Patent Application
20190293561
The present invention relates to an apparatus and a method for optically exciting fluorescence and detecting fluorescence.
Fluorescence sensing is increasingly being used in wide variety of applications including environmental monitoring and clinical diagnostics.
Fluorescence sensing is particularly useful in biological applications in which it is generally desirable to inspect a sample without destroying or damaging it. Although proteins, antibodies, DNA molecules and other forms of biological substances are generally not naturally fluorescent, fluorescence sensing can still be used. For example, a sample may be labelled with a fluorescent molecule, such as a fluorophore.
It is desirable to maximise absorption of excitation light by a fluorescent substance. This can help not only increase the number of photons emitted by the fluorescent substance, but also decrease the amount of unabsorbed excitation light thereby increasing the signal-to-noise ratio.
One way of helping to increase absorption of excitation light is to employ a waveguide. An optical mode guided within a waveguide may generate an evanescent field within a sensing layer in proximity to the waveguide. An overlap of the evanescent field with a fluorescent probe disposed within the sensing layer or on the surface of the waveguide may cause excitation of the fluorescent probe, followed by emission of a photon.
Examples of waveguide-based fluorescence sensors are described in US 2006/0147147 A1 and in R. Badugu et al.: “Fluorescence Spectroscopy with Metal-Dielectric Waveguides”, Journal of Physical Chemistry C, volume 119, pages 16245 to 16255 (2015) which describes a metal-dielectric waveguide structure which includes a thin metal film coated with a dielectric layer.
Such devices tend to use external coherent light sources to provide light under tightly-controlled conditions, such as at a specific angle of incidence. Moreover, if more than one type of fluorophore is employed, each having a different excitation wavelength, then multiple light sources may be required, each requiring precise alignment.
According to a first aspect of the present invention there is provided apparatus comprising a device for optically exciting fluorescence. The device comprises a transparent substrate having first and second opposite faces and a multilayer stack disposed on the second face of the substrate. The multilayer stack comprises a first layer having first and second opposite faces and a first refractive index and a second layer having first and second opposite faces and a second refractive index. The first face of the first layer is disposed on the second face of the substrate and the first face of the second layer is disposed on the second face of the first layer such that the first layer is interposed between the second layer and the substrate. The substrate has a third refractive index and the first refractive index is less than the second refractive index and the third refractive index. A light source is carried by the first face of the substrate and arranged to emit light towards the first face of the first layer. The apparatus comprises a detector directed at the substrate, the substrate interposed between the multilayer stack and the detector for detecting fluorescence.
Thus, light emitted by a light source can be coupled into a waveguide mode without the need for precise alignment of the light source. The light source is integrated into the device and so the device is compact. By choosing a light source with an appropriate emission spectrum, more than one type of fluorophore may be excited by the same light source.
According to a second aspect of the present invention, there is provided a device for optically exciting fluorescence. The device comprises a transparent substrate having first and second opposite faces and a multilayer stack disposed on the second face of the substrate. The multilayer stack comprises a first layer having first and second opposite faces and a first refractive index and a second layer having first and second opposite faces and a second refractive index. The first face of the first layer is disposed on the second face of the substrate and the first face of the second layer is disposed on the second face of the first layer such that the first layer is interposed between the second layer and the substrate. The substrate has a third refractive index and the first refractive index is less than the second refractive index and the third refractive index. A light source is carried by the first face of the substrate and arranged to emit light towards the first face of the first layer.
The light source may be disposed on the substrate. Alternatively, the substrate may comprise a first substrate and the light source may be disposed on a second substrate, and the second substrate may be bonded to the first substrate.
The light source may comprise a layer structure which includes a light-emitting layer. The light-emitting layer may comprise a layer of organic material. The organic material may comprise a polymer.
The light source may have a light emitting area which is rectangular.
The light source may be configured so as to emit light anisotropically into the substrate. The light source may be configured such that the intensity of light emitted within an angular range centred at a first angle between a central axis or plane which is perpendicular to an interface between the substrate and light source is different to the intensity of light emitted within the same angular range centred at a second, different angle between the central axis or plane and light source.
The device may comprise at least two light sources. The device may comprise an array of light sources.
The second refractive index may be equal to or greater than the third refractive index.
The first layer may comprise a dielectric material. The first layer may comprise a metal. If the first layer comprises a metal, the refractive index of the first layer consists of the real part of the complex refractive index of the first layer.
The second layer may have a thickness such that a single mode is supported, for example, the TEo mode. The single mode may be a waveguide mode. The single mode may be a surface plasmon mode supported at an interface between the first layer and the second layer. The single mode may be a fundamental surface plasmon mode.
The second layer may have a thickness such that at least two modes are supported. The at least two modes may comprise at least one waveguide mode and at least one surface plasmon mode. The at least two modes may comprise at least two waveguide modes, for example, the TEo mode and the TMo mode. The at least two modes may comprise at least two surface plasmon modes, for example, a fundamental mode and a higher-order mode.
The second layer may comprise a dielectric material.
The light emitted from the light source may comprise a first portion emitted within an angular range about a central axis or plane, and second portion emitted outside the angular range. The device may further comprise a light stop arranged to block the first portion of the light. The device may comprise a further light stop arranged to block a sub-range of the second portion of light.
The light stop/s may be embedded in the substrate. The light stop/s may be disposed between the substrate and the first layer.
The device may comprise at least one region of fluorescent material carried by the second face of the second layer. The device may comprise a layer of receptors for binding to a specific analyte carried by the second face. The receptors may comprise a fluorescent material.
At least a portion of the multilayer stack may be disposed in a ridge.
The second face of the second layer may have a patterned surface including at least one feature. The patterned surface may comprise a periodic feature. The patterned surface may comprise at least one ridge. The patterned surface may comprise at least one step.
The feature may have a lateral characteristic dimension, for example, the width of a step or the period of a grating, of between 1 μm and 10 mm.
The feature may have a vertical characteristic dimension, for example, the height of a step or ridge, of between 1 nm and 300 nm.
The device may comprise a circuit carried by the substrate which is in communication with the light source. The circuit may include a monolithic integrated circuit. The circuit may include a circuit portion comprising solution-processable transistors.
The detector may comprise a layer of light-sensitive organic material. The detector may comprise an annular light-sensitive region which is concentric with an optical axis or a parallel pair of light-sensitive regions having a mid-line which is collinear with an optical plane.
According to a third aspect of the present invention there is provided a lab-on-a-chip device comprising apparatus according to the first aspect of the present invention and a fluidic circuit including a port for providing a sample in fluid communication with a channel, wherein at least a portion of the channel is arranged so as to present the sample to the second face of the second layer or to a region over the second face of the second layer.
The lab-on-a-chip device may comprise control apparatus configured to cause the light source to emit light and to process a signal received from the detector.
The lab-on-a-chip device may be portable. The lab-on-a-chip device may be adapted to be hand-holdable. The lab-on-a-chip device may be adapted to be implantable, for example, in vivo.
According to a fourth aspect of the present invention, there is provided a method of operating apparatus according to the first aspect of the presentation or a lab-on-a-chip device according to the third aspect of the present invention, the method comprising causing a sample to be presented to the second face of the second layer, and causing the light source to emit light.
The method may further comprise receiving an input signal from the detector, processing the input signal to identify a characteristic feature of the input signal and, in response to identifying the characteristic feature of the input signal, outputting an indictor signal.
The method may further comprise receiving the input signal or a series of input signals from the detector over a given period and processing the input signal or series of input signals so as to identify time-dependent changes in the input signal or series of input signals.
Certain embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which:
In the following, like parts are denoted by like reference numerals.
Referring to
The fluorescence-exciting device 2 (herein also referred to as an “optical platform”) includes a light source 6, in the form of an organic light-emitting diode, a transparent substrate 7 having first and second opposite faces 22, 23 and a waveguide 8 having first and second surfaces 9, 10. The light source 6 is attached to the substrate 7 and is arranged to emit excitation light 11 (or “excitation radiation”) at a predetermined wavelength, λexc, into the substrate 7. The excitation light 11 passes through the substrate 7 and into the waveguide 8. The excitation light 11 is incident at the first surface 9 of the waveguide 8 and may couple into a guided mode 12 of the waveguide 8. The guided mode 12 generates an evanescent field 13 which extends from the second surface 10 and into a sample 3 which is placed on or near to (for example, within 300 nm) the second surface 10 of the waveguide 8.
The sample 3 contains, is in direct contact with or is proximate to at least one region 14 of a fluorescent material (or “fluorophore”) having a characteristic absorption wavelength, λab, and emission wavelength, λem. A fluorescent region 14 may take the form of, among other things, a molecule, particle or layer (or “film”). If the evanescent field 13 overlaps with the fluorescent region 14, the fluorescent region 14 absorbs the excitation light 11 and re-emits fluorescence emission 15. Fluorescence emission 15 may be coupled into the waveguide 8 and subsequently into the substrate 7.
As will be explained in more detail later, the fluorescent region 14 may form part of the fluorescence-exciting device 2. In particular, the fluorescent region 14 may be provided on the second surface 10 of the waveguide 8.
The sample 3 may be a liquid, a solid or a gas, or a mixture, such as a suspension, gel or aerosol. As will be explained in more detail later, the sample 3 may be taken from a biological system, such as animal or plant, chemical system or other form of system such as an environmental system. The sample 3 may be unprocessed, for example fresh whole blood or water sample taken from a river or reservoir, or processed, for example, filtered fresh whole blood or filtered water. The fluorescent material may take the form of an organic material, such as a protein, DNA or other organic molecule, or an inorganic material, such as an inorganic semiconductor.
The (or each) detector 4 may take the form of a photodiode and is disposed such that the fluorescence-exciting device 2 is interposed between the sample 3 and the detector 4 and is directed at first face 22 of the substrate 7 so as to collect fluorescence emission 15.
Referring also to
The sensing system 1 may be portable (for example, handheld) and/or be remotely-locatable (for example, in a process plant or in the field).
Referring to
The substrate 7 is generally layer-like and a refractive index, ns. The substrate 7 comprises a substantially optically transparent material, such as polyethylene terephthalate (PET) or polyethylene naphthalate (PEN).
The substrate 7 is preferably flexible, for example, capable of being reversibly bent through an angle of 90° or more. The substrate 7 has a thickness, tsub, which may be, for example, less 1 mm or less than 0.5 mm.
The waveguide 8 comprises a multilayer stack 24 (herein also referred to simply as the “stack”) disposed directly on the second face 23 of the substrate 7. The multilayer stack 24 includes a first layer 25 having first and second opposite faces 26, 27 disposed directly on the second face 23 of the substrate 7 and a second layer 28 having first and second opposite surfaces 29, 30 disposed directly on the second face 27 of the first layer 25. The first face 26 of the first layer 25 and the second face 30 of the second layer provide the first and second surfaces 9, 10 (
The multilayer stack 13 may include additional layers (not shown). The multilayer stack 24 preferably is free of (that is, does not include) a plasmon-creating structure, such as a layer of metal.
The first layer 25 has a refractive index n1 which is less than the refractive index ns of the planar substrate 7, i.e. n1<ns. The second layer 28 has a refractive index n2 which is greater than the refractive index n1 of the first layer 25, i.e. n2>n1.
The first layer 25 may comprise a first substantially optically-transparent material, such as silicon dioxide (SiO2), and may have a thickness, t1, of, for example, between 50 and 500 nm. Alternatively, the first layer 25 may comprise a metal such as silver (Ag) or gold (Au) with a thickness t1 of, for example, between 10 and 50 nm. When the first layer 25 comprises a metal, references to the refractive index of the first layer 25 should be considered to be referring to the real part of the complex refractive index of the metal.
The second layer 28 comprises a second substantially optically-transparent material, such as, for example, tantalum pentoxide or titanium dioxide, and has a thickness, t2, of, for example, between 50 and 500 nm.
The fluorescence-exciting device 2 may include a layer of fluorescent material 14 disposed on the second face 30 of the second layer 28.
The fluorescence-exciting device 2 is preferably flexible, for example, capable of being reversibly bent through an angle of 90° or more.
The light source 6 takes the form of a light-emitting layer structure 31 disposed on the first face 22 of the substrate 7.
The substrate 7 may comprise first and second substrates (not shown) which are optically connected (for example, using index-matching epoxy) with the light-emitting layer structure 31 supported on the first substrate (not shown) and the multilayer stack 24 disposed on the second substrate (not shown). Thus, the multilayer stack 13 and the light-emitting layer structure 31 may be fabricated separately and combined to form a unitary substrate 7.
The light-emitting layer structure 31 comprises an organic light-emitting diode (OLED) or polymer light-emitting diode (PLED). At least a portion of the light-emitting layer structure 31 is preferably fabricated using solution-processable materials. The light-emitting layer structure 31 may comprise a light-emitting diode chip bonded to the substrate 7.
The light-emitting layer structure 31 is able to emit excitation light 11 (
Before describing propagation of light within the fluorescence-exciting device 2, propagation of light through different media will first be described.
Total Internal Reflection
Referring to
n
i sin (θi)=nf sin (θf) (o)
In the arrangement shown in
Although no travelling wave exists within the second medium 43, an evanescent wave (not shown) is generated. The evanescent wave (not shown) is a solution to the wave equation which satisfies the boundary conditions at the interface. The evanescent wave has an amplitude which decays exponentially in the direction normal to the interface.
The evanescent wave (not shown) is not a travelling wave. However, if a third medium 45 is brought into close proximity with the second medium 43, the third medium 45 having a refractive index, nt, which is greater than the refractive index, n2, of the second medium 43, then the evanescent wave (not shown) may tunnel through the second medium 43 and a travelling wave 46 may resume in the third medium 45. This is called frustrated total internal reflection and is shown in
Thus an evanescent wave in a first medium, located close to a boundary with a second medium having a greater refractive index than the first medium, can give rise to a travelling wave in the first medium.
Polarisation
Light incident at a surface may be categorised as S-polarised or P-polarised.
A plane of incidence is defined by the vector along the propagation direction of the incident light and the vector perpendicular to the plane of the surface at which the light is incident. S-polarised light has its electric field component perpendicular to the plane of incidence. P-polarised light has its electric field component parallel to the plane of incidence.
Light propagation through multilayer structures will now be described. The discussion first addresses structures in which the first layer comprises a dielectric material, before describing propagation through structures in which the first layer comprises a metal.
Light Propagation Through Multilayer Structures: Dielectric First Layer
Referring to
The third refractive index n3 is less than the second refractive index n2 and the substrate refractive index, i.e. n3<n2, ns. In this example, the second refractive index n2 is equal to the substrate refractive index ns, i.e. n2=ns. However, the second refractive index n2 may be greater than the substrate refractive index ns, i.e. n2>ns. The first refractive index n1 is smaller than the second refractive index n3, i.e. n1<n2.
First, second and third interfaces 71, 72, 73 are formed between the substrate 62 and the first layer 67, between the first layer 67 and the second layer 68 and between the second layer 68 and the medium 70.
A first light ray 661 is incident at the first interface 71 at a first angle θ1 to the normal to interface 71. The first angle θ1 is smaller than the critical angle at the first interface 71. The first ray 661 is refracted at the first interface 71 and bends away from the normal in the first layer 67. At the second interface 72 between the first layer 67 and the second layer 68, the ray 661 is again refracted and bends towards the normal in the second layer 68. At the third interface 72 between the second layer 68 and the medium 70, a second angle of incidence θ2 of the first light ray 661 is smaller than the critical angle at third interface 72. The first light ray 661 is refracted and bends away from the normal. Thus, the first light ray 661 propagates through the substrate 62 and the first and second layers 67, 68 and into the medium 70.
A second light ray 662 is incident at the first interface 71 at a third angle θ3 to the normal to first interface 71. The third angle θ3 is greater than the critical angle at interface 71. The ray 662 undergoes total internal reflection at the first interface 71. A first evanescent wave 74 is generated in the first layer 67. The thickness of the first layer 67 is such that the first evanescent wave 74 tunnels through the first layer 67 and a first travelling wave 75 resumes in the second layer 68.
The angle to the normal made by the first travelling wave 75 in the second layer 68 is equal to the angle which would be made if the first layer 67 were not present and the substrate 62 were in direct contact with the second layer 68. The second refractive index n2 is equal to the substrate refractive index and so a fourth angle of incidence θ4 of the first travelling wave 75 at interface third interface 73 is equal to the third angle θ3.
The third angle θ3 is less than the critical angle at the third interface 73 and so the first travelling wave 75 does not undergo total internal reflection. The first travelling wave 75 refracts and bends away from the normal. Thus, second light ray 662 propagates through the substrate 62 and the first and second layers 67, 68 and into the medium 70.
The second light 662 follows this path when the third angle θ3 satisfies the inequality (n1/ns)<sin θ3<(n3/ns). This occurs when n1<n3, i.e. when the first refractive index n1 is smaller than the third refractive index.
A third light ray 663 is incident at the first interface 71 at a fifth angle θ5 to the normal to the first interface 71. The fifth angle θ5 is greater than the critical angle at the first interface 71. The third light ray 663 undergoes total internal reflection at the first interface 71. A second evanescent wave 76 is generated in the first layer 67. The thickness of the first layer 67 is such that the second evanescent wave 76 tunnels through the first layer 67 and a second travelling wave 77 resumes in the second layer 68.
The angle to the normal made by the second travelling wave 77 in the second layer 68 is equal to the angle which would be made if the first layer 67 were not present and the substrate 62 were in direct contact with the second layer 68. The second refractive index n2 is equal to the substrate refractive index ns and so a sixth angle of incidence θ6 of the travelling wave 77 at second interface 72 is equal to the fifth angle θ5.
When total internal reflection occurs at an interface, the angles of incidence and reflection are equal. Thus, all subsequent angles of incidence at second and third interfaces 72, 73 are equal to the sixth angle of incidence θ6.
For the second travelling wave 77, the sixth angle θ6 is greater than the critical angle at third interface 73 and the second travelling wave 77 undergoes total internal reflection. Since the first refractive index n1 is less than the third refractive index n3, the critical angle at interface second interface 72 is smaller than the critical angle at third interface 73. Therefore, the second travelling wave 77 is subsequently reflected at second interface 72. Since all subsequent angles of incidence at the second and third interfaces 72, 73 are equal to the sixth angle θ6 which is greater than the critical angles at the second and third interfaces 72, 73, the second travelling wave 77 is guided by multiple total internal reflections within the second layer 68.
The guided mode 77 is shown for only one angle of incidence, namely the fifth angle of incidence θ5. It will be appreciated, however, that other angles of incidence which excite guided modes within the second layer 68 will have points of total internal reflection at different locations on the second face 69.
Referring to
A fourth light ray 664 with a seventh angle of incidence θ7 at the first interface 71 smaller than the critical angle at the second interface 71 does not undergo total internal reflection at the second interface 71. An eighth angle of incidence θ8 of the fourth light ray 664 at the second interface 72 is smaller than the critical angle at the second interface 72. Thus, the fourth light ray 664 propagates through the substrate 62 and the first and second layers 67, 68 and into the medium 70.
A fifth light ray 665 having a ninth angle of incidence θ9 at the first interface 71′ which is smaller than the critical angle at the first interface 71′ does not undergo total internal reflection at the first interface 71′. A tenth angle of incidence θ10 of fifth light ray 665 at the third interface 73′ is greater than the critical angle at the third interface 73′ and, thus, the fifth light ray 665 is reflected at the third interface 73′.
For the fifth light ray 665, the tenth angle θ10 is less than the critical angle at the second interface 72′ and so the fifth light ray 665 does not undergo total internal reflection at the second interface 72′. Thus, the fifth light ray 665 propagates back into the substrate 62.
The fifth light 665 follows this path when the tenth angle θ10 satisfies the inequality (n3/ns)<sin θ10<(n1/ns). This occurs when n3<n1, i.e. when the third refractive index n3 is smaller than the first refractive index n1.
A sixth light ray 666 is incident at the first interface 71 at an eleventh angle θ11 to the normal to the first interface 71. The eleventh angle θ11 is greater than the critical angle at the first interface 71. The sixth light ray 666 undergoes total internal reflection at first interface 71. A third evanescent wave 78 is generated in the first layer 67. The thickness of the first layer 67 is such that the third evanescent wave 78 tunnels through the first layer 67 and a third travelling wave 79 resumes in the second layer 68.
The angle to the normal made by the third travelling wave 79 in the second layer 68 is equal to the angle which would be made if the first layer 67 were not present and the substrate 62 were in direct contact with the second layer 68. The second refractive index n2 is equal to the substrate refractive index ns and so a twelfth angle of incidence θ12 of the travelling wave 79 at the third interface 68 is equal to the eleventh angle θ11.
When total internal reflection occurs the angles of incidence and reflection are equal. Thus, all subsequent angles of incidence at second and third interfaces 71, 73 are equal to the twelfth angle θ12.
For the third travelling wave 79, the twelfth angle θ12 is greater than the critical angle at the third interface 73 and the travelling wave 79 undergoes total internal reflection. When the first refractive index n1 is greater than the third refractive index n3, the condition for total internal reflection at second interface 72 is equal to the condition for total internal reflection at first interface 71. Thus, any evanescently-coupled travelling wave which undergoes total internal reflection at third interface 73 will subsequently undergo total internal reflection at the second interface 72.
Since all subsequent angles of incidence at the second and third interfaces 72, 73 are equal to the twelfth angle θ12 which is greater than the critical angles at the second and third interfaces 72, 73, the third travelling wave 79 is guided by multiple total internal reflections within the second layer 68.
The guided mode 79 is shown for only the eleventh angle of incidence θ1. It will be appreciated that other angles of incidence which excite guided modes within the second layer 68 will have points of total internal reflection at different locations on the second face 69.
Conditions for Guided Modes in the Second Layer 68
Referring still to
ns sin θ>n3 (1)
and the condition for subsequent total internal reflection at second interface 72 is
ns sin θ>n1 (2)
For values of incidence angle θ which satisfy both inequalities, light 66 is coupled into a guided mode in the second layer 67.
Waveguide Modes
A single-mode waveguide is a waveguide which supports only one guided mode per wavelength. Typically, a single-mode waveguide has a dimension in the confining direction which is less than the wavelength of the light coupled into the waveguide.
Referring to
For a fluorescence-exciting device 2 in which the second layer 28 has a thickness greater than the thickness required to support only the fundamental TEo guided mode, more than one guided mode may exist. For example, a fundamental P-polarised mode may exist, also referred to as the TMo mode.
Light Propagation Through Multilayer Structures: Metal First Layer
A plasmon is a collective oscillation of an electron gas density. A surface plasmon is a plasmon which exists at a boundary between two media wherein the real part of the dielectric function changes sign across the boundary. The boundary tends to be a dielectric-metal interface.
An oscillating charge radiates energy. Thus a surface plasmon has an associated electromagnetic wave, and the term ‘surface plasmon polariton’ (“SPP”) denotes the combined charge oscillation and associated electromagnetic wave. The intensity of the associated electromagnetic wave decays exponentially in the direction perpendicular to the boundary, and this wave is evanescent. The surface plasmon polariton propagates along the boundary and is thus guided.
Surface plasmon polaritons can be excited by evanescent waves. Due to the momentum-matching condition for surface plasmon excitation, only P-polarised light can excite surface plasmon polaritons.
Referring to
The third refractive index n3 is less than the second refractive index n2 and the substrate refractive index, i.e. n3<n2, ns. In this example, the second refractive index n2 is equal to the substrate refractive index ns, i.e. n2=ns. However, the second refractive index n2 may be greater than the substrate refractive index ns, i.e. n2>ns. The first refractive index n1′ is smaller than the second refractive index n3, i.e. n1′<n2.
Fourth, fifth, and sixth interfaces 71′, 72′, 73′ are formed between the substrate 62 and the modified first layer 67′, between the modified first layer 67′ and the second layer 68 and between the second layer 68 and the medium 70.
A seventh light ray 667 is incident at the fourth interface 71′ at a thirteenth angle θ13 to the normal to fourth interface 71′. A first portion 6671 of the seventh light ray 667 propagates through the substrate 62, the modified first layer 67′, and the second layer 68, and into the medium 70. A second portion 6672 of the seventh light ray 667 is reflected at the fourth interface 71′. The amount of light in each portion 6671, 6672 is dependent upon the wavelength of the light and the properties of the materials which the substrate 62 and modified first layer 67′ comprise.
An eighth light ray 668 is incident at the fourth interface 71′ between the substrate 62 and the modified first layer 67′ at a fifteenth angleθ15 to the normal to the fourth interface 71′. The fifteenth angle θ15 is equal to a first resonance angle at the fourth interface 71′, wherein the first resonance angle is an angle of incidence at the fourth interface 71′ required to excite a waveguide mode in the second layer 68. The eighth light ray 668 undergoes total internal reflection at the fourth interface 71′. A fourth evanescent wave 80 is generated in the modified first layer 67′. The thickness of the modified first layer 67′ is such that the fourth evanescent wave 80 tunnels through the modified first layer 67′ and a fourth travelling wave 81 resumes in the second layer 68.
A sixteenth angle of incidence θ16 of the travelling wave 81 at second interface 73 is greater than the critical angle at interface 73′ and the critical angle at interface 72′. When total internal reflection occurs at an interface, the angles of incidence and reflection are equal. Thus, all subsequent angles of incidence at interfaces 72′, 73′ are equal to the sixteenth angle of incidence θ16. The fourth travelling wave 81 is guided by multiple total internal reflections within the second layer 68.
The guided mode 81 is shown for only one angle of incidence, namely the fifteenth angle of incidence θ15. It will be appreciated, however, that other angles of incidence which excite waveguide modes within the second layer 68 will have points of total internal reflection at different locations on the second face 69.
A ninth light ray 669 is incident at the fourth interface 71′ between the substrate 62 and the modified first layer 67′ at a seventeenth angle θ17 to the normal to the fourth interface 71′. The seventeenth angle θ17 is equal to a second resonance angle at the fourth interface 71′, wherein the second resonance angle is an angle of incidence at the fourth interface 71′ required to excite a surface plasmon polariton mode at the fifth interface 72′. The ninth light ray 669 undergoes total internal reflection at the fourth interface 71′. A fifth evanescent wave 82 is generated in the modified first layer 67′.
The fifth evanescent wave 82 excites a surface plasmon polariton mode 83 at the fifth interface 72′. The surface plasmon polariton 83 propagates along the fifth interface 72′ (that is, along the x axis). The intensity of the surface plasmon polariton 83 decays exponentially in the z and the x directions, that is, in the direction of the normal to the fifth interface 72′ and in the direction which is perpendicular to the normal and in the plane of incidence. For clarity, only the exponential decay in the x direction is shown.
The surface plasmon polariton mode 83 is described for only one angle of incidence, namely the seventeenth angle of incidence θ17. It will be appreciated, however, that there are other angles of incidence which excite surface plasmon polariton modes at the fifth interface 72.
The plot shows the reflectance of light incident at the fourth interface 71′ at various angles (horizontal axis) and with various energies (vertical axis). The reflectance is indicated by grayscale value, with darker shades (low numbers) indicating higher reflectance values and lighter shades (high numbers) indicating lower reflectance values. For example, it can be seen that incident light having energy greater than 4.5 eV (electron volts) is substantially transmitted by the multilayer device 613 at incidence angles less than approximately 65°.
The low reflectivity spots denoted by reference numeral 84 show combinations of angle of incidence and incident light energy at which excitation of fundamental S-polarised (TEo) guided modes within the second layer occurs. These guided modes are excited by coupling from an evanescent wave produced when total internal reflection occurs at the fourth interface 71′. These modes are guided by multiple total internal reflections at the fifth and sixth interfaces 72′ and 73′.
The low reflectivity spots denoted by reference numeral 85 show combinations of angle of incidence and incident light energy at which excitation of fundamental P-polarised surface plasmon (“SPo”) modes occurs.
It is seen from
Thus by choosing appropriate combination(s) of angles of incidence and wavelength(s) of incident light, more than one type of guided mode may be excited.
The multilayer device for which the dispersion plot of 7b is calculated has a second layer 68 with a thickness such that only a fundamental surface plasmon mode (in this case, the SPo mode) is supported at the interface between the first layer 67′ and the second layer 68. Other values for the thickness of the second layer 68 are possible and such values may allow higher order surface plasmon modes to be supported.
Evanescent Wave-Excited Fluorescence
Referring to
The fluorescence-exciting device 2 includes a fluorescent region 14 in the form of a fluorescent layer disposed on the second face 30 of the second layer 28. The first layer 25 comprises a dielectric material. However, the first layer 25 may comprise a metal. The second layer 28 has a thickness t2 (
The optical detector 4 is carried by the second face 22 of the substrate 7. The detector 4 is shaped in the form of a ring which is which is concentric with the central axis 32. However, in other embodiments, the detector may comprise, for example, a parallel pair of light-sensitive regions having a mid-line which is collinear with an optical plane.
In the manner hereinbefore described, the light-emitting structure 31 emits light 11 which is guided by the multilayer structure 24 having first, second and third interfaces 34, 35, 36 between the substrate 7 and the first layer 25, the first layer 25 and the second layer 28 and the second layer 28 and the fluorescent region 14 respectively.
First and second sets of light rays 111, 112 are schematically shown.
Light rays 111 emitted at an angle less than the resonant angle of incidence θres do not couple to a guided mode within the second layer 28. Light rays 112 emitted at the resonant angle of incidence θres result in evanescent waves 37 being generated within the first layer 25 which give rise to travelling waves 38 which are guided within the second layer 28.
The travelling waves 38 have points of total internal reflection at the third interface 36. At each point of total internal reflection, an evanescent wave (not shown) is generated. The evanescent wave generated by a mode in the single-mode waveguide decays such that there is substantially no intensity at a distance greater than half a wavelength of the light from the waveguide interface. The evanescent wave extends into the fluorescent layer 14 and may overlap with a fluorophore. The fluorophore may absorb a photon from the evanescent wave and subsequently emit a photon as fluorescence 15. The emitted photon may be emitted in any direction.
The detector 4 detects emitted fluorescence which propagates through the multilayer structure 24 and the substrate 7, as will be described later.
The detector 4 may comprise a layer of light-sensitive organic material. The detector 4 may comprise a spectrometer. The detector 4 may comprise a photodiode or charge-coupled device (CCD).
The absorption of a photon by a fluorophore removes energy from travelling waves 38. Thus, after each total internal reflection at the third interface 36, the intensities of travelling waves 38 are reduced. The intensity of the fluorescence 15 decreases with increasing distance away from the light-emitting layer structure 31.
Fluorescence Emitted into Waveguide
Referring to
As described previously, both near- and far-field radiation from a fluorophore in close proximity to an interface with a medium of higher refractive index may be coupled to travelling waves within the medium of higher refractive index. Near-field radiation propagates at angles greater than the critical angle at the interface; far-field radiation propagates at angles smaller than the critical angle at the interface.
A single guided mode exists within second layer 28 of device 2. Fluorescence emitted by fluorophores in proximity to the third interface 36 may couple into this guided mode. The wavelength of the emitted fluorescence is not necessarily equal to the wavelength of the light emitted by light-emitting layer structure 31. The single guided mode at the wavelength of the fluorescence may have a different resonant angle of incidence to the single guided mode at the wavelength of the light emitted by light-emitting layer structure 31.
Travelling waves 401, 402 which are coupled from evanescent waves emitted by fluorophores in close proximity to point A are shown. Travelling waves 401, 402 propagate within the second layer 28 at an angle which is greater than the critical angle at third interface 36. Travelling waves 401, 402 subsequently undergo first total internal reflections at interface 35 and evanescent waves (not shown) are generated within the first layer 25. Travelling waves 411, 412 resume in substrate 7 resulting from evanescent coupling through first layer 25, and may subsequently be incident on detector 4.
Energy is lost from travelling waves 401, 402 at each total internal reflection which results in evanescent coupling to a travelling wave in the substrate 7, and thus the intensity of travelling waves 401, 402 decreases as their propagation distance within second layer 28 from point A increases. Accordingly, the intensity of travelling waves 421, 422 is less than the intensity of travelling waves 411, 412.
The ring-shaped detector 4 provides an integrated fluorescence excitation and detection system. The system may require no alignment of the light source or light detector after manufacture. The system can be used by a person not having skills or knowledge of operation or alignment of lasers or light sources.
Reduction of Back Reflections
Referring again to
Referring to
Referring to
The modified fluorescence-exciting device 2′ is the same as the fluorescence-exciting device 2 shown in
The light stop 83 may be placed at other positions. For example, the light stop may be interposed between the substrate 7 and the multilayer stack 24. It will be appreciated that the lateral extent of the light stop is adjusted according to separation from the light-emitting layer structure 31 so as to provide appropriate angular coverage.
Metal First Layer
As explained hereinbefore, when the first layer 25 comprises a metal, guided surface plasmon polariton modes may exist at the interface 35 between the first layer 25 and the second layer 28, in addition to modes which are guided within the second layer 28 by multiple total internal reflections. These surface plasmon polariton modes include an evanescent electromagnetic wave component.
The evanescent field of the surface plasmon polariton mode can excite a fluorophore in a manner similar to the excitation of a fluorophore by the evanescent field of a waveguide mode, as described in the previous section. The evanescent, near field emission of a fluorophore can couple to a surface plasmon polariton mode in a manner similar to the excitation of a waveguide mode by the near field emission of a fluorophore, as described in the previous section. Thus the skilled person will readily appreciate that the methods and devices described in the preceding and following sections apply to devices in which the first layer 25 comprises a dielectric material and to devices in which the first layer 25 comprises a metal. In particular, any reference to a first layer 25 comprising a dielectric material or having the characteristics of a dielectric material is not to be considered to exclude devices or methods in which the first layer 25 comprises a metal.
Analyte-Specific Sensor
Referring again to
To provide specificity, an analyte-specific receptor may be provided to which the analyte may bind. The receptor may include a fluorescent label. Additionally or alternatively, the analyte may include a fluorescent label. The fluorescence 15 emitted by the fluorescent label may be modified, for example in wavelength or intensity, when the analyte binds to the receptor.
By monitoring fluorescence 15, the properties of the sample under test, the analyte and the receptor may be determined. These properties may include, but are not limited to, the presence of the analyte within the sample, the concentration of the analyte within the sample, the rate at which the analyte binds to the receptor.
Examples of analytes and suitable receptors include antibodies which may bind to antigens and immunoglobins which may bind to binding proteins.
A suitable fluorescent label may comprise a fluorophore, a quantum dot, a protein, a fluorescent dye.
Referring to
A layer of receptors 92 is disposed on the second face 30 of the second layer 28. The receptors 92 include fluorescent labels (not shown) thereby providing fluorescent regions 14 (
The fluorescence-exciting device 2′ is coupled to a device 94 for holding and presenting the sample 3 to fluorescence-exciting device 2. The device 94 is arranged to allow the sample 3 to flow continuously past the fluorescence-exciting device 2, i.e. takes the form of a flow cell. The device 94, however, may hold and present a fixed or static volume of sample 3.
The device 94 comprises a housing 95 which provides a channel having an aperture 96 which allows the sample 3 to be brought into direct contact with the second face 30 of the second layer 28. The device includes first and second ports 97, 98 in fluid communication with the channel 95 for providing inlet and outlet respectively.
An evanescent field generated by a guided mode within second layer 28 overlaps with the receptors 92. Fluorescent labels 14 (
When an analyte particle 931 passes close to a receptor 921, the analyte particle 931 may bind to the receptor 921. During and/or after binding, the fluorescence emitted by the fluorescent label of the receptor 921 is modified, for example, in wavelength and/or intensity.
Thus, monitoring of the intensity of light received by the detector 4′ can give information on, for example, the presence of an analyte 93, the concentration of analyte 93 and binding rate of analyte 93 and receptor 92.
Such information can be used to determine the concentration of a biomarker indicating a disease. For example, a concentration of myoglobin in blood greater than a predetermined value may indicate acute myocardial infarction.
As described previously, the evanescent field decays such that there is substantially no intensity at a distance greater than half a wavelength of the light from the waveguide interface. This can allow spatially selective sensing, wherein analyte particles 92 which bind to receptors 92 on second face 20 of second layer 28 may be detected without interference from contaminants in the bulk of the sample 3.
Referring to
First and second detectors 41′, 42′ are shaped in the form of rings which are concentric with the central axis 32. First and second detectors 41′, 42′ have outer diameters a1, a2 respectively, and inner diameters b1, b2 respectively.
Outer diameters a1, a2 and inner diameters b1, b2 are chosen such that light of a first wavelength which is coupled from waveguide to substrate is detected by first detector 41′ and light of a second wavelength which is coupled from waveguide to substrate is detected by second detector 42′. The first wavelength may be the wavelength of the light emitted by light-emitting layer structure 31 and the second wavelength may be the wavelength of fluorescence emitted by the fluorescent label of the receptor 921. This can allow the intensity of the light emitted by light-emitting layer structure 31 to be monitored.
The waveguide-based fluorescence sensing system 1 and sensors 91, 91′ including the waveguide-based fluorescence sensing system 1 can have one or more advantages. For example, the fluorescence-exciting device 2 can be fabricated using materials and processes which allow the device to be shaped, e.g. to fit with another structure (such as a pipe) or a body (such as a plant), and/or to be easily and quickly produced in large quantities. An integrated light source 6 can make it easier to align the light source 6 with the rest of device. The waveguide-based fluorescence sensing system 1 does not require external light sources and may be battery powered, allowing the system to be portable and/or hand held.
The sensors 91, 91′ including ring-shaped detector(s) can provide improved fluorescence collection efficiency with low levels of background from the light source 6.
A fluorescence-exciting device 2 having a light source 6 with an emission spectrum broad enough to substantially excite more than one type of fluorescent marker can allow simultaneous monitoring of different markers.
The rapid decay of the evanescent field 13 within the sample can allow selective excitation of target molecules close to the second layer without substantial scattering of excitation light by non-target molecules. This can provide an improved signal to noise ratio.
Organic Light-Emitting Diode
Referring again to
The light-emitting layer structure 31 may comprise an organic light-emitting diode (OLED).
Referring to
The OLED 64 comprises a cathode 65, an anode 67 and a light-emitting layer 66 between the cathode 65 and anode 67. The device 64 is supported on a substrate 68, for example a glass or plastic substrate.
One or more further layers may be provided between the cathode 65 and anode 67 including, without limitation, hole injection, hole transport, electron injection, electron transport, hole blocking and electron blocking layers. The hole injection layer may comprise, for example, PEDT. Further examples of materials for use in such layers are given in “Charge Carrier Transporting Molecular Materials and Their Applications in Devices”, Y Shirota and H Kageyama, Chem. Rev., 2007, 107 (4), pp 953-1010, the contents of which are incorporated herein by reference.
The device structure may be selected from:
Anode/Hole-injection layer/Light-emitting layer/Cathode
Anode/Hole transporting layer/Light-emitting layer/Cathode
Anode/Hole-injection layer/Hole-transporting layer/Light-emitting layer/Cathode
Anode/Hole-injection layer/Hole-transporting layer/Light-emitting layer/Electron-transporting layer/Cathode
Light-emitting layer 66 contains at least one light-emitting material. Light-emitting material 66 may consist of a single light-emitting compound or may be a mixture of more than one compound, optionally a host doped with one or more light-emitting dopants. Light-emitting layer 66 may contain at least one light-emitting material that emits phosphorescent light when the device is in operation, or at least one light-emitting material that emits fluorescent light when the device is in operation. Light-emitting layer 66 may contain at least one phosphorescent light-emitting material and at least one fluorescent light-emitting material. Examples of light-emitting materials are given in “Organic Light-Emitting Materials and Devices”, CRC Press, 2007, the contents of which are incorporated herein by reference.
The cathode 65 may consist of a single layer of a conductive material, optionally a layer of metal such as a layer of aluminium, or it may consist of a plurality of layers of conductive materials such as metals, optionally a bilayer of a low work function material and a high work function material such as calcium and aluminium, for example as disclosed in WO 98/10621, the contents of which are incorporated herein by reference. The cathode 65 may comprise a 1-5 nm thick layer of metal compound, optionally an oxide or fluoride of an alkali or alkali earth metal, between the organic layers of the device and one or more conductive cathode layers, optionally one or more metal layers, for example lithium fluoride as disclosed in WO 00/48258, the contents of which are incorporated herein by reference.
“Low work function” of a conductive material as described herein means a work function of less than than 3.5 eV, optionally no more than 3.2 eV, from vacuum. “High work function” of a conductive material as described herein means a work function of at least 3.5 eV, optionally at least 3.7 eV or at least 4 eV, from vacuum. Work functions of metals are as given in CRC Handbook of Chemistry and Physics, 87th Edition, 2007, p. 12-114, published by CRC Press, edited by David R. Lide.
In use, light is emitted through the anode 65 and/or cathode 67. Preferably, one of the anode and cathode is transparent and the other of the anode and cathode is opaque. Optionally, the opaque electrode is reflective.
The anode 67 may be a single layer or may consist of two or more layers. In the case where light is emitted through the anode, it is optionally a layer of indium tin oxide (ITO) or indium zinc oxide (IZO). The anode 67 may comprise a thin metal layer, for example a layer of silver (Ag) with a thickness of 20 nm. This can allow control of the direction of light emission.
Detector
The detector 4 may comprise an organic photodetector. The organic photodetector may comprise a layer structure such as that described hereinbefore.
Static Reactor
Referring to
The device 94′ comprises a housing 95′ which provides a static reactor or ‘bath’ having an aperture 96′ which allows the sample 3 to be brought into direct contact with the second face 30 of the second layer 28. The device includes a first port 97′ in fluid communication with the channel 95 for providing an inlet. The first port 97′ may also provide an outlet. At least a portion of the housing 95′ is transparent so as to allow fluorescence to reach the detector 4.
Control Circuit
Referring again to
Referring to
The circuit 120 may be bonded to the substrate 7. The circuit 120 may be formed on another substrate (not shown) mounted to the substrate 7.
Fabrication
Referring to
The substrate 7 is provided (step S1). A first layer 25 is formed on the substrate 7, for example, using a printing process, a chemical deposition process or a physical deposition process (step S2). A second layer 28 is formed on first layer 25, for example, using a printing process, a chemical deposition process or a physical deposition process (step S3). Optionally, a fluorescent layer 14 (or a layer which contains fluorescent material) may be formed, for example, by a solution-based process (step S4).
The light-emitting layer structure is provided on the substrate (step S5). It will be appreciated that the light-emitting layer structure may be provided first, before the first and second layers 25, 28 are provided. Furthermore, the light-emitting layer structure may be formed separately and then attached to the substrate 7.
Optionally, the fluorescent layer 14 (or a layer which contains fluorescent material) may be formed once the rest of the device 2 has been formed (step S6).
Referring to
The first substrate layer 71 is provided (step S11). The light stop 88 is formed on a first face of the first substrate layer 71 (step S12). The second substrate layer 72 is provided (step S13). The first face of the first substrate layer 71 is bonded to a first face of the second substrate layer 72 using, for example, index-matching epoxy, such that the light stop 88 is interposed between the first and second layers 71, 72 (step S14).
A first layer 25 is formed on a second face of the second substrate layer 72, for example, using a printing process, a chemical deposition process or a physical deposition process (step S15). The second face of the second substrate layer 72 is opposite the first face of the second substrate layer 72. A second layer 28 is formed on first layer 25, for example, using a printing process, a chemical deposition process or a physical deposition process (step S16). Optionally, a fluorescent layer 14 (or a layer which contains fluorescent material) may be formed, for example, by a solution-based process (step S17).
The light-emitting layer structure is provided on the second face of the first substrate layer 71 (step S18). The second face of the first substrate layer 71 is opposite to the first face of the first substrate layer 71. It will be appreciated that the light-emitting layer structure may be provided first, before the first and second layers 25, 28 are provided. Furthermore, the light-emitting layer structure may be formed separately and then attached to the first substrate layer 71.
Optionally, the fluorescent layer 14 (or a layer which contains fluorescent material) may be formed once the rest of the device 2′ has been formed (step S19).
The detector 4 is provided on the second face of the first substrate layer 71 (step S20). The device 94 for holding and presenting the sample to fluorescence-exciting device 2′ is provided (step S21). The device 94 is attached to the device 2′ (step S22).
Alternatively, the detector 4 may be formed separately and then attached to the substrate 7.
Modifications
It will be appreciated that various modifications may be made to the embodiments hereinbefore described. Such modifications may involve equivalent and other features which are already known in the design, manufacture and use of waveguides, detectors and/or light-emitting diodes and which may be used instead of or in addition to features already described herein. Features of one embodiment may be replaced or supplemented by features of another embodiment.
For example, the device may be immersed in the sample and the receptacle may not be required.
Although claims have been formulated in this application to particular combinations of features, it should be understood that the scope of the disclosure of the present invention also includes any novel features or any novel combination of features disclosed herein either explicitly or implicitly or any generalization thereof, whether or not it relates to the same invention as presently claimed in any claim and whether or not it mitigates any or all of the same technical problems as does the present invention. The applicants hereby give notice that new claims may be formulated to such features and/or combinations of such features during the prosecution of the present application or of any further application derived therefrom.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1608682.9 | May 2016 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2017/051376 | 5/17/2017 | WO | 00 |
