WO2011120169A1

WO2011120169A1 - Microfluidic device and system with optical evanescent coupling element

Info

Publication number: WO2011120169A1
Application number: PCT/CA2011/050176
Authority: WO
Inventors: Jesse Greener; Eugenia Kumacheva
Original assignee: University of Toronto
Current assignee: University of Toronto
Priority date: 2010-03-31
Filing date: 2011-03-31
Publication date: 2011-10-06
Anticipated expiration: 2012-09-30

Abstract

A microfluidic device is provided for the local measurement of attenuated total reflection from a microfluidic channel. The device comprises a substrate in which a microfluidic channel is formed and sealed by a sealing layer adhered to the substrate, and further comprises a fluidic inlet and outlet. A portion of the sealing layer is removed, exposing the channel and forming an aperture, to which a coupling surface of an optical evanescent coupling element is affixed. The coupling surface seals the channel within the aperture and enables the optical probing of a fluid within the channel by the evanescent field of an optical beam reflected from the coupling surface. The device is preferably used in a system comprising a spectrometer for the measurement of an attenuated total reflection spectrum.

Description

M!CROFLUIDIC DEVICE AND SYSTEM WITH OPTICAL EVANESCENT

COUPLING ELEMENT

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Application No.

61/319,328, titled "MICROFLUIDIC DEVICE AND SYSTEM WITH OPTICAL EVANESCENT COUPLING ELEMENT" and filed on March 31 , 2010, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to microfluidic devices and systems, and more particularly, the invention relates to the use of microfluidics for the optical probing of fluids and fluidic reactions. BACKGROUND OF THE INVENTION

Microfluidic synthesis offers new research opportunities in synthetic chemistry, owing to excellent control of reaction conditions, reduced consumption of reagents, the ability to conduct continuous multi-step reactions without exposure of reactive intermediates to ambient conditions, and the capability to carry out many reactions in a parallel manner.^{1 "4} Rapid optimization of

formulations can be achieved by screening the effect of reaction variables, e.g., reagent concentrations, types of catalysts, or the amount of energy applied to the system.^4"8 These applications require in situ (on-chip) characterization of the concentration of reactants or products at a particular time of the reaction.

Currently, chemical characterization is typically conducted off-chip using methods such as mass spectroscopy and liquid or gas chromatography^9"12.

Relatively few techniques, such as fluorescence spectroscopy and thermal lens microscopy, monitor chemical changes on-chip.^13,14 These methods are material- specific and require either strong localized heating, or the addition of reporter molecules (e.g. dyes). On-chip NMR has been demonstrated, but is still being developed.^15,16 On-chip Raman spectroscopy needs long data acquisition times,¹⁷ although attempts have been made to improve this method via

Resonance Raman Scattering (RRS),¹⁸ Surface Enhancement Raman Scattering (SERS)¹⁹ and the combination of the two methods.²⁰

SUMMARY OF THE INVENTION

In a first aspect, there is provided a microfluidic apparatus comprising a substrate having formed therein a microfluidic channel, a sealing layer adhered to the substrate, the sealing layer forming an upper surface of the microfluidic channel, an inlet and outlet for flowing a fluid in the channel, an aperture formed within a portion of the sealing layer exposing a portion of the channel, and an optical evanescent coupling element adhered within the aperture, the optical evanescent coupling element comprising a planar coupling surface, wherein the planar coupling surface contacts the substrate and seals the channel within the aperture.

The optical evanescent coupling element preferably comprises an attenuated total reflection crystal, surface plasmon resonance element, or diffractive optic. The planar coupling surface may be functionalized with receptors, or may comprise a hydrophobic or hydrophilic surface. The planar coupling surface may be coated with a material having an index of refraction that is different from a refractive index of the optical evanescent coupling element for controlling a refractive index contrast.

The optical evanescent coupling element may be permanently adhered within the aperture. Alternatively, optical evanescent coupling element is removably adhered within the aperture by a clamping means, where the clamping means preferably provides a force that counteracts a force applied to the planar coupling surface by a pressure within the channel, and more preferably supports a flow rate within the channel in excess of 20 ml/h.

The optical evanescent coupling element is preferably supported and protrudes from a base plate, and wherein the clamping means applies a clamping force to the optical evanescent coupling element through the base plate, wherein a thickness of the sealing layer is selected to contact the base plate with the sealing layer under application of the clamping force. Preferably, the thickness of the sealing layer substantially equals a distance over which the optical evanescent coupling element protrudes from the base plate, or is less than a distance over which the optical evanescent coupling element protrudes from the base plate, and wherein the base plate contacts the sealing layer indirectly through a compressible layer provided between the base plate and the sealing layer. The clamping means may comprise a ring clamp, wherein the ring clamp applies a force between the base plate and a back side of the substrate; a vacuum clamp, wherein the base plate is held against the sealing layer a pressure gradient applied between holes extending through the sealing layer and the substrate; and a magnetic clamp, wherein electromagnetic coils or

permanent magnets are integrated within the substrate, and wherein the coils couple with magnetic materials or additional electromagnetic coils connected to the base plate.

The channel is preferably directed through one or more bends beneath the aperture. Alternatively, the channel traverses beneath the aperture in two or more locations. The channel preferably contacts the planar coupling surface over at least 40% of an area of the planar coupling surface.

In another aspect, a system is provided for measuring an optical signal from a microfluidic channel, the system comprising: an optical source, optical detector, a microfluidic device comprising: a substrate having formed therein a microfluidic channel; a sealing layer adhered to the substrate, the sealing layer forming an upper surface of the microfluidic channel; an inlet and outlet for flowing a fluid in the channel; an aperture formed within a portion of the sealing layer exposing a portion of the channel; and an optical evanescent coupling element adhered within the aperture, the optical evanescent coupling element comprising a planar coupling surface, wherein the planar coupling surface contacts the substrate and seals the channel within the aperture, and optical coupling means for directing an incident optical beam from said optical source onto said optical evanescent coupling element and for directing one of a reflected beam and fluorescence emission from said optical evanescent coupling element onto said detector.

The system preferably further comprises a control unit for controlling at least one of said optical source and optical detector, and also preferably comprises a wavelength selective means, wherein said controller further controls said wavelength selective means. The system preferably comprises a

spectrometer that is preferably a Fourier-transform infrared spectrometer, and the detector preferably comprises a non-imaging detector.

In yet another aspect, there is provided a method of measuring attenuated total reflection from a fluid within a microfluidic device, the microfluidic device comprising: a substrate having formed therein a microfluidic channel; a sealing layer adhered to the substrate, the sealing layer forming an upper surface of the microfluidic channel; an inlet and outlet for flowing a fluid in the channel; an aperture formed within a portion of the sealing layer exposing a portion of the channel; and an optical evanescent coupling element adhered within the aperture, the optical evanescent coupling element comprising a planar coupling surface, wherein the planar coupling surface contacts the substrate and seals the channel within the aperture; the method comprising the steps of: directing an incident optical beam onto the optical evanescent coupling element, wherein the optical beam is totally reflected by the planar coupling surface and attenuated by the fluid within the channel; and directing the reflected optical beam onto a detector and measuring a signal; determining the attenuated total reflection by relating the signal to pre-determined calibration value. The pre-determined calibration value preferably comprises a signal obtained when the incident optical beam is directed onto the detector in the absence of the microfluidic device, or a signal obtained from a reference material within the channel.

A clamping force is preferably applied to adhere the optical evanescent coupling element within the aperture, and the clamping force is preferably provided with sufficient force to counteract a force applied to the planar coupling surface by a pressure within the channel. An attenuated total reflection spectrum may be measured by varying a wavelength of the incident optical beam.

Additional optical measurements may be obtained by varying the angle of incidence and/or the polarization of the incident beam.

In yet another aspect, there is provided a method of measuring total internal reflection fluorescence from a fluid within a microfluidic device, said microfluidic device comprising: a substrate having formed therein a microfluidic channel; a sealing layer adhered to said substrate, said sealing layer forming an upper surface of said microfluidic channel; an inlet and outlet for flowing a fluid in said channel; an aperture formed within a portion of said sealing layer exposing a portion of said channel; and an optical evanescent coupling element adhered within said aperture, said optical evanescent coupling element comprising a planar coupling surface, wherein said planar coupling surface contacts said substrate and seals said channel within said aperture; said method comprising the steps of: directing an incident optical beam onto said optical evanescent coupling element, wherein said incident optical beam optically excites a fluorescent species within said channel; and directing fluorescence emission from said fluorescent species onto a detector and measuring a signal.

A further understanding of the functional and advantageous aspects of the invention can be realized by reference to the following detailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the drawings, in which:

Figure 1 illustrates a microfluidic device having an aperture for housing an optical evanescent coupling element, where (a) shows an overhead view of the device, (b) shows a cross-sectional detailed view of the aperture, and (c) shows an optical evanescent coupling element housed within the aperture, thereby sealing the microfluidic channel.

Figure 2 provides a schematic of an ATR-FTIR system including a microfluidic device.

Figure 3 shows a schematic of a microfluidic device incorporating a T- junction, a reaction compartment, and an ATR characterization region. A zoomed inset of the ATR-FTIR characterization region shows the geometry of the channel and the circular ATR crystal beneath it. Arrows show the direction of flow of the liquid.

Figure 4 is a photograph showing a top view of a fabricated microfluidic device similar to the device illustrated in Figure 2 (but features 2 fluidic inputs), where the device is shown connected to inlet and outlet tubing and interfaced with an ATR crystal apparatus.

Figure 5 is a side-view of an ATR crystal interfaced with a single microchannel.

Figure 6 plots (a) an ATR-FTIR spectrum acquired from the adhesive side of the adhesive film, and (b) a spectrum acquired from TX-100 solution (C_Tx- mM) flowing through MF device (Q=3.0 imL/h) sealed with the adhesive film. The spectrum in (b) was acquired 10 minutes after flow was established through the device. Both spectra (a) and (b) were collected using 16 scans at 10 kHz and 4 cm^"1 spectral resolution.

Figure 7 (a) plots IR absorption spectra acquired for the solutions of PNIPAm (top spectrum) and PEG (bottom spectrum) at flow rate of the liquids of 3 mL/h. For each solution the presented spectra are the result of averaging five spectra. The concentration of each polymer solution, C_p0i, was 4 wt%,

corresponding to 1 .96 mM and 4.14 mM for PNIPAm and PEG, respectively. Figure 7(b) plots the variation in the concentration-dependent intensity of the absorption peaks of PNIPAm (1560 cm^"1) (open circles) and PEG (1085 cm^"1) (solid squares). The dashed lines are the result of linear fitting. The inset shows absorbance of the solutions at C_p0i < 0.5 wt%. All spectra, except those obtained for Cpoi = 0.01 wt%, were collected using 16 scans at 10 kHz and 4 cm^"1 spectral resolution. The data points collected at C_p0i = 0.01 wt% were collected using 64 scans. Error bars were obtained by measuring the standard deviation in data points from five independent experiments. Figure 8 (a) provides composite spectra acquired for the mixed aqueous solution of PNIPAm and PEG.

Absorption peaks of PNIPAm and PEG are shown in the spectral regions 1700-1500 cm^"1 and 1200-1000 cm^"1, respectively. Arrows show the monitored peaks at 1560 cm^"1 (PNIPAm) and 1085 cm^"1 (PEG). The ratios of flow rates of the solution of PNIPAm to the solution of PEG were 0/3 (1 ), 0.5/2.5 (2), 1/2 (3), 1 .5/1 .5 (4), 2/1 (5), 2.5/0.5 (6), 3/0 (7). Spectra 1→ 7 show the change in spectral characteristics of the mixture as the concentration of PNIPAm in the solution increased and the concentration of PEG in the solution decreased. The concentrations of the individual solutions of PNIPAm and PEG that were introduced in the microfluidic device are 1 .96 mM and 4.14 mM, respectively. Figure 8 (b) plots concentration-dependent variation in absorbance of PEG (solid squares) and PNIPAm (open circles). Absorbance was measured for peaks shown in (a). Spectra were collected using 16 scans at 10 kHz and 4 cm^"1 spectral resolution. Error bars were obtained by determining the standard deviation in absorbance in five independent measurements at each flow rate.

Figure 9 plots in-flow absorbance of 0.48mM (1 .0 wt%) PNIPAm (open circles) and 1 .04 mM (1 .0 wt%) PEG (solid squares) solutions measured for the bands at 1560 cm^"1 and 1085 cm^"1 , respectively as flow rate (Q) is modulated. Spectra were collected using 16 scans at 10 kHz and 4 cm^"1 spectral resolution. Error bars were obtained by determining the standard deviations in absorbance of five independent measurements for each flow rate of the liquids. Dotted lines are the results of linear fitting of the data. Figure 10 plots (a) the IR absorption spectrum acquired for the C_Tx-i oo = 32 mM aqueous solution of TX-100 as it flows through the microchannel at flow rate 6 mL/h, and (b) the variation in absorbance of the 1 100 cm^"1 peak as a function of the concentration. The dotted line is the linear fit for C_Tx-i oo≤ 65. The inset shows absorbance in the low concentration region (C_TX-_{I OO}≤ 10 mM). The dotted line in the inset is the same linear fit from the main figure. Spectra were collected using 16 scans at 10 kHz and 4 cm^"1 spectral resolution. Error bars were obtained by measuring the standard deviation from five independent experiments.

Figure 11 provides a molecular diagram for (a) the monomer NIPAm and

(b) the repeat unit of the polymer poly-NIPAm.

Figure 12 is a schematic of another embodiment of a microfluidic device incorporating a T-junction, a reaction compartment, and an ATR characterization region (scale bar is 1 cm).

Figure 13 plots (a) layered spectra acquired during the redox

polymerization of NIPAm showing changes to 975 cm-1 peak (C=C) over reaction times t=4 to t= 50 s, and (b) ln([M]/[Mo]) vs. time for different

concentrations of APS/TEMED initiation mixture.

Figure 14 plots (a) the vibrational spectrum collected on-chip showing C0₂ (2346 cm^"1) and HC0₃ ^" (1365 cm^"1) bands, where the C0₃ ^2" band in pH 12 aqueous phase is shown in (b) and the reappearance of C0₂ and HC0₃ ^" at lower pH is shown in (c). DETAILED DESCRIPTION OF THE INVENTION

As required, embodiments of the present invention are disclosed herein. However, the disclosed embodiments are merely exemplary, and it should be understood that the invention may be embodied in many various and alternative forms. The Figures are not to scale and some features may be exaggerated or minimized to show details of particular elements while related elements may have been eliminated to prevent obscuring novel aspects. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention. For purposes of teaching and not limitation, the illustrated embodiments are directed to microfluidic devices and systems incorporating an attenuated total reflection crystal.

As used herein, the terms, "comprises" and "comprising" are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in this specification including claims, the terms, "comprises" and "comprising" and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

As used herein, the term "exemplary" means "serving as an example, instance, or illustration," and should not necessarily be construed as preferred or advantageous over other configurations disclosed herein.

As used herein, the terms "about" and "approximately, when used in conjunction with ranges of dimensions of particles, compositions of mixtures or other physical properties or characteristics, is meant to cover slight variations that may exist in the upper and lower limits of the ranges of dimensions so as to not exclude embodiments where on average most of the dimensions are satisfied but where statistically dimensions may exist outside this region. It is not the intention to exclude embodiments such as these from the present invention.

As used herein, the term "fluid" means any fluid comprising a liquid component, including, but not limited to, liquids, mixtures, solvents, suspensions, colloids, and heterogeneous multi-phase systems including bubbles.

A microfluidic device illustrating a preferred embodiment of the invention is shown in Figures 1 (a)-(c). Figure 1 (a) provides an overhead view of microfluidic device 10, which comprises inlet 15, outlet 20, and microfluidic channel 25.

Channel 25 includes a channel bend 30, which is located below an aperture 35 that locally exposes the channel to the external environment. As shown in Figure 1 (b), which provides a cross-sectional view through microfluidic device 10, channel 25 is formed in substrate 50 and is recessed beneath a sealing layer 55 that is locally removed to form aperture 35 above the two channel segments 40 and 45 of bend 30.

As shown in Figure 1 (c), an optical evanescent coupling element 60 is housed within aperture 35. Optical evanescent coupling element 60 seals channel 25 from the external environment and enables the local optical interrogation of the channel by the evanescent field of an incident optical beam. The optical coupling element is preferably an attenuated total reflection (ATR) crystal. The ATR crystal may comprise a single or multi-bounce crystal.

As shown in Figure 1 (c), optical coupling element 60 seals microfluidic channel 25 from the external environment. This is achieved by tailoring the area and geometry of aperture 35 to conform to the geometry of optical coupling element 60. Optical coupling element 60 may comprise a wide variety of shapes, provided that inner surface 65 is planar and forms an upper surface for channel 25 within aperture 35.

In order to achieve robust fluidic seal, the geometry of the aperture side wall 80 preferably matches that of optical coupling element 60. While it is preferable that the entire aperture is sealed by the coupling element, in some embodiments, the aperture may be sealed by the optical coupling element only for sides of the aperture where the channel crosses the aperture. Although Figure 1 (c) illustrates an optical coupling element having vertical side walls contacting the sealing layer 55, optical coupling element 60 may have an angled surface. Accordingly, the slope of side wall 80 may be beveled to accommodate such an angled surface. A beveled side wall may be achieved by numerous methods known in the art, including micromachining, molding, and embossing. Additionally, sealing means known in the art such as o-rings may be provided within aperture 35 to further assist in the sealing of channel 25 by optical coupling element 60.

The optical coupling element allows for the optical evanescent probing of liquid flowing through channel 25 without distorting or altering the local fluidic environment. The attenuated total reflection may be measured by directing an incident optical beam 70 from the optical source onto the optical coupling element 60 such that the incident optical beam is totally internally reflected at the interface between the coupling element while being subjected to absorption via evanescent coupling to the fluid within channel 25. The reflected optical beam 75 is then directed onto the detector, the attenuated total reflection may be determined by normalizing the detected signal to a known calibration value, such as the signal obtained in the absence of the sample when the optical beam is directly incident on the detector (or reflected using a standard reflector), or the signal obtained from a reference material within the channel.

Since the optical coupling element forms the upper surface of channel 25 within the aperture 35 without altering the channel dimensions, the properties of the fluid (such as Reynolds number) are unchanged by the presence of the optical coupling element. This is highly beneficial for probing in situ reaction kinetics and/or flow characteristics (in particular, this is believed to be the source of the flow rate independence of absorption intensity shown in Figure 9).

In another preferred embodiment, the optical coupling element may be coated with a thin (less than the penetration depth of the evanescent light) layer of the material from which the sealing layer is comprised, whereby the channel material is preserved in the detection region in addition to the channel dimensions.

The channel geometry within the aperture region may comprise a straight channel, but is preferably selected to provide a large relative surface area for optical interrogation. In Figure 1 , this is achieved with a single bend. It is to be understood that the optimization of the channel geometry may be achieved by a wide range of channel geometries. In one non-limiting embodiment, the channel geometry may include a serpentine structure comprising multiple bends. In another non-limiting embodiment, the channel geometry may include several closely spaced parallel channels, with bends external to the aperture region, to provide a high channel packing fraction within the aperture region.

Embodiments of the present invention provide the benefit of only requiring the optical probing of a microfluidic device locally, at the location of the aperture. This important benefit allows for the collection of an optical signal from a desired location with optimal signal to noise ratio. For example, the location probed may be downstream from a reaction process or reaction component of the microfluidic device, where it is desirable to collect an optical signal with high sensitivity.

Furthermore, since the optical coupling element only probes a specific region on microfluidic device, the remaining areas of the microfluidic device are available for the integration of other active fluidic elements, such as fluidic injection ports or valves.

While microfluidic device 10 shown in Figure 1 comprises only a single channel, those skilled in the art will readily appreciate that the microfluidic device may comprises any number of additional fluidic components, including, but not limited to, additional inlets and outlets, mixing networks and chambers, junctions such as T-junctions and flow focusing regions, and valves. A specific example of a microfluidic device with additional fluidic components is provided in the example below. Furthermore, the microfluidic device may comprise more than one aperture and optical evanescent coupling element for probing more than one location on the microfluidic device.

In the embodiment shown in Figure 1 , the optical coupling element 60 has an inner surface 65 that is larger in width than the width of the microfluidic channel 25. It is, however, to be understood that the optical coupling element may comprise a distal geometry that may protrude into the channel, thereby sealing the channel. In a non-limiting example, such an embodiment may be achieved with a linear channel and an optical coupling element having a shape in the form of a triangular prism, where one longitudinal vertex of the prism protrudes into and seals the channel.

In one embodiment, optical coupling element 60 may be permanently adhered to microfluidic device 10 by an adhesive or other retaining mechanism. In a preferred embodiment, optical coupling element 60 is removably adhered to microfluidic device 10 within aperture 35 by a clamping mechanism that counteracts the force of fluid flowing within the channel 25 on the inner surface 65 of optical coupling element 60. Accordingly, provided that a sufficiently high clamping force is provided, high flow rates of fluid through the channel may be achieved without leakage. Various clamping mechanisms may be employed to achieve the desired retaining force. Preferred clamping mechanisms include, but are not limited to, ring clamps, an external lever clamp, magnetic clamping, and vacuum clamping.

In a preferred embodiment, further illustrated in the example below, optical coupling element is housed in or on a support having a base plate, in which the base plate has a bottom surface that is parallel to the inner surface 65 of the optical coupling element. The base plate thus provides a broad surface over which pressure can be applied to the optical coupling element. Preferably, the optical coupling element protrudes over a short (e.g. sub-millimeter) offset distance from the base plate, and the thickness of the sealing layer 55 is chosen to be equal to the offset distance. This enables the base plate surface to contact the outer surface of the sealing layer when the coupling element is secured within the aperture. Alternatively, the thickness of sealing layer 55 may be chosen to be less than the offset distance, and an additional compressible layer may be included between the base plate and sealing layer 55 in order to obtain a consistent and reliable seal.

Materials that can be used to form substrate 50 housing the channels and sealing layer 55 include elastomers, but are preferably, materials with sufficient hardness to resist the distortion of the microfluidic channel when optical coupling element 60 is clamped to microfluidic device 10, such as, hard plastics, metals, ceramics, glasses.

In a preferred embodiment, substrate 50 may be imprinted with the fluidic features and can form a bond between the imprinted and sealing 55 layers.

Imprinting can be accomplished by techniques including, but not limited to, hot embossing, injection molding, laser ablation and end milling. Bonding to a sealing layer, typically but not necessarily of the same material as the imprinted device, has been achieved by low temperature bonding after exposure of the imprinted layer and/or the sealing layer to liquid-phase solvents or plasma gas, as described in J. Greener, W. Li, J. Ren, D. Voicu, V. Pakharenko, T. Tang and E. Kumacheva, Lab Chip, 2010, 10, 522-524, which is incorporated herein by reference in its entirety. Other methods include exposure of imprinted layer and/or sealing layer to solvent vapours or UV light to achieve surface activation and subsequent low-temperature bonding or purely thermal bonding at temperatures very close to the glass transition temperature of the thermoplastic materials.

The optical properties of microfluidic device 10 and the optical coupling element 60 are preferably selected such that light reflected back to the detector has minimized interaction with material. This may be accomplished by reducing the difference between the index of refraction of the optical coupling element and substrate 50, and/or modifying the angle of incidence of light incident on optical coupling element 60, such that light passing into substrate 50 as opposed to light passing into liquid filled microchannels, escapes from the microfluidic device and does not propagate to the detector.

Alternatively, this may be achieved by increasing the index of refraction contrast and/or modifying the angle of incidence of light incident on optical coupling element 60 such that light passing into substrate 50 (as opposed to light passing into liquid filled microchannels) is reflected to the detector with as little interaction with material as possible. The index contrast may be tailored by applying a coating to either the optical coupling element or the microfluidic device material.

In a preferred embodiment, the apparatus shown in Figure 1 forms a component of a measurement system such as an attenuated total reflection measurement system that further comprises an optical source 100, optical detector 1 10, and preferably further includes a control unit 120 as shown in Figure 2 for processing signals detected by the optical detector. The system also includes optical elements for directing the incident beam from the source to the optical coupling element (not shown), and optical components for receiving a reflected or emitted beam and directing the reflected or emitted beam to the detector (not shown). Exemplary but non-limited optical components include lenses, mirrors, and fiber optics. In another embodiment, the system comprises a total internal reflection fluorescence system, in which incident light excites a fluorescent species within the channel, and the emitted fluorescence is collected and directed to the detector.

The system may further comprise a spectral device for the measurement of an optical spectrum . Exemplary yet non-limiting spectral devices include monochromators, diffraction gratings, and scanning interferometers. Control 120 unit may include a processor, and may comprise an internal processor within a spectroscopy system or may additionally or alternatively comprise an external computer. The optical beam is may be delivered to and from the optical coupling element 60 by optical fibers (not shown). In one embodiment, optical detector 1 10 is a single detector that integrates the net signal from the received optical beam. In a preferred embodiment, the system comprises a spectrometer-ATR system 130, such as an ATR-FTIR (Fourier Transform Infrared Spectrometer) in which the optical source 100, optical detector 1 10 and control unit 120 comprise an FTIR system 130.

Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) is a well-established analytical tool applicable to spectral

characterization of chemical and biological species without particular sample preparation. Acquisition of absorption spectra enables monitoring of the appearance of reaction products or consumption of reactants. The reaction kinetics can be studied by using the Beer-Lambert law as:

A = cx lxe (1 )

where c is the analyte concentration (M), / is the path length (cm) and ε is the wavelength-dependant molar extinction coefficient (M^~1 cm^"1).

The path length for ATR-FTIR is typically only a few microns in the mid-IR spectral range, with its exact value being determined by the wavelength of light, the angle of incidence and the indices of refraction of the ATR crystal and the medium being probed. Thus, ATR overcomes the major limitation of conventional transmission FTIR: the dramatic reduction of transmission for strongly absorbing carrier-phase liquids (e.g., water) for path lengths exceeding 10-20 μητι. The ability to align the ATR-FTIR probe with the walls of microchannel also eliminates the need for fabrication of microfluidic devices from IR-transparent materials. Further increases in the sensitivity of the device can be achieved in several ways, namely, by increasing the ATR-FTIR path length, e.g., by using a lower index of refraction ATR crystal, by interfacing a larger, multiple reflection, ATR crystal with the microfluidic device (thereby probing a longer segment of the microchannel), and by implementing a purge gas or vacuum system to reduce interference from water vapour and C0₂ along the optical path within the spectrometer.

Although the preceding discussion of the exemplary embodiments was provided within the context of total attenuated reflection, it is to be understood that optical coupling element may support alternative detection methods. In one embodiment, optical coupling element 60 may alternatively be a surface plasmon resonance crystal. In another embodiment, optical coupling element 60 may be a diffractive optical element.

In another embodiment, an optical beam incident on optical coupling element 60 may comprise additional frequencies of light that are selected to pass through the coupling element (for example, frequencies in the UV/Vis/NIR range), whereby such additional frequencies may be useful in probing different properties of the material(s) in the channel (i.e., vibrational vs. electronic) and/or at different penetration depths due to the frequency-dependence of the evanescent field depth.

The aforementioned embodiments can be used for characterisation of adsorption of chemical and biological species adsorbed on the surface of the coupling element under flow. For example, an internal surface of optical coupling element 60, namely the surface in fluidic contact with liquid contained within channel 25, may further comprise an activated or functionalized surface layer. For example, the internal surface may be activated with a species or

functionalized with a thin layer of material to control the hydrophobic or

hydrophilic nature of the surface. In another non-limiting embodiment, the internal surface may be functionalized with a receptor such as, but not limited to, an antibody, nucleic acid probe, or aptamer.

The following examples are presented to enable those skilled in the art to understand and to practice the present invention. They should not be considered as a limitation on the scope of the invention, but merely as being illustrative and representative thereof.

EXAMPLES

Example 1 : Microfluidic System with Integrated ATR Probe

An exemplary yet non-limiting embodiment is shown in Figures 3 and 4, which provide a schematic and a photograph (top view), respectively, of a microfluidic device 200 with a built-in ATR-probe. Referring to Figure 3, the device consists of inlets 205 and 210 for the supply of a one-phase liquid medium, or multiple miscible or immiscible liquid phases, a mixing and/or reaction compartment 215, the ATR probe zone 220, and outlet 240. Exemplary designs of the inlets include a T-junction (shown at 225) or a flow-focusing geometry, as shown in Figures 3 and 4, respectively, so that droplets or bubbles can be introduced in the microfluidic device. The microchannel 250 in the ATR zone 220 includes a serpentine geometry in order to maximise the area of interface between the crystal and the liquid phase (approximately 45% surface coverage of the crystal versus 15% for a straight channel).

The entire microfluidic device 200 may be fabricated by an imprinting process in which the fluidic features are imprinted in a cycloolefin polymer (COP) sheet by hot embossing using imprint templates fabricated from photoresist on a metal base as described in co-pending Patent Cooperation Treaty Application No. PCT/CA2010/000144, titled "Method of Producing a Stamp for Hot

Embossing", filed February 3, 2010, the entire contents of which are incorporated herein by reference, and the publication J. Greener, J., W. Li Ren, D. Voicu, V. Pakharenko, T. Tang and E. Kumacheva, Lab Chip, 2010, 10, 522-524, the entire contents of which are incorporated herein by reference.

Figure 5 shows a schematic (not to scale) of the ATR region including microfluidic channel 300, which is formed within the device substrate 305, and passing over the ATR crystal 315. Note that for simplicity, only a single channel is shown and additional channels within the serpentine geometry are not shown. A planar (non-patterned) sealing sheet 320 encloses the microchannel everywhere, except at a circular hole forming an aperture having a diameter equal to that of the ATR crystal (which was 1 .8 mm in the present example). The patterned COP sheet was sealed with either a non-patterned COP sealing sheet using low-temperature thermal bonding or with an adhesive film (HDCIear, Henkel Corp.). The ATR crystal was aligned with the bottom wall of the microchannel, thereby sealing the bottom-side of the microfluidic device. A thin compressible rubbery mat 325 (thickness 100 mm), located between the sealed device and the ATR base-plate 330 assembly, was optionally used to conform to the space between the sealing layer and the ATR base-plate.

The ATR crystal 315 was clamped to the device, sealing the channel 300, by a ring clamp 335 containing an internal thread matching an external thread of the ATR base plate 330. The clamp enabled the application of sufficient pressure to the ATR crystal to provide a robust seal, allowing for high flow rates

(exceeding 20 ml/h) within the microfluidic channel, as discussed further below. By selecting the thickness of the sealing sheet 320 to be less than the protrusion distance of the ATR crystal from the base plate, and by including the

compressible layer 325, the base plate was supported under pressure by the compression of the compressible layer between the base plate and the sealing sheet.

As noted above, alternative clamping mechanisms may be used to apply pressure between the ATR crystal and the microfluidic device. For example, an external lever may be employed, or holes may be introduced through the microfluidic device for vacuum clamping to secure the base plate (holes may further extend through the base plate for the application of a vacuum clamping force by an external member). Alternatively, magnetic clamping may be incorporated by integrating electromagnetic coils into the microfluidic device that couple with magnetic materials or electromagnetic coils on or near the base plate. In another example, a threaded connector could be integrated into the microfluidic device, into which the ATR base plate could be threaded. Those skilled in the art will readily appreciate that a wide variety of clamping

mechanisms may be used within the scope of the invention.

ATR-FTIR measurements were conducted using a single reflection Ge

ATR crystal accessory (PIKE Tech.) attached to Vertex 70 FTIR spectrometer (Bruker Inc.). Opus 6.5 software was used for data acquisition, manipulation and analysis. Following thermo-embossing, the imprinted polymer sheets were sealed by low-temperature bonding or with an adhesive film. In the case of sealing via adhesive film, it was ensured that the exposed adhesive coating in the channel did not leach into the solution phase during the timeframe of the experiments (ca. 10 minutes) by monitoring the ATR-FTIR spectra acquired from the flowing solution in the microchannels. Figure 6(a) plots an ATR-FTIR spectrum acquired from the adhesive side of the adhesive film, and (b) a spectrum acquired from TX-100 solution

mM) flowing through MF device (0=3.0 mL/h) sealed with the adhesive film. The spectrum in (b) was acquired 10 minutes after flow was established through the device. Both spectra (a) and (b) were collected using 16 scans at 10 kHz and 4 cm^"1 spectral resolution. The strongest and most isolated absorbance peak (1730 cm^"1) was used to determine the presence of dislodged or dissolved adhesive. No signal from the adhesive could be detected in the TX-100 solutions acquired for this work. Figure 7 is a typical spectrum of a TX-100 solution flowing through the MF device which was sealed with an adhesive film.

As exemplary solutes, two polymers PEG and PNIPAm were used. Both the polymers have important biomedical applications. To test the applicability of the method to the characterisation of amphiphilic molecules, a solution of a non- ionic surfactant Triton X-100 (TX-100) was also used. Aqueous solutions were introduced into the microfluidic device, which was interfaced with the ATR crystal. The volumetric flow rate (Q) of solutions was in the range 0 < Q < 20 ml_/h, corresponding to their linear velocity in the range from 0 to 28 cm/s. The liquids were supplied to the microfluidic device using a syringe pump (PHD 2000, Harvard Apparatus). All experiments were conducted at room temperature, that is, below the lower critical solution temperature of PNIPAm.

The IR spectra were collected using water as a background. Absorbance was calculated as A(v) = -log(l_s(v)/l_b(v)), where l_s(v) and l_b(v) are the intensities of the single beam FTIR spectra at the wavenumber v for the solution and the pure water, respectively. Figure 7(a) shows typical spectra acquired for PNIPAm (top) and PEG (bottom) in the spectral range 1700-900 cm^"1. A strong absorption peak of water at 1640 cm^"1 (corresponding to the OH bend), is not seen because water was used as a background. Each polymer solution had distinct vibrational bands marked with arrows in Figure 7(a): CO (1625 cm^"1) and NH (1560 cm^"1) for PNIPAm and COC (1085 cm^"1) for PEG.

Figure 7(b) shows the variation in intensity of the COC absorption peak (for PEG) and the NH absorption peak (for PNIPAm) for solutions with varying polymer concentrations, C_p0i. For each polymer solution, the variation in absorbance was linear down to the lowest C_p0i = 0.01 wt%, corresponding to 5 and 10 mM for PNIPAm and PEG, respectively. The graphs presented in Figure 7(b) can be used as calibration graphs, so that the concentration of PEG or PNIPAm is determined from measured absorbance values. Second, molar extinction coefficients can be determined by replacing A/c in eqn (1 ) with the slopes of the calibration graph in Figure 7(b) (with concentration units changed to mM). By setting / equal to the penetration depth at the wavelength at which the absorption peak is located (that is, 0.43 and 0.62 mm for the NH modes in PNIPAm and COC modes in PEG, respectively), extinction coefficients of PNIPAm and PEG were determined to be spNiPAm = 1 1.9x10⁴ M^"1cm^"1 and SPEG = 5.3x10⁴ M^"1cm^"1, respectively.

Next, the applicability of the ATR-FTIR method to the mixtures of polymer solutions was examined. The concentrations of polymers in the mixture were controlled by varying the relative flow rates of individual polymer solutions as

CpEGjn - CpEG ^X QpEG ' ' QT (^³) C PNIPAm . m ~ ^ PNIPA ^ QpNlPA .m ' ' QT (2b) where CpEG.m and Cp iPAm,m are the concentrations of PEG and PNIPAm, respectively, in the mixed solution; CPEG and Cp iPAm are the concentrations of PEG and PNIPAm, respectively, in the individual solutions supplied to the corresponding inlets, CPEG = 4.14 mM, CpNiPAm = 1 -96 mM; Q_PEG and ΟΡ ΙΡΑΓΠ are the flow rates of the PEG and PNIPAm solutions, respectively, and QT is the total flow rate of the system, Q_T = 3 mL/h. Thus with increasing the flow rate of the PNIPAm solution, the concentration of this polymer in the mixed solution increased and the concentration of PEG in the mixture decreased.

The present study focused on two spectral regions, namely, 1700-1500 cm¹ and 1200-1000 cm¹ , in which the dominant peaks of the polymers (at 1560 cm^"1 and 1085 cm^"1) do not overlap. Figure 8(a) shows IR spectra acquired for the mixed polymer solutions with varying concentrations of PNIPAm and PEG. Two monitored peaks are indicated with arrows. Spectra 1→ 7 show, as expected, that progressive growth in the intensity of absorption of PNIPAm was accompanied by the reduction in absorbance of PEG. Figure 8(b) shows the variation in absorbances of PNIPAm and PEG (corresponding to the spectra in Figure 8(a)), with the polymer concentrations in the mixture, C_PEG,m and C_PNiPAm,m- For each polymer, the relationship between absorbance and polymer concentration remained linear. Furthermore, for each polymer in the mixed solution, the slopes of the variation of absorbance vs.

concentration were identical to those measured for solutions of individual polymers (shown in Figure 7(b)). The molar extinction coefficients of the polymers in the mixture were also identical to those calculated for polymers in the individual solutions. Thus, it was concluded that the calibration graphs plotted for PEG and PNIPAm in Figure 7(a) could be used to determine the concentration of these polymers in their mixed aqueous solutions. It is noted that the ability to analyse polymer mixtures benefited from the existence of distinct absorption peaks of PEG and PNIPAm (labeled with arrows in Figure 7(a)), however, when absorption bands overlap, deconvolution techniques can be used to determine the spectral contributions of individual components.

The effect of the flow rates, Q_PEG and ΟΡ ΙΡΑ_ΓΠ, on the measurements of absorbance of the individual solutions of PEG and PNIPAm were examined. Each solution was introduced in the microfluidic device at a flow rate varying from 0 to 1 0 mlJh. Figure 9 shows that in this range of flow rates of the liquids, no change in absorbance of the polymers was recorded within the sensitivity of the technique. This result has important implications for the applicability of this technique to studies of reaction kinetics. In such experiments, reaction time t can be modulated by varying the flow rates of the liquids as: t = D x A/Q (3)

where D and A are the length and cross-sectional area, respectively, of microchannel in which the reaction takes place and Q (mL/s) is the volumetric flow rate of the liquid.

Since the ATR-FTIR is a technique inherently sensitive to the adsorption of solutes to the surface of the ATR crystal, the applicability of the described method to the differentiation of adsorbed and dissolved molecules was examined. This was achieved by monitoring the concentration-dependent absorbance of the aqueous solution of a non-ionic surfactant Triton X-100 (TX- 100).

Figure 10(a) shows the absorption spectrum in the region of 1400-900 cm^"1. The peaks at 1 100 cm^"1 and 1250 cm^"1 correspond to the anti-symmetric and symmetric vibrational modes of the COC groups, respectively and the peak at 950 cm^"1 is from the C=C bonds in its phenyl ring. Figure 10(b) focuses on the dominant band at 1 100 cm^"1 and plots the variation of its absorbance vs.

concentration of TX-100 (CTX-100) in the solution. For CTX-100 > 5 mM the absorbance of this band linearly increased with increasing solute concentration (Figure 10(b)). Using the slope of this graph, one estimates the molar extinction coefficient for the 1 100 cm^"1 band to be ε_Τχ-ι οο = 1 .6 x 10³ M^"1 cm^"1.

Concentration-dependent variation in absorbance in the lower concentration range, that is, for 0.16mM < C_Tx-i oo≤ 10mM, is given in the inset in Figure 7(b). The deviation from linearity at C_TX-_{I OO} < 5 mM, well above the critical micelle concentration for TX-100 (CMC_Tx-ioo = 0.2 mM), was presumably the result of the contribution of the adsorbed layer of TX-100 at the ATR crystal-solution interface. The presence of the TX-100 layer was supported by the existence of a weak peak at 1 100 cm^"1 after the crystal was washed with pure water and dried.

It is noted that within the sensitivity of the technique, no such residual absorption peaks were observed for PNIPAm or PEG after washing the ATR crystal, despite their significantly larger molar extinction coefficients. Whereas the deviation from linearity for absorbance of surface active molecules in their dilute solutions needs further investigation, without wanted to be limited by theory, one can speculate that this effect can be used to conduct studies of adsorption of amphiphilic molecules in flow.

Example 2: Monitoring of In-Situ Polymerization In this example, embodiments of the present disclosure are adapted for the in-situ (on-chip) monitoring of the kinetics of polymerization reaction, where the optical monitoring was performed using infrared (IR) spectroscopy. The present study involved an exemplary polymerization reaction of /V-isopropylamide in water, which was initiated by ammonium persulfate (APS) and Ν,Ν,Ν',Ν'- tetramethylethylenediamine (TEMED) to form poly-NIPAm. The results presented below demonstrate the ability to rapidly examine the effect of the concentration of APS, TEMED and NIPAm and the effect of pH of the solution on the kinetics of polymerization. As shown in Figure 1 1 , in which a molecular diagram is provided for (a) the monomer NIPAm and (b) the repeat unit of the polymer poly-NIPAm, polymerization results in the loss of C=C double bonds.

Figure 12 show schematic of the microfluidic device 400 that incorporates a mixing region 410, reaction region 420 and detection region 430. Inlets 435- 450 carry reagents APS, TEMED, NIPAm and water (for dilutions). Reaction time was controlled by modulating the total volumetric flow rate of the reagent streams while keeping their flow rate ratios constant. ATR-FTIR spectra that were acquired in detection region 430 after different times on chip are show in Figure 13(a).

Based on the observed rate of chain polymerization, the following relationship is derived:

ln([M]/[M₀]) = -k_p- (f ^■ ki/k_t)^½■ [R]^½■ t (2) where [M₀] is the initial concentration of the NIPAm, [M] and [R] are the concentrations of the monomer and radical species at time t, respectively; k_p, k,, k_t are the rate constants governing polymerization, radical initiation and radical termination, respectively; and f is the yield of the initiation step. The applicability of chain polymerization model to the chemical initiation of APS by TEMED was verified by showing that the plot of ln([M]/M₀) vs. time is linear, as shown in Figure 13(b).

Example 3: On-line Characterization of Reaction of C0₂ with Water

In another example, the reaction of carbon dioxide (C0₂) with water was studied using an apparatus according to the preceding embodiments. Dissolved C0₂ can react with water to form products including bi-carbonate (HC0₃ ^~), carbonate (C0₃ ^2~), carbonic acid (H₂C0₃). The present example demonstrates the ability to differentiate between these states using an integrated ATR-FTIR microfluidic apparatus. C0₂ was introduced onto a microfluidic device (similar to that shown in Figure 12) through one inlet at a pressure of 15 PSI and water (pH 8) in another inlet at flow rate Q = 14 mL/hr. Bubbles were formed at the point of mixing.

During the time between mixing and characterization using ATR-FTIR, some of the C0₂ (g) dissolved into the aqueous phase.⁸ Once dissolved, some CO₂ becomes hydrated. At pH values between 6-10, the hydrated CO₂ is primarily in the form of HCO₃ ^". The remaining CO₂ (g) bubbles float at the top of the channel, whereas the water phase passes along the surface of the ATR crystal in the detection region.

Figure 14(a) shows the bands associated with aqueous CO₂ (2343 cm^"1) and the distinctive asymmetric bicarbonate band (1365 cm^"1). In this experiment the magnitude of the absorbance peaks and published values of extinction coefficients²² were employed to obtain quantitative concentration measurements of [CO₂] (aq) = 71 mM and [HCO₃ ] (aq) = 315 mM.

In a subsequent experiment, CO₂ (g) was introduced into the microfluidic device as described above, but flowed with water containing NaOH, resulting in a pH of 12. At this pH only carbonate (CO₃ ² ) exists in solution. Accordingly, Figure 14(b) shows only the symmetric absorption band for carbonate at 1380 cm^"1.

Using the same process outlined above, quantitative measurements of [CO3² ] were determined and it was found that [CO₃ ² ] = 5.6 mM. Upon

decreasing the flow rate of the aqueous phase to Q = 7 mlJhr, CO₂ was introduced at a relative higher concentration and the pH of the liquid decreased to 8.5 (due to the increased formation of carbonic acid), resulting the carbonate species converting to C0₂ (aq) and bicarbonate (Figure 14(c)). The

concentrations were determined to be [C0₂] = 2.8 mM and [HC0₃ ^~] = 7.6 mM. The approximate doubling in measured concentrations of species in Figure 14(b) vs. that of Figure 14(c) was interpreted to be the result of the higher

concentration of C0₂ that was introduced to the chip.

The foregoing description of the preferred embodiments of the invention has been presented to illustrate the principles of the invention and not to limit the invention to the particular embodiment illustrated. It is intended that the scope of the invention be defined by all of the embodiments encompassed within the following claims and their equivalents.

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Claims

THEREFORE WHAT IS CLAIMED IS:

1 . A microfluidic apparatus comprising:

a substrate having formed therein a microfluidic channel;

an inlet and outlet for flowing a fluid in said channel;

an aperture formed within a portion of said substrate layer exposing a portion of said channel; and

an optical coupling element adhered within said aperture, said optical coupling element comprising a planar coupling surface, wherein said planar coupling surface contacts said substrate and seals said channel within said aperture.

2. The apparatus according to claim 1 wherein said optical coupling element is an evanescent coupling element.

3. The apparatus according to claim 1 or 2 wherein said optical coupling element comprises an attenuated total reflection element.

4. The apparatus according to claim 1 or 2wherein said optical coupling element comprises a surface plasmon resonance element.

5. The apparatus according to claim 1 or 2 wherein said optical coupling element comprises a diffractive optic.

6. The apparatus according to any one of claims 1 to 5 wherein said substrate further comprises sealing layer, said sealing layer forming an upper surface of said microfluidic channel, wherein said aperture is formed in said sealing layer.

7. The apparatus according to any one of claims 1 to 6 wherein said planar coupling surface is functionalized with receptors.

8. The apparatus according to any one of claims 1 to 7 wherein said planar coupling surface comprises one of a hydrophobic surface and a hydrophilic surface.

9. The apparatus according to any one of claims 1 to 8 wherein a refractive index of said substrate is selected to reduce an amount of reflection of a light beam incident on said substrate, wherein said reflection is produced at an interface of said optical coupling element and said substrate.

10. The apparatus according to any one of claims 1 to 9 wherein a refractive index of said substrate is selected to reduce an interaction of a light beam with said substrate when said light beam is incident on said substrate from said optical coupling element.

1 1 . The apparatus according to any one of claims 1 to 10 wherein said optical coupling element is permanently adhered within said aperture.

12. The apparatus according to any one of claims 1 to 10 wherein said optical coupling element is removably clamped to said aperture.

13. The apparatus according to any one of claims 1 to 12 further comprising a clamping means for adhering said optical coupling element to said aperture.

14. The apparatus according to claim 13 wherein said clamping means is configured to provide a force that counteracts a force applied to said planar coupling surface by a pressure within said channel.

15. The apparatus according to any one of claims 1 to 14 wherein said optical coupling element is adhered to said aperture such that a flow rate within said channel may exceed 20 ml/h.

16. The apparatus according to any one of claims 1 to 15 wherein said optical coupling element protrudes from a base plate, and wherein said optical coupling element is adhered to said aperture by a force applied through said base plate, wherein a depth of said aperture is selected to contact said base plate with an external surface of said substrate under application of said force.

17. The apparatus according to claim 16 wherein said depth of said aperture substantially equals a distance over which said optical coupling element protrudes from said base plate.

18. The apparatus according to claim 16 wherein said depth of said aperture is than a distance over which said optical coupling element protrudes from said base plate, and wherein said base plate contacts an external surface of said substrate indirectly through a compressible layer provided between said base plate and said substrate.

19. The apparatus according to claim 13 wherein said optical coupling element protrudes from a base plate.

20. The apparatus according to claim 19 wherein said clamping means comprises a ring clamp, and wherein said ring clamp applies a force between said base plate and a back side of said substrate.

21 . The apparatus according to claim 19 wherein said clamping means comprises a vacuum clamp, wherein said base plate is held against an external surface of said substrate by a pressure gradient applied between holes extending through substrate and said base plate.

22. The apparatus according to claim 19 wherein said clamping means comprises a magnetic clamp for clamping said base plate to said substrate.

23. The apparatus according to any one of claims 1 to 22 wherein said channel is directed through one or more bends beneath said aperture.

24. The apparatus according to any one of claims 1 to 23 wherein said channel traverses beneath said aperture in two or more locations.

25. The apparatus according to any one of claims 1 to 24 wherein said channel contacts said planar coupling surface over at least 40% of an area of said planar coupling surface.

26. The apparatus according to any one of claims 1 to 25 wherein said planar coupling surface is coated with a material having an index of refraction that is different from a refractive index of said optical coupling element for controlling a refractive index contrast.

27. A system for measuring an optical signal from a microfluidic channel, said system comprising:

a microfluidic apparatus according to any one of claims 1 to 26;

an optical source for providing an incident optical beam, wherein said incident optical beam is directed onto said optical coupling element; and

an optical detector for detecting a reflected beam reflected through said optical coupling element.

28. The system according to claim 27 further comprising a control unit for processing signals detected by said optical detector.

29. The system according to claim 27 or 28 further comprising spectral device configured to spectrally resolve said reflected beam.

30. The system according to any one of claims 27 to 29 further comprising a Fourier-transform infrared spectrometer.

31 . The system according to any one of claims 27 to 30 wherein said detector is a non-imaging detector.

32. A method of measuring attenuated total reflection from a fluid within a microfluidic device, said method comprising the steps of:

providing a microfluidic device according to any one of claims 1 to 26; directing an incident optical beam onto said optical coupling element such said incident optical beam is totally internally reflected and attenuated through evanescent coupling at an interface between said optical coupling element and said fluid;

directing a reflected optical beam onto a detector and measuring a signal; and determining said attenuated total reflection by relating said signal to predetermined calibration value.

33. The method according to claim 32 wherein said pre-determined calibration value comprises a signal obtained when said incident optical beam is directed onto said detector in absence of said microfluidic device.

34. The method according to claim 32 wherein said pre-determined calibration values comprises a signal obtained from a reference material within said channel.

35. The method according to claim 32 further comprising the step of applying a clamping force to adhere said optical coupling element within said aperture.

36. The method according to claim 35 further comprising the step of applying said clamping force with sufficient force to counteract a pressure within said channel.

37. The method according to any one of claims 32 to 36 further comprising the step of measuring an attenuated total reflection spectrum.

38. The method according to any one of claims 32 to 37 further comprising the step of varying one of a polarization of said incident optical beam, angle of incidence of said incident optical beam upon said optical coupling element, and a combination thereof.

39. A method of measuring total internal reflection fluorescence from a fluid within a microfluidic device, said fluid comprising a fluorescent species, said method comprising the steps of:

providing a microfluidic device according to any one of claims 1 to 26; providing an incident optical beam having a wavelength selected to excite said fluorescent species;

directing said optical beam onto said optical coupling element such said incident optical beam excites said fluorescent species; and

directing fluorescence emitted from said fluorescent species with a detector.