samples in the system buffer is less than 0.1 % solubility, the samples may not be purged away completely by the system buffer; however, if the system buffer wets the interior of the printhead well, and is highly immiscible in water, then the purging process may be sufficiently effective.

In embodiments of the present invention, examples of suitable system buffers include, but are not limited to, a polydimethylsiloxane such as Dow's silicone oil DC200 5cS, and perfluohnated organic solvents such as perflurodecalin, Fluorinert FC-43 (made by 3M) or Galden HT200 or HT300 (made by Solvay Solexis), for example.

A variant of the immiscible system buffer is to select one which is slightly miscible with water (0.1 -10% solubility). During aspiration of samples there will not be sufficient time for the system buffer to dissolve to a significant extent in the samples, so contamination of the printed spots is not a concern. During purging, most of the samples will be displaced from the printhead, establishing an excess of system buffer over sample which allows the system buffer to dissolve the remaining traces of sample. It is desirable that the amount of water which can dissolve in the system buffer exceeds the amount of system buffer which can dissolve in water. Suitable buffers with low and unsymmethcal miscibility with water include 2- ethylhexyl benzoate, dibutyl adipate, polyethylene glycol dibutyl ether, dipropylene glycol n-butyl ether, coasol (a mixture of c20% diisobutyl succinate, c60% diisobutyl glutarate and c20% diisobutyl adipate), isobutyrate of 2,2,4-thmethylpentane-1 ,3-diol and the corresponding diisobutyrate. Even more suitable, but compromised by safety considerations, would be ethylene glycol monohexyl ether, ethylene glycol mono-2ethylhexyl ether and diethylene glycol monohexyl ether.

Various of the above candidate buffers have been tested by way of example of a method using an immiscible system buffer, and aspiration of water-based samples into it. In such examples, the printing instrument was ran in the normal way, however samples were labelled with Cy3 fluorescent dye, and the system buffer was unlabelled, so scanning of the printed substrates shows when sample had been aspirated into nozzles. The majority of printed spots were fluorescent, indicating successful aspiration, and a minority of non-fluorescent spots indicated that some nozzles had not aspirated correctly.

When fluorescently labelled water-based samples are aspirated into standard water-based system buffer, the printed spots reveal that the drops ejected by each nozzle are relatively undiluted at first, but that as more drops are ejected dilution increases slowly at first and then more rapidly. In use, software is operable to protect the user by limiting the number of drops that are ejected from each nozzle; typically this limit is set at 500 drops out of a total of 1600 drops within the channel behind each nozzle.

In an example in which the immiscible buffer tested is coasol, about 95% of the nozzles of the phnthead aspirated sample. The number of drops which could be ejected from a given nozzle without significant dilution ranged from zero to 2000, and was around 1000 for a large number of nozzles. Both 2-ethylhexyl benzoate and dibutyl adipate were also useful examples of buffers.

Noticeably, during test printing runs it was found that, unlike with conventional printing where there is a gradual increase in dilution as the number of drops of sample printed increase, with the present embodiments there is no noticeable decrease in print quality as the number of printed drops increases until a sudden failure when a certain threshold of drops is reached. Therefore, where with conventional printing only approximately 500 drops or 1/3 of the chamber behind the nozzle are printed, the present invention allows printing of approximately 1000 drops or 2/3 of the chamber behind the nozzle to be printed without a loss in quality.

In some non-contact phntheads, the problem of mixing of sample and system buffer is addressed by interposing a barrier between them. After the printhead has been purged with system buffer, but before the samples are aspirated, a small amount of air is deliberately aspirated into the printhead via the nozzles, displacing system buffer backwards through the printhead. Now when samples are aspirated, they displace the air further backwards and the air barrier prevents the two liquid buffers from mixing. This addresses many of the difficulties described above, and also allows unprinted sample to be returned to the source after printing. However, the presence of air in an inkjet device is a considerable source of unreliability; it is preferable to use a liquid barrier.

According to a second aspect of the method according to the present invention, a barrier buffer is aspirated into the printhead prior to the aspiration of the samples, wherein the barrier buffer is a liquid which is substantially immiscible with the sample buffer.

Preferably the barrier buffer is an oil.

The barrier buffer is preferably immiscible with either the system buffer and/or the sample buffer therefore preventing any mixing therebetween. In the way, the need to optimise the sample and system buffers to work together is reduced or even negated as there will be no or limited mixing of the buffers in the printhead. In embodi merits of the second aspect of the invention, one or more of the system buffer, the barrier buffer and the sample buffer may contain one or more components to prevent the formation of emulsion.

In embodiments of the second aspect of the present invention, the barrier buffer provides a barrier between the samples and the system buffer, wherein the barrier buffer is preferably immiscible with both the sample buffer and the system buffer. In use, system buffer is used to purge the printhead (and the connection block, if present), then a small amount of barrier buffer is aspirated, followed by the samples. The barrier buffer maintains separation of the samples and the system buffer, but at no stage is air introduced into the printhead. The samples can be printed subsequently without dilution or contamination by components from the system buffer. As a result a larger proportion of the aspirated volume of each sample can be used than is possible with miscible buffers.

This scheme is more complicated than the first aspect of the invention in that there are three buffers involved, and there are two boundaries between liquids. With immiscible liquids, there is a danger that an emulsion may form, so some of the buffers might contain components to prevent this.

There is also a danger that samples and/or barrier buffer may not be purged away completely by the system buffer, and may therefore accumulate. This can be addressed by various combinations of miscibility and immiscibility: the system buffer may be miscible, partially miscible or immiscible with the sample buffer and with the barrier buffer (though these are immiscible with each other).

The second aspect of the present invention requires the barrier buffer to be a liquid which is immiscible with water, safe and not too flammable (although only small amounts are used); of which there are many suitable examples. If, in addition, the liquid is volatile and less dense than water, there is the option of returning unphnted sample to the source plate, accompanied by some barrier buffer. In such embodiments, the barrier buffer will float on top of the samples in the wells, and evaporate over time, leaving the samples unaffected. A suitable buffer might be hexane; volatile solvents of low density tend to be flammable, but the amount used in the method of the present invention would be very small, therefore mitigating the risk. Another possibility is to use a barrier buffer which is denser than water, but partially miscible with water and highly volatile but not flammable, for example dichloromethane; then the barrier buffer returned with each unprinted sample sinks at first, but over time mixes with the sample and evaporates away. A third variant is to use a non-volatile, non-flammable, low-density immiscible barrier buffer such as silicone oil; this would float on top of the samples and remain there, but it would serve to prevent evaporation of the samples from the wells prior to subsequent use.

Preferably, in any aspect of the present invention, the system buffer has a viscosity in the range 4 cP to 20 cP, more preferably 5 cP to 2OcP and even more preferably 6 cP to 10 cP.

Preferably, in any aspect of the present invention, the system buffer has a surface tension no more than 30 mN/m.

Claims

1. A method of printing samples utilising a phnthead with one or more nozzles capable of spotting the samples onto the substrates, comprising the steps of; preparing one or more samples in a sample buffer; purging the one or more nozzles in a forwards direction with a system buffer; aspirating one or more samples into the printhead, flowing in a backwards direction through the nozzles and displacing at least a portion of the system buffer in the printhead without mixing with it; and printing the samples through the nozzles in a forwards direction, onto the substrates, without contact between the printhead and the substrates.

2. A method according to claim 1 , wherein the steps of purging, aspiration and printing are repeated with further samples.

3. A method according to claim 1 or claim 2, further comprising the step of purging the system buffer from the one or more nozzles of the printhead using a shutdown buffer prior to finishing printing.

4. A method according to any one of claims 1 to 3, wherein the system buffer is substantially immiscible with the sample buffer.

5. A method according to claim 4, wherein the sample buffer is aqueous.

6. A method according to claim 4 or claim 5, wherein the system buffer is substantially immiscible with water.

7. A method according to any one of claims 4 to 6, wherein the system buffer and/or the sample buffer contains one or more components to prevent the formation of an emulsion.

8. A method according to any one of claims 4 to 6, wherein the samples and the system buffer are substantially immiscible with one another.

9. A method according to any one of claims 4 to 8, wherein the solubility of the system buffer in water and the solubility of water in the system buffer are both less than 0.1 % and the system buffer wets the interior of the printhead well.

10. A method according to claim 9. wherein the system buffer contains polydimethylsiloxane, perfluohnated organic solvents, or a mixture thereof.

11.A method according to any one of claims 4 to 8, wherein the solubility of the system buffer in water and the solubility of water in the system buffer are both in the range 0.1 -10%.

12. A method according to claim 11 , wherein the solubility of the system buffer in water is less than the solubility of water in the system buffer.

13.A method according to claim 11 or claim 12, wherein the system buffer contains 2-ethylhexyl benzoate, dibutyl adipate, polyethylene glycol dibutyl ether, dipropylene glycol n-butyl ether, coasol (a mixture of c20% diisobutyl succinate, c60% diisobutyl glutarate and c20% diisobutyl adipate), ethylene glycol monohexyl ether, ethylene glycol mono-2ethylhexyl ether, diethylene glycol monohexyl ether, either the isobutyrate or the diisobutyrate of 2,2,4-thmethylpentane- 1 ,3-diol, or a mixture thereof

14.A method according to any one of claims 1 to 3, wherein a barrier buffer is aspirated into the printhead prior to the aspiration of the samples, and the barrier buffer is a liquid which is substantially immiscible with the sample buffer.

15. A method according to claim 14, wherein the barrier buffer is an oil.

16.A method according to claim 14 or claim 15, wherein one or more of the system buffer, the barrier buffer and the sample buffer contain one or more components to prevent the formation of emulsion.

17.A method according to any one of claims 14 to 16, wherein the barrier buffer is substantially immiscible with the system buffer.

18.A method according to any of claims 14 to 17, wherein the system buffer is substantially miscible with the sample buffer.

19.A method according to claims 14 to 16 or claim 18, wherein the system buffer is substantially miscible with the barrier buffer.

20. A method according to any of claims 14 to 19, wherein after printing of the samples, unused samples are returned to the places from which they were aspirated.

21. A method according to claim 20, wherein the barrier buffer is less dense than water and volatile.

22. A method according to claim 21 , wherein the barrier buffer is hexane.

23.A method according to claim 20, wherein the barrier buffer is partially miscible with water, denser than water, volatile and not flammable.

24.A method according to claim 23, wherein the barrier buffer is dichloromethane

25.A method according to claim 20, wherein the barrier buffer is less dense than water and non-volatile.

26.A method according to claim 25, wherein the barrier buffer is polydimethylsiloxane^

27.A method according to any of claims 1 to 26, wherein the viscosity of the system buffer is in the range 4 to 20 cP

28.A method according to claim 27, wherein the viscosity of the system buffer is in the range 6 to10 cP.

29.A method according to any of claims 1 to 28, wherein the system buffer contains one or more surfactants to reduce its surface to no more than 30 mN/m.

30. A method according to any of claims 1 to 29, wherein the nozzles are replaced by tips which make contact with the substrates during spotting.