GB2535969A - Solid Phase Synthesis and Products Obtained thereby - Google Patents

Abstract

Described is a method for preparing or modifying an array surface having a multiplicity of different polymeric species arranged thereon. The method comprises exposing the surface to a plasma or glow discharge so as to form or modify the properties of the multiplicity of polymeric species. The surface roughness can be modified by the plasma. The reactivity of groups on the surface of the polymeric species can also be modified to allow attachment of additional functionality. The plasma can also be used to effect the polymerisation of a monomer mixture in order to form the polymeric species, via plasma polymerisation. Exemplified is the use of plasma polymerisation on low vapour pressure amino acids to generate a peptide array. Also described are microarrays produced by the method.

Description

Solid Phase Synthesis and Products obtained thereby The present invention relates to methods for use in connection with solid phase synthesis, in particular using combinatorial chemistry methods, so as to generate for example, microarrays and the like, as well as to arrays obtained using these methods.

For many biological or biochemical assays or procedures, direct immobilisation of biomolecules, and in particular polymeric molecules such as nucleic acids (e.g. DNA or RNA), or peptides or proteins onto surfaces is required. In many cases, where these are involved in experimental research, a combinatorial chemistry approach is adopted in order to generate a complex and variable population of polymeric biomolecules on the surface.

In one particular case, chemicals, each of which represents a single unit or elements of the desired polymeric array are mixed together in a liquid medium and applied to a substrate, such as silicon or glass substrate or a suitable synthetic membrane, and then "cured" using for example photolithography or uv irradiation. This can lead to an array of different polymer structures on the surface. For example, monomer units can be mixed to produce hundreds of different polymer species on the surface, and examples of 480 different polymers have been reported.

A review of microarray deposition technology is found for example G. McHale Analyst, 2007 132(3) 192-5.

Microarrays produced in this way have a wide variety of uses, which include many high throughput screening applications to measure a particular response of interest. In most cases, accuracy of results is very important, and therefore any factors which may impede this can be damaging.

In particular, surface roughness can be an important consideration in the functioning of certain microarrays. In some instances, the precise level of surface roughness can have an effect on the efficient operation of the microarray, for example when optimising the degree of cell binding to a surface. However, conventional photolithography techniques are unable to impact on this feature.

The applicants have found that plasma activation or polymerisation/curing may provide a useful and effective technique in the production of microarrays with the desired characteristics. In particular, it may be used to carry out the polymerisation, in particular in a manner in which surface roughness is controlled, or it may be used to roughen a preformed microarray surface.

According to the present invention there is provided a method for preparing or modifying an array surface having a multiplicity of different polymeric species arranged thereon, said method comprising exposing said surface to a plasma so as to form, or modify the properties of, a multiplicity of polymeric species.

In a particular embodiment, the plasma is used to modify the roughness of a surface comprising a multiplicity of different polymeric species, such as a preformed microarray. This may be achieved by using inert gas plasmas, for example comprising helium, argon of neon and or active gas plasmas comprising oxygen or nitrogen or a mixture thereof. The power of the plasma will be adjusted so that the desired roughening effect is achieved. For example by changing the delivered power, flow rate and operating pressure, the desired surface roughness effect can be achieved.

Where active plasmas are used in the procedure for modifying the roughness of the surface, they may also have the effect of altering the reactivity or nature of the polymeric species at the surface. This may be desirable in some instances, for example, where additional functionality is required at the surface in addition to the base polymers, for example to further encourage specific binding over non-specific binding for bio-markers or bio-filtration.

In fact, active plasmas used in this way may be used to carry out modification of the surface functional groups of a microarray alone, without impacting on the roughness, depending upon the conditions used in the plasma, and this procedure forms one aspect of the invention.

In a particular embodiment however, the plasma is used to effect the polymerisation itself. Thus, a surface having an appropriate monomer mix deposited thereon is exposed to a suitable plasma, for example one comprising a mixture of gases, under conditions in which the monomers polymerise in manner on the surface to form a random mix of polymers. The monomers may be predeposited using for example, conventional liquid deposition techniques.

It may be possible also, at the same time, to control factors such as the surface roughness. However, generally, one may carry out an initial polymerisation or curing step, followed by a suitable roughening treatment. These two procedures could be conducted-sequentially in the same chamber, without the need 30 for removing the product.

Thus, by utilising plasma polymerisation as a means of forming the polymers from the monomer mix in the array, close control of the resultant polymer structure can be maintained as well as the roughness of the surface. The polymerisation mode can be controlled and varied for instance by controlling the power of the plasma, whether or not the plasma is continuous or pulsed, the polymerisation or activation time, the concentration of the gas, the flow rates etc. By controlling these factors, the degree of polymerisation and surface activation can be selected, so that the random polymers formed on the surface have the desired physical characteristics such as length etc. The general nature of the polymers may even be varied as the polymer is formed, for example by supplying different monomer mixtures at different regions or times.

Shadowing, in which areas of the surface are masked or covered for some of all the activation process, making it more difficult for the activating gas particles to be present in those regions, may also be used to produce a gradient effect, so that polymers in one area of the array are generally modified or polymerised differently, for example are roughened to a different degree and thereby display different properties.

In this way, a random array having the desired predetermined density and orientation as well as physical properties, in particular roughness, may be achieved. Where either both the polymer curing and the roughness control or just the roughness control is carried out using a plasma, the surface may be formed and modified in one place, without changing chambers, thus simplifying the procedure.

Using this method for polymerisation purposes, plasmas are generated from molecules, which are subjected to an electrical field. When this is done in the presence of a substrate, the radicals of the compound in the monomer mix on the substrate, in this case the microarray, polymerise. Conventional polymer synthesis tends to produce structures containing repeat units that bear a strong resemblance to the monomer species, whereas a polymer network generated using a plasma in this way can be extremely complex. The properties of the resultant polymer can depend upon the nature of the substrate as well as the nature of the monomer used and conditions under which it is polymerised. By operating the plasma in specific modes, it is possible to create plasma polymers that resemble conventional polymerised polymers and variants thereof.

The nature of the monomer mixture applied to the substrate before polymerisation in any particular case, will be determined by the desired end use. For instance, the nature of the mixture of monomers used will depend upon the type of array required. For protein or peptide arrays for example, the monomers will comprise mixtures of amino acids whereas for nucleic acid arrays, mixtures of nucleotides will be used.

Any compounds, including saturated compounds may be polymerised using the plasma technique, as the activation converts the monomer compounds into radicals which are able to combine together to form polymeric moieties. The conditions within the plasma, as discussed above, may be controlled so as to minimise any unwanted degradation of the monomer structure during the process.

In the method, in general, the microarray to be treated is placed within a plasma chamber. In some cases, this will comprise a polymerised array, where the polymers have been formed using convention photolithography, uv radiation or the like. However, the surface may also carry a mixture of monomers which are able to generate the target polymeric types, before curing, so that they may be cured in the chamber. Depending on the approach taken, the target chemicals will either be in an essentially liquid or solid state, a glow discharge is ignited within the chamber by a suitable applied voltage to effect polymerisation and/or roughening.

Preferably, any vapour of the monomer mixtures in the microarry, should be limited or even eliminated in order to prevent any cross contamination across the array.

The plasma used may be continuous wave or pulsed depending upon the required result. Using pulsed plasmas, in which low average powers can be achieved, a highly controllable surface covering can be obtained with minimal deterioration of monomer, which is particularly important when retention of the monomer structure in the target polymer is required. However, where roughness is being adjusted, or the nature of surface groups modified using an active gas plasma (such as oxygen, nitrogen or air), continuous wave polymerisation may be used. In the case where the plasma is utilised to both effect polymerisation and then introduce a degree or roughening, then two different processes maybe used.

The applied fields are suitably of power of from 2 to 500W, suitably at about 20 to 200W peak power. When applied as a pulsed field, the pulses are suitably applied in a sequence which yields very low average powers, for example in a sequence in which the ratio of the time on: time off is in the range of from 1:3 to 1:1500, depending upon the nature of the monomer gas employed. Although for monomers which may be difficult to polymerise, time on: time off ranges may be at the lower end of this range, for example from 1:3 to 1:5, many polmerisations can take place with a time on:time off range of 1:500 to 1:1500. Particular examples of such sequence are sequences where power is on for 20-50ps, for example about 30ps, and off for from 1000ps to 30000ps, in particular about 20000ps.

Typical average powers obtained in this way are 0.01W. however 30 it may be required to use much higher powers in a continuous mode to ensure rapid polymerisation.

The fields are suitably applied from 30 seconds to 90 minutes, preferably from 5 to 60 minutes, depending upon the nature of 35 the monomer mix and complexity of microarray and gradient required.

Suitable plasmas for use in the method of the invention include non-equilibrium plasmas such as those generated by radiofrequencies (Rf), microwaves or direct current (DC). They may operate at atmospheric or sub-atmospheric pressures as are known in the art. In particular however, they are generated by radiofrequencies (Rf).

Various forms of equipment may be used to generate gaseous plasmas. Generally these comprise containers or plasma chambers in which plasmas may be generated. Particular examples of such equipment are described for instance in W02005/089961 and W002/28548, but many other conventional plasma generating apparatus are available.

In all cases, a glow discharge is suitably ignited by applying a high frequency voltage, for example at 13.56MHz. This is applied using electrodes, which may be internal or external to the chamber, but in the case of larger chambers are internal.

Suitably the gas, or gas mixture is supplied at a rate of at least 1 standard cubic centimetre per minute (scan) and preferably in the range of from 1 to 100sccm.

Gases may be drawn or flowed into the plasma region. In particular, where a plasma chamber is used, gases may be drawn into the chamber as a result of a reduction in the pressure within the chamber, caused by use of an evacuating pump, or by any other means suitable for introducing gases into a vacuum chamber.

A particularly suitable apparatus and method for the polymerisation or roughening of the arrays in accordance with the invention is described in W02005/089961, the content of 35 which is hereby incorporated by reference, however many different chamber set-ups are available with the most appropriate one dependant on the required through-put.

Precise conditions under which the plasma polymerization or modification takes place in an effective manner will vary depending upon factors such as the nature of the polymer being formed, as well as the nature of the microarray and will be determined using routine methods and/or other techniques.

The dimensions of the chamber will be selected so as to accommodate the particular array substrate being treated. The chamber may be a sealable container, to allow for batch processes, or it may comprise inlets and outlets for the substrates, to allow it to be utilised in a continuous process as an in-line system. In particular in the latter case, the pressure conditions necessary for creating a plasma discharge within the chamber are maintained using high volume pumps, as is conventional for example in a device with a "whistling leak". However it will also be possible to process arrays at atmospheric pressure, or close to, negating the need for "whistling leaks".

The thickness of the deposited array or degree of roughening or activation applied using the method of the invention will depend upon the nature of the product. The preformed array or the thickness of the pre-cured monomer mix is selected to allow for this. In some cases, the thickness of the array will be uniform, so as to ensure that target assay and any binding takes place evenly all over the surface of the array. However, as mentioned above, shadowing techniques may be used to form gradients within the array if this is required.

Factors which may be used to control the polymerisation of modification include the length of exposure to the plasma and 35 the pattern of the pulsing, as well as the pressure, flow rate and nature of the monomer.

Generally, a coating of random polymers which is up to 5000A thick, for example from 1-2000A is applied for most array purposes, however much smaller thicknesses may be adequate 5 depending on the subsequent analysis required.

Using the method of the invention, a variety of microarrays with functionalised surfaces may be prepared on suitable substrates which include glass, silica or membranes such as nitrocellulose membranes, or preformed microarrays may be modified as required, as described above.

These may be subsequently treated to prepare them for use if necessary.

Thus in particular, the invention provides a microarray having a multiplicity of different polymeric species thereon.

Example 1 -Preparation of Peptide microarray An array substrate such as a glass or silicon carrying a varying mix of low vapour pressure polymerisable amino acids in solid or liquid form is placed inside the appropriate sized vacuum chamber and evacuated to low pressure -5 mtorr. On reaching base pressure and ensuring minimal out-gassing rate / vapour pressure. Argon is introduced at a flow rate of 100 sccm to a pressure of 40 mtorr. On reaching the required operating pressure a pulsed plasma is struck using a radio frequency source at 100W peak power at a pulse on-time of 50 ms and an off time of 250 ms and the polymerisation of all chemical mixtures in the microarray ensues. The process runs for 20 mins after which the RE, and gases are turned off and the system is evacuated to base pressure. The resulting polymerised array is then ready for analysis.

Example 2 -Adjustment of surface roughness A microarray, using a monomer mix with a high vapour pressures may be unsuitable for polymerising using the plasma as described in Example 1 and so may be UV initiated to form the polymers in the microarray. The microarray is placed inside the appropriate sized vacuum chamber and evacuated to low pressure -5 x mtorr. On reaching base pressure and ensuring the out-gassing rate is satisfactory, a gas selected from Argon or Helium is introduced to a pressure of 80 mtorr. On reaching the required operating pressure a continuous plasma is struck using a radio frequency source at 40W peak power until the desired level of surface rouchness is achieved, which is generally about 20 mins after which the RF, and gases are turned off and the system is evacuated to base pressure.

Claims (5)

1. Claims 1. A method for preparing or modifying an array surface having a multiplicity of different polymeric species arranged thereon, said method comprising exposing said surface to a plasma so as to form, or modify the properties of, a multiplicity of polymeric species.
2. 2. A method according to claim I wherein the plasma is used to modify the roughness of a surface comprising a multiplicity of different polymeric species.
3. 3. A method according to claim 1 or claim 2 wherein the plasma is an active plasma which has the effect of modifying 15 the reactivity of groups on the surface of the polymeric species.
4. 4. A method according to any one of the preceding claims wherein the plasma is used to effect the polymerisation of a 20 mixture of monomers so as to generate the multiplicity of polymeric species.
5. 5. A microarray formed or modified using a process according to any one of the preceding claims.