Patent Application
20080277606
The present invention relates to a microfluidic device comprising a monolithically integrated dye-doped polydimethyl siloxane (PDMS) optical filter and its use in disposable on-chip fluorescence detection
Microfluidic devices have had a tremendous impact on the development of miniaturized and micro total analysis systems (μTAS) in chemical engineering and molecular biology. These systems have potential for wide application in clinical point-of-care diagnostics, genetic analysis, drug discovery, food industry, environmental monitoring, forensic investigation and chemical/biological warfare defense. Miniaturization of devices leads to decreased time of analysis, reduced consumption of reagents and analytes, increased separation efficiency, high throughput screening, portability and disposability.
A major goal of microfluidics research is the development of integrated systems that successfully incorporate all stages of a complete chemical or biological analysis into a single device. Typical stages in an analysis include sampling, pre-treatment, chemical reactions, analytical separations, and analyte detection. The final step is often the most challenging due to the small quantities of analyte present and the consequent need for high sensitivity detection. In practice, optical techniques are often the only ones that provide adequate sensitivity, and considerable efforts have therefore been devoted to developing integrated optical components for use in microfluidic devices. In this regard efforts have been made to integrate amongst other things microlenses, filters, mirrors, gratings, waveguides, light sources and photodetectors.
Detection of analytes in micro total analysis systems can be achieved by a number of methods, of which fluorescence based detection is the most widely used. Fluorescence based detection is of particular use for example in the detection of DNA due to its high sensitivity, ease of automation and real time detection. Many current biochemistry protocols, such as Sanger sequencing and the polymerase chain reaction (PCR) have been adapted to fluorescent labelling methods. The incorporation of such detection methods into chip based assays is therefore an important development in the field and fluorescence detection can be used in a variety of applications on an analysis chip.
Fluorescence detection sensitivity is severely compromised by background signals, which may originate from endogenous sample constituents or from unbound or nonspecifically bound probes (reagent background) scattered light from the excitation light source or direct excitation of the detector by the light source. Detection of autofluorescence and scattered or direct excitation light can be minimized by selecting filters to absorb an unwanted signal and transmit a desired signal. In this way, the signal-to-noise ratio can be enhanced greatly. Therefore, the use of filters plays an important role in fluorescence detection.
Conventional fluorescence-sensing systems use bulky and discrete components that are expensive and non-portable. The deployment of such conventional optical systems in combination with a micro total analysis system is therefore inconvenient. Miniaturization of such systems is not straightforward since it involves the design of compact light sources, filters, and sensitive on-chip photodetectors.
Functional integration of optical components within monolithic substrates in the micro total analysis system and lab-on-a-chip field has recently been the subject of significant research and development activity. In fluorescence based detection, optical short pass filters are used for sharpening of the excitation light and long-pass filters are generally employed to prevent excitation light from reaching the detector. Often these filters are used as external components, even in otherwise integrated optical microchips.
In the context of microfluidic devices, optical long-pass filters play a particularly important role. In conventional fluorescence detection, the excitation source and detector are usually arranged orthogonally to one another to prevent direct illumination of the detector by the excitation source. This orthogonal geometry, however, is difficult to implement in a microfluidic environment since it requires the production of optical grade side-surfaces and the non-facile integration of optical components onto the side-surfaces of the microfluidic chip. Light sources and detectors are most conveniently located on the upper and lower faces of the microfluidic chip in a co-linear geometry, but this would ordinarily flood the detector with direct light from the excitation source, masking the typically weak fluorescence signal from the analyte. The key to achieving effective discrimination of the excitation and emission light in this ‘head-on’ configuration is the use of a long-pass filter in front of the detector, which blocks the excitation light and passes only the longer wavelength emission signal. The use of long-pass filters for this purpose is well established but relies on discrete stand-alone filters—an approach which yields satisfactory optical performance but prevents monolithic integration and increases the distance between the microchannel and the detector, leading to inefficient collection of the fluorescence signal.
In assembled hybrid devices, a commercial ZnS/YF3 interference filter has been used in a backside illumination geometry to block out scattered excitation light. Similarly, commercial long-pass and band-pass filters have been used in the hybrid epi-fluorescence detection module of a hand-held protein analyzer. Combined holographic notch plus and interference filters have been attached to a glass microchip via a PDMS layer. In addition a 80-micrometre-thick yellow polycarbonate long-pass filter sandwiched between the PDMS microfluidic layer and a second PDMS plate comprising a microavalanche photodiode has been employed. However in this assembly, a 5-times higher background signal was still observed with the light source turned on, indicating inefficient blocking. Such hybrid assembly approaches suffer from large distances between microchannel, filter and detector, resulting in inefficient collection of the fluorescent signal and limited detection sensitivity. Furthermore, the background signal obtained in such hybrid assemblies is too high for sensitive detection.
A monolithically integrated multilayer interference filter fabricated on top of a PIN silicon photodiode has been developed for silicon based microchips. The filters typically comprise up to 40 alternating SiO2/TiO2 layers and exhibit 5%-transmittance at 490 nm and 90%-transmittance at 510 nm. However, these integrated DNA analysis microdevices are currently unsuitable for disposable diagnostic tests given the complex fabrication and associated high cost.
A CdS thin-film filter has been developed which exhibits strong blocking of the excitation light for most incident angles. However, only ˜40% of the emission signal is transmitted, resulting in low sensitivity. Long-pass filters based on doped AlGaAs or multi-layer Fabry-Perot cavities have also been reported to suffer from side illumination and low transmittance, respectively.
Microfluidic systems can be manufactured in glass, oxidized silicon or in a polymer such as polydimethylsiloxane. Polydimethylsiloxane is particularly favoured for use in microfluidic devices as it allows the reproduction of features on the micron and submicron scale with high fidelity by replica moulding, has good optical transparency down to 280 nm, cures at low temperature, is non-toxic, can be deformed reversibly, seals reversibly to itself and other materials or can seal irreversibly after exposure to air or oxygen plasma by formation of covalent bonds.
The interference-filters and CdS-filters described above could in principle be straightforwardly integrated with glass microfluidic chips, but they are unsuitable for conformable elastomeric materials such as PDMS since polycrystalline materials such as CdS, TiO2 and SiO2 are typically deposited at relatively high temperatures (>300° C.) and have a tendency to crack when the substrate is flexed.
Polydimethyl siloxane has previously been modified for biomedical applications by changing its mechanical properties. In addition stained polydimethylsiloxane produced by post-polymerization staining with Sudan Red has been used as a photothermal detector. Self-assembly in mesoscale systems was investigated by post-polymerisation staining of polydimethylsiloxane with perfluorodecalin, perfluoromethyldecalin, crystal violet and Sudan Red 7B. However, such staining processes are diffusion based and thus slow, dye incorporation is often shallow and non-uniform and the dye load is difficult to control.
The use of dye doped polydimethylsiloxane layers as filters have been suggested. However, problems associated with incorporation of the dye into the polydimethylsiloxane have prevented the realisation of this suggestion, and currently only an example of an external unstructured post-polymerisation stained polydimethylsiloxane layer for use as a filter has been disclosed.
There is a need in the art for an improved microfluidic device for use in optically based detection system which allow sensitive detection in a low cost device. The present invention provides a process for the production of an optical filter comprising a dye-doped polydimethylsiloxane microfluidic layer and its incorporation into an integrated microfluidic device.
The first aspect of the invention provides a microfluidic device comprising a zone for receiving a sample, an excitation source and a detector, characterised in that a portion of the zone allows transmission of energy within a required wavelength range and prevents transmission of energy at all other wavelengths.
In particular, the portion of the zone is composed of a composition comprising polydimethylsiloxane and a dye, wherein said dye allows transmission of energy at a required wavelength range and prevents transmission of energy at all other wavelengths.
The microfluidic device is further provided wherein the zone allows transmission of energy at a required wavelength range and prevents transmission of energy at all other wavelengths. In particular, the zone is composed of a composition comprising polydimethylsiloxane and a dye, wherein said dye allows transmission of energy at a required wavelength range and prevents transmission of energy at all other wavelengths.
The zone for receiving the sample can therefore be provided as a monolithic structure, that is the zone is an integrated part of the microfluidic device.
The zone may be bounded by one or more walls. As discussed above the walls of the zone are an integrated part of the device. One or more of the walls or a portion of one or more of the walls can comprise the composition as defined above (i.e. a wall or a portion thereof can allow transmission of energy at a required wavelength range and prevent transmission of energy at all other wavelengths). Preferably all walls bounding the zone comprise the composition as described above. Preferably the wall or portion of wall comprising the composition as described above is arranged between the sample and the detector, between the sample and the light source or between the sample and the detector and between the sample and the light source. Preferably the microfluidic device comprises the polydimethylsiloxane composition as defined above. In particular, the polydimethylsiloxane composition can be used to produce part of a wall section, or a plate section sealing the zone.
It will be appreciated that the use of a composition comprising polydimethylsiloxane and a dye to produce at least part of a zone, avoids the need for a separate internal or external filter. Excitation of a sample within the zone will cause the sample to transmit energy. The presence of the polydimethylsiloxane composition in the microfluidic device and more particularly in the walls of the zone will allow transmission of energy at a required wavelength range while preventing transmission of energy at all other wavelengths. The microfluidic device and in particular the zone will act as a filter. It will be appreciated that the use of the polydimethylsiloxane composition in the manufacture of the microfluidic device allows the reduction and/or prevention of transmission of energy from a sample at undesirable wavelengths (i.e. it reduces the background noise from scattering of excitation light). The polydimethylsiloxane composition may further reduce ground noise from autofluorescence of a fluorescent and/or phosphorescent sample.
The device of the first aspect does not require an additional filter. Instead the incorporation of the composition into the microfluidic device of the first aspect allows the device to act as a short pass excitation light filter, a band pass filter, a long pass detection filter, or an absorbing filter to minimise scattering from the background to prevent direct or scattered light from reaching the detector.
The zone preferably has a depth of about 1 to 999 μm, preferably about 10 to about 500 μm, more preferably 20 to 100 μm. For optical detection it is sometimes useful to use deeper zones, for example 600-800 μm. The excitation source preferably comprises one or more light sources which can be integrated into the microfluidic device and/or which are external thereto. The excitation source may be a conventional lamp, a laser, a laser diode, an inorganic light emitting diode (LED) or an organic light emitting diode (OLED). The detector can be integrated into the microfluidic device and/or external thereto. The detector may be a photomuliplier tube or an inorganic or organic photodiode. The microfluidic device preferably comprises a substrate, for example a substrate chip, said zone being integrated into and/or supported thereon. The substrate may comprise the polydimethyl siloxane composition or any other material conventionally used in the art.
Any components of the device can be manufactured from the composition defined above. Preferably any components directly or indirectly in the pathway of the energy transmitted from the sample after excitation thereof are manufactured from the composition.
The device may comprise one or more layers of the polydimethylsiloxane composition. The layers can be 3 mm or less thick, preferably 2 mm or less thick, preferably 1 mm thick or less, preferably 100 micrometres thick or less, preferably 10 micrometres thick or less or preferably 1 micrometre thick or less. The layers can be stacked on top of each other and can be fused together reversibly by for example intrinsic adhesion or irreversibly by for example treatment with oxygen or air plasma or unfused. The use of such layers minimises the distance between the zones and a detector enabling high efficacy collection of the energy.
The microfluidic device therefore allows more sensitive detection of an analyte. The use of the device of the first aspect further allows superior light collection as the distance from the zone to the filter is minimised. The size of the device can also be kept to a minimum due to the reduction in additional components including additional external components.
The microfluidic device may be used for fluorescence detection of an analyte. The analyte may be a fluorescent and/or a phosphorescent analyte. Alternatively, the analyte may have been modified with a fluorophore and/or a phosphore to allow detection (for example, labelled protein for example in an immunoassay or labelled nucleic acid for example in a DNA hybridisation assay). In particular, an analyte comprising a fluorophore and/or a phosphore may be introduced into the microfluidic device and optionally modified (by a biological, physical or chemical process). Alternatively, the analyte may have been modified prior to its introduction into the microfluidic device. The fluorophore and/or phosphore undergoes detection in the device by excitation of the fluorophore and/or phosphore by a light source, the output of which is optionally sharpened using a short pass filter. The excited fluorophore and/or phosphore reemits light, said reemitted light being detected by a detector either integrated into the microfluidic device or external to the microfluidic device. The polydimethylsiloxane composition in the microfluidic device blocks excitation light at unwanted wavelengths, thereby enhancing the signal to noise ratio of the reemitted light and allowing more sensitive detection.
The microfluidic device may comprise one or more additional filters either internal and/or external to the microfluidic device. The additional filter can act as a short pass excitation light filter, a band pass filter, a long pass detection filter or an absorbing filter to minimising scattering from the background to prevent direct or scattered excitation light from reaching the detector. The microfluidic device preferably comprises an inorganic or organic semi-conductor based light source and/or an inorganic or organic semi-conductor based photodiode.
For the purposes of this invention, the dye is a low fluorescent, photostable compound which allows transmission of energy at a required wavelength range and prevents transmission of energy at all other wavelengths. Preferably, the dye is a lysochrome dye (i.e. a fat soluble dye). Examples of such lysochrome dyes include azo dyes which have undergone molecular rearrangement and are no longer able to ionise. It will be appreciated that selection of the dye will determine the wavelength range at which energy can be transmitted and the width of the wavelength range. The dye can therefore be used to tune the composition to allow transmission at the required wavelength range with the required or desired degree of selectivity and sensitivity.
Preferably, the dye comprises an aromatic system comprising one or more aromatic rings such as phenyl rings. More preferably, the dye comprises a conjugated aromatic system comprising two or more fused phenyl rings. The two or more fused rings may be directly fused to each other or may be fused to an intermediate moiety such as an unsaturated or partially saturated ring such as a cyclohexyl ring. In one feature of the first aspect of the invention, the dye comprises a compound of formula (I)
The compound of formula (I) may be substituted at one or more positions on the phenyl or napthalene ring with one or more of hydroxy, halo, C1-4 alkyl.
For the purposes of the invention, the dye may comprise one or more selected from the Sudan dye family such as Sudan Blue II (Solvent Blue 35), Sudan Black B (Solvent Black), Sudan I (Solvent Yellow 14), Sudan II (Solvent Orange 7), Sudan III (Solvent Red 23) and Suden IV (Solvent Red 24), Solvent Blue 37, Solvent Blue 38, Solvent Blue 59 and Solvent Green 3 (Quinzarine Green 55). More preferably, the dye is selected from one or more of Sudan II,
and/or Sudan IV
In an alternative or additional feature of the first aspect, the dye may be Sudan Blue II.
Alternatively the dye can be a porphyrin or a derivative thereof, pigment, colourant, or a nanoparticle such as CdS. It will be appreciated that two or more dyes can be incorporated into the polydimethylsiloxane composition to allow transmission of energy at two or more wavelengths (i.e. to create band pass filtration).
The dye may be provided in the composition in an admixture with the polydimethylsiloxane. Alternatively some or all of the dye may be reacted with polydimethylsiloxane monomers to form a co-polymer.
In particular, the microfluidic device comprises a composition comprising polydimethylsiloxane and Sudan II wherein said polydimethylsiloxane composition allows transmission at a wavelength of 570 nm or above and prevents transmission at a wavelength of below 520 nm.
The composition acts as a filter to allow transmission of energy, preferably light, at a wavelength or within a range of wavelengths. The transmission of energy at the specified wavelength or range of wavelengths is sufficient to allow detection of the transmitted energy. The composition further prevents transmission of energy (i.e. light) at all other wavelengths (i.e. at undesired or non-specified wavelengths). The composition does not necessarily prevent the transmission of 100% percent of the energy at an undesired wavelength. Instead the polydimethylsiloxane composition reduces the transmission of energy at an undesired wavelength sufficiently so that it does not interfere with the detection of energy at a desired wavelength or range of wavelengths.
The width of the range of wavelengths will depend on the dye or dyes used. In particular, transmission of energy will occur over a range of 100-300 nm.
The transition phase from transmission at a desired wavelength to non-transmission is preferably 100 nm or less, more preferably 50 nm or less. The composition of the invention allows broadband filtration for example the use of a composition comprising polydimethylsiloxane and Sudan Blue II in the manufacture of a microfluidic device allows transmission from 400 to 500 nm but prevents transmission at wavelengths less than 300 nm or greater than 550 nm. Alternatively the composition may allow narrow band filtration i.e. transmission over a wavelength range of 50 nm or less, preferably 20 nm or less, preferably 5 nm or less, preferably 1 nm or less.
It will be appreciated by a person skilled in the art that the incorporation of the dye into the composition determines the wavelength at which energy, for example light, can be transmitted through the polydimethylsiloxane composition. The composition can therefore be tuned according to the spectra of the excitation source (for example the excitation light source) and detection signal as required by the particular method of detection.
Preferably, the composition allows greater than 80% transmission of energy at a required wavelength, more preferably greater than 90% transmission of energy at a required wavelength, most preferably greater than 95% or above transmission of energy at a required wavelength. Furthermore, the composition preferably allows less than 10% transmission of energy at an undesired wavelength, preferably less than 5% transmission of energy at an undesired wavelength, preferably less than 2% or below transmission of energy at an undesired wavelength. Preferably 1% or below transmission of energy at an undesired wavelength, preferably 0.1% or below transmission of energy at an undesired wavelength, preferably 0.01% or below transmission at an undesired wavelength.
Preferably, the composition allows the transmission of light. Preferably the light is detectable as fluorescence or phosphorescence.
The microfluidic device can be produced at particularly low cost. Incorporation of the polydimethylsiloxane-dye composition into the device removes the need for separate conventional filters, which can be costly and time consuming to incorporate. In particular, interference filters require complex silicon based multilayer fabrication while gelatin filters require incorporation of a dye into liquid gelatin, coating of the gelatin-dye mixture onto glass, drying of the mixture, stripping of the mixture from the glass and coating with lacquer. These conventional filters then need to be incorporated into a microfabricated device.
The device of the present invention can be provided as a disposable device due to its low cost production. The production of the device is particularly cost effective when compared to the production of devices known in the art incorporating conventional filters, for example silicon based interference filters.
The use of the polydimethylsiloxane composition of the present invention allow the production of filters or microfluidic devices which exhibit low autofluorescence, negligible leaching with aqueous solutions and limited light induced degradation.
The spectral characteristics of the composition compare favourably with commercially available Schott glass long-pass filters, indicating that the invention allows the integration of high quality optical filters into the form of PDMS microfluidic chips. The use of the composition of the invention allows the production of filters which are robust in use, showing only slight degradation after extended illumination and negligible dye leaching after prolonged exposure to aqueous solutions. The present invention allows the provision of low cost high quality integrated filters and represents a key step towards the development of high-sensitivity disposable microfluidic devices for point-of-care diagnostics.
The second aspect of the invention relates to a process for the production of a composition comprising polydimethylsiloxane and a dye, comprising dissolving the dye in an apolar solvent, admixing the solubilised dye with a polydimethylsiloxane monomer and polymerising the polydimethylsiloxane monomer in the presence of the dye.
The polymerisation of the polydimethylsiloxane monomers in the presence of the dye allows precise dye concentration control and uniform dye incorporation.
The apolar solvent is preferably toluene, xylene or hexane. The process for producing the polydimethylsiloxane composition optionally includes adding a hardener to the polydimethylsiloxane monomers prior to polymerisation. In addition, the pre-polymerisation mixture may optionally be degassed prior to polymerisation.
The process of the second aspect allows the production of the composition in a reliable manner, thereby providing a material which can be used to manufacture a microfluidic device as disclosed in the first aspect.
The third aspect of the invention relates to a method of producing a microfluidic device as claimed in the first aspect of the invention. The composition comprises dissolving a dye in a non-polar solvent, admixing the solubilised dye and polydimethylsiloxane monomers, forming a predetermined object and curing the polymeric mixture. The device is preferably formed by the introduction of the solubilised dye and polydimethylsiloxane monomers into a mould, said final product being removed from the mould after curing.
The method may optionally comprise adding a hardener to the solubilised dye and polydimethylsiloxane monomers admixture. The device may be preferably formed by the introduction of the solubilised dye, the polydimethylsiloxane monomers and a hardener into a mould, said final product being removed from the mould after curing.
Curing of the polymeric mixture may be performed by baking the mixture in an oven at for example 95° for up to 2 hours or 65° C. for 4 hours, preferably 6 hours, more preferably 8 hours. Alternatively, the mixture can be left in the air to cure (for example for 24 hours or above at room temperature). The device can be prepared by casting or injection moulding. Where a portion of the zone for example one or more walls or a portion of one or more walls comprises the polydimethylsiloxane-dye composition the portion can be manufacture as discussed above and attached to a second polydimethylsiloxane portion which does not contain the dye such as a substrate plate.
The fourth aspect of the invention provides a composition comprising polydimethylsiloxane and a dye.
Preferably, the dye comprises an aromatic system comprising one or more aromatic rings such as phenyl rings. More preferably, the dye comprises a conjugated aromatic system comprising two or more fused phenyl rings. In one feature of the first aspect of the invention, the dye comprises a compound of formula (I)
The compound of formula (I) may be substituted at one or more positions on the phenyl or napthalene ring with one or more of hydroxy, halo, C1-4 alkyl.
For the purposes of the invention, the dye may comprise one or more selected from the Sudan dye family such as Sudan Blue II (Solvent Blue 35), Sudan Black B (Solvent Black), Sudan I (Solvent Yellow 14), Sudan II (Solvent Orange 7), Sudan III (Solvent Red 23), Sudan IV (Solvent Red 24), Solvent Blue 37, Solvent Blue 38, Solvent Blue 59 and Solvent Green 3 (Quinizarine Green 55). More preferably, the dye is selected from one or more of Sudan II,
and/or Sudan IV
In an alternative or additional feature of the first aspect, the dye may be Sudan Blue (II).
Alternatively the dye can be a porphyrin or a derivative thereof, colourant, pigment or a nanoparticle such as CdS. It will be appreciated that two or more dyes can be incorporated into the polydimethylsiloxane composition to allow transmission of energy at two or more wavelengths (i.e. to create band pass filtration).
The dyes of the fourth aspect are soluble in apolar solvents such as toluene, xylene or hexane. The dyes further exhibit a low tendency for aggregation.
The composition can be used to produce optical components for incorporation into a device for example a device of the first aspect of the invention. In particular, the composition can be incorporated into or used to produce a filter, lens, prism, microlens, reaction zone, sensor, substrate plate, etc.
The fifth aspect relates to a diagnostic test comprising introducing a sample into a microfluidic device of the first aspect of the invention optionally modifying the sample by a chemical, physical or biological process, exciting the sample (for example by illumination) and detecting the emitted energy. In particular, the test of the fifth aspect allows fluorescence or phosphorescence detection, preferably fluorescence detection of an analyte. The analyte may be a fluorescent and/or phosphorescent analyte. Alternatively the analyte may be modified by a fluorophore or a phosphore. For example an analyte may be a fluorescently or phosphorescently labelled protein (for example in an immunoassay) or nucleic acid (for example in a DNA hybridisation assay). The analyte comprising a fluorophore and/or a phosphore is introduced into the microfluidic device and optionally modified. Alternatively a non-fluorophore or phosphorescent analyte is introduced into the microfluidic device and is modified such that is fluoresces and/or phosphoresces. The fluorophore and/or phosphore is excited by a light source and the light re-emitted from the fluorophore and/or phosphore is detected.
The fluorophore or phosphore is preferably a labelled protein, nucleic acid or a portion or derivative thereof.
The diagnostic test preferably comprises a test for detecting the presence or absence of an analyte, preferably by fluorescence and/or phosphorescence.
The sixth aspect of the invention relates to a kit for carrying out a diagnostic test comprising a microfluidic device of the first aspect of the invention and instructions relating to the diagnostic test to be carried out.
All preferred features of each of the aspects of the invention apply to all other aspects mutatis mutandis.
The invention may be put into practice in various ways and a number of specific embodiments will be described by way of example to illustrate the invention with reference to the accompanying drawings, in which:
The present invention will now be illustrated by reference to one or more of the following non-limiting examples.
Optical long-pass filters were fabricated by pre-polymerization doping of polydimethylesiloxane (PDMS). Sudan II, III, IV and Sudan Blue II dyes (Sigma-Aldrich, Gillingham, UK) were dissolved in 1 mL toluene and than added to 16.5 mL of PDMS monomer and hardener pre-mixed at a ratio of 10:1 v/v (Sylgard 184 Silicone Elastomer kit, Dow Corning, Coventry, UK). Vigorous manual mixing was continued until a uniform PDMS colouring was obtained. The doped PDMS was then poured into plastic Petri dishes for fabrication of unstructured filters or over an SU-8 master for molding of the monolithically integrated microfluidic/filter layer. The two-level SU-8 master was fabricated on a silicon substrate at the Centre of Integrated Photonics (Ipswich, UK) using standard SU-8 processing protocols. Filter thickness was controlled via the poured PDMS volume, with a standard filter thickness of 3 mm. For filter performance optimization the thickness was later reduced to 2 mm and 1 mm, with adjusted dye load. PDMS curing was performed at room temperature for 48 hours. For high Sudan II dye loads, a 4 hour 65° C. curing step was added.
Filters were prepared at various thicknesses (1-3 mm) and dye-loadings (60-1800 μg/mL). The highest dye loads necessitated an increase of the toluene content in order to achieve full dissolution of the dye molecules and uniform dispersion in the final matrix. However, excessively high solvent content resulted in incomplete polymerization even after prolonged curing. The overall solvent content in the doped PDMS was thus limited to below 10 v/v/-%. For high dye loads, a 4 hour 65° C. curing step was added to ensure complete hardening of the PDMS.
Transmittance of the dye-doped PDMS filters was measured on a UV-VIS Spectrometer V-560 (Jasco, Great Dunmow, UK) equipped with a customized thin-film holder. Spectra were baseline corrected and referenced against air. Typical scans were performed from 250 to 850 nm at 400 nm/min with 2 nm line width. Autofluorescence of the Sudan filter range was measured with a 200 mW Ar Ion laser excitation source and a USB 2000 CCD spectrometer (Ocean Optics, Duiven, The Netherlands) using an integration sphere.
Filter stability against continuous light exposure was tested by illuminating the filters with UV light (200 W mercury arc lamp) or storing the filters on a laboratory bench and exposing them to ambient light for 4 weeks. Chemical stability was tested by filters immersed in water or ethanol being ultrasonicated at 33 kHz (300 W) for 5 min, 10 min, 1 hour, 3 hours and 5 hours (Sonomatic S1000, Langford Electronics, Birmingham, UK). The resulting optical properties were then compared to those of untreated PDMS filters.
For the fabrication of monolithically integrated microfluidic/filter layers, the Sudan II doped PDMS monomer/hardener solution was poured over an SU-8 master prior to curing. The SU-8 master was fabricated on a silicon substrate using standard SU-8 processing protocols (Centre for Integrated Photonics, Ipswich, UK). The cured PDMS layer, shown in
For use of the monolithically integrated microfluidic/filter layers in on-chip fluorescence detection, access holes are punched at the channel ends with a glass Pasteur pipette, resulting in ˜2-mm-diameter holes, followed by reversible sealing against a 1-mm-thick unstructured PDMS slab. Rhodamine B solutions of different concentration are then hydrodynamically pumped through the microchip inlet(s). In a collinear detection geometry, a blue OLED and an organic photodiode can be used as the excitation source and detector, respectively (
A more rigorous measurement of filter autofluorescence may be obtained using an integrating sphere—a hollow sphere that has its inner surface coated with a diffusely reflecting material. When a light-source—in this case an autofluorescent filter—is placed inside an ideal integrating sphere the light is redistributed isotropically over the sphere's interior-surface, irrespective of the angular distribution of the emission. Hence, if NΩ photons of a given wavelength are detected over a solid angle Ω, the total number N of emitted photons is given by N=kλNΩ(4π/Ω) where kλ is a wavelength dependent constant that accounts for reflection losses in the sphere. The use of an integrating sphere therefore enables the total number of photons emitted via autofluorescence to be determined from a measurement at a single location on the sphere wall, obviating the need to map out the full angular distribution of the emission. The PDMS filters were placed inside an 18 cm diameter sphere (Labsphere) and illuminated with the 2 mm diameter beam output from a 200 mW 488 nm Ar Ion laser (43 Series Ar Ion laser, Melles Griot) entering through a small inlet port. The emitted light was detected using a fibre-optic coupled CCD spectrometer (USB 2000 CCD spectrometer with 600 μm diameter fiber, Ocean Optics) inserted into a second port, oriented at 90 degrees relative to the laser beam. The autofluorescence characteristics of the Sudan II PDMS filters were measured as well as those of a commercially available Schott glass long-pass filter with a cut-on point at 583 nm. The Schott filter showed a small amount of autofluorescence in the range 570 to 950 nm but no fluorescence was measurable from the PDMS filter, confirming the high quality of the Sudan II/PDMS filters (
Optimization of Chromophore Incorporation into the PDMS Host Matrix.
For comparative purposes, post-polymerization staining with polar dyes as previously reported by Whitesides was performed with Rhodamine B and Rhodamine 640. While in both cases staining was observed after immersion in aqueous dye solution for 48 hours, a cross-sectional cut revealed heavy staining only near the surfaces. This is consistent with a diffusional staining process. Attempts to increase the dye intake by plasma treatment of the PDMS surface to enhance wettability, or by increasing the monomer/hardener ratio from 10:1 to 50:1 v/v to enlarge pore size were unsuccessful.
Pre-polymerization doping was tested by adding a chromophore to the monomer/hardener mix prior to curing. For the PDMS host matrix, this required apolar chromophores since the Sylgard monomer is dissolved in hexane and xylene. Addition of polar dyes dissolved in water or ethanol resulted in emulsive non-uniform chromophore incorporation and incomplete polymerization. In contrast, apolar Sudan dyes dissolved in small volumes of hexane or toluene and thoroughly mixed with PDMS monomer/hardener were uniformly incorporated. The applied solvent volume was minimized such that the overall solvent content in the doped PDMS was below 10 v/v-%. Higher solvent contents often resulted in incomplete polymerization even after prolonged curing.
The optical properties of Sudan doped PDMS layers after curing are depicted in
Since in fluorescence detection a sharp cut-on between excitation light blocking and transmission of emitted fluorescence is crucial, Sudan II doped layers with typical transition phases <50 nm were selected for further optimization. Thin-film filters are beneficial for efficient collection of fluorophore emission and as such the effect of filter thickness was subsequently investigated.
Autofluoresence of the filters was tested using a 200 mW Ar Ion laser, an integration sphere and a CCD spectrometer equipped with an optical fibre. The barium sulfate coated integration sphere diffuses all light uniformly throughout the sphere and enhances collection of isotropically emitted fluorescence. The PDMS filters were placed inside the sphere and illuminated with the 2 mm diam. laser beam entering through a sphere orifice. Detection of any emitted autofluorescence originating from the filter was performed with the CCD spectrometer fibre inserted into a second orifice, oriented at 90 degrees relative to the laser beam. The autofluorescence of optimised 1-mm and 2-mm-thick PDMS layers doped with 1200 and 900 mg/mL Sudan II, respectively, and of a commercial 2-mm-thick Schott filter are depicted in
To demonstrate the excellent selective blocking characteristics of the presented filters they were illuminated with monochromatic light mimicking excitation and emission wavelengths typically used for the model fluorophore Rhodamine B (
For the optimized 2 mm Sudan II doped filters, stability against chemical solvents and prolonged light exposure was subsequently investigated, which are important parameter for industrial applications (
For shortpass or bandpass filter applications, Sudan Blue II was dissolved in toluene to form different concentration solutions with 15 microg/ml, 10 microg/ml, 5 microg/ml, respectively. It was necessary to determine that the dyes were fully dissolved. One volume liquid solution was mixed with 10 volumes of PDMS monomer and one volume hardener, the mixture was then poured into a pre-shaped mould to form defined shape components (i.e. plate, chip, or lens). The mixture was degassed to remove air bubbles trapped in the mixture before curing the monomer. Curing was carried out by leaving the mixture in the air at the room temperature. Preshaped rubber like flexible PDMS components can be obtained.
The transmission spectra of Sudan Blue II doped into PDMS formed plate with 2 mm thickness were taken by UV-VIS spectrophotometer shown in
Filters can therefore be made from the PDMS composition and can be used for fluorescence detection. The composition can be introduced directly into a microfluidic device either as one part of a channel section or as a plate section for sealing the chip (as shown in
The ideal longpass filter should provide excellent attenuation and transmission either side of the cut-on, and we therefore used the convenient figure of merit Q=T(λtransmit)/T(λblock) to assess the filter performance, where T(λ) is the percentage transmission at a wavelength λ, and λtransmit and λblock correspond to convenient wavelengths either side of the filter cut-on. Q corresponds to the expected improvement in sensitivity when clear PDMS is replaced by the dye-doped PDMS if the following criteria are met: (i) the spectrum of the light source lies entirely in the attenuation region of the long-pass filter; (ii) the analyte emission spectrum lies entirely in the transmissive region of the long-pass filter; (iii) the limit-of-detection is determined by the background due to the excitation light rather than detector noise or other electronic noise; and (iv) the autofluorescence from the filter is negligible. If these criteria are satisfied then the optimal filter performance is obtained at the dye loading that maximises Q.
In preliminary tests, to assess the relative performance of the three dye molecules, 3-mm-thick filters were fabricated using Sudan II, Sudan III and Sudan IV dye loadings in the range 60 to 720 μg/mL. The concentration dependence of the Q-values for the three dyes are shown in
A sharp roll-on between attenuation and transmission is crucial in fluorescence detection, so Sudan II filters, which have a typical roll-on transition width <50 nm, were selected for further optimization. In order to ensure efficient collection of fluorophore emission in a microfluidic environment, the distance between the fluorophore and detector should be kept small, and subsequent tests were therefore conducted on 1 mm filters, which is typical of the substrate thicknesses employed in microfluidic devices. In
where c is the dye concentration, d is the filter thickness, and εlong and εshort represent the extinction coefficients of the dye molecule at the long and short wavelengths respectively. The extinction coefficients of Sudan II were measured in a 100 μM solution in toluene, yielding values of 24280 and 170 M−1cm−1 at 500 and 600 nm respectively. This corresponds to a maximum theoretical Q-value of 18260 for 1200 μg/mL (˜4 mM) Sudan II in a 1-mm layer, substantially higher than the value actually obtained in our PDMS filters. The ideal Q value as a function of dye concentration is shown as a dotted line in
High quality monolithically integrated disposable PDMS based microfluidic layers were fabricated with optical long-pass filter characteristics. The use of Sudan II and Sudan IV dyes showed good solubility and yielded high quality filters with cut-on wavelengths of 550 and 580 nm respectively. For instance, 3-mm-thick filters with relatively low dye-loadings of 600 μg/mL yielded short wavelength optical densities of 3.3 and 4.3 for Sudan II and Sudan IV, respectively. Using Sudan II as a test dye, high quality 1 mm PDMS layers were fabricated with dye-loadings up to 1200 μg/mL. The resultant filters had excellent optical characteristics, e.g. <0.01%-transmission at 500 nm and >80%-transmission above 570 nm. Importantly, the filters showed negligible autofluorescence, allowing them to be effectively employed in microchip-based fluorescence detection. The filters proved robust in use, undergoing only negligible leaching in aqueous solution and marginal photodegradation. Patterning of the PDMS was unaffected by the dye doping, allowing for the fabrication of coloured substrates that serve concurrently as channel medium and optical filter.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0523079.2 | Nov 2005 | GB | national |
| 0604799.7 | Mar 2006 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2006/004198 | 11/9/2006 | WO | 00 | 5/9/2008 |
