Prior Art Polycrystalline diamond is currently one of the most interesting materials under consideration for microstructure technology. It can be chemical vapour deposited (CVD), typically on silicon, at low cost.

Interest has been shown in using diamond microstructures such as microcapillaries in the biochemical and pharmaceutical industries for synthesis and analysis applications. This is because of extremely good material properties of diamond which include the rare combination of high thermal conductivity, high electrical insulating capability (when pure), high mechanical strength, chemical inertness, high wear-resistance, transparency to electromagnetic radiation in a very broad waveband and high refractive index. In the case of chromatography or electrophoresis these properties allow the use of high field strengths and high pressures and could lead to shortened analysis times.

A method for producing diamond microstructures is known from the article"Diamond replicas from microstructed silicon masters"by H. Björkman, P. Rangsten, P. Hollman, K.

Hjort published in"Sensors and Actuators"73 (1999) pages 24-29, Elsevier Science S. A. It describes how to produce microstructures such as capillaries in diamond, using silicon microstructuring and diamond replication. However the resulting structures were not suitable for use in biochemical and pharmaceutical processes owing to the hydrophobic nature of the surface of the CVD. A CVD diamond surface is typically hydrogen-terminated from the atomic hydrogen in the CVD process. The hydrogen-termination that arises from the diamond deposition process is stable in air at room temperature and gives a very hydrophobic surface.

In most analytical methods involving separation of biomolecules, a hydrophilic (and non-

charged) surface of the column wall or stationary phase is desirable. Generally, such a surface simplifies liquid handling (often aqueous buffers) and shows less non-specific interaction with the analyte, which favours rapid adsorption-desorption kinetics and minimises sample losses. Therefore there is a need for diamond microstructures which exhibit hydrophilic surfaces.

Obiect of the invention The objects of the present invention relate to diamond microstructure devices suitable for use in biochemical and pharmaceutical synthesis and analysis, methods for producing such diamond microstructures and instruments using such devices.

Summary of the Invention The above problems with the prior art diamond microstructure devices are solved by means of a method in accordance with the characterising part of claim 1. Microstructures suitable for use in biochemical and pharmaceutical synthesis and analysis have the features stated in claims 6 and 8.

Brief description of the Drawings Figure 1 shows schematically a droplet on a surface.

Figure 2 is a table showing contact angle against oxidisation temperature for diamond.

Detailed Description of Embodiments Illustrating the Invention The present invention will be illustrated by non-limiting examples below.

Diamond films were deposited on silicon substrates, preferably (100) silicon substrates, using Hot Filament Chemical Vapour Deposition (HFCVD). Naturally any other form of diamond deposition could have been used. Ultrasonic agitation of the silicon substrates with nano- cluster diamond suspended in ethanol was used to enhance the nucleation density of diamond.

The diamond films were grown in a hydrogen atmosphere with 1.5 % methane at 35 mbar.

Eight 10 cm long 0.5 mm diameter tantalum filaments arranged in an array with a power

consumption of 2800 W, at a distance of 8 mm from the substrate, were used. During diamond deposition, the temperatures of the silicon substrates were about 850-900°C.

Wetting experiments were performed on the smooth side of polycrystalline diamond films after the silicon substrates had been removed by sacrificial etching HF: HN03 (3: 7) at 80 °C.

For comparison, commercial optical grade CVD diamond from Drukker International B. V.

Beverstraat 20,5431 SH Cuijk, The Netherlands, with a surface roughness below 30 um was also used.

To determine the surface wetting ability droplets of water with a known volume were put on the diamond surfaces where after the droplet diameter was measured. The diameter was then converted to the corresponding surface contact angle by using the equation below.

V is the droplet volume, d is the droplet diameter, and a is the contact angle-see Fig. 1. A large contact angle a implies a hydrophobic surface and a small contact angle implies a hydrophilic surface.

The samples used for the experimental investigation of diamond wetting properties were thick (20 to 100 m) polycrystalline diamond films deposited on smooth (100) n-type silicon wafers. The silicon was sacrificially etched away in HF: HN03 (7: 3) at 70°C, revealing smooth diamond surfaces for the measurements. Additional etching for 25 minutes ensured clean diamond surfaces. These surfaces were then made even more hydrophobic by placing them in a hot filament CVD reactor with a pure hydrogen atmosphere, a filament temperature of about 2000°C, a substrate temperature of about 800°C, and a pressure of 35 mbar for 20 minutes.

After this treatment, the diamond samples were placed in a wet-oxidation furnace (Koyo- Lindberg ut-6) at different temperatures ranging from 200°C to 700°C for 15 minutes each time. The gases used were equal amounts of O2 and H2 that were burnt by a torch at 850°C, converting the gases to 67 atomic% H20 (g) and 33 atomic% 02 (g) before entering the furnace.

To further investigate the wetting properties, the experiment was repeated on samples that were placed in a dry-oxidation furnace (only 02 (g)) for 15 minutes at different temperatures ranging from 200°C to 500°C.

Some initial tests of the long-term stability of the surface-terminations were also performed by putting the surfaces in air (21°C 1°C and 43 % 3 % humidity) for a few days and measuring the contact angle before and after storage.

Hydrophilicity inside diamond channels was investigated by comparing the capillary forces of as-deposited, dry- (450°C for 15 minutes), and wet-oxidised (450°C for 15 minutes) diamond channels by optical inspection Diamond channels were made as follows : Thick bonded SOI wafers of (100) orientation were used as starting material. a) The wafer was patterned using standard lithography. b) The device layer was structured by dry or wet etching. The buried oxide layer was used as etch stop. The exposed oxide was removed. c) Diamond was deposited a first time. d) The remains of the buried oxide were used as a stop layer while the bulk silicon was etched away from the backside of the wafer by wet etching. The now-revealed oxide was then removed. e) This left a planar surface over the whole chip, and diamond was deposited a second time. f) The ends of the diamond capillaries were opened, exposing the enclosed silicon, and the silicon was sacrificially etched away leaving hollow diamond structures.

Before changing the hydrophilicity of the surface of a diamond microstructure, either after it has been manufactured or after it has been used and become blocked, it is desirable to ensure that any hollow structures are free of blockages or contaminants. Therefore, before oxidising the diamond surfaces it is preferable to perform a cleaning step g) by immersing it in, or otherwise exposing it to, a cleaning solvent or corrosive fluid which does not attack diamond but which dissolves or corrodes contaminants, for a sufficiently long time in order to ensure that all hollow structures are unblocked. The choice of solvent or corrosive fluid is naturally dependent on the history of the diamond microstructure being cleaned-a new microstructure needs to have any remaining silicon removed while a microstructure which has been used for biochemical purposes needs to be treated with a suitable solvent or strong acid or alkali.

In a first embodiment of a method in accordance with the invention to fabricate long capillaries for use as, for example, chromatography columns, SOI wafers with a 46 am device layer were used. Micromachining of meander-shaped v-ridges (73 am wide at the base and 13 am wide at the top) was performed with an anisotropic potassium hydroxide solution (70 g KOH/100 ml H20) at 80°C using the slow-etching (111) planes and the buried oxide layer as etch stops. Diamond was deposited to a thickness of about 20 um. After diamond deposition, the carrier wafers were removed in a potassium hydroxide solution (40 g KOH/100 ml H20) at 90°C, using the remains of the oxide layers as etch stop. The remains of the oxide layers were then removed by HF: H20 (1: 4). This was followed by a second diamond deposition to a thicknesses of about 20 am. Finally, the diamond microchips were cleaved, exposing the enclosed silicon cores, which were sacrificially etched in HF: HN03 (3: 7) at 80°C. The microchips were wet-oxidised (as described in more detail below) at 450°C to make the surfaces hydrophilic.

In a second embodiment of a method in accordance with the invention to fabricate long capillaries for use as, for example, chromatography columns, the diamond microchips for chromatography were fabricated in a similar way as the capillaries above. Two different kinds of SOI wafers were used; wafers with 40 um and 70 am device layer thicknesses. The micromachining of the straight ridges with 100 am wide rectangular x-sections were performed in a Plasma Therm Deep Etch System (ICP) using the Bosch process (cycling C4Fg, SF6, and Ar). The ridges were anisotropically etched using silicon dioxide as masking material and the silicon dioxide interlayer as etch stop. The exposed oxide was removed by RF plasma etching (20 seem 02, 20 mbar, 100 W) to avoid under-etching. 10-20 am thick diamond films were deposited, as described above. After diamond deposition, the carrier wafers were removed in a potassium hydroxide solution (40 g KOH/100 ml H20) at 90°C using the remains of the oxide layers as etch stop. The remains of the oxide layers were then removed by HF (1: 4). This was followed by a second diamond deposition to thicknesses between 10-20 am. Then the diamond chips were cleaved, exposing the enclosed silicon cores on both sides, which were sacrificially etched in HF: HN03 (3: 7) at 80°C. Finally, the microchips were wet-oxidised (as described in more detail below) in the oxidation furnace at 450°C to make the surfaces hydrophilic.

After the silicon had been sacrif cially etched away revealing a free-standing diamond film, the contact angle was measured and found to be 47°, which is regarded as hydrophobic. When the diamond surface had been put in the hydrogen furnace for 15 minutes, the contact angle had risen to 65°, reflecting an increase in hydrophobicity.

In the wet-oxidation method for producing hydrophilic surfaces, a hydrogen-terminated diamond surface was put in the oxidation furnace at different temperatures ranging from 200 to 700°C for 15 minutes. At 200°C, there was no detectable change in hydrophilicity. Between 200°C and 400°C, the surfaces started to change from hydrophobic to more hydrophilic properties.

The hydrophilicity of the surface was adjusted by choosing the temperature at which the oxidation is performed. Figure 2 shows a table showing contact angle a against oxidation temperature T with results for wet oxidation shown with circles and dry oxidiation by triangles. Using wet oxidation, a temperature of 250 °C gave a contact angle of about 32°, a temperature of 300 °C gave an angle of about 15°, a temperature of 350°C gave an angle of about 13°, a temperature of 400°C gave an angle of about 7°, a temperature of 450°C gave an angle of about 6°. The lowest contact angle of 5° was achieved at 463 °C and higher temperatures did not decrease the contact angle. Intermediate temperatures gave intermediate contact angles. The present invention therefore makes it possible to accurately provide a surface with a desired contact angle/hydrophilicity by wet oxidising. At 463°C, the surface had reached its maximum hydrophilicity resulting in a contact angle of 5°. No etching was detected up to 527°C. Normally, etching of the grain bounderies between the crystals of the polycrystalline diamond is expected to start in an oxygen atmosphere at 500-550°C. However, the presence of water vapour retards the etching possibly because the OH-group terminates the surface. Although the grain bounderies were notably etched at 700°C, a contact angle of 5° could still be measured.

In the dry-oxidation method for producing hydrophilic diamond surfaces, a hydrogen- terminated diamond surface was put in the oxidation furnace at different temperatures ranging from 200-500°C for 15 minutes. At 200°C, there was a small change in contact angle.

Between 200°C and 450°C, the surfaces started to show clear changes from hydrophobic to more hydrophilic properties. Using dry oxidation, a temperature of 250 °C gave a contact angle of about 48°, a temperature of 300 °C gave an angle of about 36°, a temperature of 350°C gave an angle of about 20°, a temperature of 400°C gave an angle of about 18°, a temperature of 450°C gave an angle of about 7°, and a temperature of 500°C gave an angle of about 4°. At 500°C, the surface had reached its maximum hydrophilicity within the temperature range tested, resulting in a contact angle of 4°. However, this hydrophilic surface- termination was quite unstable. After only a few minutes, the contact angle had risen to around 10°. No etching was detected. Higher temperatures did not decrease the contact angle.

Intermediate temperatures gave intermediate contact angles. The present invention therefore makes it possible to accurately provide a surface with a desired contact angle/hydrophilicity by dry oxidising.

No difference in contact angles between the polycrystalline diamond films deposited on silicon and the commercial CVD diamond film were detected for similar surface treatments.

The microchannels produced with hydrophilic surfaces could be used for chromatography (and electrochromatography) in the gradient mode as well as the in situ preparation of the continuous bed in microchips. The diamond chip was mounted on a supporting plate of black Plexiglas having a slit for UV-detection. Fluid transfer fittings were attached to the chip by means of specially designed screw clamps.

Two samples, composed of different proteins, were separated by pressure-driven gradient elution on an anion-exchange diamond column having a hydrophilic surface produced by a method in accordance with the present invention. The preparation of the continuous beds was as follows. A monomer solution of low viscosity was pressed into the chip channel (as a consequence thereof, there is no chromatographic restriction as to the width of the channel or its configuration). During the subsequent polymerisation the polymer chains aggregate due to hydrophobic interactions promoted by salt, which results in the formation of channels between the aggregates large enough to permit hydrodynamic flow. The bed can be regarded as a polymer rod consisting of covalently linked 0.1-0.4 um particles. These particles form in

turn larger aggregates, depending on the polymerisation conditions. The bed was mechanically rigid and additional structures to support the bed were not needed. A bed derivatised with ammonium groups for anion-exchange chromatography was prepared as follows. A set of selected monomers (piperazine diacrylamide, methacryl amide and dimethyl diallyl ammonium chloride) and a salt (ammonium sulphate) were dissolved in a sodium phosphate buffer (50 mM, pH 7.0). This monomer mixture was degassed with a stream of nitrogen and supplemented with an aqueous solution of ammonium persulfate and N, N, N', N'- tetramethylethylendiamine (TEMED) before being pressed into the microchip channel. The polymerisation proceeded for 24 hours with both ends of the chip column covered with rubber lids. UV-detection was performed through a segment without a bed at the end of the chip channel. Before use, the bed was washed with water and then equilibrated with the starting buffer.

In the investigations of whether the hydrophilic properties of a diamond capillary could be changed by wet-oxidation, a hydrogen-terminated diamond capillary had been put in the oxidation furnace at 450°C for 15 minutes in analogy with the process described above. Dry- oxidation of capillaries for 15 minutes at 450°C, with all other conditions in analogy with what has previously been described, was also performed. Estimation of capillary forces, which was performed by optical inspection for these treated capillaries and an as-deposited capillary as control, revealed much stronger forces inside both treated capillaries than in the control. This fact indicates that the oxidation works also inside capillary channels.

Diamond microchips for liquid chromatography were fabricated using the method described above. The straight columns were 40 mm long, 100 um wide, and 40 um or 70 um high. The walls were smooth on the inside and rough on the outside. All surfaces were made hydrophilic using wet-oxidation, although dry-oxidation could equally well have been used.

A continuous chromatography bed was prepared in a straight chip column of 40 um i. d.

(height) x 100 um i. d. (width) with a total length of 40 mm (the effective length of the continuous bed was 32 mm). Two examples of rapid chromatography were tested. Four acidic proteins (myoglobin (horse), conalbumin, ovalbumin (chicken), and trypsin inhibitor

(soybean)) with isoelectric points ranging from 4.5 to 6.9 were separated by gradient elution in less than 30 s. A sample of Hemoglobin Ao and p-lactoglobulin A was separated in about 20 s on the same column. Following 10 to 20 s equilibration with the starting buffer, the chip was immediately ready for another analysis. For both chromatographic runs, the sample was injected by applying a pressure of 1 bar for 10 s. During the runs, the pressure was 9 bar and the composition of the mobile phase was changed from 20 mM phosphate buffer, pH 7.8, to 0.4 M sodium chloride in 20 mM phosphate buffer, pH 7.8 over a period of 30 s.

The present invention relates to the conversion from hydrophobic to hydrophilic of the properties of diamond surfaces based on a mixture of H20 and 02 in a wet-oxidation furnace, The wet-oxidation method of the present invention is particularly efficient at temperatures higher than about 400°C. In addition, the present invention relates to dry-oxidation with pure 02 in a dry-oxidation furnace, wherein the conversion from hydrophobic to hydrophilic properties of the same kind of diamond surfaces is particularly efficient at temperatures higher than about 450°C. Using the present method it is possible to tailor the contact angle of a diamond surface between 5 and 65°, easily and robustly, depending on what the particular application requires.

In all chromatographic techniques, it is of great importance to minimise the"wall-effect", which means that the stationary phase should be uniform also close to the inner wall of the separation column. Otherwise zone-broadening may occur due to, for instance, an increase in Eddy diffusion or unspecific interactions between the analyte and the inner wall. These problems are especially pronounced for proteins that have relatively low diffusion coefficients and multiple interaction sites. However, the columns of the present invention had a homogenous continuous bed also close to the wall, which was confirmed by examination by light microscopy and by the fact that the protein peaks were symmetrical. Moreover, the resolution of the chromatograms is in accordance with previous separations on continuous beds synthesised in both capillaries and microchips. This indicates that also the diamond chips may be used with advantage for other types of microscale chromatography.

Using the present invention to dry-and wet-oxidise polycrystalline diamond surfaces so that they become hydrophilic means that it is possible to tailor the contact angle to a chosen value

between 5 and 65°. After a week of ageing, a hydrophilic diamond surface's contact angle changes from 5° to nearly 25°, at which point the contact angle stabilises.

Long and narrow capillaries were fabricated in diamond, using SOI wafers as substrates, to investigate the limitations of silicon etching in long diamond channels. With cross-sections of 2000 llm2, it was possible to etch silicon enclosed in polycrystalline diamond deeper than 30 mm. The sacrificial etching of silicon inside capillaries was very fast in the beginning. After a few mm of etching, the etch-rate began to slow down drastically. The reason for this decrease in etch-rate is most likely limited diffusion of fresh etchants and reaction products in the narrow capillaries.

Diamond microchips intended for chromatography were fabricated. The straight columns were 40 mm long, 100 am wide, and 40 um or 70 um high. The walls were smooth on the inside and rough on the outside. By using wet-or dry-oxidation, it was possible to change the hydrophilicity of diamond surfaces inside columns. The diamond microchips were used for fast liquid chromatography on continuous beds of two proteins: ( (A) 1, myoglobin (horse); 2, conalbumin; 3, ovalbumin (chicken); 4, trypsin inhibitor (soybean), (B) 1, Hemoglobin Ao; 2, ß-lactoglobulin A). The chromatography was fast and easy to perform, and the resolutions of the chromatograms were in accordance with separations on continuous beds synthesised in both capillaries and microchips using conventional column materials.

The chemical inertness and high strength of diamond make diamond microstructures particularly useful in microscale analysis and synthesis procedures. This is because microstructures often become contaminated or blocked during use and previously there has not been any satisfactory way of cleaning then or unblocking them. However it is easy to clean and unblock diamond microstructures by merely immersing them in a solvent or corrosive solution suitable for dissolving or corroding the contaminant or blocking substance until the contaminant or blocking substance has been completely dissolved. The inert diamond surfaces remain unaffected by the immersion and can be used again as if they were new. The cleaning process can be accelerated by agitation.

While the present invention has been illustrated by examples for making diamond-based hydrophilic liquid chromatography columns it is equally applicable to, and also encompasses, other diamond-based microstructures, such as reaction chambers, gel beds, transport pipes and the like, for use in analysis and synthesis processes.