TECHNICAL FIELD

[0001] The present invention relates to long-range surface plasmon- polariton biosensors, and in particular, systems, apparatus and methods for detecting one or more analytes in a biological sample.

BACKGROUND

[0002] Current technologies for identifying various pathogens include immunoassays that detect viral antigen or polyrherase chain reaction (PCR) amplification and fluorescence-based oligonucleotide detection that detect production of virus genes. These methods require laboratory facilities, expertise in culturing methods, expertise in interpretation of results, and long incubation periods. Fluorescence-based sensors have also been developed in a variety of formats, including planar, evanescent sensors. Fluorescence is currently used in antibody-antigen detection, as well as in the detection of nucleic acids (gene chips). Unfortunately, the need for fluorescent tags limits the utility of these sensors.

[0003] Present technologies for detecting one or more analytes have several limitations including low sensitivity, low specificity, require large sample volumes, inability to simultaneously analyze large numbers of analytes, require labeling, and/or the inability to detect binding events in real-time.

[0004] Surface plasmon polaritons (SPPs) are transverse magnetic

(TM) polarized optical surface waves that propagate, typically, along a metal-dielectric interface. A single-interface SPP exhibits interesting and useful properties such as an energy asymptote in its dispersion curve, high surface and bulk sensitivities, and subwavelength confinement near its energy asymptote. [0005] Sensors utilizing SPPs have been used for measuring binding- induced changes in the local index of the sensors. SPPs however, are characterized by a high attenuation. Due to the high attenuation and asymmetrical field distribution of single-interface SPPs, they cannot be easily excited by butt-coupling a polarisation-maintaining single-mode optical fibre (PM-SMF) to the input waveguide which precludes miniaturization.

[0006] One way of reducing the SPP attenuation is to use a thin metal film or stripe bounded on all sides by the same dielectric and operating the structure in the so-called long-range SPP (LRSPP) mode. Exemplary LRSPP waveguides are disclosed in US Patent Nos. 6,442,321; 6,614,960;

6,914,999; 6,801,691 and 6,741,782 and are herein incorporated by reference in their entirety.

[0007] Long-range surface plasmon polaritons (LRSPPs) are optical surface waves excited by TM polarized incident light propagating along a thin metal stripe or slab bounded on all sides by the same dielectric cladding. "Long-range" is taken to mean that the LRSPP attenuation is at least a factor of 2 to 3 lower than that of the single-interface SPP, resulting in propagation over a longer distance. Indeed, attenuation reduction factors, or

equivalently, range extension factors, greater than 100 can be achieved with the LRSPP. The range extension mitigates an important limitation of the single-interface SPP and the increased propagation length of LRSPPs enables a better overall sensitivity due to increased optical interaction length.

[0008] There is a need to develop label-free optical sensing that has high sensitivity, overcomes the need for tagging and unnecessary chemical manipulation of the analyte (and/or receptor molecules), requires very low working volumes, does not have electromagnetic interference, can determine kinetic, specificity, affinity and concentration in one step, and provides real time and simultaneous detection of a plurality of biomolecules based on LRSPP waveguides and biosensors comprising LRSPP waveguides and fluidic channel assemblies. SUMMARY OF THE INVENTION:

[0009] It is an embodiment of the present invention to provide long- range surface plasmon-polariton (LRSPP) biosensors, and in particular, systems, apparatus and methods for detecting one or more analytes in a biological sample.

[0010] The LRSPP biosensors of the present invention can be applied to various sensing applications such as medical diagnostics, environmental monitoring, and food safety and security.

[0011] The LRSPP biosensors and waveguides of the present invention are readily adaptable for and are particularly suitable for performing a plurality of different forms of simultaneous detection by using different waveguides in combination with different fluidic channel designs.

[0012] The detection strategies discussed below illustrate generally examples of analyte detection that can be done using the LRSPP biosensors according to various embodiments described herein. The LRSPP biosensors of the present invention may be useful for the detection of various diseases which require at least two combination tests for reliable diagnosis.

[0013] According to an aspect, the present disclosure provides a biosensor for detecting a plurality of analytes in a biological sample, the biosensor comprising a LRSPP waveguide including a plurality of branches. At least one of the branches being a sensing branch capable of having an adlayer deposited thereon for immobilizing a ligand for binding to at least one of the analytes to be detected. At least one of the branches being a reference branch. The waveguide is surrounded with a dielectric cladding. Etched in the dielectric cladding is at least one microfluidic channel. The channel configured to move fluids towards and away from the sensing branch. [0014] In one aspect, the present invention relates to a biosensor comprising : a waveguide for receiving and propagating an optical radiation along the length of the waveguide as a long range surface plasmon-polariton (LRSPP) wave with its transverse electric field substantially perpendicular to the width of the waveguide, the waveguide comprising : an input region for receiving the optical radiation at one end; an output region at an opposed end for emitting the propagated optical radiation away from the waveguide and towards a detector; and a sensing region between the input and output region; a dielectric cladding surrounding the waveguide; and at least one fluidic channel formed in the dielectric cladding for moving a fluid towards and away from the sensing region.

[0015] According to an aspect, the waveguide further comprises: a splitter region downstream from and optically coupled to the input region; and a plurality of branches downstream from the splitter region for propagating the optical radiation along the length of the waveguide.

[0016] According to an aspect, the biosensor further comprising an adlayer adsorbed onto the sensing region, the adlayer for immobilizing a ligand.

[0017] According to an aspect, the waveguide comprises a plurality of units along the length of the waveguide, the units optically coupled and arranged in an end-to-end relationship with adjacent units, the units being separated from the adjacent units by a gap.

[0018] According to an aspect, at least one branch further comprises a bypass branch optically coupled thereto.

[0019] According to an aspect, the bypass branch is separated from the daughter branch by a gap. [0020] According to an aspect, the waveguide comprises a metallic strip, the strip comprising gold, silver, copper or aluminum.

[0021] According to an aspect, the cladding is glass, quartz, or a low- index UV or thermal curing polymer.

[0022] According to an aspect, the waveguide has a thickness from about 20 to about 60 nm, preferably about 35 nm and a width from about 1 to about 12 μιτι, preferably about 5 Mm.

[0023] According to an aspect, the at least one branch further comprises curved portions having a radius of curvature from about 3 to about 7 mm.

[0024] According to an aspect, the at least one branch is surrounded by the dielectric cladding along its entire length thereof.

[0025] According to an aspect, the sensing region comprises a length of l/(2a _s (0)), where a _s (0) is the field attenuation coefficient across the branch in the absence of an adlayer.

[0026] According to an aspect, the sensing region is from about 0.5 to about 4 mm in length.

[0027] According to an aspect, the fluidic channels comprise curved portions.

[0028] According to an aspect, the index of refraction of the cladding is substantially similar to the index of refraction of the fluid.

[0029] According to an aspect, the biosensor further comprising: a base for supporting the biosensor thereon and a cover secured over the biosensor and the base. [0030] According to an aspect, the cover comprises: a fluid inlet in fluid communication with the fluidic channel for directing fluid downwards and towards the sensing region and a fluid outlet for removing fluid from the fluidic channel upwards and away from the fluidic channel.

[0031] According to an aspect, the base comprises: a sidewall surrounding at least a portion of the dielectric cladding; a fluid inlet formed in the sidewall, the inlet in fluid communication with the fluidic channel for directing fluids laterally and towards the sensing region; and a fluid outlet formed in the sidewall, the outlet for removing fluid from the fluidic channel laterally and away from the sensing region.

[0032] In one aspect, the present invention relates to a method of detecting one or more analytes in a biological sample comprising : providing biosensor comprising: a waveguide for receiving and propagating an optical radiation along the length of the

waveguide as a plasmon-polariton wave with its transverse

electric field substantially perpendicular to the width of the

waveguide, the waveguide comprising:

an input port for receiving the optical radiation;

a plurality of branches for propagating the optical radiation along the length of the waveguide;

an output port at the end of each branch for

emitting the propagated optical radiation away

from the waveguide and towards a detector; and a sensing region associated with at least one

branch;

a dielectric cladding surrounding the waveguide; and at least one fluidic channel formed in the dielectric cladding for moving fluids towards and away from the sensing region; forming an adiayer onto the sensing region, the adiayer for immobilizing a ligand;

introducing a biological sample suspected of containing an

analyte onto the sensing region;

transmitting an optical radiation into the waveguide through the

input port;

monitoring the changes in the power levels of the propagated

optical radiation emitted from the output port using the detector, wherein a change in the power levels of the propagated optical radiation is indicative of the presence of the analyte in the biological sample through the binding of the analyte to the ligand.

[0033] According to an aspect, the method further comprising ;

monitoring the changes in the power levels of the propagated optical radiation emitted from the output port of a reference branch using the detector, wherein the reference branch is surrounded by the dielectric cladding along the entire length of the reference branch; and comparing the power levels from the reference branch to at least one other branch.

[0034] According to an aspect, the adiayer is a self-assembled monolayer.

[0035] According to an aspect, the ligand is an antibody or an antigen and the biological sample is blood.

[0036] According to an aspect, the waveguide comprising at least two branches, wherein a first adiayer is formed on a first branch for binding a first ligand and a second adiayer is formed on a second branch for binding a second ligand.

[0037] The detailed description herein is only intended to provide examples and representative embodiments of the invention and is not intended to limit the scope of the invention. The full scope of the invention is presented in the specification as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Fig. 1(a) is a schematic diagram of an optical arrangement for power attenuation-based sensing according to an aspect of the present invention;

[0039] Fig. 1(b) is a perspective view of a biosensor including a LRSPP waveguide according to an aspect of the present invention ;

[0040] Fig. 1(c) is cross sectional view along the line lc of the biosensor of Fig. 1(b);

[0041] Fig. 2(a) is a plan view of a linear waveguide with a blunted sensor facet;

[0042] Fig. 2(b) is a plan view of a linear waveguide with a tapered sensor facet formed using a trapezoidal waveguide;

[0043] Fig. 2(c) is a plan view of a fluidic channel design configured to span the width of a plurality of waveguides;

[0044] Fig. 2(d) is a plan view of configured to span the width of one waveguide;

[0045] Fig. 3 is a graph showing a computed mode power attenuation (MPA) and the real part of effective refractive index Re{neff} of the SPP mode supported by a metal strip for three different sensing indices (n _c ) ;

[0046] Fig. 4(a) is a perspective view of a biosensor including a LRSPP waveguide and a fluidic assembly for top-access microfluidic channels according to another aspect of the present invention; [0047] Fig. 4(b) is a perspective view of a biosensor including a LRSPP waveguide and a fluidic assembly for top-access microfluidic channels according to another aspect of the present invention;

[0048] Fig. 4(c) is a perspective view of a biosensor including a fluidic assembly for side-access microfluidic channels according to another aspect of the present invention;

[0049] Fig. 5(a) is a plan view of a waveguide including a splitter region formed by mirroring and overlapping curved sections according to another aspect of the present invention;

[0050] Fig. 5(b) is a plan view of a top-access microfluidic channel design for a waveguide including a splitter region, consisting of a single channel exposing one sensing region;

[0051] Fig. 5(c) is a plan view of a top-access microfluidic channel design for a waveguide including a splitter region, consisting a double channel exposing two sensing regions;

[0052] Fig. 5(d) is a plan view of side-access microfluidic channels for a waveguide including a splitter region, consisting a double channel exposing two sensing regions;

[0053] Fig. 6(a) is a plan view of a triple coupler waveguide comprising three linear segments downstream of the input region and three branches according to another aspect of the present invention;

[0054] Fig. 6(b) is a plan view of a top-access microfluidic channel for the waveguide in Fig. 6(a);

[0055] Fig. 6(c) is a plan view of a side-access microfluidic channel for the waveguide in Fig. 6(a); [0056] Fig. 6(d) shows the computed normalized power distribution as a function of coupling lengths of the waveguide in Fig. 6(a);

[0057] Fig. 7(a) is a plan view of an advanced triple coupler where a Y- junction is cascaded to each lateral branch according to another aspect of the present invention;

[0058] Fig. 7(b) is a plan view of top-access and side-access microfluidic channel designs for the waveguide structure in Fig. 7(a);

[0059] Fig. 7(c) is a plan view of side-access microfluidic channel designs for the waveguide structure in Fig. 7(a);

[0060] Fig. 8(a) is a plan view of another embodiment of a branched waveguide according to another aspect of the present invention;

[0061] Fig. 8(b) is a plan view of top-access microfluidic channel designs for the waveguide structure in Fig. 8(a);

[0062] Fig. 8(c) is a plan view of side-access microfluidic channel designs for the waveguide structure in Fig. 8(a);

[0063] Fig. 9 is a graph showing the normalized power distribution as a function of coupling length between two outputs from a coupler in Fig. 8(a);

[0064] Fig. 10(a) shows the response of a Y-junction sensor comprising the waveguide of Fig. 5(a) in response to five different solutions for bulk sensing;

[0065] Fig. 10(b) shows the power ratio of the sensing branch to the reference branch of the Y-junction sensor of Fig. 10(a); [0066] Fig. 11(a) shows the response of a Y-junction sensor comprising the waveguide of Fig. 5(a) in response to five different solutions for protein sensing;

[0067] Fig. 11(b) shows the power ratio of a sensing branch to a reference branch of the Y-junction sensor of Fig. 11(a);

[0068] Fig. 12 is a graph showing the antibody titre/viremia levels over different phases of a Dengue virus infection;

[0069] Fig. 13(a) shows the detection scheme for dengue-specific antibodies in a blood sample;

[0070] Fig. 13(b) shows the detection scheme of dengue NS1 antigen in a blood sample;

[0071] Fig. 13(c) shows the detection scheme of dengue-specific IgM antibody in a blood sample;

[0072] Fig. 13(d) shows the detection scheme of dengue-specific IgG antibody in a blood sample using anti-human IgG antibody; and

[0073] Fig. 13(e) shows the detection scheme of dengue-specific IgG antibody in a blood sample using anti-human IgG antibody.

DETAILED DESCRIPTION

[0074] Reference will be made below in detail to exemplary

embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numerals used throughout the drawings refer to the same or like parts.

[0075] Long-range surface plasmon polaritons (LRSPPs) are optical surface waves excited by transverse magnetic TM polarized incident light propagating along a thin metal stripe or slab bounded on all sides by the same dielectric cladding. "Long-range" is taken to mean that the LRSPP attenuation is at least a factor of 2 to 3 lower than that of the single- interface SPP, resulting in propagation over a longer distance. Indeed, attenuation reduction factors, or equivalently, range extension factors, greater than 100 can be achieved with the LRSPP. The range extension mitigates an important limitation of the single-interface SPP and the increased propagation length of LRSPPs enables a better overall sensitivity due to increased optical interaction length.

[0076] LRSPP waveguides are sensitive to bulk and surface changes because the mode is bound to the surface of the metal, has fields that peak thereon, and propagates mostly in the background dielectric. Any minor change along the metal surface will affect the mode, changing the

waveguide's transmittance.

[0077] While LRSPPs may, in certain environments, be less confined and less surface sensitive than single-interface SPPs, they propagate much farther so long-interaction length sensors make it possible to achieve greater adlayer sensitivity and lower limits of detection. Also, the sensing depth is greater (~1 pm vs. ~200 nm) so greater protein loading is possible. LRSPPs may therefore be useful for sensing large biological entities such as cells which cause strong scattering of loosely bound LRSPPs into radiative modes.

[0078] Analyte detection may be accomplished using an optical arrangement 10 for power attenuation-based sensing comprising an optical biosensor such as shown in Fig. 1(a). A polarized laser diode (LD) 12 may be butt-coupled via a polarization-maintaining single-mode optical fiber (PM- SMF) 14 to the input of an optical biosensor 16. The polarization of the light emitted by the LD 12 may be aligned with the PM-SMF 14 to ensure that the light incident onto the biosensor 16 is p-polarized. A broad range of suitable free-space operating wavelength may be used. In some embodiments, free- space operating wavelengths of 850, 1310, or 1550 nm, or therebetween can be used. In one preferred embodiment, the free-space operating wavelength is 1310 nm.

[0079] Optical radiation is received by the optical biosensor 16 and propagated downstream and along the length of a waveguide (not shown).

[0080] The output of the optical biosensor 16 is collimated by a microscope objective lens (OB) 18 and passed through an aperture (A) 20 to reduce the background light.

[0081] In the case of single channel biosensor 16, the output signal is split using a beam splitter (BS) 22 into two portions: one portion may be directed to an infrared camera (CCD) 24 to visually monitor the emerging mode for ease of alignment, and another portion may be directed to a photodetector (PD) 26 which is connected to a power meter to record realtime changes in the output power. For multiple-channel sensors 16 (which have the ability to perform single or simultaneous analyte detection and analysis), the output signal may be sent directly to CCD 24 where the mode images are recorded and post- processed using analysis software.

[0082] An intervening material (instead of air) could be inserted between the PM-SMF 14 and the biosensor 16, such as an optical bonding material, or such an intervening material could be placed in physical contact with the sensor. Optical input and output configurations may include generally-available apparatus and techniques used in the optical technical domain to manage confined light, such as, for example, optical fibers, or free-space beams such as coupled to or from the sensors described herein using lenses or beamsplitters. A Gaussian beam, emerging from a lens system or from an optical fiber, for example, is suitable for use as an input.

[0083] Shown in Figs. 1(b) and 1(c) is an embodiment of a biosensor 16 comprising a plurality of waveguides 100. The waveguides according to the present invention are polarization sensitive in that the plasmon-polariton wave is highly linearly polarised in the vertical direction, i.e. perpendicular to the plane of the metallic strip. Hence, it may serve as a polarisation filter, whereby substantially only a vertical polarised mode (aligned along the y- axis as defined in Fig. 1(b) of the incident light is guided. Therefore, the waveguides of the present invention are configured for receiving and propagating an optical radiation along the length of the waveguide as a long range surface plasmon-polariton (LRSPP) wave with its transverse electric field substantially perpendicular to the width of the waveguide.

[0084] Waveguide 100 comprises an input region 102 for receiving the optical radiation at one end and an output region 104 at an opposed end for emitting the propagated optical radiation away from waveguide 100 and towards a detector (e.g. PD and/or CCD). A sensing region 106 may be located between input region 102 and output region 104 for reasons to be discussed below.

[0085] Waveguide 100 may comprise a metallic strip of thickness t, width w, and permittivity ε2. The thickness and the width of the strip are selected such that the waveguide 100 supports a long-range surface plasmon-polariton (LRSPP) mode at the free-space operating wavelength of interest. Suitable materials for the waveguide include (but are not limited to) gold, silver, copper, and aluminum.

[0086] A dielectric cladding 108 surrounds waveguide 100. In an embodiment, cladding 108 surrounds all the sides of waveguide 100.

Cladding 108 may be comprised of a homogenous dielectric of permittivity ε3. Suitable materials for the dielectric include (but are not limited to) glass, quartz, and polymers. Particularly suitable combinations of materials for sensing applications include Au for the strip and low-index UV or thermal curing optical polymer, CYTOP or Teflon for the cladding. The waveguide 100 and cladding 108 may be supported by support 110 comprising, for example, a silicon wafer. [0087] Suitable dimensions for Au waveguides 100 in a CYTOP cladding

108 at a free-space operating wavelength of 1310 nm is from about 20 to 60 nm thickness t and from about 1 to about 12 pm for the width w. In a preferred embodiment, waveguide 100 has thickness of about 35 nm and width of about 5 pm.

[0088] As shown in Figs. 1(b) and 1(c), biosensor 16 comprises at least one fluidic channel 112 for containing a sensing solution 114 of index n _c . Channel 112 etched into the cladding 108 and therefore exposes at least a portion of the sensing region 106 of waveguide 100 to any solutions 114 that may be contained in channel 112. Channel 112 is configured for moving solution 114 towards and away from sensing region 106.

[0089] Waveguides 100 which are not exposed to channel 112, and hence do not have sensing region 106, may be used as reference

waveguides useful for any noise cancellation.

[0090] The sensing region 106 is suitable for depositing an adlayer 116 thereon. For example, various biomolecules can be attached to the surface of the sensing region 106 using various methods including, but are not limited to, simple physisorption; binding using a protein A or protein G linker; binding using a streptavidin or avidin-biotin linker; or binding using covalent attachment.

[0091] In some embodiments, the sensing region 106 is prepared by forming a suitable self-assembled monolayer (SAM) thereon. Alkanethiols, where a thiol is covalently linked to a longer hydrocarbon chain, are commonly used SAM molecules. A suitable alkanethiol is 16- Mercaptohexadecanioc acid (16-MHA). In an embodiment, the SAM is assembled by incubation of the alkanethiol in an appropriate solvent over the metallic surface, for example Au. [0092] Once assembled, the SAM can then be functionalised as desired. One functionalization method is using carbodiimide coupling (i.e. using l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N- Hydroxysuccinimide (NHS) chemistry for protein coupling) of the

biomolecules to the carboxylated metallic surface. Other methods of forming an adlayer at the sensing region of the waveguide suitable for detecting other biomolecules (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), etc ..) known to those of ordinary skill in the art may also be used.

[0093] As shown in Figs. 2(a) and 2(b), the input region 102 may include a sensor facet 118 that is blunted (2(a)) or tapered (2(b)). The tapered shape may be formed using a trapezoidal waveguide which may improve the coupling efficiency between the input fibre 14 and sensor facet 118. The tapered input can have a sensor facet of width w _t , which can be about 2 μιτι.

[0094] As shown in Figs. 2(c) and 2(d) channel 112 having a channel width W _s may be configured to span the width of a plurality of waveguides (2(c)) or be dedicated to one waveguide (2(d)). Channel width W _s , length L _f , and overall shape may be adapted as required and described below. As shown in Fig. 2(c), channel 112 may be substantially oval with radius of curvatures Rl and R2.

[0095] Fig. 3 is a graph showing the optical performance of a cross- section of the LRSPP waveguide modeled using a 2-dimensional finite element method (FEM). For the model, the free-space operating wavelength was set, for illustrative purposes, to λ ₀ = 1310 nm, Au (ε2 = -86.06 - j8.322) was selected as the material of the metal waveguide and CYTOP was selected as the surrounding material (ε3 = 1.3348 ² = 1.7817). The width of the metal strip was set to 5 μιτι, the thickness t was varied from about 20 to 60 nm, and the refractive index n _c of the sensing solution was taken as 1.3303 (n _c 2 < ε3), 1.3348 (n _c 2 = ε3) and 1.3393 (n _c 2 > ε3). As shown, both the computed mode power attenuation (MPA) and the real part of effective refractive index (Re{neff}) of the LRSPP mode supported by the LRSPP waveguide increased as t increases, which indicates increasing field confinement as t increases.

[0096] As will be described further, the biosensors and waveguides according to various embodiments as described herein are readily adaptable for and are particularly suitable for performing a plurality of different forms of simultaneous detection by using different waveguides in combination with different fluidic channel designs.

[0097] Fig. 4(a) shows another embodiment of the biosensor 16. In this embodiment, biosensor 16 comprises a waveguide 200, cladding 108 surrounding the waveguide, and two fluidic channels 112 etched into the cladding. A base 120 supports waveguide 200 and cladding 108 and a lid 122 is secured over top of the base. The lid 122 may be removably secured or permanently secured by wafer bonding, for example. This configuration may be termed "top-access" because a fluidic inlet 124 and a fluidic outlet 126 are formed in lid 122 where fluidic inlet 124 and outlet 126 are in fluid communication with fluidic channels 112 such that fluid inlet 124 directs fluid downwards and towards sensing region 106 and fluid outlet 126 directs fluid upwards and away from sensing region 106.

[0098] As shown in the embodiment of Fig. 4(a), waveguide 200 comprises three sensing regions 106. Since two fluidic channels 112 share the same fluidic inlet 124 and outlet 126, sensing regions 106 are exposed to same sensing solution 114.

[0099] Fig. 4(b) shows another embodiment of a top-access biosensor 16 where more than one pair of fluidic inlets 124 and outlets 126 are formed in the lid 122. In this embodiment, different solutions 114 can be directed to the different sensing regions 106 to allow for simultaneous analysis and detection of a plurality of different analytes. [00100] Fig. 4(c) shows another embodiment of a biosensor 16. In this embodiment, base 120 comprises a sidewall 128 that may surround at least a portion of cladding 108. Fluid inlet 124 and fluid outlet 126 are formed in sidewall 128 and are in fluid communication with channel 112. In this configuration, which may be termed "side-access", the channel 112 directs fluids laterally towards and away from sensing region(s) 106 of waveguide 200. A housing 130 may further be included provide structural support for fluid inlet 124 and outlet 126 in the side-access configuration.

[00101] Fig. 5(a) shows an example of a waveguide 300 comprising a splitter region 132 downstream from and optically coupled to input region 102. As shown, splitter region 132 acts as a power splitter or a Y-junction. Splitter region 132 can be formed by mirroring and overlapping a pair of curved sections 134. Splitter region 132 therefore splits the received optical radiation into separate branches 136 along the remaining length of waveguide 300.

[00102] Optically coupled to the downstream end of the two first-level curved sections 134 are two additional curved sections 134 which bend in opposite directions to form an "S" bend on each side of the splitter region 132. To minimize radiation loss at the bends, an exemplary radius of curvature (Rl, R2, R3, R4) for the curved sections 134 is in the range of about 3 mm to about 7 mm, where width w = 5 pm and t = 35 nm. In aspects, the radius of curvature (Rl, R2, R3, R4) for the curved sections 134 can be in the range of about 4 mm to about 5.5 mm.

[00103] The width of branches 136 at the output end 104 can be adjusted to vary the ratio of the output powers. The number of branches 136 can be greater than two. For example, the biosensor 16 can comprise a plurality of input waveguides to form an N x N divider.

[00104] Figs. 5(b) and 5(c) show plan views of a biosensor configured for top-access microfluidic comprising a Y-junction upstream of a single dedicated channel 112 for one branch 136 (5(b)) and a double-width channel 112 dimensioned for two branches 136 (5(c)). A typical width for the single dedicated channel w _s is about 80 pm and a typical width for the double channel w _d is about 210 pm. However, any suitable width of channel that eases the wetting of the waveguide 300 is contemplated. The length L _f of channel 112 can be in the range from about 0.5 mm to about 4 mm so that it has a sufficient length that will allow radiative modes to spread if induced by uncoupled light at the input end of the sensing waveguide. In aspects, the length L _f of channel 112 can be in the range from about 1 mm to about 2 mm. More particularly, the length L _f of the channel can be from about 1.5 mm to 2 mm. It will be understood that the surface sensitivity of the sensing channel can be maximized by choosing an optimal sensing length L _opt = l/(2as(0)) where as(0) is the LRSPP field attenuation coefficient along the sensing waveguide without any adlayer formed thereon.

[00105] Shown in Fig. 5(d) is a plan view of biosensor 16 configured for side-access channels including waveguide 300 comprising a Y-junction 132. As shown, biosensor comprises two separate side-access fluidic channels 112 having curved sections 138 to avoid "dead volumes" during solution flow and it is symmetrical along the axis of symmetry. In this embodiment, the waveguide 300 comprises two branches 136 each branch includes one sensing region 106. There are no reference branches for noise cancellation in this embodiment. Given that there are two sensing regions 106, two different solutions can simultaneously be used with waveguide 300 in this biosensor. A typical width for the horizontal channels (wl, w2, w3, w4) is about 250 pm and for the channels that expose sensing regions 106 (w5, w6) is about 80 pm. Any value smaller or larger than the typical widths is possible for the channel design.

[00106] Fig. 6(a) shows an example of a triple coupler waveguide 400 comprising three linear segments 140 downstream of the input region. The three linear segments 140 are arranged in parallel and in close proximity to each other over a length L _c where the linear segments 140 are coupled. The separation distance Sc between the linear segments 140 can be up to about 20 pm, and in some embodiments from about 1 pm to 20 pm. The coupling length L _c can be from a few microns to a few dozen millimeters depending on the separation S _c , width and thickness of the waveguide, the materials used, the operating wavelength, and the level of coupling desired.

[00107] In the embodiment shown in Fig. 6(a), when optical radiation is directed into the input region 102 of waveguide 400, the optical radiation is then split in the region where the linear segments 140 are coupled. The optical radiation is then is propagated along the remaining length of the waveguide 400 in separate directions as branches 136 dictated by the direction of each of linear segments 140. In some embodiments, the lateral branches 136 can include curved sections 134 and the lateral branches 136 can be distanced from the central branch 136 a distance large enough so that any optical coupling between the branches 136 is negligible.

[00108] Figs. 6(b) and 6(c) show plan views of a biosensor 16

configured for top-access and side-access channels, respectively comprising the waveguide of Fig. 6(a). As shown in Figs. 6(b) and 6(c), the lateral branches 136 of the waveguide serve as the sensing branches since it is these branches that are exposed to channels 112. The central branch 136 of the waveguide serves as the reference branch since this branch is entirely surrounded by cladding 108 along its entire length.

[00109] Shown in Fig. 6(d) is an example of the computed normalized power distribution as a function of coupling lengths L _c for the three outputs (PI, P2, P3) from the waveguide of Fig. 6(a). The linear segments 140 are separated by S _c = 2 pm. In this example, the central branch is used as a reference branch (P2), the power distributions for the triple coupler can be arranged such that most of the power is transferred to the sensing branches (PI and P3). A suitable coupling length L _c where most of the power can be transferred to the sensing branches is in the range from 400 pm to 700 pm. [00110] Fig. 7(a) is a plan view of another embodiment of a triple coupler waveguide 500. In this embodiment, the waveguide comprises three linear segments 140 downstream of the input region. Each linear segment then forms into a branch 136 for propagating the received optical radiation into different directions. The lateral branches 136 each further comprise a splitter region 132 which from there, each lateral branch 136 diverges to form two daughter branches 142.

[00111] Shown in Figs. 7(b) and7(c) are plan views of top-access and side-access channel designs, respectively for the waveguide structure in Fig. 7(a). The four daughter branches 142of the waveguide 500 serve as the sensing branches since it is these branches that are exposed to the channels 112. The central branch 136 of the waveguide 500 serves as the reference branch since this branch is entirely surrounded by cladding 108 along its entire length.

[00112] Fig. 8(a) is a plan view of view of another embodiment of a branched waveguide 600. In this embodiment, waveguide 600 comprises a splitter region 132 downstream from and optically coupled to input region 102. Splitter region 132 acts as a power splitter or a Y-junction and splits the received optical radiation into two separate trunk branches 136.

Waveguide 600 may further comprise one or more bypass branches 144 optically coupled to one or more of the trunk branches at a coupling region having a coupling length L _c . At the coupling region, the trunk branch 136 and its bypass branch 144 may be separated by distance S _c of about 2 μιτι and have a coupling length L _c of about 200 μητι. Bypass branches 144 may comprise one or more curved sections 134 (R5, R6, R7, R8, R9, R10).

[00113] Each trunk branch 136 may further comprise a splitter region 132 which from there, each trunk branch 136 diverges to form two daughter branches 142. [00114] The distance (di, d ₂ , d ₃ , d ₄ , d ₅ ) between the output ends 104 of each of the branches can be arranged such that this structure can be beneficially used as a Young's interferometer in a far-field measurement setup.

[00115] Figs. 8(b) and 8(c) are plan views of top-access and side-access channel designs, respectively for waveguide 600 in Fig. 8(a). The four daughter branches 142 of waveguide 600 serve as the sensing branches since it is these branches that are exposed to channels 112. The bypass branches 144 serve as reference branches since these branches are entirely surrounded by cladding 108 along their entire length.

[00116] As shown in Fig. 8(c), the side-access channels have curved sections to avoid "dead volumes". A typical width for the horizontal channels (wl, w2, w3, w4) is about 250 μητι and for the channels (w5, w6) which expose sensing regions 106 is about 210 pm.

[00117] Fig. 9 shows the computed power distributions as a function of coupling length between two outputs from a coupler in Fig. 8(a) with separation S _c = 2 μιτι in between. Since most of the power is required for the sensing branches, a coupling length L _c less than about 200 μιη may be chosen.

[00118] The waveguides according to the present disclosure propagate an optical radiation along the entire length of the waveguides (100, 200, 300, 400, 500, 600) from input region 102 to the output region 104. In some embodiments, the waveguides according to the present disclosure can be one continuous strip. In other embodiments, the waveguides may comprise a plurality of units aligned along the length of the waveguide, the units optically coupled and arranged in an end-to-end relationship with adjacent units, the units being separated from the adjacent units by a gap. According to an aspect, the gap distance may be from about 1 μι ^η to about 2 μιη. [00119] In use, there is provided a method of detecting one or more analytes in a biological sample using the biosensor of the present disclosure. The biosensor comprising a sensing branch and a reference branch. An adiayer is formed onto the sensing region of one branch of the waveguide of the biosensor. The adiayer is configured to immobilize a ligand for a target analyte. The immobilized ligand may be an antibody for an antigen or be an antigen for a target antibody. A biological sample suspected of containing the target analyte is deposited onto the sensing region via one or more channels. Optical radiation is transmitted into the waveguide through the input port and the optical radiation is propagated downstream along the length of the waveguide and emitted from the output port. The changes in the power levels of the propagated optical radiation emitted from the output port is monitored using the detector, wherein a change in the power levels of the propagated optical radiation is indicative of the presence of the analyte(s) in the biological sample through the binding of the analyte to the ligand on the surface of the sensing branch of the waveguide. According to an aspect, the power levels from the sensing branch is compared to the power levels from the reference branch to minimize noise.

[00120] Example 1 :

[00121] Figs. 10(a) and 10(b) and 11(a) and 11(b) show illustrative experimental results for bulk refractive index and protein sensing

experiments, respectively, using a Y-junction biosensor including an Au waveguide comprising two branches (i.e. waveguide 300 shown in Fig 5(a) comprising a Y-junction) : one sensing branch exposed to a fluidic channel and solution contained therein and one reference branch fully surrounded by CYTOP cladding. The waveguide had a thickness of t = 35 nm, width of w = 5 pm, "S" bends were designed with a radius of curvature R of 5.5 mm, and the distance between each output end of the branches was 160 pm. The CYTOP cladding was about 8 pm thick from top to bottom. The channel was 80 pm wide, and 1.65 mm long. The biosensor was designed to operate at a wavelength of 1310 nm. The waveguide was incubated in 1 mM IPA solution of 16-MHA overnight to allow for the formation of an adiayer of self- assembled monolayer (SAM).

[00122] For the bulk refractive index sensing experiment as shown in Figs. 10(a) and 10(b), five PBS/Gly mixtures were prepared with an index increment of 2 x 10-3 RIU near the refractive index of CYTOP (nCYTOP = 1.3348). The sensing indices were carefully adjusted so that the measured values using a prism-coupler based instrument read n _c = 1.330, 1.332, 1.334, 1.336 and 1.338 at λ ₀ = 1310 nm.

[00123] The maximum power ratio is observed for the solution with n _c = 1.334, which is closest to the refractive index of CYTOP (nCYTOP = 1.3348).

[00124] As shown in Fig. 10(b), a power ratio Pout,F/Pout,C was formed by dividing the output power from the sensing arm by that from the reference branch. Forming the power ratio eliminates the common drift and noise in the system. The change in the power ratio is not directly

proportional to the step change in refractive index. The largest change in power ratio of A(Pout,F/Pout,C) = 0.74 is observed for the step from solution 4 to 5 (n _c = 1.336 to 1.338), and the standard deviation of the power ratio in this region is σ = 0.006, yielding a signal-to-noise (SNR) of

A(Pout,F/Pout,C)/o = 0.74/0.006 = 123.

[00125] Time-averaging was carried out by computing the average power ratio over blocks of 10 subsequent time points. The standard deviation of the time-averaged result is improved by a factor of 3 to σ = 0.002 and the corresponding SNR to A(Pout,F/Pout,C)/o = 0.74/0.002 = 370. The bulk detection limit of the referenced Y-junction is 5.4 x 10-6 RIU.

[00126] The protein sensing experiment of Figs. 11(a) and 11(b) was designed to demonstrate the performance of the biosensor through BSA adsorption on a carboxyl-terminated surface. A sensing buffer of n _c = 1.338 was chosen to obtain sensitive response for protein binding 100 pg/rnl of BSA is used to minimize the bulk refractive index change in the buffer.

Figure 11(a) shows the output powers from the sensing and reference arms of the Y-junction during the BSA physisorption experiment.

[00127] When the power ratio Pout,F/Pout,C (computed timepoint-by- timepoint) is plotted in Fig. 11(b), the drift and common fluctuations in the signals are eliminated, and the binding response due to BSA physisorbing on the surface becomes evident. The physisorption of BSA on the carboxyl- terminated surface produces a change in power ratio of A(Pout,F/Pout,C) = 0.11 with a signal-to-noise ratio of A(Pout,F/Pout,C)/o = 16 for σ = 0.007 based on the raw data. The raw data was time-averaged and is shown in Fig. 11(b). The standard deviation of the time-averaged power ratio is σ = 0.0038, which is almost half that of the raw power ratio. The signal-to-noise ratio is then improved to 29. The detection limit for protein sensing in terms of surface mass density is estimated as ΔΓ = 90 pg/mm ² .

[00128] Example 2 :

[00129] The detection strategies discussed below illustrate generally examples of detection that can be done using a biosensor having a plurality of branches and fluidic channels according to embodiments of the present disclosure. The biosensors of the present disclosure are especially useful for the detection of diseases that require at least two different corroborative tests to ensure a reliable diagnosis. An example is the detection of dengue infection, which is a major health problem in tropical and subtropical countries around the world. Different detection strategies for dengue detection using a multi-channel sensor are summarized in Table 1.

[00130] Detection strategy 1

[00131] During the infection of dengue disease, a patient's blood sample contains dengue non-structural 1 (NS1) antigen and dengue-specific antibodies (IgM and IgG) with levels varying over time as illustrated in Fig. 12. To improve the overall sensitivity of dengue detection, the strategy employ a combination test based on the detection of both dengue NSl antigen and dengue-specific antibodies (i.e. the analytes). The detection scheme of dengue-specific antibodies and dengue NSl antigen in blood sample are illustrated in Figs. 13(a) and 13(b), respectively.

[00132] A biosensor comprising a waveguide including a plurality of branches is first incubated in an n-alkanethiol (CH ₃ (CH ₂ ) _n SH) solution for more than 2 hours to form a SAM on the metallic surface. Next, a biological sample (blood sample) suspected of containing the analytes is attached to the surfaces through carbodiimide coupling. In this strategy, dengue virus is passed through one microfluidic channel to bind to any dengue-specific antibodies while anti-NSl monoclonal antibody is passed through the other channel to detect the presence of dengue NSl antigen. The outputs from the two channels will give a result for positive or negative dengue infection.

[00133] Detection strategy 2

[00134] To distinguish a positive result from a negative, an additional control test can be carried out during the detection. The detection scheme of dengue-specific antibodies is illustrated in Fig. 13(a). A biosensor comprising a waveguide including a plurality of branches is first incubated in an n-alkanethiol solution for more than 2 hours to form a SAM on the metal strips. In this strategy, a blood sample and a control sample are passed through the microfluidic channels respectively and attached to the surfaces through carbodiimide coupling. Next, dengue virus is injected over the functionalized surfaces to detect the presence of dengue-specific antibodies. A test sample is considered positive if the time-averaged surface mass density is greater than twice the value of the negative control. [00135] Detection strategy 3

[00136] To distinguish a positive result from a negative, an additional control test can be carried out during the detection. The detection scheme of dengue NS1 antigen is illustrated in Fig. 13(b). A biosensor comprising a waveguide including a plurality of branches is first incubated in an n- alkanethiol solution for more than 2 hours to form a SAM on the metal strips. In this strategy, a blood sample and a control sample are passed through the microfluidic channels respectively and attached to the surfaces through carbodiimide coupling. Next, anti-NSl monoclonal antibody is injected over the functionalized surfaces to detect the presence of dengue NS1 antigen. A test sample is considered positive if the time-averaged surface mass density is greater than twice the value of the negative control.

[00137] Detecti o n strateg y 4

[00138] An advanced detection of dengue infection includes the quantification of dengue-specific IgM and IgG antibodies in a blood sample to determine the stage of infection. As shown in Fig. 12, the level of IgM and IgG antibodies in a blood sample is distinctively different for primary and secondary dengue infection. The detection scheme of dengue IgM in a blood sample is illustrated in Fig. 13(c). The detection of dengue IgG antibody in a blood sample can be done using either anti-human IgG antibody or protein G, as illustrated in Figs. 13(d) and 13(e), respectively.

[00139] A biosensor comprising a waveguide including a plurality of branches is first incubated in an n-alkanethiol solution for 2 or more hours to form a SAM on the metallic surface. In this strategy, anti-human IgM antibody is passed through one microfluidic channel to capture IgM

antibodies in a blood sample while anti-human IgG antibody or protein G is passed through the other channel to capture IgG antibodies in a blood sample. Next, a biological sample (test sample) is injected over the functionalized surfaces. Then dengue virus is passed through the microfluidic channels to detect the presence of dengue-specific IgM and IgG antibodies. The ratio of responses for dengue-specific IgM antibody to dengue-specific IgG antibody will give a result for primary or secondary dengue infection.

[00140] Table 1 : Detection strategies for dengue infection using a multichannel sensor.

[00141] The embodiments of the present application described above are intended to be examples only. Those of skill in the art may effect alterations, modifications and variations to the particular embodiments without departing from the intended scope of the present application. In particular, features from one or more of the above-described embodiments may be selected to create alternate embodiments comprised of a

subcombination of features which may not be explicitly described above. In addition, features from one or more of the above-described embodiments may be selected and combined to create alternate embodiments comprised of a combination of features which may not be explicitly described above. Features suitable for such combinations and subcombinations would be readily apparent to persons skilled in the art upon review of the present application as a whole. Any dimensions provided in the drawings are provided for illustrative purposes only and are not intended to be limiting on the scope of the invention. The subject matter described herein and in the recited claims intends to cover and embrace all suitable changes in technology.