AN OPTICAL SUBSTRATE AND SENSING CHIP

The present invention is directed to a method for the manufacture of an optical substrate for use in optical sensing applications and with optical sensing chips containing the same.

The recent increase in the prevalence of antibiotic-resistant bacteria and risk of biological warfare or terrorism has emphasised the need for rapid and cost effective determination of pathogens in both military and civilian environment.

Optical sensors are particularly suitable for that determination in that they allow real time monitoring of an environment according to changes in an optical property of a sample. Such sensors include, for example, surface plasmon resonance sensor (SPR) or leaky waveguide (LW) sensors, such as resonant mirror (RM) sensors.

Typically, these sensors comprise a glass prism upon which, for example, a metal- coated glass slide (or "chip") is located by an intervening layer of optically matched liquid.

For SPR, light incident the boundary surface of the prism at certain "resonant" angles is coupled to the electron cloud of the metal layer. The resultant surface optical mode has an evanescent electrical field which can report changes in the optical environment in a sensing layer provided on the metal layer by shifting the angle necessary for resonance. The shift is detected by a detector monitoring light reflected from the boundary surface.

LW sensors, such as those described in international patent application WO 99/44042 A2, rely on coupling of light into a sensing layer.

However, most of these sensors lack sensitivity toward detection of particles because they require a large change in refractive index of the sensing layer or because the particles do not fully penetrate into the sensing layer. Typically, therefore, the detection of particulate analytes, as opposed to soluble analytes, by these sensors relies upon light scattered or emitted by particles in or above the sensing layer.

For example, international patent application WO 01/42768 Al describes an SPR sensor adapted for the detection of particles by scattering of light.

International patent application WO 2004/074819 Al describes a metal clad LW sensor (MCLW) which is particularly adapted for the detection of particles by an evanescent field from an optical mode to the sensing layer. The sensor leads to a three-fold increase in the intensity of scattered light as compared to a SPR sensor.

However, all of these sensors are expensive in that they rely on complex, high quality optical arrangements which demand skilled operators. They do not, therefore, lend themselves to unattended operation or deployment across a large area.

The present invention generally seeks to overcome these problems by enabling a simple, low cost sensor which requires minimum operator attendance.

Polymer waveguide structures have attracted much recent attention for optical interconnects and communication systems due to their low cost and high process ability (Chuang, W-C et al. at pp 93 to 95 in Progress in Electromagnetics Research Symposium, August 2005, Hangzhou, China).

A polystyrene planar waveguide structure integrally formed with lens has also been described as suitable for evanescent detection of particles by fluorescence (Sipe, D. M.

et al., in Proceedings of SPIE 2000, 3913 (In- Vitro Diagnostics Instrumentation), 215-

222.

A dye-clad LW (DCLW) chip based on a polymer waveguide structure integrally formed with a diffraction grating coupler has recently been reported (Zourob, M. et al., in Lab Chip, 2005, 5, 772-777). The chip is, however, unsuitable for the detection of particles by scatter because of stray light from the grating.

The present invention is directed to an optical substrate which can form the basis for an optical sensing chip having an integrally formed optical coupler other than a lens or diffraction grating.

Accordingly, in a first aspect, the present invention provides a method for the manufacture of an optical substrate for optical sensing applications comprising curing a curable polymer in a mould providing that the substrate is wholly or partly formed from a cured polymer shaped so as to be capable of directing light to an internal surface of the substrate, or of a layer provided thereto, at an angle equal to or greater than the critical angle for total internal reflection thereat.

Those skilled in the art will appreciate that the method provides a substrate that can be directly provided with one or more layers suitable for optical sensing and that the shaped polymer can act so as to couple light from an external source to the resultant "chip" in the necessary way for sensing by evanescent and bulk optical modes therein.

The curing may be earned out by irradiation with light and/or by heating or chemically according to any method or conditions which minimises birefringence in the optical substrate. In one embodiment a thermally and UV-curable polymer and is cast cured

by irradiation with UV-light followed by removal of the substrate from the mould and hardening of the polymer by heating.

A suitable mould comprises a material which is rigid enough for retention of the shape of the mould when loaded with the curable polymer but flexible enough to permit easy removal of the polymer substrate.

The material of the mould may also be chosen for low adhesion of the mould to the polymer substrate - but it is possible that the mould can be treated or coated so as to otherwise impart the necessary optical smoothness to the substrate.

In preferred embodiments, the mould comprises a material having Shore A hardness between 40 and 70 and low surface tension (below 30 mN/m).

The material may also be chosen so that the mould is transparent to UV-light and may, for example, comprise a thermosetting polymer such as fluorinated polymer or a silicone, for example, Teflon® or polytrifluoroethylene or polydimethylsiloxane. In a preferred embodiment, the mould comprises Sylgard® 184 (Dow Corning®) and has Shore A hardness about 50 and surface tension about 20 mN/m.

Of course, a suitable mould must be shaped such that the cured polymer is shaped so as to be capable of directing light to an internal surface of the substrate, or of a layer provided thereto, at an angle equal to or greater than the critical angle for total internal reflection thereat.

The mould may comprise a cavity providing for an optical substrate wholly comprising a cured polymer. The cavity may, in particular, provide that the polymer substrate

simply comprises a prism or hemisphere or that it comprises a generally planar part carrying one or more prismatic or hemi-spherical shaped bosses thereon.

The mould may, in addition, comprise more than one such cavity permitting manufacture of more than one such polymer substrates, for example 2 to 20 polymer substrates and, in particular, 14 or 16 polymer substrates.

The mould may, however, be comprised by separable parts and, in particular, a main body including one or more such cavities and a lid or lids.

A single lid preferably comprises a thermosetting polymer as mentioned above because its flexibility permits unrolling of the lid across the main body whereby to better exclude air from the mould and the cured polymer substrate.

Alternatively, the lid may comprise a metal or glass plate which is polished to a standard optical finish (scratch/dig ratio 60/40).

The mould may, in the case of manufacture of more than one polymer substrate, comprise more than one polymer, glass or metal lid whereby to better exclude air from the mould and the cured polymer substrate.

The mould may be manufactured by casting a thermosetting polymer such as mentioned above on a template or templates. Suitable templates comprise rigid materials such as metal or glass which are preferably finished, for example, by polishing whereby to tend to deliver an optical finish to the mould (and thence the polymer substrate).

However, the mould may itself be finished to a standard optical finish by, for example, mechanical polishing or electro-polishing after its removal from the template.

Of course, the shape of the template should generally correspond to the shape of the mould or part mould. A template may, therefore, comprise a prism or hemisphere or a plate defining one or more generally planar platforms including prismatic or hemispherical shaped bosses or embossments, for example, 14 or 16 such platforms. An accompanying template may simply comprise a polished glass plate from which a polymer lid or lids can be cast.

In one embodiment, a template is comprised by a glass backing plate to which one or more, for example 2 to 20 and, in particular, 14 or 16, polished glass slides are glued - each slide having an optical grade prism glued thereto.

A suitable curable polymer comprises a polymer which has low birefringence when cured and is transparent to light at wavelengths for optical sensing application. The polymer will also be chosen for low surface tension and/or for suitable refractive index (from 1.49 to 1.59) when cured.

In preferred embodiments, the polymer is both thermally and UV-curable. The polymer may, in particular, comprise an epoxy resin such as the optical adhesives N61 or N68 (both Norland) or UVlO (Masterbond).

In these embodiments, the mould cavity or cavities may be part filled by epoxy resin and part filled by a transparent lid comprising another polymer, for example, polycarbonate, whereby the curing leads to a polymer substrate comprised by an epoxy resin prism or hemisphere bonded to the lid. Suitable polymer materials for the lid have refractive index for optical sensing applications matched to the cured epoxy resin.

In similar embodiments, a glass slide is used as the Hd whereby curing leads to a part polymer substrate comprised by an epoxy resin prism or hemisphere bonded to the lid.

Of course, the glass slide will have a refractive index for optical sensing applications which is matched to the cured epoxy resin.

In some embodiments, the glass slide or polymer lid may be provided with one or more layers providing for optical sensing of a soluble or particulate analyte on an opposite surface to the bonding surface - viz. the lid may be a conventional optical sensing chip.

In all these embodiments, the curing of the polymer in the mould may be achieved by irradiation with, for example, a suitably positioned (for example, at 25 cm from mould) standard 200 W/inch ² mercury lamp for a predetermined time.

Of course, different polymers will require irradiation for different times, for example 90 to 100 seconds for adhesives N61 and N68 as compared to 30 to 40 seconds for adhesive UVlO under the aforementioned conditions.

The hardening of the cured polymer substrate or substrates may be achieved by heating in an oven at a suitable temperature for a predetermined time.

For adhesives N61, N68 and UVlO the hardening may comprise heating in an oven between about 4O ⁰ C and 6O ⁰ C during 12 hours.

It will be appreciated that both the mould and the polymer substrate should be formed with minimal trapping of air bubbles so as to better provide an optical substrate suitable for optical sensing applications.

The method may, therefore, be particularly adapted for loading of the polymer to the template or the mould so as to tend to exclude the formation of air bubbles and/or for

removing bubbles by, for example, pricking and/or standing for a predetermined time with or without vacuum.

In any embodiment, the lid or lids may be adapted, for example, by etching, to provide markers to the upper surface of the optical substrate for locating a sensing area thereon and/or for auto-focusing a camera or similar detector and/or for providing information about particle size.

In a second aspect, the present invention provides an optical substrate obtainable or obtained according to the first aspect of the invention. Specific embodiments of the substrate will be apparent from the foregoing description and the claims appended hereto.

In a third aspect, the present invention provides a mould for use in a method of manufacture according to the first aspect of the invention. Specific embodiments of the mould will be apparent from the foregoing description and the claims appended hereto.

In a fourth aspect, the present invention provides a template for use in obtaining the mould according to the third aspect of the invention. Specific embodiments of the template will also be apparent from the foregoing description.

In a fifth aspect the present invention provides for a method of manufacture of an optical sensing chip comprising providing an optical substrate according to the second aspect of the invention or providing one or more layers to the substrate providing for optical sensing a soluble and/or a particulate analyte.

In one embodiment, the method is directed to manufacture of an SPR chip and comprises coating the optical substrate with a metal layer and, in particular, a gold or silver layer.

In another embodiment, the method is directed to manufacture of a LW chip such as that mentioned in WO 2004/074819 Al and comprises coating the optical substrate with a metal layer or dye-layer, for example, an aluminium, chromium, tantalum, titanium, or zirconium layer, followed by coating with a silica sol gel layer.

In both embodiments, the method may also comprise the step of coating the metal or silica sol gel layer with a sensing layer such as a dextran including, or adapted for inclusion of, a specific recognition element for a target solute or particle.

Those skilled in the art will understand, however, that the method may be directed to the manufacture of any known type of optical sensing chip - especially those types described in the references mentioned above.

In a sixth aspect, the present invention provides an optical sensing chip obtainable or obtained according to the fifth aspect of the invention. Specific embodiments of the chip will be apparent from the foregoing description and references as well as the claims attached hereto.

It will be appreciated that the present invention provides a cheap and reliable method for the manufacture of optical substrates which can be used directly for the manufacture of many types of optical sensing chip.

The optical substrate and optical sensing chips are, therefore, disposable and because they are integrally formed with, or bonded to, a polymer optical coupler they avoid the

need for location on an expensive glass prism by an intervening layer of optically matched liquid.

The optical substrate and optical sensing chips enable optical sensors which are cheaper and require less operator attendance than presently available optical sensors so facilitating wide area deployment of multiple optical sensors.

The present invention will now be described with reference to the following embodiment, examples and drawings in which:

Figure 1 shows a photograph of a template for casting a polydimethylsiloxane mould for use in a preferred embodiment of the method of the present invention;

Figure 2 shows a photograph of the detail of the template of Fig. 1;

Figure 3 shows a photograph of the polydimethylsiloxane mould cast from the template of Fig. 1 ;

Figure 4 shows a photograph of the polydimethylsiloxane mould loaded with epoxy resin and a large polydimethylsiloxane cover lid;

Figure 5 shows a photograph of the polydimethylsiloxane mould loaded with epoxy resin and a plurality of polydimethylsiloxane disc lids;

Figures 6 a) to c) show photographs of optical substrates obtained from the mould shown in Figure 5;

Figures 7 a) to d) show photographs of birefringence in a conventional optical arrangement based on a glass prism and glass slide as compared to the optical substrate and sensing chips including the optical substrates obtained according to the invention;

Figure 8 shows a cross-sectional view of a LW chip including an optical substrate obtained according to the present invention;

Figure 9 shows graphs reporting optical modes in a LW chip including an optical substrate obtained according to the present invention; and

Figure 10 shows a graph of the results of interrogation of glycerol solutions of varying concentration by a LW chip including an optical substrate obtained according to the present invention.

Production of Template

Referring now to Figures 1 and 2, there is shown a template 10 suitable for casting a mould according to a preferred embodiment of the method of the present invention.

The template 10 is formed by bonding an optical grade, trapezoidal prism 11 (3 mm depth; upper surface width 10 mm; lower surface width 7 mm) to the centre of one side of a polished square (2.5 cm x 2.5 cm), glass slide 12 (thickness 1 mm) with an adhesive and bonding the opposite side of the slide to one side of a circular, glass back plate 13 (thickness 6 mm) and repeating these steps so as to obtain an array of fourteen approximately equally spaced slides on the plate.

Production of Mould

The template 10 is provided with a circumferential wall to the surface carrying the array by bonding a strip of polycarbonate (thickness about 1 to 2 cm) to its side wall with an adhesive.

To the well so obtained is added a pre-prepared mixture of Sylgard® 184 base and its curing agent (500 g: 50 g) and the well is placed in a vacuum oven (10 mbar or below)

at room temperature until most air bubbles are removed from the mixture. The vacuum is discharged and the well left to stand at room temperature until the mixture is fully cast to a mould (36 hours). Finally, the polycarbonate strip is cut away and the mould is mechanically detached from the template.

As may be seen from Figure 3, the cast mould 14 comprises an array of fourteen cavities 15 having a trapezoidal prism portion (3 mm depth) centrally positioned within a square portion (1 mm depth).

For the production of the optical substrate, the mould is conveniently supported by a circular dish 16 having a circumferential lip and a circumferential wall 17 for receipt of a lid is provided by an aluminium strip.

Production of Optical Substrate

Example 1: To each cavity 15 of the mould 14 is added optical adhesive Norland N61 drop- wise until it is filled (about 1. 2 ml). The dish is placed in a vacuum oven (10 mbar or below) at room temperature for a period of about 10 minutes to remove any air bubbles from the adhesive. After removal from the oven, to the mould is laid a similarly sized circular lid 18 of a polydimethylsiloxane membrane attached to a polycarbonate ring and the membrane gently pressed into contact with the mould (Figure 4).

A 200 W/inch ² mercury lamp positioned at a distance of 25 cm above the mould 14 is operated during a period of 90 to 100 seconds. The dish is placed in an oven heated to 4O ⁰ C to 60 ⁰ C. After 12 hours the dish is removed from the oven, the lid 18 detached from the mould 14 and the polymer optical substrates 19 pressed from the mould.

Example 2: To each cavity 15 of the mould 14 used in Example 1 is added optical adhesive Norland N61 drop-wise until it is filled. The dish is placed in a vacuum oven for a period of about 10 minutes to remove any air bubbles from the adhesive. After removal from the oven, to the mould 14 is laid a number of polydimethylsiloxane disc lids 20 of diameter and thickness about 2.5 cm so to cover each cavity 15 (Figure 5).

A 200 W/inch ^" mercury lamp positioned at a distance of 25 cm above the mould 14 is operated during a period of 90 to 100 seconds. The dish is placed in an oven heated to 4O ⁰ C to 60°C. After 12 hours the dish is removed from the oven and the lids 20 are detached from the mould 14 and the polymer optical substrates 19 peeled from the lids 20.

Example 3: To each cavity of the mould used in Example 1 is added optical adhesive Norland N61 drop-wise until the prism portion is filled. The dish is placed in a vacuum oven for a period of about 10 minutes to remove any air bubbles from the adhesive. The square portion of the cavity is filled by a polished glass plate.

A 200 W/inch ² mercury lamp positioned at a distance of 25 cm above the mould is operated during a period of 90 to 100 seconds. The dish is placed in an oven heated to 4O ⁰ C to 6O ⁰ C. After 12 hours the mould is removed from the oven and the part polymer optical substrates pressed from the mould.

Referring now to Figures 6 a) to c), there is shown respectively optical substrates 19 obtained by the method of the present invention for the different epoxy resins UVlO, N68 and N61.

All of the polymer substrates 19 are substantially free from trapped air but those comprising UVlO and N68 show slight yellow colour and prism shrinkage compared

to the template. Although these distortions do not interfere with most optical sensing applications, the substrate 19 comprising N61 is best in that it is transparent and shows very little prism shrinkage as compared with the template.

Birefringence of Optical Substrate and Sensing Chips

A comparative study of birefringence in a conventionally located glass prism - slide arrangement, a moulded epoxy resin prism bonded to a commercially available LW chip, a moulded epoxy prism on another LW chip and an all epoxy substrate of Example 1 or 2 was undertaken.

The results shown respectively in Figures 7 a) to d) report that birefringence is highest in the all epoxy substrate but low enough so as to not affect the resonance peak. In addition, the birefringence in the epoxy substrate is mostly confined to the corners of the prism.

Production of LW Sensing Chip Including Substrate

Example 4: Three MCLW chips 20 (Figure 8) based on an optical substrate 19 obtained by the method of the present invention were obtained by applying a thin layer 21 (9 nm) of titanium metal to the planar surface of an N61 optical substrate 19 by vacuum deposition and apply to this metal layer a layer 22 (300 nm) of a silica sol gel by spin-coating.

The chips were tested using a conventional waveguide instrument for its ability to support one or more optical modes. A plot (Figure 9) of intensity against angle of incidence for each chip shows that optical sensing chips including an optical substrate obtained by the method of the present invention can support good optical modes.

The chips also show goods resonance peaks as compared to conventional leaky waveguide chips.

Use of an LW Sensing Chip Including Optical Substrate

Aqueous glycerol solutions of concentrations (1 to 10 % w/w) were passed in a flow cell over the top of a MCLW chip of Example 4.

The resonance peak positions were monitored during the passage of each solution (25 seconds; 125 peaks) and averaged together when the signal stabilised. The peak position for a control solution of water was subtracted from the peak position of each aqueous glycerol solution. A plot (Figure 10) of peak angle against refractive index of each of the ten solutions reveals a linear relationship.

The graph shows the utility of optical sensing chips for sensing soluble and particulate analytes by monitoring resonance peak angle.

Other embodiments and examples of the present invention will be apparent from the foregoing description - particularly relating to automation of the claimed methods and to other optical sensing chips.

References to directed or external light herein are references to light of wavelengths suitable for optical sensing applications, including IR and UV wavelengths.

References to "transparent" optical substrates herein are references to substrates which permit passage of light from an external light sources of suitable wavelength for optical sensing applications, including IR and UV light sources.

References to a "particulate" analytc herein are references to an analyte which comprises particles and/or particle agglomerates.