Field of the invention The present invention pertains to the field of biochemistry. More particularly, the present invention pertains to a system and method for detecting the presence and/or absence of chemical or biological substances.

Technical background Light, as any electromagnetic radiation, is elastically scattered by small particles, so called scatterers, of arbitrary material and shape. If the size of the scatterers is much smaller than the wavelength of light, the scattering process is defined as Rayleigh scattering and is characterized by the certain intensity and particular spatial distribution of the scattered light. In the case of particles comparable to the wavelength of light the scattering process is known as Mie scattering and is described by the generalized Lorentz-Mie theory, though it only applies correctly if a particle is a sphere, an infinite cylinder or an oblate or prolate spheroid. The Mie theory describes the scattering intensities of these particles types. The scattering from non-metallic particles in the gas or liquid phases and thus the appearance of various liquids or gases (for example, atmospheric aerosols, clouds or interstellar dust) can be monitored for quality and composition control.

In addition to the general scattering at small particles, light experiences strong absorption and scattering at metal nanometer sized particles due to effectively coupling to the collective oscillations of the free electrons in these particles called localized surface plasmons (LSP). The universality of this phenomenon implies that any metallic nanoscopic structure should support LSP resonances in some energy range since all metals contain free charge carriers, i.e. electrons. However, from the application point of view resonances in the visible-near infrared (IR) range are considered most important - this is were the classic plasmonic materials, like Au and Ag, have pronounced light extinction resonances when confined on the nanoscopic scale. Around irradiated metallic nanostructures strongly enhanced electromagnetic fields are induced which are affected by the change of the environment of the nanostructures. This effect can be used to detect biological and chemical molecular species that adsorb, i.e. physically or chemically bind to the nanostructures, by the change of transmitted scattered and/or absorbed light. Elastic light scattering either from metallic or non-metallic particles can be monitored both in forward scattering and in reflection modes. Forward scattering constitutes direct transmission in the propagation direction of the incoming light or scattering at a certain angle in respect to the direction of the incoming light. The former is excluded from the following discussion since in nanoplasmonic/optics research the changes in directly transmitted light are monitored as the changes in light extinction, which is a combined product of light scattering and light absorption. From hereon, the discussion of the prior art is limited to the scattering in the forward direction at an angle in respect to the incoming light. In turn, reflection mode can be either a) specular, when scattering is detected in a mirror- like fashion - i.e. by detecting directly reflected light, or b) diffuse - i.e. when other components of the reflected light excluding directly reflected one are detected. For example, in case of non-metallic particles, suspended in solution, their size can be tracked by static and/or dynamic light scattering using transmitted (i.e. forward scattered) light. In biomedical applications, forward scattered light can be used to detect specific dense protein aggregates of several tens of micrometers in the tissue - as in the case of the detection of neuritic plaques and neurofibrillary tangles in the brain tissue, characteristic for the development of neurodegenerative disorders such as Alzheimer disease. However, such methods use a commercial research-grade spectrophotometer and an integrating sphere to collect scattered light from the tissue samples, which is mostly unsuitable for the 'on-the-spot' clinical applications / clinical diagnostics and requires personnel highly qualified in optics research rather than in medical sciences.

In a similar fashion, the (polarized) forward scattered light can probe individual cells in solution under a laminar flow with a method called elastic scattering spectroscopy (ESS) where forward scattered light is detected.

Special case of the label-free optical interrogation of intermolecular interactions with a scattered light is represented by interferometric methods such as back- scattering interferometry, where the comparison between interferometric light patterns before and after molecular binding is made. The method however lacks the ability to detect intermolecular interactions in real physiological fluids such as urine, saliva or whole blood samples, where various impurities, often comparable in size with the microfluidic channel employed, are abundant and thus would strongly and non- systematically affect the detected interference pattern. As a consequence, the method, though having intrinsically very high sensitivity of detection, is not suitable for clinical diagnostics. The detection of forward scattered light from metallic nanoparticles for the evaluation of biomolecular interactions and the construction of various configurations of biochemosensing instrumental setups are wide fields or research and development. E.g., metallic nanosized colloids-based wavelength-ratiometric and polarization-based scattering sensors frequently combined with angular-dependent metal-enhanced fluorescence sensors have been developed, which operate by measuring the ratio of scattered intensities upon colloidal aggregation at two incident wavelengths thus making the method independent of the total colloid concentration. The method is solution-based and employs cross-linking of colloidal particles, precovered by protein layers and thus bringing the method away form the label-free sensors, by the complementary proteins/antiboides present in the analyte solutions.

Another method refers to biomolecular recognition based on single gold nanoparticle light scattering. The method is essentially using dark-field illumination mode, i.e., illumination in transmission when directly transmitted light at a grazing incidence is discarded in favour of scattered light propagating normal to the surface of the studied sample. The method obviously suffers from the complication of the experimental setup essentially available in a purely research environment and is hardy adaptable for the clinical diagnostics purposes. Similarly, detection of light scattering using dark-field microscopy is used to follow the adsorption of small-molecule analytes with extremely high (zemptomolar) sensitivity. Further, various schemes for the experimental setup capable of producing the incoming light scattering with efficient background suppression have been reported. However, all proposed schemes utilize either illumination, or detection (with microscope objective), or both through the analyte solution, which, in case of considering real- world clinical applications, is problematic due to the presence of various microscopic impurities in the realistic analytes solutions/samples, affecting the incoming or scattered light.

Dark-field illumination/detection mode can as well be developed into a highspeed multispectral array imaging, though suffering from already mentioned limitation that the illumination is done through the analyte solution, which makes the technique most likely unsuitable for clinical applications.

Other methods focus on the detection of the diffuse scattering in reflection mode. As another example, total internal reflection illumination with the prism directly adjacent to the back side of the glass sample has been employed in order to excite single metallic nanoparticles on the surface of the sample by the evanescent field. With this method optical scattering spectra can be recorded and the change of the refractive index of the medium surrounding metal nanoparticles can be monitored.

In US 6,180,415 illumination and detection is done from the same front side of the sample surface where plasmon resonant particles (PRE) are positioned. Spectral emission of light (light scattering) from the sample surface is directed through the lens to an optical detector. Similarly to US 6,214,560, no change in the scattering spectrum is detected due to the change of the surrounding refractive index of the plasmonic particles and thus due to the adsorption of the analyte molecules onto their surface. Consequently, the plasmonic particles and their scattering are used purely as the analyte' s marker.

The detection of scattered light from noble metal nanoparticles can be used for the colorimetric detection of the biomolecular binding. E.g., DNA hybridization can be detected when nucleic acid targets are recognized by the DNA-modified gold probes. The latter change their visual appearance detected as scattered light, which is excited by the evanescent field propagating from the light passing through a glass waveguide. Again, the method falls into the group of labelled biosensing as plasmonic particles, attached to the DNA, are used as markers, which change their scattering properties when brought in close contact with each other.

Further methods use the detection of specularly-reflected scattering light for bio- and refractive index sensing. Both illumination and detection are done from the solution-side of the sample and in addition diffractive effects can be used for interferometric sensing as the metal nanostructures on the samples are arranged in the periodic pattern. Though providing high sensitivity and a potential for the construction of palm-sized biodetection system, such reported schemes suffer from the necessity to cross the analyte solution with the excitation/scattered light.

The combination of the total internal reflection (TIR) and diffraction grating (DG) can be used to probe the presence of the analytes in a solution by the evanescent field created in the proximity of the DG. This is an indirect method, as the probed analyte liquid does not come in contact with DG, composed of nanostructures. The refractive index of the analyte liquid can be evaluated with these methods. Related techniques are grating light reflection spectroscopy (GLRS) and total internal reflection- diffraction grating (TIR-DG).

Yet a further method is scattering (specular reflection)-based label-free biochemodetection method. In this method, a photonic crystal surface is used to resonantly reflect the incoming light so that binding of the analytes to the surface creates detectable shift in the resonant reflection. Though using the resonant scattering, the method is conceptually different to those previously described as it uses single- wavelength light source (a laser) and strictly periodic pattern of the nano structures forming a photonic crystal surface. A common drawback of the described biochemosensing methods using the detection of diffuse forward or reflected scattered light is that, as mentioned above, despite high sensitivity these methods are not suitable for the applications in the (point- of-care) diagnostics in the clinical settings, where the analytes comprise human physiological fluids and frequently contain large amount of microscopic impurities. Another potential issue is the multiplexed detection - i.e. the detection of multiple analytes simultaneously, which is a necessity for clinical diagnostics. Finally, sensor chip structure, optical properties and on-chip fluids handling are the issues that are not addressed in their interdependency in the mentioned research-grade apparatuses.

In US 2007/0222995, an artifact is disclosed having a textured metal surface with nanometer scale features. The artifact is used to detect the presence or absence of analytes bound to the substrate, functionalized with biomolecules. In this disclosure, a light beam is incident on the back side of the artifact, propagates through the substrate, and is reflected by the metal/substrate interface before detection. The need for exciting the propagating SPR and the use of angular-resolved detection, makes the setup of this system very complex. Further, the requirement of using the evanescent field to launch the propagating SPR involves the need for the prism and/or total internal reflection configuration to be used, further reducing the simplicity of the setup.

US 2005/0265648 describes an evanescent wave sensor having a porous nanostructure layer bound to a surface of a substrate. The wave sensor is used to detect binding of an analyte to capture agents by evaluating changes in reflected light angle. Similarly to the disadvantages of US 2007/0222995, this system is very complex.

Another system for examining thin layer structures on a surface for differences in respect of optical thickness is disclosed in US Patent No. 6493097. With the present invention, better spectroscopic resolution can be achieved as it uses white light source (as opposed to the 'wavelength- scanned', i.e., discrete number of the incoming wavelengths), used in the above mentioned patent. However, as the system requires total internal or external reflection for illuminating the substrate, a complex setup is used. Summary of the invention

Accordingly, the present invention preferably seeks to mitigate, alleviate or eliminate one or more of the above-identified deficiencies in the art and disadvantages singly or in any combination and solves at least the above-mentioned problems by providing a system, and method according to the appended patent claims.

An object of the invention is to overcome the disadvantages and problems of prior art. Another object is to provide multiplexed biochemodetection of various analytes, including those present in human physiological fluids.

An idea according to an embodiment is to detect the change in electromagnetic radiation being scattered by nanoscopic structures that support localized surface plasmons affected by chemical or biological substances.

Another idea is to detect the electromagnetic radiation being scattered by the chemical or biological substances themselves.

Another idea is to detect nanoscopic structures-enhanced scattered radiation when the chemical or biological substances are bound to the substrate with or without fabricated nanoscopic structures that support localized surface plasmons.

A further idea according to an embodiment is to determine the presence and/or absence of chemical and/or biological substances by detecting a change in electromagnetic radiation, before and after the substrate has been exposed to the chemical and/or biological substances.

A yet further idea according to an embodiment is to provide a device and a method for detecting presence or absence of substances using a very simple setup, being cost-effective while still providing fast and accurate measurements.

According to an aspect of the invention, a system is provided. The system comprises a substrate having at least one portion being translucent to electromagnetic radiation, the portion having a first and a second surface on opposite sides of the substrate, a light source configured to emit electromagnetic radiation incident on the second surface of the substrate at a first fixed angle and propagating through the at least one portion of the substrate, a plurality of objects arranged on the first surface, the objects being configured to provide scattering of electromagnetic radiation, and wherein the plurality of objects comprises a plurality of structures for providing localized surface plasmonic resonance when the plurality of structures are exposed to electromagnetic radiation that is incident on the second surface of the substrate and transmitted through the substrate. The system further comprises a detector facing the second surface of the substrate and configured to receive electromagnetic radiation scattered by the plurality of objects at a second fixed angle, and configured to detect a change in scattering by analyzing spectral change.

According to a further aspect of the invention, a method for detecting the presence and/or absence of chemical or biological substances is provided. The method comprises the steps of providing a substrate having at least one portion being translucent to electromagnetic radiation, the portion having a first and a second surface on opposite sides of the substrate, arranging a light source configured to emit electromagnetic radiation incident on the second surface of the substrate at a first fixed angle and propagating through the at least one portion of the substrate, and arranging a plurality of objects on the first surface, wherein the plurality of objects comprises a plurality of structures for providing localized surface plasmonic resonance when the plurality of structures are exposed to electromagnetic radiation that is incident on the second surface of the substrate and transmitted through the substrate. The method further comprises the steps of providing scattered electromagnetic radiation by exposing the plurality of objects to electromagnetic radiation emitted from the light source, detecting the scattered electromagnetic radiation by means of a detector facing the second surface of the substrate at a second fixed angle and being configured to receive electromagnetic radiation scattered by the plurality of objects, and determining the presence and/or absence of a chemical or biological substance by means of detecting a change in scattering by analyzing spectral change.

Brief description of drawings

These and other aspects, features and advantages of which the invention is capable of will be apparent and elucidated from the following description of embodiments of the present invention, reference being made to the accompanying drawings, in which:

Fig. 1 shows a schematic diagram of a system according to an embodiment of the invention; Fig. 2a is a perspective view of a substrate according to an embodiment of a device;

Fig. 2b is a perspective view of a substrate according to an embodiment of a device;

Fig. 2c is a top view of the substrate shown in Fig. 2a; Fig. 2d is a top view of the substrate shown in Fig. 2b; Fig. 2e is a top view of a substrate according to an embodiment of a device;

Fig. 2f is a top view of a substrate according to an embodiment of a device;

Fig. 3a is a top view of a substrate according to an embodiment of a device;

Fig. 3b is a top view of a substrate according to an embodiment of a device; Fig. 3c is a top view of a substrate according to an embodiment of a device;

Fig. 3d is a top view of a substrate according to an embodiment of a device;

Fig. 4 shows a schematic drawing of a number of fibres for detecting electromagnetic radiation of a system according to an embodiment;

Fig. 5a-c shows a device according to an embodiment; Fig 6 is a flow chart of a method according to an embodiment;

Fig. 7a shows a schematic view of resonant diffuse scattering as detected by a photo detector;

Fig. 7b shows a schematic view of the changes in resonant diffuse scattering, as detected by a spectrophotometer; Fig. 8a shows the injection of an analyte fluid into a system according to an embodiment;

Fig. 8b shows a schematic drawing of the system shown in Fig. 6a; and

Fig. 9a-c shows the binding of chemical or biological substances to a substrate of a device according to an embodiment.

Detailed description of embodiments

Several embodiments of the present invention will be described in more detail below with reference to the accompanying drawings in order for those skilled in the art to be able to carry out the invention. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The embodiments do not limit the invention, but the invention is only limited by the appended patent claims. Furthermore, the terminology used in the detailed description of the particular embodiments illustrated in the accompanying drawings is not intended to be limiting of the invention.

Embodiments of the invention include a generalized method for the detection of the diffuse and/or specularly reflected scattered light from metallic or non-metallic objects, when the latter are positioned on the translucent support. In the case of non- metallic objects they can be protein aggregates, cells, bacteria, viruses or other entities, whose size is either comparable or smaller (and much smaller) than the wavelength of the incident light. These entities are adsorbed at the front side of surface of the translucent support either physically, i.e. without forming a chemical bond with the support or with the molecular capture agents, pre-deposited on the support (i.e. physisorption), or chemically, i.e. chemically binding to the molecular capture agents (i.e. chemisorption). In case of physisorption the entities might adsorb on the surface by capillary, van der Waals or other forces, thus being adsorbed non- selectively. In the case of chemisorption, binding to the capture agents, pre-deposited at the surface of the support, will provide a binding selectivity, so that only selected entities from the analyte fluid, complementary to the molecular capturing agents or prone to binding to them, will be left on the surface of the translucent support after the analyte fluid is evacuated or even in the presence of the analyte fluid.

Both illumination (transmitting light) and diffuse and/or specular scattering detection is done from the backside of the translucent support, so that neither excitation light nor emitted diffuse and/or specular scattered light is transmitted through the analyte fluid. With the proper selection of the excitation light high sensitivity can be reached for the label-free detection of the micro- and nanoscopic biological and chemical entities, thus providing simple and rapid means for the diagnostics of the human (or other) physiological fluids for the presence or absence of certain biological and chemical entities, including protein aggregates characteristic for neurodegenerative diseases, plurality of bacteria and viruses.

The diffuse and / or specularly reflected scattering both from biological or chemical micro- and nanoscopic entities and from metallic nano- and micro-objects is excited and detected with the same system, shown on Fig. 1. The apparatus consists of a light source 1, which can be either a source of monochromatic light (a laser, diode etc.) or a white-light source (a lamp, a diode etc.), covering a UV-visual-IR spectral range or parts of it. The light source might contain an exit slit. The light from the source is directed into the optical fibre 2, which is mounted directly at the exit of the light source. The optical fibre 2 transfers the excitation light from the light source 1 to the collimating lens 3. Collimated excitation light is then focused by the focusing lens 4 on the first surface of a translucent substrate 5, entering it from the backside. The possible simplification includes direct illumination of the translucent substrate from the light source 1, where all or some elements among 2, 3, 4, are excluded. A polarizer can be inserted between lenses 3 and 4 to polarize the excitation light. In case of the white-light excitation source, a monochromator can be as well inserted between lenses 3 and 4 to select specific excitation wavelength or a sequence of excitation wavelengths in the situation when monochromator comprises several filters or a moving mirror able to dynamically change the wavelength of the outcoming light. On the first surface, i.e. the front side of the translucent substrate 5, the excitation light is focused on a portion with fabricated nanostructures 6, functionalized with molecular capture agents. Directly transmitted light 7 is captured by appropriate filtering (not shown) to avoid the signal background with high brightness inside the apparatus due to multiple reflections/scattering. The diffuse scattered light 9 and/or the specularly scattered light 8 emitted towards the backside of the translucent substrate, are the detected signals in the apparatus. The diffuse scattered light 9 and/or the specularly scattered light 8 is further captured by the collimating lens 10 and forwarded to the beam- splitting mirror 11. A polarizing filter can be inserted between collimating lens 10 and beam- splitting mirror 11. Further, a diffraction grating can be inserted between collimating lens 10 and beam- splitting mirror 11 or instead of collimating lens 10, where the image of the first surface of the substrate is formed. The beam- splitting mirror 11 further separates the collected and collimated diffuse scattered light in two portions, one of which is directed to a photodetector, such as a CCD 12 and another portion to the focusing lens 13. The focusing lens 13 focuses light onto a fibre bundle 14, where each fibre corresponds to the capture agents-functionalized portion on the first surface of the translucent support 5. Further the light in each fibre in the bundle is directed to the spectrophotometer 15 or an array of spectrophotometers. Possible simplification might include direct collection of the diffuse and/or reflected scattered light 9 by the CCD, positioned in the place of the collimating lens 10. The sequence of optical elements 10-15 can be positioned to capture specularly scattered light 8, simultaneously or independently of capturing the diffuse scattered light 9.

The incident light may be incident on the second surface of the substrate at a first fixed angle, and the detector may receive scattered electromagnetic radiation at a second fixed angle.

In an embodiment, the emitted electromagnetic radiation is incident on the second surface at a first angle with respect to the normal of the substrate, and the detector receives diffusely scattered electromagnetic radiation at a second angle.

In an embodiment, the emitted electromagnetic radiation is incident on the second surface at a first angle with respect to the normal of the substrate, and the detector receives specularly scattered electromagnetic radiation at the first angle. In an embodiment, the first angle is 0°, and in another embodiment, the first angle is a Brewster angle.

In an embodiment, the emitted electromagnetic radiation is p-polarized. Special case of the detection of the specularly scattered light 8 is when the illumination of the translucent substrate 5 is done at a Brewster angle (also called

'polarization angle', i.e. the angle of incidence at which light with defined polarization is transmitted without losses (reflections) through the surface). For example, if the incoming light is p-polarized and is coming from the air at the backside of the translucent substrate 5, it will be transmitted through the translucent support (glass) without losses (reflections) if incident at the angle of approximately 56 deg. to the normal. It will be further delivered to the fabricated nanostructures 6, which in turn will scatter it in the specular direction 8. This is advantageous in that all emitted p-polarized electromagnetic radiation incident on the second surface is transmitted without losses to the first surface, where it excites localized surface plasmon resonance in the nanostructures. Further, background noise derived from scattered light that is not emitted by the nanoscopic particles is reduced. Such background radiation also includes stray radiation in the surrounding. This advantage is due to the fact that the incident p- polarized light is not scattered at the air-glass interface, i.e. the second surface. In an embodiment, the plurality of objects comprises biological and / or chemical nanoscopic and / or microscopic entities (bacteria, cells and similar) that can scatter electromagnetic radiation when bound on the second surface of the substrate. This is advantageous in that direct detection of the analyte is possible by detecting scattered electromagnetic radiation (Rayleigh or Mie scattering).

In an embodiment, the plurality of objects comprises biological and / or chemical substances that do not sufficiently scatter provided electromagnetic radiation by Rayleigh or Mie scattering when bound to the second surface of the substrate, so the plurality of nanostructures are bound further to the biological and / or chemical substances for providing localized surface plasmonic resonance when the plurality of nanostructures are exposed to electromagnetic radiation that is incident on the second surface of the substrate and transmitted through the substrate. This is advantageous in that a native Rayleigh or Mie scattering can be amplified and a further amount of scattered light can be used to detect the presence of the biological and / or chemical substances.

In an embodiment, the plurality of objects comprises plurality of structures for providing localized surface plasmonic resonance when the plurality of structures are exposed to electromagnetic radiation that is incident on the second surface of the substrate and transmitted through the substrate. This is advantageous in that the character of scattered light (its resonance / color) can be changed when biological and/or chemical substances are adsorbed on the surface of the plurality of structures and such change can be monitored to detect the presence and/or absence of the biological and/or chemical substances in the investigated sample fluid.

In an embodiment, the plurality of objects comprises a plurality of structures for providing localized surface plasmonic resonance when the plurality of structures are exposed to electromagnetic radiation that is incident on the second surface of the substrate and transmitted through the substrate. When the detected biological and / or chemical substances are bound to the the plurality of structures, the plurality of structures are bound further to the biological and / or chemical substances for providing localized surface plasmonic resonance when the plurality of structures are exposed to electromagnetic radiation that is incident on the second surface of the substrate and transmitted through the substrate. This is advantageous in that the character of scattered light (its resonance/color) can be changed when biological and/or chemical substances are adsorbed on the surface of the plurality of structures and such change can be monitored to detect the presence and/or absence of the biological and/or chemical substances in the investigated sample fluid. In the situation when LSP metal nano structures are employed as the scattering entities, they are fabricated on the first surface of the translucent substrate 5. In particular, metals for the nanostructures are e.g. gold (Au), silver (Ag), copper (Cu), platinum (Pt), palladium (Pd), nickel (Ni), iron (Fe), cobalt (Co), and aluminium (Al). Various combinations of these metals can be employed. The combination of these metals with their oxides (native or specially deposited) can be used to produce layered or core-shell type of nanostructures. The combination of these metals with other oxides, in particular silicon dioxide (silica, SiO2) and titanium dioxide (titania, TiO2), can be used to produce complex metal-dielectric nanostructures.

In an embodiment, the dimension of the structures is less than one micron. Hence, the dimensions of the structures are sub-wavelength, resulting in a Mie or Rayleigh scattering profile.

The size of these nanostructures can be ranging between lnm and 1000 nm. Typically, the size of the nanoparticles (average diameter of the outer circumference) is between 50 nm and 500 nm. The translucent substrate 5 can be pre-covered with thin metal film (like gold or others) while still retaining its (partial) transparency prior to the fabrication of the nanoparticles on its front surface.

Nanoparticles to be bound to the detected biological and / or chemical substances may be produced by a plurality of methods. These include, but are not limited to, the solution-based chemical synthesis methods, lithographic methods followed by the particles lift off from the substrate. The nanoparticles are solution- delivered (injected) after nanoscopic and microscopic biological entities are adsorbed on the first surface.

Nanoparticles on the support are fabricated by the plurality of methods. These include, but are not limited to, colloidal self-assembly-based templating methods (i.e., colloidal lithography, nanosphere lithography, hole-mask colloidal lithography and similar), photolithography, electron-beam lithography combined with nanoimprint lithography, focused ion beam etching.

The shape of individual nanostructures, fabricated from the mentioned above materials, can be, but not limited to, spherical, disk-like, cone-like, trapezoidal etc. Special shape can be used to provide the property of directional diffuse scattering towards the second surface, i.e. the backside of the translucent substrate, where nanostructures are fabricated. In the latter case the diffuse scattering towards to backside of the support dominates the total diffuse scattering, produced by the nanostructures on the first surface, i.e. the front side of the substrate. Nanostructures can have strongly anisotropic shape - for example, be of elliptical shape, with the longitudinal (long) axis of all particles oriented identically on the whole surface of the support. In case of anisotropic nanoparticles shape, the illumination of the nanoparticles with the polarized excitation light when the polarizing filter is inserted between lenses 3 and 4 in Fig. 1 is done in a way that the polarization of the grazing excitation light coincides with the orientation of the longitudinal axis of the anisotropic nanoparticles. Consequently, the detected reflected diffuse and/or specular scattered light can be filtered by the polarizer, oriented perpendicularly to the orientation of the longitudinal axis of the anisotropic nanoparticles (cross-polarization scheme) to reduce the background noise.

Alternatively, the detected reflected diffuse and/or specular scattered light can be filtered by the polarizer, and thus oriented to match the orientation of the longitudinal axis of the anisotropic nanoparticles.

The details of the fabricated nanoparticles arrangement on the front side of the translucent substrate are shown in Fig. 2a-f. In the following, nanoparticles are schematically represented by the disk- shaped structures. Their diameter is shown not to scale with the thickness or lateral dimensions of the substrate. In Fig. 2a a schematic side view of the disordered arrangement of nanoparticles 17, fabricated on the front side of the translucent substrate 16, is shown. Ordered arrangement of nanoparticles 19, fabricated on the front side of the translucent substrate 18, is shown in Fig. 2b. Fig. 2c schematically shows the top view of the disordered arrangement of the nanostructures 17 on the substrate 16. Fig. 2d schematically shows the top view of the ordered arrangement of the nanostructures 19 on the substrate 18. Finally, nanostructures on the translucent substrate might be arranged in the N x N array format. Fig. 2e shows arrangements of disordered nanoparticles 17, fabricated in the 3 x 3 array format. Fig. 2f shows arrangements of ordered nanoparticles 19, fabricated in the 3 x 3 array format.

In an embodiment, the first surface of the substrate is functionalized by molecular capture agents. This implies that the first surface has an ability, or functionality, to bind analyte substances, which is advantageous in that a specific chemical or biological substance may be bound to the first surface.

In an embodiment, the first surface has fabricated nanoparticles that support localized surface plasmons. The nanoparticles are further functionalized by molecular capture agents.

In an embodiment, the substrate comprises a plurality of portions, each first surfaces of the portions are functionalized by different molecular capture agents. This is advantageous in that different specific chemical or biological substances are bound the first surfaces of specific portions. Thus, a single substrate may be used to detect the presence and/or absence of several different chemical or biological substances.

In an embodiment, the substrate comprises a further transluscent portion, the first surface of the further portion not being functionalized by molecular capture agents. Hence, the further portion may be used as a reference of background noise and nonspecific binding of chemical or biological substances.

The functionalization of the fabricated nanoparticles, and/or the nanoparticles further delivered after biological and/or chemical substances to be detected are bound, is done by molecular capture agents - i.e., molecular biological or chemical species, which have specific affinity to the target species of the analyte solution/fluid. The molecular capture agents and the target species of the analyte solution can be any biological or chemical entities, including, but not limited to, nucleic acids (RNA, DNA), proteins, polypeptides, oligonucleotides, chemical compounds, drugs, bacteria, viruses, etc. The molecular capture agents can be directly bound to the nanostructures by physisorption or chemisorption, or bound through the linking chemistry, employing, for example, functionalized thiol monolayers, whose functional groups are activated by intermediate chemical agents, later to bind, for example, specific antibodies, which have affinity towards antigens, present in the analyte solution/fluid. Functionalization of the nanostructures arrays is done in solution with subsequent drying so functionalized patches in N x N format are present on the surface of the translucent substrate. Usually one spot is left unfunctionalized for the reference measurements of the unspecific binding of the target analyte species to the surface of the unfunctionalized (i.e., not carrying any molecular capture agents) nanoparticles. Fig. 3a schematically shows the functionalization of the disordered arrangement of nanostructures in 3 x 3 array format - the solution of molecular capture agents 22 is pipetted onto the first surface of the translucent substrate 23, covered by the nanoparticles 24. The (3;3) spot 25 is left clean (unfunctionalized) for the reference measurements. Fig. 3b schematically shows the functionalization of the ordered arrangement of nanostructures 24 in a 3 x 3 array format, leaving the spot (3;3) unfunctionalized. Fig. 3c schematically shows the disordered arrangement of nanostructures 24 in a 3 x 3 array format, in turn functionalized in a 3 x 3 arrays format, leaving the spot (3; 3) unfunctionalized. Fig. 3d schematically shows the ordered arrangement of nanostructures 24 in a 3 x 3 array format, functionalized in a 3 x 3 arrays format, leaving the spot (3; 3) unfunctionalized.

In case of using the optical fibres bundle for the detection of the reflected diffuse scattering from the backside of the translucent substrate as shown in Fig.l, the fibres in the bundle are arranged in accordance to the functionalization array format of the substrate. For example, if the substrate with nanostructures is functionalized in the 3 x 3 array format, the fibre bundle consists correspondingly of 9 fibres, arranged in the 3 x 3 array format, identical to the functionalization array. This principle is schematically illustrated in Fig. 4, where individual optical fibres 26, coupled to the optical detector (spectrometer) 27, are fixed on the supporting plate 28 in 3 x 3 array format.

In an embodiment, the system further comprises a transporting means having a fluid inlet, and a fluid outlet connected to the inlet by means of a flowing area, wherein the transporting means is arranged adjacent to the first surface of the substrate such that, when a fluid is present in the flowing area, the first surface is exposed to the fluid.

In an embodiment, the fluid inlet of the transporting means extends from an upper part of the transporting means to a lower part of the transporting means, the fluid outlet of the transporting means extends from the lower part to the upper part, and the flowing area is arranged at the lower part. This is advantageous in that the transporting means is a robust construction, easily manufactured by standard moulding techniques.

In an embodiment, the flowing area of the transporting means comprises a plurality of fluid channels, which is advantageous in a homogenous flow through the complete flowing area.

In an embodiment, the fluid outlet is connected to a pump means for providing a fluid flow through the flowing area, which is advantageous in that the fluid of chemical or biological substance is continuously flown through the flowing area. In an embodiment, the detector comprises a photo detector and/or a spectrometer. Hence, standard equipment is used to detect the scattered light.

In an embodiment, the electromagnetic radiation emitted from the light source is guided by means of optical fibres to the second surface of the substrate. Hence, the incident light will be collected and a less amount of light will be lost.

The analytical device according to one embodiment consists of several components, whose schematic assembly is shown in Fig. 5a-c. In Fig. 5a the translucent substrate 29, on which nanoparticles are fabricated and appropriately functionalized, is attached to a non-translucent frame 30 with the central opening on which the translucent substrate is residing so that its backside is covering the opening. Further, as shown on Fig. 5b, the non-translucent frame 30 with the fixed translucent substrate has an opening at the backside so the excitation light 32 can reach the backside of the translucent substrate. On the front side of the non-translucent frame with the fixed translucent substrate a soft or rigid stamp 33 acting as a transporting means is tightly attached, containing the system of channels for the analyte fluid to reach the front side of the translucent substrate with appropriately functionalized nanostructures. The stamp contains an inlet 34 and an outlet 35 to deliver and evacuate the analyte fluid. For example, the material of the stamp can be polydimethylsiloxane (PDMS), which will provide biocompatibility, possibility to sterilize the stamp, ease of mass-fabrication, and assure the tight leak-proof contact of the stamp to the front surface of the translucent substrate. The inner structure of the fluidic channels of the stamp can be similar to the one schematically depicted in Fig. 5c. There the channels are etched in the body of the stamp 36, the entrance into the fluidic system 38 delivers the incoming liquid further into the system of ramifying channels 40 to bring it to the flowing area or central chamber 41, which is positioned directly over the arrangement of the functionalized nanoparticles at the front side of the translucent substrate. After getting in contact with functionalized nanostructures, the liquid is directed into the exit of the channels system 39 and is evacuated through the outlet 37.

The system of ramifying channels inside the stamp provide the possibility to homogeneously deliver the analyte fluid in contact with the whole arrangement of nanostructures on the surface of the translucent substrate.

Upon the delivery of the analyte fluid to the functionalized nanostructures, their reflected diffuse and/or specular scattering will be changed depending on whether the target analyte species bind to the molecular capture agents. The reflected diffuse and/or specular scattering of the nanoparticles can be characterized by the color and intensity, if the CCD camera does the detection, or a spectrum, if the detection is done by a fibre connected to the spectrometer. Consequently, upon the binding of the target analyte species the color and the intensity of the reflected scattered light will be changed, if detected by the CCD camera, or the spectrum will be shifted towards the longer (or shorter) wavelengths region. With reference to Fig. 6, embodiments of a method 100 for determining the presence and/or absence of chemical and/or biological substances will be described.

In an embodiment, the method 100 comprises providing 110 a substrate having at least one portion being translucent to electromagnetic radiation, the portion having a first and a second surface arranged on opposite sides of the substrate. The method further comprises arranging 120 a light source configured to emit electromagnetic radiation incident on the second surface of the substrate and propagating through the at least one portion of the substrate. Moreover, the method comprises arranging 130 a plurality of objects arranged on the first surface, and providing 140 scattered electromagnetic radiation by exposing the plurality of objects to electromagnetic radiation emitted from the light source. The method also comprises detecting 150 the scattered electromagnetic radiation by means of a detector facing the second surface of the substrate and configured to receive electromagnetic radiation scattered by the plurality of objects, and determining 160 the presence and/or absence of chemical or biological substances by means of the detected scattered electromagnetic radiation. In an embodiment, the method further comprises the step of functionalizing

112 the first surface with molecular capture agents. Moreover, the step of arranging 130 a plurality of objects comprises providing a plurality of biological and/or chemical entities, and the step of determining 160 the presence and/or absence of chemical or biological substances comprises comparing detected electromagnetic radiation 122 before binding of the entities to the first surface, with detected electromagnetic radiation 150 after binding of the objects to the first surface.

Hence, the change in scattered light may be used as a detection method if light- scattering nanoscopic or microscopic biological and/or chemical entities are adsorbed on the first surface, functionalized with capture molecules.

In an embodiment, the step of arranging 130 a plurality of objects comprises providing 132 a plurality of structures, functionalizing 134 the structures with molecular capture agents, and further providing 136 a plurality of biological and/or chemical entities adsorbed by the plurality of structures. The step of providing 140 scattered electromagnetic radiation further comprises exciting 142 localized surface plasmonic resonance by means of the plurality of structures, and the step of determining 160 the presence and/or absence of chemical or biological substances further comprises comparing detected electromagnetic radiation 135, 150 before and after the plurality of functionalized structures is exposed to the biological and/or chemical entities. In an embodiment, the method further comprises the step of arranging 152 a plurality of nanoparticles functionalized by means of molecular capture agents. Further, the step of providing 140 scattered electromagnetic radiation further comprises exciting 154 localized surface plasmonic resonance by means of the plurality of nanoparticles, and the step of determining 160 the presence and/or absence of chemical or biological substances further comprises comparing detected electromagnetic radiation 122, 135,

150, 154 before and after the plurality of functionalized nanoparticles is adsorbed on the biological and/or chemical entities.

In a yet further embodiment, the method further comprises the step of injecting a contrast agent adapted to bind to the plurality of a specific biological and/or chemical entity already bound to the molecular capture agent, wherein the contrast agent is an antibody, a pre-functionalized colloidal particle, or any other entity that allows binding to the specific biological and/or chemical entity.

The detection strategy is schematically presented in Fig. 7a-b. In Fig. 7a, which models the detection by the CCD, exemplary arrangement of the nanoparticles on the translucent substrate 42 is functionalized by the molecular capture agents in the 3 x 3 array format. Consequently, the reflected diffuse and/or specular scattering from the functionalized areas will be different from the rest of the surface. In case when the whole surface of the substrate is covered by the arrangement of the nanoparticles, the background extraction is done to eliminate the high background signal, coming from the non-functionalized areas of the surface with nanostructures, as one spot in N x N array format is always left unfunctionalized, thus providing the constant reference of reflected diffuse and/or specular scattering from unfunctionalized nano structures. In case of nanostructures arrangement fabrication already in N x N array format, the unfunctionalized surface of the substrate will give only small amount of background signal as there are no scattering entities (nanostructures) present on those areas. Again, one spot in the N x N array will be left unfunctionalized to provide constant referencing of the unfunctionalized nanoparticles reflected diffuse and/or specular scattering and unspecific binding of the target analyte species. In Fig. 7a, the scattering spots 43, arranged in 3 x 3 array format, are shown as detected by the CCD camera. Upon the target analyte binding, the color and the intensity of (some) spots will be changed, which, compared to the rest of the spots, will reveal which molecular capture agent was involved in binding, thus defining the nature of the target analyte. In case of the use of dynamic filtering of the excitation monochromatic light so that the light of only one wavelength will irradiate the nanostructures at a time, a sequence of CCD imaged can be recorded, each corresponding to a single irradiation wavelength. Such sequence represents a spectrum (i.e., each wavelength corresponds to the reflected diffuse and/or specular scattering intensity), when properly analysed. Thus the change in reflected diffuse and/or specular scattering upon the target analyte binding to the molecular capture agents can be analysed in terms of spectral change - i.e., by comparison of spectrum before and after the binding event. In the similar way, such binding essay in array format can be done by using direct detection of the spectrum by fibre-coupled spectrometer. If fibres are arranged in the imaging plane of the reflected diffuse and/or specular scattering light in the bundle, reproducing the array format of the functionalized chip, each spot of the chip can be identified by its respective spectrum, which is schematically shown in Fig. 7b. Each functionalized spot in the 3 x 3 array format is characterized by the reflected diffuse and/or specular scattering spectrum before binding of any species from the analyte solution 44, and after such binding 45. Most of the spots will only show certain unspecific binding, whereas the spot functionalized with the molecular capture agent that has particular affinity to the target analyte will show the most pronounced change in the reflected diffuse and/or specular scattering spectrum. In Fig. 7b such spot is the spot (1; 3) of the array. Consequently, the identification of the spot leads to the identification of the capture agent and thus the identification of the target analyte with particular affinity to the capture agent.

The assembly and operation of the analytical device is depicted in Fig. 8a. The rigid frame 46 carrying the translucent substrate and the soft or rigid fluidic stamp 48 is enclosed in the rigid housing 47. The entrance to the fhiidic system 51 is tightly connected to the entrance filter 49. The entrance filter provides initial filtering of the analyte fluid from the macroscopic inclusions/impurities. The analyte fluid 53 is injected to the entrance filter by the standard pipette 54. Further on, the analyte liquid is delivered to the functionalized nanostructures chamber inside the fluidic system. The excess of the analyte fluid is later evacuated through the exit 52, tightly connected to the exit filter/stopper 50. The purpose of the exit filter/stopper is to prevent the excess of the analyte fluid to enter the fluidic system of the apparatus, partially depicted in Fig. 8b. There upon the locking of the entirety of the assembled analytical device into the apparatus by the lock 55, the fluidic system of the analytical device gets tightly connected to the fluids pumping system of the apparatus, consisting of the connector tubing 56, directly attached to the exit of the fluidic system of the analytical device, and (peristaltic or any other) pump 57, providing the constant pressure gradient in the fluidic system of the analytical device and, in particular, in the stopping filter 50 at the exit of the fluidic system.

Example 1 - Detection of the bacteria in the human physiological fluid sample (blood, saliva, urine, lungs fluid etc.) by the direct detection of their reflected diffuse and/or specular scattering. This example comprises the use of a system according to one embodiment. The front surface of the translucent substrate is covered with poly(ethylene glycol) (PEG) silane (or a mixture of carboxyl-PEG silane and PEG silane) and functionalized with bio tin, which further binds streptavidin and biotinilated capture antibodies. The latter act as molecular capture agents for the antigens, which are present on the outer membrane of the target bacteria or virus. The target analyte fluid is introduced into the analytical device and delivered to the functionalized surface of the translucent substrate inside the fluidic system. The light from the light source is directed to the backside of the translucent substrate and reflected diffuse and/or specular scattered light from bacteria, bound at the surface, is collected. The detection of bacteria presence is done by comparison of the collected reflected diffuse and/or specular scattering signal from the backside of the translucent substrate before and after the binding event.

As a further extension, additional injection of antibodies-functionalized nanoparticles can be done, which bind to the immobilized bacteria (viruses) and induce strong reflected diffuse and/or specular scattering. Example 2 - Detection of the specific antigens in human physiological fluid sample

(blood, saliva, urine, lungs fluid etc.).

This example comprises the use of a system of Example 1, and a similar detection method. Depicted in Fig. 9a-c, the fabricated nanoparticles 59 on the translucent substrate 58 are made of Au and the translucent substrate is silica (SiO2).

The gold nanostructures are functionalized with highly specific biorecognition molecules (capture agents) such as antibodies. Antibody could be functionalized by any of the following methods:

(i) Bare gold nanostructures are functionalized by 16-mercaptohexadecanoic acid or any thiolated carboxylic acid or a mixture of any thiolated carboxylic acid and any thiolated polyethylene glycol) (PEG), e.g., 16-mercaptohexadecanoic-acid and Methylene - glycol-mono- 11 -mercapto-undecyl 61.

(ii) Antibodies 62 amine-couple to the surface modified according to (i).

(iii) Protein A or protein G 63 amine-couples to the surface modified according to (i) and is used to bind the capturing agent (antibodies). The method provides proper orientation of the antibodies to capture the target antigen.

(iv) Surface modified with any homofunctinal thiolated alkanes or a mixture of any homofunctional thiolated alkanes and thiolated polyethylene glycol. Fab fragments of the capture antibodies are bund to the functionalized surface by the thiol disulphide exchange.

(v) Fab fragments 64 of antibodies are used and are functionalized on the fabricated gold nanostructures directly or together with a mixture of thiolated PEG.

In the described cases (i) - (v) the front surface of the translucent substrate (silica), that is not occupied by the fabricated nanostructures, is protected with non- fouling molecules such as:

(a) Silanized PEG 60 with molecular weight between 400 Da and 3000 Da.

(b) Silanized mPEG-amine.

(c) Silanized mPEG urea. (d) Poly(L-lysine)-g- polyethylene glycol) (PLL-g-PEG). Any combination of (i)-(v) and (a)-(d) can be used.

Example 3 - Detection of the bacteria in the human physiological fluid sample (blood, saliva, urine, lungs fluid etc.) by binding of the bacteria to the plasmonic nanostructures, fabricated on the front side of the translucent substrate. The plasmonic nanostructures have the LSPR in the UV-visible-IR range and are functionalized by the molecular capture agents as described in Example 2.

Upon the injection of the analyte fluid, containing target bacteria, the latter are selectively bound to the functionalized nanostructures. The detection is performed by comparison of the reflected diffuse and/or specular scattering from the functionalized nanostructures before and after binding event by direct monitoring of the scattered light (with a CCD camera) from the N x N array of functionalized portions or by collecting the spectra from the N x N array of functionalized portions.

It will be appreciated that embodiments of the invention described in the foregoing are susceptible to being modified without departing from the scope of the invention as defined by the appended patent claims.