WO2022207794A1 - Cuvette with biorecognition elements and method for measuring refractive index in a spectrophotometer - Google Patents

Abstract

There is presented a cuvette for use in determining a refractive index of a matter sample in a spectrophotometer, comprising a container for holding the matter sample, the container having an entry window that allows input radiation to reach the matter sample, the container furthermore having an exit window that allows a part of the input radiation to exit the container part, the entry window and the exit window defining a radiation path, a photonic crystal rigidly attached to a side of the container or integrally formed in a side of the container and arranged in the radiation path, the photonic crystal having a grating part causing a reflectance spectrum of the photonic crystal to exhibit a resonance, wherein the cuvette further comprises a plurality of biorecognition elements being immobilized on and/or at the photonic crystal.

Description

CUVETTE WITH BIORECOGNITION ELEMENTS AND METHOD FOR MEASURING REFRACTIVE INDEX IN A SPECTROPHOTOMETER

FIELD OF THE INVENTION

The present invention relates to apparatus for facilitating determining an optical characteristic of matter sample, and more particularly a cuvette with a photonic crystal and biorecognition elements, and furthermore a corresponding method for determining an optical characteristic of a matter sample and a method for preparing a cuvette.

BACKGROUND OF THE INVENTION

Spectrophotometers are ubiquitous in science, technology, education and medicine, and can generally measure absorbance of for instance a liquid as a function of wavelength. The liquid is contained in a so-called cuvette, which can be made for instance from polymer, glass or quartz. The cuvette is placed into a slot in the spectrophotometer. By correlating the resulting absorption spectrum to a known calibration curve for a specific compound, the concentration of that compound can easily be calculated using Lambert-Beers law. Cuvettes and spectrophotometers can be considered widespread, accessible, simple and cost-efficient.

However, for non-absorbing compounds, spectrophotometers are not usually suitable for determining concentrations. An alternative method to determining concentrations uses a refracto meter, which measures refractive index of the liquid. However, such equipment can be expensive and is much less common. In particular, refracto meters that can measure at multiple wavelengths are prohibitively expensive. Since concentration of a specific compound in e.g. a liquid sample is often correlated with the index of refraction of the liquid sample including the compound, a calibration for that specific compound in the liquid can be used to obtain the concentration based on a measurement of the index of refraction.

Additionally, there is generally a risk of interference. Even if interference might be addressed in alternative methods, such as Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance (QCM), these methods rely on intricate and costly technology.

Therefore, there is a need for an improved device and method, such as a simple device and method being simple, cost-effective and/or enabling reducing, minimizing or eliminating a risk of interference, such as a device and method enabling detection and/or obtaining concentration of a specific compound in a simple and/or cost-efficient manner. SUMMARY OF THE INVENTION

An object of the present invention is to provide an improved device, such as a cuvette, and method overcoming at least some of the disadvantages of known devices and methods, and in particular a device and method enabling in a simple and/or cost-efficient manner detection and/or and obtaining concentration of a specific compound, while reducing, minimizing or eliminating a risk of interference.

According to a first aspect of the invention, there is presented a cuvette for use in determining a refractive index of a matter sample in a spectrophotometer, comprising a. a container for holding the matter sample, such as the container being for holding a fluid (such as gas or liquid) sample, the container having i. an entry window that allows input radiation to reach the matter sample, the container furthermore having ii. an exit window that allows a part of the input radiation to exit the container part, the entry window and the exit window defining a radiation path, b. a photonic crystal rigidly attached to a side of the container or integrally formed in a side of the container and arranged in the radiation path, such as the photonic crystal being arranged in the radiation path to enable the input radiation to interact with the photonic crystal and the matter sample, the photonic crystal having i. a grating part causing a reflectance spectrum of the photonic crystal to exhibit a resonance, such as the grating part causing a resonance wavelength of the photonic crystal to shift dependent on the matter sample, wherein the cuvette further comprises c. a plurality of biorecognition elements being immobilized on and/or at the photonic crystal.

It has been found by the present inventors that a simple and cost-effective device for enabling reducing, minimizing or eliminating interference may be provided by a cuvette according to the first aspect. It may in particular be considered an insight of the present inventors, that providing a cuvette (which can be considered a simple and cost-efficient technology) with a photonic crystal (enabling determining refractive index and hence in turn enabling determining a concentration of non-absorbing compounds) may additionally be provided with a plurality of biorecognition elements being immobilized on and/or at the photonic crystal, which serve to yield specificity and reduce, minimize or eliminate a risk of interference. Cuvettes have traditionally been employed in spectrophotometers for the purpose of performing assays in bulk solution, wherein bulk properties, such as bulk absorption in a volume, such as in a liquid, in the cuvette has been measured. The invention may render it possible to utilize existing equipment, such as a spectrophotometer, which is traditionally used for investigating bulk properties, and use it for surface sensitive measurements, such as specific surface sensitive measurements. Thus, the invention may enable existing equipment, which has traditionally been employed for doing bulk measurements, such as a spectrophotometer, to do surface sensitive measurements, such as specific surface sensitive measurements, and optionally double function both as bulk measuring equipment and (specific) surface sensitive equipment. The present invention may thus effectively lower the bar (such as the initial costs, in particular for the numerous laboratories already having a spectrophotometer), costs and/or resources required for doing surface sensitive measurements, such as specific surface sensitive measurements.

Another possible advantage of the invention may be that it enables reducing the cost of preparing and/or conducting measurements. More particularly, it may be relatively costly and/or resource demanding to provide the components, such as, e.g., molecules, for doing bulk solution measurements. The present invention may be seen as advantageous in that it enables performing a surface sensitive measurement, such as a specific surface sensitive measurement, in a cuvette, which may require less components (such as less components for surface functionalization compared to an amount of components for doing bulk analysis), and which may be utilized in spectrophotometers (which may be considered as widespread and accessible in, e.g., a high number of laboratories, and which may be considered to be relatively simple and/or cost-effective). The present invention may thus effectively lower the costs and/or resources required for doing cuvette measurements in a spectrophotometer.

A cuvette as such (without the biorecognition elements) may be provided, prepared and operated as described in WO 2017/129196 Al, which is hereby included by reference in entirety (and the description of the cuvette in the first aspect and Figs. 1-2 and corresponding description, the method of manufacture in the sixth aspect, and Figs. 9-10 and the corresponding description, and the methods of determining, respectively, refractive index and a concentration of a material in a matter sample in the third aspect, Fig. 8 and the corresponding description and the fourth aspect).

The cuvette (and corresponding spectrophotometer and method for determining an optical characteristic) may be applicable within, e.g., any one or more of the fields of biotech, pharma, clinical, education, academia/universities, research and development (R8iD), government, environment and food and beverage (F8iB). A 'cuvette' may be understood to be a small (e.g., wherein the container has a volume of equal to or less than 100 mL, such as equal to or smaller than 10 mL, such as equal to or smaller than 5 mL, such as equal to or smaller than 2 mL, such as equal to or smaller than 1 mL), optionally tube-like, vessel with a container, optionally having with straight sides and/or a circular or square cross section. It may be sealed at one end, and made of a clear, transparent material such as plastic, glass, or fused quartz or at last comprise regions capable of functioning as entry- and exit-windows. Cuvettes may be designed to hold samples for spectroscopic measurement, where a beam of light is passed through the sample within the cuvette to measure the absorbance, transmittance, fluorescence intensity, fluorescence polarization, or fluorescence lifetime of the sample. This measurement is done with a spectrophotometer. In some embodiments, the cuvette has a footprint of 12.5 mm x 12.5 mm, such as wherein the footprint of the cuvette is quadratic. In some embodiments, the length of the optical path from entry window to exit window is 10 mm.

It may generally be understood that the cuvette may have any shape, such as the container defining an axis and wherein the shape of the cuvette in a cross-sectional plane around this axis being any one of polygonal, such as quadrilateral, such as a parallelogram- or trapezium-shaped or rectangular, such as square-shaped, or ellipsoidal, such as circular. In embodiments, the cuvette has a set of parallel sides (such as the entry- and exit-windows being on opposing and parallel sides), which may be advantageous for enabling normal or near-normal incidence, such as for enabling transmission measurements through the photonic crystal between the entry- and exit-windows, where light traverses each interface (at the entry-window and the exit-window) at normal or near-normal incidence. It may furthermore be an advantage, that the light need not traverse a liquid-gas interface (such as the light can instead traverse, both upon entry and exit, a solid-liquid or solid-gas, interface), which may be beneficial for the transmission at said interfaces.

In embodiments, the cuvette is arranged so that upon orientating the cuvette so as to enable the container to hold (such as hold by gravity) a maximum amount of liquid, the optical path is substantially horizontal (such as horizontal +/-30⁰, such as horizontal +/-20⁰, such as horizontal +/-10⁰), such as horizontal. An advantage of this may be that it enables measurements to be carried out with the often-used horizontally aligned optics (e.g., in spectrophotometers) while the orientation of the cuvette is optimal for holding a maximum amount of liquid.

The optical path may be distanced from the bottom, such as a local or global minimum in said container when oriented so as to enable the container to hold (such as hold by gravity) a maximum amount of liquid, such as distanced at least 0.1 mm, such as at least 1 mm, such as at least 2 mm, such as at least 5 mm. A possible advantage of this may be that a result of settling (the falling of suspended particles through a liquid), such as sedimentation (the final result of the settling process), which may arrive at said bottom, does not end up being intersected by the optical path, where it could interfere with measurements.

In embodiments, the cuvette is rectangular, such as square-shaped. A shape may in this context be defined in a cross-sectional plane being orthogonal to an axis of a cavity for holding a sample for spectroscopic measurements, such as said cross-sectional plane being intersected by and/or comprising the optical path (such as, in everyday language, said shape may be the shape as seen "from above" during normal use).

In embodiments, the cuvette shape is circular, which may be advantageous due to the circular symmetry, which may render the operation of the cuvette robust (which may alternatively be referred to as fool proof) with respect to angular orientation. It is generally understood that any cuvette, including ellipsoidal and circular cuvettes, has an entry window and an exit window and one or more sides, at least due to different parts of the cuvette being at least conceptually divided into these parts.

It may be understood that the 'refractive index of a matter sample' is in the present context the refractive index at (the surface of) the photonic crystal. More particularly, the 'refractive index of a matter sample' may be understood to be the 'surface refractive index' (such as the surface refractive index at the surface of the photonic crystal at the photonic crystal- container interface). The 'surface refractive index' may be understood to be the refractive index close to the surface of the photonic crystal inside the cuvette. The 'surface refractive index' may be understood to be the refractive index (primarily) within 1 evanescent decay length (with respect to the wavelength of the probing light) from the surface. The 'surface refractive index' may be understood to be the refractive index within a distance from the photonic crystal surface (and into the container) of equal to or less than 1000 nm, such as equal to or less than 700 nm, such a equal to or less than 600 nm, such as equal to or less than 500 nm, such as equal to or less than 400 nm, such as equal to or less than 200 nm, such as equal to or less than 100 nm, such as equal to or less than 50 nm, such as equal to or less than 10 nm (depending on the design of the photonic crystal). In the case of a homogenous sample with no surface layers, the surface refractive index will equal the bulk refractive index of the sample. An advantage of the 'refractive index of a matter sample' in the present context being the refractive index at (the surface of) the photonic crystal may be that it enables that the biorecognition elements (BREs) being immobilized on and/or at the photonic crystal to bind complementary compounds - if present in the matter sample - which will change the refractive index on or at the photonic crystal, which is measured and which in turn enables detection of or determining concentration of the complementary compound. Thus, even if there are other compounds in the matter sample, which could affect the refractive index at the photonic crystal, they will not yield a (false positive) signal, because they will not be bound by the biorecognition elements and immobilized on or at the photonic crystal. Advantageously, the cuvette may allow for optimization of the photonic crystal to minimize the influence of any changes in the background sample on the measurement of the (surface) refractive index.

A 'spectrophotometer' is generally understood to be an instrument for measuring the wavelength-dependent transmittance or reflectance of solutions or solid objects.

It may be understood that a 'spectrophotometer' includes a broadband light (inherently non coherent), enabling that some or all of the light (such as broadband light or narrow-band or monochromatic light) is transmitted through the matter sample and received by a detector or a spectrometer. According to one ("diode array") embodiment broadband light is transmitted through the matter sample and the transmitted light is measured in a spectrally resolving manner by a spectrometer (such as via a grating or prism in combination with a diode array). According to another ("scanning") embodiment, a narrow spectral range of the broadband light is selected (such as via a grating or a prism in combination with a slit) and the resulting narrowband or monochromatic light is transmitted through the matter sample and the transmitted light is detected by a (not necessarily spectrally resolving) detector. It is understood that 'broadband light source' encompasses an embodiment with a (broadband) light source comprising a plurality of narrow-band or monochromatic light sources, such as wherein the selection of a narrowband or monochromatic light comprises switching on (only) the corresponding narrowband or monochromatic light source. The spectrometer determines intensities of the different wavelengths of the received light by use of a wavelength dispersion device such as a prism or a grating. This implies that the cuvette must be suited for transmitting broadband light through the cuvette and without altering the propagation direction of different wavelengths.

It may be understood that a "photonic crystal" is made from at least two materials arranged to provide a periodic variation of refractive index. Due to the transparency requirement (the photonic crystal is arranged in radiation path between opposite entry and exit windows), the materials must be dielectric materials, and certainly exclude metals which inherently have a low transmission.

It may be understood that entry and exit windows are opposite and in different walls for the measurement light. It may then follow that the photonic crystal is arranged in the light path between the entry and exit windows.

An advantage of having the photonic crystal rigidly attached to a side of the container or integrally formed in a side of the container and arranged in the radiation path, may be that at least some precipitates in a matter sample can settle at the bottom of the cuvette where they do not interfere with input radiation, whereby they in turn do not influence measurements. Cuvettes having a photonic crystal arranged at the bottom of their container part are unable to provide this very important effect.

The grating part may have a uniform grating. Alternatively or additionally, the grating is pseudo-periodic. Alternatively or additionally, the grating part has a chirped grating part. In some embodiments, the grating part has two or more sections having different grating structures. For instance, a first section has a first grating period and a second section has a second grating period different from the first grating period. The grating part may have further sections. Having multiple sections allows for a method of determining a dispersion of a compound, comprising: measuring a refractive index at least at a first and a second resonance wavelength in a photonic crystal comprising at least the first and second sections, and optionally further sections, just described.

In some embodiments, the grating part comprises a one-dimensional grating. Some embodiments comprise a two-dimensional grating in the grating part. Some embodiments comprise a three-dimensional grating in the grating part.

In some embodiments, the grating part is formed at least partially from a polymer material. The polymer could be PMMA, EFiRon, or HI01XP, or equivalent or similar polymers. Some embodiments of the first aspect are suitable for existing spectrophotometers, the container is preferably of a size suitable for existing spectrophotometers. In some embodiments, the cuvette has a footprint of 12.5 mm x 12.5 mm. In other embodiments, the cuvette is cylindrical to suit cuvette receptacles in spectrophotometers having a cylindrical receptacle. Preferably the cuvette has a square footprint, since this allows the cuvette to accurately be inserted in two ways in the spectrophotometer: 1) The photonic crystal is in the radiation path of the spectrophotometer light source, 2) The photonic crystal is not in the radiation path of the spectrophotometer light source. This is obtained simply by rotating the cuvette with respect to the spectrophotometer's cuvette receptacle. In some embodiments, absorption and refractive index can be determined simultaneously in one measurement.

In some embodiments, the grating part is a planar grating and the photonic crystal is arranged so that the planar grating is normal to the radiation path. This enables normal radiation incidence, which often provides the most efficient coupling of the radiation into the matter sample.

Some embodiments comprise a polarization filter at the entry window. Some embodiments comprise polarization filter at the exit window. This may for instance prevent mixing of TE and TM polarization specific features such as resonances, in the spectrum, making them simpler to interpret. In some embodiments, the grating part is a planar grating and the photonic crystal is arranged so that a normal to the planar grating is within 10 degrees of a normal to a surface part of the container to which the photonic crystal is attached. In some embodiments, the normal is within 20 degrees. In some embodiments, the normal is within 45 degrees.

In some embodiments, the cuvette is configured so that the radiation path coincides with a path followed by radiation from a spectrophotometer light source in a spectrophotometer in which the cuvette is suitable.

A surface refractive index scanning system for characterization of a sample is described in WO 15/169324 Al, which is hereby incorporated by reference in entirety.

By a 'biorecognition element' may be understood an element, such as a biomolecule, which enables specifically recognizing a specific biomolecule or a specific group of biomolecules, or (directly) viruses, bacteria or cells. By biomolecule is generally understood a molecule falling within carbohydrates, lipids, nucleic acids, or proteins, such as enzymes, antibodies, and aptamers or synthetic materials, such as molecularly imprinted polymers or peptide nucleic acids (PNA). Said recognition might result in a change, e.g., due to binding, release (e.g., of a third entity) or a conformational change. Said change may be measurable, e.g., as a change in refractive index. Recognition of a specific biomolecule or a specific group of biomolecules may be based on the biorecognition element being complimentary. In embodiments, the biorecognition element may be a nucleic acid (such as DNA, LNA, PNA or RNA), an aptamer, an enzyme, an antibody, or a molecularly imprinted polymer (MIP). The biorecognition element may be synthetic or natural.

By 'being immobilized on and/or at the photonic crystal' may be understood that the biorecognition elements are not free to diffuse around in the container, such as when the container is filled with an aqueous liquid, such as water. In embodiments each recognition element is chemically bound to the photonic crystal, such as each recognition element being bound by one or more covalent bonds to the photonic crystal.

According to an embodiment, there is presented a cuvette, wherein the photonic crystal comprises: a substrate part and a grating part on top of the substrate part forming a low index of refraction (nL) layer, wherein the grating part also comprises a high index of refraction (nH) material, and a layer of the high index of refraction material above the grating part.

According to an embodiment, there is presented a cuvette, wherein the grating part is defined by a periodically varying modulation of the refractive index.

According to an embodiment, there is presented a cuvette, wherein the photonic crystal is in contact with air when the cuvette is empty.

According to an embodiment, there is presented a cuvette, further comprising a monolayer, such as an organophosphonate monolayer (such as an aminophosphonate monolayer) or a silane monolayer, which acts as a coupling agent between the photonic crystal and the biorecognition elements, such as wherein the biorecognition elements are covalently bonded to the photonic crystal via the monolayer.

According to an embodiment, there is presented a cuvette, further comprising a silane monolayer which acts as a coupling agent between the photonic crystal and the biorecognition elements, such as wherein the biorecognition elements are covalently bonded to the photonic crystal via the silane monolayer. A possible advantage may be that silanization chemistry may be utilized and/or that strong bonds are formed, such as the biorecognition elements being securely immobilized on or at the photonic crystal. Another possible advantage of a silane monolayer may be that silanes are relatively simple to put on, e.g., an oxide surface, and may therefore be an advantageous choice of grafting layer for (covalent) attachment of biomolecules. By 'on or at the photonic crystal' may be understood that the biorecognition elements are chemically and/or physically bound to the photonic crystal and/or that they are immobilized within a certain distance, such as within 1 mm, such as within 0.1 mm, such as within 10 micrometer, such as within 1 micrometer, such as within 100 nanometer, from the surface of the photonic crystal.

According to an embodiment, there is presented a cuvette, further comprising an aminophosphonate monolayer, which acts as a coupling agent between the photonic crystal and the biorecognition elements, such as wherein the biorecognition elements are covalently bonded to the photonic crystal via the aminophosphonate monolayer. As an example of an aminophosphonate monolayer, reference is made to the academic article "Anchoring of Aminophosphonates on Titanium Oxide for Biomolecular Coupling", Canepa, et al., J. Phys. Chem. C 2019, 123, 16843-16850, which article is hereby included by reference in entirety.

According to an embodiment, there is presented a cuvette, wherein the biorecognition elements are antibodies, such as oriented antibodies. An advantage of antibodies may be that they enable detecting and/or quantifying a concentration of a range of pathogens and associated toxins. By 'oriented antibodies' may be understood that the immobilized antibodies have a preferred orientation, such as most of the antibodies having a similar orientation.

According to an embodiment, there is presented a cuvette, wherein material of the cuvette traversed by the radiation path consists of non-metallic material.

According to an embodiment, there is presented a cuvette, wherein material of the cuvette traversed by the radiation path consists of semiconducting material, electrically isolating material and/or dielectric material.

According to an embodiment, there is presented a cuvette, wherein material of the cuvette traversed by the radiation path consists of electrically isolating material and/or dielectric material.

Each of a 'metallic material' and 'semi-conducting material' is understood as is common in the art, and a 'metallic material' may in particular be understood to comprise and consist of materials capable of conducting electricity at a temperature of absolute zero. Alternatively, a 'metallic material' may in particular be understood to comprise and consist of materials having a volume resistivity less than 10⁵ Ohm-m at 20 degrees Celsius. Alternatively, a 'metallic material' may in particular be understood to comprise and consist of materials for which an electric field within the bulk material is substantially or exactly zero due to freely moving electrons (moving in electron bands) cancelling any electric (external) fields. An 'electrically isolating material' is understood as is common in the art, such as a material having a volume resistivity above 10⁹ Ohm-cm at 20 degrees Celsius. A 'dielectric material' is understood as is common in the art, such as an insulator (such as a material having a volume resistivity above 10⁹ Ohm-cm) and a real part of the relative permittivity being above 1, such as equal to or larger than 2.0, such as equal to or larger than 3.0.

According to an embodiment, there is presented a cuvette, wherein a transmittance (for light following the light path from entering the exit window to exiting the exit window) for the cuvette is at least 0.1 %, such as at least 1 %, such as at least 2 %, such as at least 5 %, such as at least 10 %, such as at least 25 %, such as at least 50 %, such as at least 75 %, such as at least 90 %.

By 'transmittance' is understood the fraction of incident electromagnetic power that is transmitted through a sample.

The transmittance is to be understood to be given for a situation wherein the container is filled with vacuum (i.e., there is an absence of material in the container) or atmospheric air. Alternatively, the transmittance is to be understood to be given for a situation wherein the container is filled with is pure water (exclusively H₂0 in liquid form). The transmittance is understood to be given for, such as measured at, a wavelength within the visible spectrum, such as within 200-1100 nm, such as within 400-700 nm, such as at a wavelength of 589 nm or 600 nm. Alternatively, the transmittance is understood to be given for, such as measured at, a wavelength similar or identical to or larger than the periodicity of the photonic crystal.

According to an embodiment, there is presented a cuvette, wherein the photonic crystal comprises a dielectric layer, such a dielectric thin-film, such as a transparent dielectric thin- film, and optionally wherein a silane monolayer is arranged to act as a coupling agent between the dielectric layer of the photonic crystal and the biorecognition elements, such as wherein the biorecognition elements are covalently bonded to the dielectric layer of the photonic crystal via the silane monolayer. A surface of the dielectric layer may form or be at an interface between the photonic crystal and the container, such as a (solid-fluid) interface between the photonic crystal and the sample matter, when the container is filled with sample matter. A possible advantage of having the photonic crystal comprising a dielectric layer may be that it enables providing a layer with a surface chemistry and/or surface physics appropriate for immobilizing biorecognition elements on or at the photonic crystal while at the same time having optical properties allowing light to be transmitted through the cuvette (incl. through the dielectric layer and interfaces on either side of the dielectric layer) to a sufficient degree for enabling optical analysis of the sample matter or compounds therein. By 'layer' is understood a structure or element having dimensions being significantly larger, such as at least 10 times, such as at least 100 times, such as at least 1000 times, such as at least 10⁴ times, such as at least 10⁵ times, such as at least 10⁶ (one million) times larger, in each of two dimensions (such as lateral dimensions) compared to a third dimension (such as thickness).

According to a further embodiment, there is presented a cuvette, wherein the dielectric layer is a metal oxide (such as any of titanium dioxide, tantalum pentoxide (Ta₂0₅), indium tin oxide (commonly abbreviated to ITO), hafnium oxide (Hf0₂)) or a ceramic (such as silicon nitride (S₃iN₄)). A possible advantage may be that metal oxides and/or ceramics may have a low attenuance and/or a high transmittance within relevant wavelengths, such as within whole or parts of the visible spectrum (400-700 nm, such as at 589 nm) and that it enables immobilization of biorecognition elements on and/or at the photonic crystal, e.g., via silanization.

According to a further embodiment, there is presented a cuvette, wherein the dielectric layer is titanium dioxide (Ti0₂). A possible advantage may be that titanium has a low attenuance and/or a high transmittance within relevant wavelengths, such as within whole or parts of the visible spectrum (400-700 nm, such as at 589 nm) and that it enables immobilization of biorecognition elements on and/or at the photonic crystal via immobilization, e.g., via silanization, at the titanium dioxide layer. Titanium dioxide may furthermore be advantageous for having a relatively high real refractive index. A relatively high refractive index may be advantageous for enabling a larger refractive index difference with respect to surrounding materials, such as a material of the cuvette or a liquid, such as a water-based liquid in the cuvette, which difference may in turn be beneficial for giving a high degree of confinement and/or creating a strong evanescent field, which may yield a high degree of interaction with the surroundings (which may in turn yield a strong signal). Furthermore, it may be beneficial for allowing a wider design-space, where, e.g., decay length can be optimized to probe (only) a specific distance from the surface. Another possible advantage of titanium dioxide may be that it has relatively low extinction coefficient (imaginary part of the refractive index), which in turn facilitates low losses (which may be advantageous for enabling a strong signal).

According to another embodiment, there is presented a cuvette, wherein within the plurality of biorecognition elements being immobilized on and/or at the photonic crystal there are biorecognition elements which are identical to each other, such as the plurality of biorecognition elements comprising biorecognition elements which are identical to other biorecognition elements within the plurality of biorecognition elements. For example, each biorecognition element may be an antibody, and the plurality of biorecognition elements may comprise, such as consist of, a plurality of antibodies (antibody molecules) which are identical to each other. A possible advantage of this may be specific detection (such as specifically recognizing a bio-element, such as the specific biomolecule, group of biomolecules, virus, bacteria, or cells, which the biorecognition element recognizes) and/or a stronger signal (e.g., due to signal arising from a plurality of recognized bio-elements).

According to another embodiment, there is presented a cuvette, wherein within the plurality of biorecognition elements being immobilized on and/or at the photonic crystal there are biorecognition elements which are different with respect to each other, such as the plurality of biorecognition elements comprising biorecognition elements which are of a first type and other biorecognition elements which are of a second type, wherein the first type is different from the second type. For example, each biorecognition element may be an antibody, and the plurality of biorecognition elements may comprise, different types of antibodies. A possible advantage of this may be the possibility of detecting different bio-elements.

According to another aspect, there is presented a spectrophotometer for characterizing a refractive index of a matter sample, comprising a cuvette receptacle configured to receive a cuvette in accordance with the first aspect. In a further embodiment, said spectrophotometer furthermore comprises a cuvette in accordance with the first aspect. According to a second aspect, there is presented a spectrophotometer for characterizing a refractive index of a matter sample, comprising a. a cuvette receptacle configured to receive a cuvette in accordance with the first aspect, b. a spectrophotometer light source arranged to provide input radiation along the radiation path of the cuvette, c. a spectrometer arranged to receive non-absorbed parts of the input radiation from the exit window and to determine a spectrum based on said non- absorbed parts, and to determine a resonance wavelength or resonance frequency or other resonance property in the spectrum, the spectrophotometer being configured to determine the refractive index by solving a set of suitable optical equations that take into account at least 1) optical and physical characteristics of the photonic crystal, 2) the determined resonance wavelength or resonance frequency or said other resonance property, the refractive index being an unknown to be solved for in said set of suitable equations.

The skilled person will readily appreciate that "a set of suitable optical equations" means a set of equations or other model or empirical formulation that allows for determining the refractive index based on the optical elements involved, including the cuvette and ultimately a matter sample held by the cuvette. The selection of equations may be selected based for instance on limitations in available computing power (more complex equations typically require more computing power), and/or be selected based on the precision required of the solution representing the refractive index (more detailed models may give a more precise result, whereas approximations and simple models may lead to a less precise result). Accordingly, many different implementations can be applied in respect of the set of suitable optical equations. Among other sources, textbooks and scientific papers are available that will readily allow the person skilled in the art to provide a set of suitable optical equations that may be used in methods and apparatuses disclosed in the present specification.

As is evident for the person skilled in the art, the set of equations may comprise an analytical or a semi-analytical expression or experimentally determined relationship between relevant parameters. In some embodiments, two or more spectral components in the spectrum are measured in the spectrophotometer and used to characterize the resonance, and in particular used as input to the set of optical equations in order to solve for the refractive index - described above - or other relevant characteristic. This allows for a determination that, to lesser or greater extent - take into account a shape of the resonance, such as a peak-to- width property determined based on the measured values. Alternatively or additionally, the model may compare an amplitude or equivalent value of each of a plurality of measured spectral components with an expected value (in relative terms) for said each spectral component, for instance as predicted by an at least partly analytical model and/or an at least partly semi-analytical model, or based at least partly on a table of expected values. An asymmetry in the resonance, determined based on measured spectral components, can in some cases be used to determine the relevant parameter, such as the refractive index, as part of the set of optical equations.

In some embodiments, the spectrophotometer further comprises a mirror system configured to guide input light reflected by the photonic crystal to the spectrometer.

The material may be a single chemical compound or mix of multiple compounds, fluids (both liquid and gaseous), polymer or polymers; biological or synthetic fluid or fluids; acid or acids; base or bases; raw or pre-processed materials, or any other material that is suitable for being a least partly characterized using a spectrophotometer. In any case, the material may be liquid or solid, or a mixture thereof, whether it comprises one or several different materials, such as the ones mentioned in the present paragraph or elsewhere in the present specification. In some cases, certain materials falling within the group of materials described above are not suitable, at least for some concentrations within the matter sample, for being characterized in a spectrophotometer. Embodiments of spectrophotometers in accordance with the present invention may or may not be able to overcome such limitations.

In some embodiments of the fifth aspect, the spectrophotometer comprises a selector unit allowing a user to select between at least two different materials or mixture of materials, and in response, the spectrophotometer determines the concentration by using the predetermined relationship corresponding to the selected material or mixture of materials. In some embodiments, the predetermined relationship for the material the concentration of which is to be determined, represents the relationship for the material mixed in a (substantially) known medium, such as but not limited to: water, alcohol, acid or acids, base or bases, or other medium, fluid or solid, or a mix of such materials.

The discussion above relating to "a set of suitable optical equations" applies equally to the fifth aspect and any other aspects that involve such a set of suitable optical equations. Persons skilled in the art will also appreciate that similar considerations apply to the "predetermined relationship".

According to a third aspect, there is presented a method for determining an optical characteristic or material characteristic of a matter sample in a spectrophotometer, comprising inserting a cuvette in accordance with the first aspect into a cuvette receptacle of a suitable spectrophotometer, the cuvette comprising the matter sample, irradiating the matter sample with input radiation along the radiation path, recording a spectrum of non- absorbed parts of the input radiation using the spectrometer, determining said characteristic of the matter sample by solving a set of suitable optical equations that take into account at least 1) optical and physical characteristics of the photonic crystal, 2) a determined resonance wavelength or resonance frequency or other resonance property of the spectrum, said characteristic being an unknown to be solved for in said set of suitable equations.

In some embodiments, the characteristic is a refractive index of the material, and the method is accordingly adapted to solve for the refractive index.

In some embodiments, the characteristic is a concentration of the material in the matter sample. The set of suitable optical equations further comprises a predetermined relationship for converting, for said material, a resonance wavelength or resonance frequency determined by the spectrophotometer into a concentration of said material.

According to an embodiment, there is presented a method, wherein the matter sample is aqueous. It may be understood that in embodiments, the matter sample comprises 'water', which in this context is understood as a sample comprising, such as predominantly comprising (such as comprising at least 50 wt%), H₂0 in liquid form, and then additionally optionally comprising other compounds, such as nucleic acids, pathogens and associated viruses, toxins, bacteria and/or cells.

According to an embodiment, there is presented a method, wherein a. the matter sample is obtained, such as directly obtained, from i. a food or beverage production, or wherein b. the matter sample comprises a food product or a beverage.

According to an embodiment, there is presented a method, wherein c. the matter sample is obtained, such as directly obtained, from i. a groundwater reservoir, or ii. an aquaculture plant, or wherein d. the matter sample comprises material obtained from the animal or human body, such as the matter sample being or comprising a body fluid, such as a human body fluid, or a part thereof.

'Body fluid' is understood as is common in the art, and may in embodiments be any one or more of whole blood, blood plasma, blood serum, urine and/or saliva. According to a third aspect, there is presented a method for preparing a cuvette according to the first aspect, said method comprising silanization on the photonic crystal, such as wherein the plurality of biorecognition elements being immobilized on and/or at the photonic crystal are immobilized via a silanization on the photonic crystal. 'Silanization' is the partial or full covering of a surface with organofunctional alkoxysilane molecules, optionally followed by biotin-streptavidin or covalent binding of biomolecules. An advantage of alkoxysilane molecules may be the relative ease of silanization. Another alternative may be given by providing a starting monolayer in the form of organophosphonates or poly(a Ikyl- phosphonates).

According to an embodiment, there is presented a method for preparing a cuvette, wherein the plurality of biorecognition elements being immobilized on and/or at the photonic crystal are immobilized or have been immobilized via a silanization, such as a silanization process, on the photonic crystal.

According to an embodiment, there is presented a method for preparing a cuvette, such as a cuvette for use in determining a refractive index of a matter sample in a spectrophotometer, wherein the method comprises: a. Providing a container for holding the matter sample, the container having i. an entry window that allows input radiation to reach the matter sample, the container furthermore having ii. an exit window that allows a part of the input radiation to exit the container part, the entry window and the exit window defining a radiation path, b. Providing and rigidly attaching a photonic crystal to a side of the container or integrally formed in a side of the container and arranged in the radiation path, the photonic crystal having i. a grating part causing a reflectance spectrum of the photonic crystal to exhibit a resonance, c. providing and immobilizing via said silanization a plurality of biorecognition elements on and/or at the photonic crystal.

The method for preparing a cuvette according to the third aspect, further comprising click- chemistry, such as copper-free click chemistry.

The first, second, third and fourth aspect of the present invention may each be combined with any of the other aspects. These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter. BRIEF DESCRIPTION OF THE FIGURES

The cuvette, spectrophotometer, method for determining an optical characteristic or material characteristic of a matter sample and method for preparing a cuvette according to the invention will now be described in more detail with regard to the accompanying figures. The figures show one way of implementing the present invention and is not to be construed as being limiting to other possible embodiments falling within the scope of the attached claim set.

Embodiments of the invention will now be further described with reference to the accompanying drawings, in which:

Figs. 1-3 illustrate a liquid container for use in an embodied method;

Figs. 4-5 illustrate a photonic crystal for use in an embodied method;

Fig. 6 shows an SDS PAGE result; and

Fig. 7 shows (a) a guided mode resonance filter treated with a molecular recognition layer to which target analytes selectively bind and (b) illustrates that the molecular binding induces a refractive index within the evanescent field of a quasi guided mode supported by the structure, which leads to a change in the exhibited resonance wavelength.

DETAILED DISCLOSURE OF THE INVENTION

Fig. 1 illustrates a liquid container 1 comprising first and second container surfaces 3,5 defining part of an interior 7 of the liquid container 1. The first and second container surfaces 3,5 comprise transparent and parallel parts 9,11 and together form part of a wall 12. The first container surface 3 comprises a photonic crystal 13. The first and second container surfaces 3,5 may be made from, e.g., polymers such as, e.g., Poly(methyl methacrylate) (PMMA), or from other transparent substantially rigid materials such as, e.g., glass. The liquid container 1 is for use in conjunction with an optical element (not shown) comprising a light source and light detector. The light may be a kind of broad spectrum light source, such as, e.g., a gas lamp, preferably with a relatively flat spectrum in the visible spectrum, possibly also in the UV spectrum. In this case, the light sensor is preferably able to measure both the intensity and wavelength of incident light. The light sensor may be able to sense light having passed through any part of the photonic crystal 13 for light having an incident angle of 85-95 degrees relative to the first plane, preferably 80-100 degrees.

The photonic crystal 13 is attached to the first container surface 3 over the transparent part 9. The photonic crystal 13 could equally well have been integrally formed with the first container surface 3. Details of the photonic crystal are illustrated more clearly in Figs. 4 and 5 showing its first grating part 17 with a first modulation period defined by the period length 19. The first grating part 17 extends in a first plane and comprises first and second overlapping layers 23,25. The two layers 23,25 both have the first modulation period, while the first layer 23 has first refractive index, and the second layer 25 has a second refractive index. The first refractive index is larger than the second refractive index. The first layer faces the interior 7 and the second layer 25 faces the first container surface 3.

In one embodiment, the liquid container 1 is a cuvette defining two sets of opposing cuvette walls, one of the sets of opposing walls comprising the first and second container surfaces 3,5. The cuvette may define a square footprint wherein the ends of the two sets of opposing walls in conjunction with the edges of a bottom wall illustrated by the dashed line 27 define the perimeter of the square footprint. Such cuvettes may be particularly suitable for use with, e.g., a commercially available spectrophotometer. In this case, the dashed line 29 of each of the illustrations of Figs. 1-3 represents an opening, which may be sealed off by a plug (not shown).

In one embodiment, the liquid container 1 forms part of a flow path wherein the liquid to be characterised flows in a flow path intercepting the light path, the light path and the direction of the light being illustrated by arrows 33 in Figs. 2 and 3. The flow path is perpendicular to the plane in which the illustrations of Figs. 1-3 are drawn. The flow of the liquid is controlled to be zero during recording of the first and second transmission spectra. This allows for characterising a change in particle composition in the liquid. Such change may occur if due to, e.g., a chemical reaction of particles in the liquid, a decay of particles in the liquid or a flow of particles relative to the liquid. These processes may be analysed by adding steps of recording consecutive sets of first and second transmission spectra at consecutive points in time and consecutive amounts of 'flow propagation'. Herein flow propagation refers to flow of liquid propagating past the first and second surfaces 3,5 intercepting the light path.

Fig. 1 furthermore shows that the photonic crystal 13 comprises a layer 40, which in the present case is Ti0 . Still further, Fig. 1 shows biorecognition elements 42 (in the present figure being ordered antibodies covalently bonded to the layer 40) immobilized on the layer 40 of the photonic crystal 13.

Fig. 2 illustrates the orientation of the first and second surfaces 3,5, the interior 7 and the photonic crystal 13 relative the light path and the direction of the light therein indicated by arrows 33. Accordingly, Fig. 2 illustrates the situation when recording a first transmission spectrum wherein part of the first and second container surfaces 3,5, the interior 7 containing liquid and the photonic crystal 13 intercept the light path, and the interior 7 intercepts the light path before the photonic crystal 13.

For clarity, the layer 40 and biorecognition elements 42 depicted in Fig. 1 are not shown in Fig. 2.

In the illustration of Fig. 3, the direction of the direction of the light in the light path indicated by arrows 33 are opposite that in Fig. 2. Accordingly, Fig. 3 illustrates the situation when recording a second transmission spectrum, wherein at least part of the first and second container surfaces 3,5, the interior 7 containing liquid and the photonic crystal 13 intercept the light path, and the photonic crystal 13 intercepts the light path before the interior 7.

A third transmission spectrum may also be recorded, wherein the light path is not intercepted by the photonic crystal. In the case of the liquid container 1 being a cuvette defining two sets of opposing cuvette walls, one of the sets of opposing walls comprising the first and second container surfaces 3,5, the other set then does not comprise a photonic crystal. The third spectrum may then be recorded by rotating the light path relative to the cuvette, or the cuvette relative to the light path, by 90 degrees in respect of the mutual orientations during recording of the first or second transmission spectrum. The third transmission spectrum allows recording of an absorption or attenuance spectrum. An attenuance spectrum represents losses due to absorbance, scattering and luminescence.

For clarity, the layer 40 and biorecognition elements 42 depicted in Fig. 1 are not shown in Fig. 3.

Immobilization of the plurality of biorecognition elements on and/or at the photonic crystal may take place according to immobilization examples 1 and 2 provided below.

Immobilization example 1: Surface chemistry protocol without click chemistry ¹'²'³'⁵'⁶

Sulfo-NHS (N-hydroxysulfosuccinimide) are used to prepare amine-reactive esters of carboxylate groups for chemical labelling, crosslinking and solid-phase immobilization applications. Carboxylates (-COOH) may be reacted to NHS or Sulfo-NHS in the presence of a carbodiimide such as EDC (1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride) , resulting in a semi-stable NHS or Sulfo-NHS ester, which may then be reacted with primary amines (-NH2) to form amide crosslinks. Silanization protocol

1. Clean a T1O2 substrate chip using an oxygen plasma (30 W and 50 seem O2 for 60 s) or UV cleaner. The plasma-cleaned chip should preferably be used promptly. This may be beneficial in order to obtain clean, oxidized surfaces for effective chemical modification in later steps.

2. Prepare silane/ethanol solution (1% v/3-Aminopropyl)triethoxysilane in ethanol/H20 (95%/5%))and allow to stand for 20 min before filtering with a 0.2 mm cut-off syringe filter.

3. Immerse the plasma-cleaned T1O2 chip in the silane/ethanol solution for 30 min. Wash the sensor chip with ethanol flow for 50 s, dry in a stream of nitrogen gas and heat at

110 °C for 10 min. The silanized surfaces should preferably be used as soon as possible after preparation, and kept under a dry nitrogen atmosphere until use if stored.

Activation of NHS-ester with EDC & sulfo-NHS in MES (2-(N-morpholino)ethanesulfonic acid) buffer NHS-ester Activation

Add 0.4mg of EDC (final concentration 2mM) directly to 1 ml_ of Protein #1 (wherein Protein #1 is a protein of interest, and where it is desirable to couple it to the surface, and which will be the antibody to couple to), which, based on a 50kDa protein, results in a 10-fold molar excess of EDC to Protein #1. Add either 0.6mg of NHS or 1.1 mg of Sulfo-NHS to the reaction (final concentration 5mM).

Amine reaction of activated NHS-ester of protein

Separate activated Protein #1 from excess EDC, EDC-byproducts, NHS and (if used) 2- mercaptoethanol using an appropriate size desalting column that has been equilibrated with PBS. If step B.5 was not performed (i.e., buffer not exchanged using a desalting column), then increase buffer pH above 7.0 using concentrated PBS or other non-amine buffer such as sodium bicarbonate.

2. Add Protein #2 (wherein Protein #2 is the silane-amine surface in this case, such as the silanized amine photonic crystal in this case) to the solution containing activated

Protein #1.

3. Mix the solution well and then allow reaction to proceed for 2 hours at room temperature.

Immobilization example 2: Copper-free click chemistry route for antibody functionalization³'⁴'⁵-⁶

Silanization Protocol

1. Clean PO2 substrate chip using an oxygen plasma: (30 W and 50 seem O2 for 60 s.) The plasma-cleaned chip should preferably be used promptly. This may be beneficial in order to obtain clean, oxidized surfaces for effective chemical modification in later steps.)

2. The Ti0₂ substrates can be dispersed in 1 ml_ of ethanol containing 50 pg of NTPA (N-Nitroso-2,3-dihydroxypropyl-2-hydroxypropylamine) and 10 pL of NH₄OH (27%).

3. The mixture to be stirred for 1 h at 25 °C. Subsequently, the resulting azide- functionalized Ti0₂ surfaces to be centrifuged and washed with ethanol and PBS several times.

The silanized surfaces should preferably be used as soon as possible after preparation, and kept under a dry nitrogen atmosphere until use if stored.

Alternative silanization protocol (gas phase)

1. Substrate coated with T1O2 to be cleaned by washing with chloroform and dried in a nitrogen stream. 2. Next, the cleaned sample to be placed in a dessicator over two small containers, one containing a 30 pi of the silane precursor (3-aminopropyltriethoxysilane, APTES), and the second 10 pi of catalyst - triethylamine - and left at room temperature for 2 h under an argon atmosphere for gas phase silanization.

4. Then, after removing the reagents from the the dessicator and the samples are left in argon ambience for 48 h for curing of the silane layer (TiC>2-NH2).

Protocol to prepare reduced antibodies with SH- groups in Fc region and bioactivity intact

1. To functionalize the antibodies with ADIBO (azadibenzocyclooctyne), thiol groups have to be derived from the disulfide bonds in antibodies keeping the bioactivity of antibodies intact.

2. Need to confirm antibody disulfide reduction goes successful by comparing 2 reducing conditions.

2.a) 0.25 ml_ of antibody in phosphate-buffered saline (PBS) solution (2 mgmL ¹), 2 pl_ of ethylenediaminetetraacetic acid (EDTA) in deionized water (DW, 0.3 M, pH 8.0), 2 mI_ sodium bicarbonate in DW (1 M), and 2.5 mI_ b-mercaptoethanol in DW (1.5 M) to be added (reaction 1).

2.b) 0.25 ml_ of antibody in phosphate-buffered saline (PBS) solution (2 mgmL-1), 2 mI_ of ethylenediaminetetraacetic acid (EDTA) in deionized water (DW, 0.3 M, pH 8.0), 2 mI_ sodium bicarbonate in DW (1 M), and 2.5 mI_ b-mercaptoethanol in DW (3 M) to be added (reaction 2).

In reactions 1 -2, the same quantity of antibody was used, while the volumes of the other reagents to be increased by 2X for harsher conditions than reaction 1. In the protocol here only 2-Mercaptoethanol concentration has been increased, rest components need to be increased subsequently with respect to reaction 1. 3. Reaction 1 and reaction 2 to be checked with SDS PAGE (sodium dodecyl sulphate- polyacrylamide gel electrophoresis) with the expected result as in Fig. 6.

4. All reaction mixtures to be incubated at 37 °C for 1 h.

5. Reduced antibody to be purified using a PD-10 column with PBS. Protocol to prepare ADIBO-antibody-conjugates

1. A 20 mM solution of ADIB0-PEG4-maleimide (with PEG being Polyethylene glycol) in DMSO (Dimethyl sulfoxide) was prepared and added to the thiol-functionalized antibody with a molar ratio of 1 :10.

2. The mixture to be incubated for 2 h at room temperature or 24 h at 4 °C in the dark. 3. After reaction, the mixture to be purified using a desalting column (such as a PD-10 column, Merck KGaA, Darmstadt, Germany) with PBS and concentrated using a 100 kDa centrifugal filter unit.

4. The final concentration of the ADIBO-functionalized antibody needs to be determined.

Protocol to prepare ADIBO-antibody-conjugates functionalized on Ti02 surface 1. An azide group has been introduced on the surface of T1O2 via silane coupling between the hydroxyl group on the T1O2 surface of the substrate and the silane group in N-[3-(triethoxysilyl)propyl]-2-azidoacetamide (NTPA).

2. To control the orientation of the antibodies, the alkyne group has been functionalized in the Fc region of the antibody by reacting the maleimide group in aza- dibenzocyclooctyne- maleimide (ADIBO-Mal) with the thiol group derived by reducing antibody with 2-mercaptoethanol. 3. Subsequently, the azide-functionalized T1O2 substrates and ADIBO-conjugated antibodies to be mixed in order to conjugate the antibodies to the PO2 surfaceat room temperature.

Since two benzene rings are conjugated to a cyclooctyne in a single ADIBO molecule, it reacts easily with the azide functional group with high efficiency and reproducibility via a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, also known as a copper- free click reaction.

For antibody conjugation, 50 pg of ADIBO-functionalized antibody in 1 ml_ of PBS for 1 h at 25 °C to be treated with PO2 azide surfaces.

4. The antibody conjugated Ti0₂ surfaces to be centrifuged and washed with PBS containing 0.1% (w/v) Tween-20 and PBS several times.

5. Lastly, the antibody conjugated T1O2 surfaces to be treated with BSA (1% (w/v) in PBS solution, pH 7.4) for 30 minutes and washed with PBS containing 0.1% (w/v) Tween-20 and PBS.

Measurement example: Specification and limit of detection for biomolecule with the cuvette according to embodiments according to the first aspect, such as the Nanocuvette™ (Copenhagen Nanosvstems ApS, Faruml

Biosensing applications with guided mode resonance filter sensors or photonic crystals, as used with the cuvette according to embodiments according to the first aspect, such as the Nanocuvette™ platform, are performed by preparing a recognition layer of immobilized biomolecules (biorecognition elements) on the high-index wave-guide layer as shown in the left-side subfigure (a) of Fig.7.[1] With any standard broadband white light illumination, the underlying structure will exhibit resonant reflection at a particular wavelength. In presence of aqueous solution containing target analytes over the surface, there will be selective binding of the receptor molecules which will increase the optical density at the surface. This results in changing the propagation constant of the resonant mode, which is manifested as a spectral shift of the resonantly reflected light, as illustrated in the right hand side subfigure (b) of Fig. 7.[1] The left side subfigure of Figure 7 shows a guided mode resonance filter treated with a molecular recognition layer to which target analytes selectively bind.

The right hand side subfigure (b) of Figure 7 shows that the molecular binding induces a refractive index within the evanescent field of a quasi guided mode supported by the structure, which leads to a change in the exhibited resonance wavelength.

The guided mode resonant sensor’s Rl sensitivity of photonic crystal can be converted into a sensitivity number for biomolecule detection. The capture of biomolecules at the sensor surface would change the Rl in the region of the sensor’s evanescent field. An analysis from Rl sensitivity to biomolecule sensitivity conversion has been conducted and confirmed with experiments for ring resonator type sensor measuring changes from glass(n_m = 1.45) to solution (n_s = 1.33). In principle, the analysis can be generalized and used for all resonant mode-based Rl sensors which involves changes in spectral shift of resonant mode wavelength due to change in sample Rl. It leads to an equation for the spectral shift of the resonant mode dl, for biomolecular detection at a bulk refractive index sensitivity S: [1,2]

Where s_r is the surface density of biomolecules, a_ex is the excess polarizability of the molecule, n_m is the Rl of the sensor material, and n_s is the Rl of the sample buffer. Using the above equation, the molecule detection limit a_min at the resonant wavelength l can be deduced where A_min is the bulk Rl detection limit of the sensor in units of RIU:

For a bulk Rl detection limit of 6 10⁷ RIU [1], s_r = 2.9 x 10¹²cm ² [3], a_ex =

(4p e₀) x (3.85 x 10^-21)cm^-2 [3], BSA (M_w 66500 g/mole), the mass detection limit is 0.5 pg/mm², which similarly to Surface Plasmon Resonance technology surpasses many competing methods. [2]

For the cuvette, such as the Nanocuvette™ platform, and a spectrophotometer, such as a spectrophotometer, the bulk refractive index sensitivity can vary from ~ 10^_5 to 10^-4, respectively, with values as low as 1.0 x 10^_6 RIU measured with custom built instrumentation and reported in a peer-reviewed publication in 2015 [1] Using values of surface density for BSA (Bovine serum albumin) [3] and refractive index of our specific materials used for the cuvette, such as the Nanocuvette™ concept, the estimated detection limit is 1.0 pg/mm² to 10 pg/mm², respectively. The typically illuminated area is 1.0 mm², meaning that it should be possible to detect between 1 pg and 10 pg in a standard cuvette form factor with a sample volume of 2 ml_. Thereby, the cuvette, such as the NanoCuvette™ concept, with antibodies can provide an estimated detection limit between 0.5 pg/†nL and 5 pg/†nL, equivalent to between 0.5 ng/L and 5 ng/L. This is similar to humans, which can detect geosmin down to 2 ng/L in water.

Geosmin (M_w 182.3 g/mole) is a lighter molecule than BSA, nevertheless the detection density will be limited by the antibodies for geosmin, i.e. protein capture molecule for geosmin and so we should expect similar limit of detection even for this small molecular weight compound.

Geosmin sensing would need to capture agents, that is anti-geosmin antibodies functionalized on the optical filter used for the cuvette, such as the Nanocuvette™ platform. In a report about enzyme-linked-immuno-assay (ELISA) for geosmin detection from 1991 [4], they detect 1000 pg/L. There is considerable ongoing research in this direction with ELISAs have reported 6.2 ± 1.5 pg/L for 2-ketogeosmin in 2014 [5] and combined with nanoparticle-based detection and chemiluminescent signal improved detection to 1100 ± 400 ng/L for 2-ketogeosmin. [5]

In conclusion, it is be possible to use the cuvette, such as the Nanocuvette™ platform, and surface functionalization with capture molecules to detect geosmin in an estimated range of 0.5 ng/L and 5 ng/L, respectively. In an embodiment, the method of Ref. [6] would be implemented for surface functionalization of the cuvette, such as the Nanocuvette™ platform, for specific geosmin detection.

Functionalization is in the context of the present application generally understood to be interchangeable with immobilization of biorecognition elements. According to embodiments, the cuvette, spectrophotometer, method for determining refractive index and/or the method for providing the cuvette comprises means (features) or steps for temperature stabilization (or compensation). This may be beneficial for measurements with the cuvette, such as the Nanocuvette™ platform, as there can be effects on refractive index due to temperature at a rate dn/dT= 1 x 10⁵ RIU per 0.1 °C. [7]

Although the present invention has been described in connection with the specified embodiments, it should not be construed as being in any way limited to the presented examples. The scope of the present invention is set out by the accompanying claim set. In the context of the claims, the terms "comprising" or "comprises" do not exclude other possible elements or steps. Also, the mentioning of references such as "a" or "an" etc. should not be construed as excluding a plurality. The use of reference signs in the claims with respect to elements indicated in the figures shall also not be construed as limiting the scope of the invention. Furthermore, individual features mentioned in different claims, may possibly be advantageously combined, and the mentioning of these features in different claims does not exclude that a combination of features is not possible and advantageous.

LIST OF REFERENCES FOR IMMOBILIZATION EXAMPLES 1-2

1. F. Patolsky et.al, "Fabrication of silicon nanowire devices for ultrasensitive, label- free, real-time detection of biological and chemical species", Nat. Prot. 2006, volume 1, pages^'! 711 -1724

2. https://www.thermofisher.com/dk/en/home/life-science/protein-biology/protein- biology-learning-center/protein-biology-resource-library/pierce-protein- methods/carbodiimide-crosslinker-chemistry.html 3. S.Jeong et.al, "Highly robust and optimized conjugation of antibodies to nanoparticles using quantitatively validated protocols", Nanoscale, 2017,9, 2548-255

4. D. Prim et. al, "ADIBO-Based "Click" Chemistry for Diagnostic Peptide Micro-Array Fabrication: Physicochemical and Assay Characteristics", Molecules 2013, 18(8), 9833-9849

5. M. Dominik et.al, "Titanium oxide thin films obtained with physical and chemical vapour deposition methods for optical biosensing purposes", Biosens & Bioelctron, 2017, Vol 93, pages 102-109.

6. A. Ebner et.al, "Comparison of different aminofunctionalization strategies for attachment of single antibodies to AFM cantilevers", Ultramicroscopy, 2007, Issues

10-11, pages 922-927.

LIST OF REFERENCES FOR THE MEASUREMENT EXAMPLE

1. Pertur Gordon Hermannsson, "Design and use of guided mode resonance filters for refractive index sensing", 2015, PhD Thesis. 2. Zhu, et.al., "Analysis of biomolecule detection with optofluidic ring resonator sensor", Optics Express, 2007, Vol 15(15), 9139.

3. White, et.al., "On the performance quantification of resonant refractive index sensors", Optics Express, 2008, Vol 16(2), 1020.

4. Arnold, et.al., "Shift of whispering-gallery modes in microspheres by protein adsorption", Optics Letters, 2003, Vol 28(4), 272.

5. Chung, et. al., "Development of an enzyme-linked immunosorbent assay for geosmin", J. Agric. Food Chem, 1991, 39, 764 6. Soo-Woo Kang, "Development of nano-particle based immuno-assays for detection of odorous volatile compounds", 2014, PhD Thesis.

7. Jahns, et. al., "Hand-held imaging photonic crystal biosensor for multiplexed, label- free protein detection", Biomed. Optics Express, 2015, Vol 6(10), 3724. 8. Kristian Tolbol Sorensen, "Photonic crystal slab sensors in microfluidics", 2018, PhD

Thesis.

Claims

1. A cuvette for use in determining a refractive index of a matter sample in a spectrophotometer, comprising a. a container for holding the matter sample, the container having i. an entry window that allows input radiation to reach the matter sample, the container furthermore having ii. an exit window that allows a part of the input radiation to exit the container part, the entry window and the exit window defining a radiation path, b. a photonic crystal rigidly attached to a side of the container or integrally formed in a side of the container and arranged in the radiation path, the photonic crystal having i. a grating part causing a reflectance spectrum of the photonic crystal to exhibit a resonance, wherein the cuvette further comprises c. a plurality of biorecognition elements being immobilized on and/or at the photonic crystal.

2. A cuvette according to any of the preceding claims, further comprising a silane monolayer which acts as a coupling agent between the photonic crystal and the biorecognition elements, such as wherein the biorecognition elements are covalently bonded to the photonic crystal via the silane monolayer.

3. A cuvette according to any of the preceding claims, wherein the biorecognition elements are antibodies, such as oriented antibodies.

4. A cuvette according to any of the preceding claims, wherein material of the cuvette traversed by the radiation path consists of non-metallic material.

5. A cuvette according to any of the preceding claims, wherein the photonic crystal comprises a dielectric layer, such a dielectric thin-film, such as a transparent dielectric thin-film, and optionally wherein a silane monolayer is arranged to act as a coupling agent between the dielectric layer of the photonic crystal and the biorecognition elements, such as wherein the biorecognition elements are covalently bonded to the dielectric layer of the photonic crystal via the silane monolayer.

6. A cuvette according to claim 5, such as according to both of claim 2 and claim 5, wherein the dielectric layer is titanium dioxide (T1O2).

7. A cuvette according to any of the preceding claims, further comprising a monolayer, such as an organophosphonate monolayer or a silane monolayer, which acts as a coupling agent between the photonic crystal and the biorecognition elements, such as wherein the biorecognition elements are covalently bonded to the photonic crystal via the monolayer.

8. A cuvette according to claim 5, such as according to both of claim 2 and claim 5, wherein the dielectric layer is a. a metal oxide, such as titanium dioxide, tantalum pentoxide, indium tin oxide, or hafnium oxide, or b. a ceramic, such as silicon nitride.

9. A cuvette according to any of the preceding claims, wherein within the plurality of biorecognition elements being immobilized on and/or at the photonic crystal there are a. biorecognition elements which are identical to each other, such as the plurality of biorecognition elements comprising biorecognition elements which are identical to other biorecognition elements within the plurality of biorecognition elements, and/or b. biorecognition elements which are different with respect to each other, such as the plurality of biorecognition elements comprising biorecognition elements which are of a first type and other biorecognition elements which are of a second type wherein the first type is different from the second type.

10. A spectrophotometer for characterizing a refractive index of a matter sample, comprising a. a cuvette receptacle configured to receive a cuvette in accordance with one of claims 1-9, b. a spectrophotometer light source arranged to provide input radiation along the radiation path of the cuvette, c. a spectrometer arranged to receive non-absorbed parts of the input radiation from the exit window and to determine a spectrum based on said non- absorbed parts, and to determine a resonance wavelength or resonance frequency or other resonance property in the spectrum, the spectrophotometer being configured to determine the refractive index by solving a set of suitable optical equations that take into account at least 1) optical and physical characteristics of the photonic crystal, 2) the determined resonance wavelength or resonance frequency or said other resonance property, the refractive index being an unknown to be solved for in said set of suitable equations.

11. A method for determining an optical characteristic or material characteristic of a matter sample in a spectrophotometer, comprising inserting a cuvette in accordance with one of claims 1-9 into a cuvette receptacle of a suitable spectrophotometer, the cuvette comprising the matter sample, irradiating the matter sample with input radiation along the radiation path, recording a spectrum of non-absorbed parts of the input radiation using the spectrometer, determining said characteristic of the matter sample by solving a set of suitable optical equations that take into account at least 1) optical and physical characteristics of the photonic crystal, 2) a determined resonance wavelength or resonance frequency or other resonance property of the spectrum, said characteristic being an unknown to be solved for in said set of suitable equations.

12. A method according to claim 11, wherein a. the matter sample is obtained, such as directly obtained, from i. a groundwater reservoir, or ii. an aquaculture plant, or wherein b. the matter sample comprises material obtained from the animal or human body, such as the matter sample being or comprising a body fluid, such as a human body fluid, or a part thereof.

13. Method for preparing a cuvette according to claims 1-9, said method comprising silanization on the photonic crystal.

14. Method for preparing a cuvette according to claim 13, wherein the plurality of biorecognition elements being immobilized on and/or at the photonic crystal are immobilized or have been immobilized via a silanization, such as a silanization process, on the photonic crystal.

15. Method for preparing a cuvette according to any of claims 13-14, wherein the method comprises: a. Providing a container for holding the matter sample, the container having i. an entry window that allows input radiation to reach the matter sample, the container furthermore having ii. an exit window that allows a part of the input radiation to exit the container part, the entry window and the exit window defining a radiation path, b. Providing and rigidly attaching a photonic crystal to a side of the container or integrally formed in a side of the container and arranged in the radiation path, the photonic crystal having i. a grating part causing a reflectance spectrum of the photonic crystal to exhibit a resonance, c. providing and immobilizing via said silanization a plurality of biorecognition elements on and/or at the photonic crystal.