WO2020260684A1

WO2020260684A1 - An optical sensor

Info

Publication number: WO2020260684A1
Application number: PCT/EP2020/068215
Authority: WO
Inventors: Thomas Just SØRENSEN
Original assignee: Københavns Universitet
Priority date: 2019-06-28
Filing date: 2020-06-29
Publication date: 2020-12-30

Abstract

Disclosed herein is sensor for emitting luminescence proportional to the concentration of a target analyte, comprising a luminescent lanthanide entity capable of interacting with a target analyte, wherein the relative luminescence emission intensity of the luminescent lanthanide entity changes, when subjected to an excitation light source, in dependency of the concentration of the target analyte, and a matrix material immobilizing the luminescent lanthanide entity. Also enclosed is a system for measuring a concentration of a target analyte and a composite comprising a layer of matrix material immobilizing a luminescent lanthanide entity and a substrate comprising a microstructure area.

Description

AN OPTICAL SENSOR

Introduction

The present invention relates to a sensor for emitting luminescence proportional to the concentration of a target analyte, a system for measuring a concentration of a target analyte, and a composite comprising a substrate and a matrix material immobilizing a luminescent lanthanide entity. The sensor and the system have a high physical integrity making it possible to collect reliable data for prolonged time.

Prior art

WO16183053 A1 pertains to methods of monitoring an

environment for the presence of a solvent by: (i) exposing the environment to a luminescent compound, where the relative luminescence emission intensity of the luminescent compound changes upon interaction with the solvent; and (ii) monitoring a change in the relative luminescence emission intensity of the luminescent compound, where the absence of the change indicates the absence of the solvent from the environment, and where the presence of the change indicates the presence of the solvent in the environment. The

luminescent compounds include a phosphorous atom with one or more carboxyl groups, where the carboxyl groups are

coordinated with one or more metallic ions (e.g., lanthanide ions and yttrium ions) .

W015075308 A1 and W015075309 A1 relate to a method for determining a scale inhibitor concentration in a sample comprising at least a first scale inhibitor, which is a synthetic organic compound comprising at least one ionised group. A scale inhibitor is used, for example, in offshore oil production for stimulation of the oil wells, for controlling and/or preventing scale depositions.

The method comprises optionally diluting and/or purifying the sample and allowing the sample to interact with a reagent comprising a lanthanide (III) ion. The sample is excited at a first excitation wavelength and a sample signal deriving from the lanthanide (III) ion is detected at a signal wavelength by using time-resolved luminescence measurement, and the concentration of the at least first scale inhibitor in the sample is determined by using the detected sample signal.

The analysis of the scale inhibitor concentrations may be performed in a fluid vessel e.g. a well, a part of a fluidic as cuvette, comprising a reagent comprising lanthanide (III) ions. The first scale inhibitor in the sample is allowed to interact with the reagent comprising lanthanide (III) ions in the first fluid vessel, and the sample is excited at the first excitation wavelength and the time-resolved

fluorescence signal is detected at the signal wavelength.

US2018284023 AA discloses a CO2 quantitative fluorescent sensing material, a preparation method and an application thereof. The preparation method for the C02 quantitative fluorescent sensing material includes dissolving 9,10- diacrylic anthracene in a solvent to prepare 5-10 mg/mL of a first solution; dissolving MnCl2 or Mn(C10₄)₂ in water to prepare 50-100 mg/mL of a second solution; mixing the first solution and the second solution; adding a diluted acid into the mixed solution; sealing and heating the mixed solution. During application, an ionic liquid produced by a reaction of CO2 gas and an amine compound improves an aggregation- induced emission of the CO2 quantitative fluorescent sensing material and a fluorescence thereof is significantly improved .

The prior art sensors have the disadvantage of having the luminescent compound only loosely attached to a platform or present in a solvent. Thus, in WO16183053 A1 the luminescent compound may be embedded in a solid structure (e.g., a test strip), and in W015075308 A1 and W015075309 A1 the

luminescent compound is present in a solvent. The

luminescent compounds of the prior art sensors may be leached from the solid structure, making the sensor less sensitive and accurate as the sensor ages. The luminescent compounds present in a solvent may not be reused and a new supply for the next measurement is necessary. The object of the present invention is to obtain a sensor having a high physical stability. The high physical stability makes it possible to collect reliable data for prolonged time.

Furthermore, it is possible to use the sensor for

applications in which leaching is to be avoided, such as production of biological materials and monitoring of patients .

Disclosure of the invention

The present invention relates to a sensor for emitting luminescence proportional to the concentration of a target analyte, comprising

a luminescent lanthanide entity capable of interacting with a target analyte, wherein the relative luminescence emission intensity of the luminescent lanthanide entity changes, when subjected to an excitation light source, in dependency of the concentration of the target analyte, and - a matrix material immobilizing the luminescent lanthanide entity.

The immobilization of the luminescent lanthanide entity allows for the preparation of a more durable sensor that can be used for prolonged time periods maintaining the sensibility and the accuracy when measuring the concentration of a target analyte, such as glucose, lactate, citrate, carbonate, bicarbonate, etc.

Usually, the luminescent lanthanide entity comprises a luminescent lanthanide chromophore capable of interacting with a target analyte and a linker for immobilization to the matrix material. Thus, the linker connects the matrix material to the lanthanide chromophore for the

immobilization to occur.

In a certain aspect of the invention the luminescent lanthanide entity has the general formula:

L2xLnlLl wherein

Lnl is a lanthanide ion;

LI is a polydentate ligand bonded to Lnl;

each L2 is the same or different and is a ligand bonded to Lnl; and

x is 0 or an integer of from 1 to 8.

More specifically, the lanthanide chromophore has the following composition:

wherein

Ln is a lanthanide ion;

each Y is the same or different and is optionally substituted C2-3 alkylene optionally substituted with a linker ;

each L4 is the same or different and is a monodentate ligand bonded to Ln;

p is zero or an integer of from 1 to 4 ;

each L6 is the same or different and is optionally substituted methylene optionally substituted with a linker ;

Ra, Rb, Rc, Rd, Re, Rf, and Rg are independently selected from H or the groups of formulae (Ila), (lib) and (lie) :

wherein

XI is 0 which 0 is also coordinated to Ln;

wherein

X2 is 0, which 0 is optionally coordinated to Ln; Rh and Ri are the same or different and are selected from H, unsubstituted or substituted Cl-6 alkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl and a linker; and

v is 0 or 1 ;

Optional linker

wherein

XI is 0, which 0 is also coordinated to Ln;

X3 is C or N, which N is optionally coordinated to Ln; and

X4 is C(=0) or P(=0) (ORx) wherein Rx is H, unsubstituted or substituted Cl-6 alkyl unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; j is 0 or 1; and

k is 0 or 1 ;

or a salt or solvate thereof.

The lanthanide chromophores have the ability of changing the emission response due to changes in the concentration of a target analyte. Thus, the emission response may either increase or decrease because of increasing amount of a target analyte in a medium in contact with the sensor. The change in response may be measured and compared to emissions for known concentrations of the target analyte for establishing the concentration of the target analyte in the medium on contact with the sensor.

In a preferred aspect of the invention, the linker of the luminescent lanthanide entity is covalently attached to the matrix material. The covalent attachment ensures that the lanthanide chromophore is maintained as an integral part of the matrix material, which is not leached or otherwise removed from the matrix material during extended use.

While the linker may be produced in a variety of chemical compositions, it is currently preferred that the linker of the luminescent lanthanide entity is a silane linker. The silane linker is especially useful when the matrix material is a sol-gel, because the silane linker can be activated with a -Si(OAlk) 3 group in which Aik means an C1-C6 straight of branched alkyl or alkenyl, for direct reaction with the polymers making up the sol-gel.

In some embodiments of the present invention, the linker has the general formula:

M-R3-NH-C (=0) -X-R3-Q wherein

R3 independently represents a group of the formula -R4- (X- R4)n-, wherein R4 independently is selected from straight or branched C2-C6 alkylene, C2 -C24 -haloalkylene ,

X is a hetero atom or a group selected among 0, S, NH, n is an integer of 0-12,

M is matrix material, and

Q independently is the lanthanide chromophore and/or the antenna dye.

As mentioned above, suitably, the matrix material is a sol- gel .

The sol-gel may be a polymerization of oxides of silicon. After the formation of the sol it may be treated by a variety of methods to obtain a film, which may be porous or mesoporous. The porous or mesoporous film ensures that the target analyte can easier diffuse through the matrix material to the lanthanide chromophore.

In an embodiment of the invention, the sensor further comprises a substrate to which the matrix material is adhered .

The substrate may have any macroscopic and microscopic form suitable for the specific application of the invention. Thus, the macroscopic form of the substrate may be strip for measuring a concentration of a target analyte by dipping the strip into the medium containing the target analyte. Alternatively, the substrate may be a container containing the medium to analyzed.

In a certain embodiment of the invention, the substrate is transparent for the excitation light as well as the emitted light. The transparency ensures that the concentration of a target analyte can be measured from the outside of the container. Thus, the transparent container may be applied the matrix material having the lanthanide chromophore attached, to the inner surface where it may come into intimate contact with the medium containing the target analyte. An excitation light may then be imposed from the outside of the container and the emitted light may then be measured for establishing the concentration of the target analyte .

In an aspect of the invention, the matrix material is deposited as a thin film on the substrate. The thin film allows for a fast diffusion of the target analyte to the lanthanide chromophore due to the short distances. The fast diffusion, in turn, ensures a short response time when a change in a medium of a target analyte occurs.

In an embodiment of the invention, the surface of the substrate has a predetermined microstructure comprising valleys and ridges. The microstructure ensures a high surface area, which in turn, increases the susceptibility of the sensor. Furthermore, a microstructure also increases the attachment force between the matrix material and the substrate .

In an implementation of the invention, the predetermined microstructure comprises a pillared structure. The pillared structure allows for obtaining an interference of the emitted light with the substrate to obtain either an amplification or an extinction of certain wavelengths.

In another implementation of the present invention, the predetermined microstructure comprises a stepped structure. A stepped microstructure offers the possibility of having a larger amount of luminescent lanthanide entities present in a small area, thus increasing the intensity and the sensibility.

In a preferred aspect of the invention, an antenna dye is present in the vicinity of the luminescent lanthanide entity. The presence of an antenna dye close to the lanthanide chromophore is useful in some applications of the invention for increasing the intensity of the emitted light, as the energy absorbed by the antenna dye may be transferred to the lanthanide chromophore. The distance between the lanthanide chromophore and the antenna dye is usually not above lOnm, such as not above 5 nm, and preferably not above 1 nm.

In an embodiment of the invention, the antenna die is chemically integrated in the luminescent lanthanide entity. The presence of the lanthanide chromophore and the antenna dye in the same chemical compound ensures that a consistent transfer of energy occurs, thus increasing the accuracy of the measurements of the concentration of the target analyte.

In an implementation of the invention, the antenna dye is an polyaromatic dye with a absorption above 300nm.

The invention also relates to system for measuring a concentration of a target analyte, comprising

- an excitation light source,

a sensor as disclosed above, producing a luminescence emission in response to an excitation light,

- a luminescence detector for measuring the intensity of the luminescence emission, and

- a computer for calculating the concentration of the target analyte by comparing the intensity of the luminescence emission with the intensity of the luminescence emission for known concentrations of the target analyte.

Short description of the drawings

Fig. 1 discloses measurement of glucose using a lanthanide entity.

Fig. 2 shows measurements for fructose using the same lanthanide entity as in Fig. 1.

Fig. 3 shows a standard curve for two different wave

lengths .

Fig. 4 discloses a system according to the invention.

Fig. 5a discloses a 2D optode, i.e. direct deposition on the substrate .

Fig. 5b discloses a 3D bulk optode, i.e. covalent attachment of a luminescent lanthanide entity to a matrix material subsequently deposited as a thin film on the substrate.

Fig. 5c shows deposition of a matrix material on a substrate having a microstructure, i.e. a pillared structure.

Fig. 5d discloses a deposition of a matrix material on a substrate having a microstructure, i.e. a stepped structure. Fig. 6 shows the (1) absence, (2) presence in the chemical structure, and (3) presence in the matrix material of the antenna dye.

Fig. 7 shows the results of the experiment of example 8 for various concentrations of carbonate.

Fig. 8 shows the intensity measurements at 615 nm for the experiment of example 8.

Fig. 9 shows the intensity measurements at 580 nm for the experiment of example 8. Fig. 10 shows the calculated ratio of the intensity

measurements at 615 nm and 580 nm.

Fig. 11 shows the results of the experiment with Eu. 1 reported in example 9 for various concentrations of

bicarbonate .

Fig. 12 shows the results of the experiment with Tb . 1 reported in example 9 for various concentrations of

bicarbonate .

Fig. 13 shows the results of the experiment with Sm. 1 reported in example 9 for various concentrations of

bicarbonate .

Fig. 14 shows the results of the experiments reported in example 10 measured at an intensity of 615 nm.

Fig. 15 shows the lifetime results of the experiments reported in example 10.

Fig. 16 shows the intensity ratio for the experiment shown in example 10.

Fig. 17 shows the results of the experiments reported in example 13 measured at an intensity of 615 nm.

Fig. 18 shows the lifetime results of the experiments reported in example 13.

Fig. 19 shows the intensity ratio for the experiment shown in example 13.

Detailed description of the invention

Luminescent lanthanide entity

Luminescent lanthanide entities are multicomponent systems, with three components: the lanthanide ( III ) ion, the

chelator, and a linker for immobilization to the matrix material. The lanthanide ( I I I ) ion and the chelator may herein commonly be referred to as the lanthanide

chromophore. The choice of each component is important to optimize the efficiency of the luminescent lanthanide entity.

The choice of lanthanide ( II I ) ion determines the

luminescence intensity, the emission wavelength and the excited state lifetime. The lanthanide ( III ) ion may be selected among gadolinium ( I II ) , europium ( I I I ) , terbium ( I I I ) , erbium (III), holmium ( I II ) , samarium ( I I I ) , ysprosium ( III ) , neodymium ( I I I ) , and ytterbium ( I I I ) . Gadolinium ( I I I ) is the most luminescent of the lanthanide ( II I ) ions, however, it emits in the UV (310 nm) and is therefore only applicable for certain applications. Europium ( II I ) and terbium(III) ions are the most studied luminescent lanthanide ( III ) ions. They display significantly higher emission intensity than erbiu ( I I I ) , holmium ( I I I ) , samariu ( I I I ) and

dysprosium ( I I I ) , which display luminescence in the visible spectrum (>405 nm) . However, only europium ( I I I ) and

terbium(III) ions have luminescence lifetimes of

milliseconds an above. In contrast, samarium ( II I ) ,

dysprosium ( I I I ) , and the near-infrared emitting

neodymium ( I I I ) and ytterbium ( I I I ) have luminescence

lifetimes in the microsecond or sub-microsecond range. Thus, in general luminescent lanthanide entities based on

europium ( I I I ) and terbium(III) ions are preferred.

The chelating part of the lanthanide luminescent sensor has two main purposes. It serves to prevent the release of the potentially toxic lanthanide ions into the environment, notably the biological environment, and it protects the lanthanide from quenching by O-H oscillators of solvent molecules. For the linear ligands the scaffold is usually made up of four or more acetate groups, which are covalently bound to a polyamine backbone e.g. EDTA and DTPA. In aqueous media, EDTA and DTPA form thermodynamically stable yet kinetically labile complexes. Preferably, the chelating part is a polyaminocarboxylate chelator in the form of a

macrocyclic ligand, thereby ensuring both thermodynamic and kinetic stability. As complexes of macrocyclic ligands often are kinetically inert, it is ensured that they remain intact in biological systems.

Selected thermodynamically stable lanthanide chromophores are indicated in the scheme below:

Lanthanide ( I I I ) ions are typically nine-coordinated in aqueous medium, e.g. with a [Ln-DOTA]- ligand occupying eight binding sites at the metal centre and water at the ninth capping—coordination site. The capping ligand will be placed at the face of the plane formed by the oxygen atoms of the acetate arms as shown in the exemplary chromophores above. Other coordination numbers than nine may be

considered.

Linker for immobilization

In an embodiment of the invention, the linker connecting the lanthanide chromophore to the matrix material is a silane linker. Use of a silane linker is particularly applicable when the matrix material is also comprising silane. The linker attaching the lanthanide chromophore to a matrix material, such as a silane matrix, may be designed in a variety of ways. In a certain embodiment the linker group having the general formula indicated below is attaching the matrix material to the lanthanide chromophore

M-R³-NH-C (=0) -X-R³-Q

wherein R³ independently represents a linker chosen from a group of the formula -R⁴- (X-R⁴ ) _n- , wherein R⁴ independently is selected from straight or branched C2-C6 alkylene, C2-C24- haloalkylene , X is a hetero atom or group selected among 0, S, NH, and n is an integer of 0-12, M is the matrix material and Q independently is the lanthanide chromophore and/or the antenna dye.

Generally, the R³ groups are connected to carbon atoms of the matrix material and the lanthanide chromophore,

respectively .

In other embodiments of the invention, the linker comprises an alkyne group capable of performing click chemistry with an azide group of a matrix material. In a still further embodiment of the invention, the linker is attached in one end to the lanthanide complex and in the other to a monomer capable of being co-polymerized with other monomers. In a specific implementation the co-polymerizable group is styrene .

Matrix material

The matrix materials of the present invention are generally of the sol-gel based type disclosed in WO 2015/058778. In one embodiment, the sol-gel based matrix is produced by preparing a first sol-gel component by polymerisation of a first alkoxysilane in the presence of an acid catalyst, preparing a second sol-gel component by polymerisation of a second alkoxysilane in the presence of an Lewis acid catalyst, and mixing the first sol-gel component and the second sol-gel

component for the preparation of the sol-gel based matrix.

The first alkoxysilane is generally of the general formula R¹-Si(OR²) 3 and the second alkoxysiloxane is of the general formula

wherein

R¹ represents a straight or branched C₁-C₆ alkyl or C₂- C₆ alkenyl, a C₃-C₆ cycloalkyl, a C_1-C6 aminoalkyl, a C₁-C6 hydroxyalkyl , a C₁-C₆ cyanoalkyl, a phenyl, a group of the formula -Y-(X-Y)_nH, wherein Y independently is selected from straight or branched C₁-C₆ alkylene, X is a hetero atom or group selected among 0, S, NH, and n is an integer of 1-5, or R¹ represents a C₁-C₆ alkyl substituted with a group

Z,

wherein Z independently is selected form the group comprising hydrogen, cyano, halogen, hydroxy, nitro, amide C₁-C₂₄ alkyl, Ci-C₂₄-haloalkyl , C₂-C₂₄-alkenyl , C₂-C₂₄-alkynyl , aryl, C₂-C₂₄-alkoxy, Ci-C₂₄-alkylsulfonyl , amino,

aminocarbonyl , aminothiocarbonyl , aminocarbonylamino , aminothiocarbonylamino , aminocarbonyloxy, aminosulfonyl , aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester comprising a C₁-C₆ alkyl alcohol moiety, (carboxyl ester) amino comprising a C₁-C₆ alkyl alcohol moiety, (carboxyl ester) oxy comprising a C₁-C₆ alkyl alcohol moiety, sulfonyl, sulfonyloxy, thiol, thiocarbonyl , C₁-C₂₄- alkylthio, 5 or 6 membered heteroaryl, or a C₃-C₇

cycloalkyl ;

R² independently represents a straight or branched C₁-C₆ alkyl; and

R³ is defined as above.

An additional alkoxysilane may be added to the first or the second alkyloxysilane, said additional alkoxysilane being of the formula:

R⁵-Si (OR²) ₃,

wherein R² is as defined above and R⁵ represents -R³-NH- C(=0)-X-R³-Q as defined above.

In other implementations of the present invention, the matrix material is a polymer such as polyvinyl alcohol or polystyrene. The lanthanide entity may be immobilized to functional groups of the polymer. Thus, the lanthanide entity may be covalently attached to a group of polyvinyl alcohol using so-called click chemistry. Specifically, the lanthanide entity may be provided with an alkyne group and the polymer matrix material may be provided with an azide group. Upon reaction between the alkyne group and the azide group a 1 , 2 , 3-triazole is formed thus connecting the

lanthanide entity with the matrix material . In another implementation of the invention, the lanthanide entity is provided with a polymerizable group such as a styrene group. Subsequently, the matrix material is formed as a co-polymer between styrene and the lanthanide entity provided with the styrene group.

Antenna dye

As direct excitation into the lanthanide centre is

inefficient, a luminescent lanthanide entity may rely on the antenna principle to obtain a higher intensity of the emitted light. The antenna unit acts as the light harvesting photosensitizer for the lanthanide, effectively increasing the absorption cross section of the complex. Thus, optical excitation of the molecule may occur with an efficiency of >10 000 hh¹ cnr¹ rather than ~1 hh¹ cnr¹. Following excitation there are multiple pathways for the energy absorbed by the antenna to migrate to the lanthanide centre. It is currently believed that the dominating energy transfer cascade is the following: a photon absorbed by the antenna chromophore promotes the chromophore from the ground state So to the first excited singlet state Si. The excitation is then followed by rapid intersystem crossing, optionally

facilitated by the lanthanide ( II I ) ion, to an accessible triplet excited state Ti. From the triplet state Ti the energy is then transferred to an isoenergetic lanthanide centred excited state.

For the energy transfer to be efficient, it is important that the triplet excited state of the antenna matches the energy level of the accepting lanthanide centred state. This is a delicate balance as the excited state must not be too high in energy, as this will result in poor overlap of the lanthanide centred state and the energy transfer will be ineffective. In such complexes the antenna fluorescence will dominate. On the other hand, if the triplet energy is too close in energy to that of the lanthanide excited state it will result in back energy transfer, and a low quantum yield of lanthanide luminescence. As a guidance for the selecting luminescent lanthanide chromophores and corresponding antenna dyes, efficient sensitisation generally requires an antenna triplet-lanthanide excited state energy gap of approximately 2.000 cnr¹ to avoid back energy transfer while maintaining a good overlap of the excited states.

In general, an efficient antenna unit has a triplet energy of more than 20.000 cm^-1, and can thus sensitize the

majority of the luminescent lanthanide ( III ) ions. Furthermore, efficient sensitisation requires that the antenna and lanthanide centre are in close proximity if not in direct contact. Generally, the distance between the antenna dye and the lanthanide centre is 10 nm or less, such as 5 nm or less and preferably 1 nm or less. The most efficient antenna chromophores are directly coordinated to the lanthanide ( I I I ) ion.

Below useful antenna units are shown with their key photo- physical properties. The antenna units are derived from organic fluorophores and have photophysical properties favorable for sensitisation of lanthanide ( III ) ions.

azaxanthone azathioxanthone acridone

k_abs = 330 nm ka_bS = 372 n kg_bs = 410 n

e = 6ΌO0 M W e = 5-800 M¹CtTf¹ e = 18-500 M ¹cm ¹

E_T = 23'500 cm-¹ E_T = 23-500 cm-¹ E_T = 21-500 cm^"1

Further specific antenna units of interest for the present invention include:

In accordance with a certain implementation of the present invention the antenna dye is a polyaromatic dye with an absorption above 300nm. suitably, the triplet excited state of the antenna dye has a triplet energy of more than 20.000 cm^-1. In a certain implementation of the invention the structure of the antenna dye is a heteroaromatic ring system comprising at least one heteroaromatic ring fused to at least one phenyl ring, said heteroaromatic ring comprising 0, S, or N as the ring atom.

Substrate for the matrix material

In one aspect, the invention relates to a composite

comprising a layer of sol-gel based matrix immobilizing the luminescent lanthanide entity and a substrate, such as a platform comprising a microstructure area, wherein the sol- gel based matrix is attached to the microstructure area. The composite provides a fast response time for the detection of analytes. The response of the composite may be detectable by detecting light or other electromagnetic radiation emitted by the sol-gel matrix, e.g. fluorescence and/or

phosphorescence, and/or the like.

The platform may comprise a plurality of microstructures, such as 2, 10, 50, 100, 1000, or more microstructures. The microstructure may be arranged in an array measuring various analytes and concentrations thereof.

For the purpose of the present description, the term

microstructure area refers to a structure having a plurality of micrometer-scale pillars, furrows, ridges, holes, indents, etc. The plurality of micrometer-scale pillars may be depressions and/or protrusions of a predetermined cross- sectional geometry, e.g. cylindrical or conical pillars. The microstructure may have a shape having an extent in at least one dimension, e.g. in two or even all three dimensions, between 0.1 pm and 50 mm, e.g. between 1 and 20 mm,

preferable between 5 and 10 mm.

The pillars may be cylindrical, cubic, or any other form.

The pillars may be arranged in a pattern, e.g. a regular pattern, in a square or hexagonal grid. However, a random pattern of pillars may be used as well. The pillars may have any size and shape. The pillars may be between 0.1 pm and 500 pm, preferably between 5 and 100 pm, more preferable between 10 and 40 pm in height.

The furrows may have a width of between 0.1 pm and 500 pm, preferably between 5 and 100 pm, more preferable between 10 and 40 pm. The pitch may be between 0.1 pm and 500 pm, preferably between 3 and 100 pm, more preferable between 5 and 10 pm. The holes in the substrate may have a pitch of between 0.1 p and 500 pm, preferably between 5 and 100 pm, more preferable between 30 and 50 pm. The indents in the

substrate may have a pitch of between 1 pm and 500 pm, preferably between 10 and 100 pm, more preferable between 20 and 40 pm. Compared to the holes, the indents have smooth corners. The ridges have a pitch of between 0.1 pm and 100 pm, preferably between 3 and 50 pm, more preferable between 5 and 10 pm.

The distance or length between each pillar, furrow, hole, indent, or ridge may be between 0.1 pm and 500 pm,

preferably between 5 and 100 pm, more preferable between 10 and 40 pm. The width or diameter of the pillars may be between 2 and 100 pm, more preferable between 5 and 40 pm.

In a preferred embodiment the distance between each pillar is between 5 and 40 pm, the height of the pillars are between 10 and 40 pm and the width of each pillar is between 5 and 40 pm. The pillars are preferably arranged in a hexagonal geometry. The advantage of this embodiment is that it allows single step manufacturing of blown molded flask and injection molded container parts with one or more microstructures and at the same time is suited for attaching the sol-gel based matrix to the microstructure because it is an optimal compromise between the rheology of the

polymer/glas s of the container and the ability to form a strong attachment with the sol-gel based matrix.

The microstructure may comprise a plurality of pillars in which the pillars, depressions and/or protrusions have different heights. The distance between the pillars, depressions and/or protrusions may be different.

The microstructure may be made of any suitable material such as a polymer, a plastic, glass, etc. Examples of suitable materials include inorganic materials, such as silicon, silicon oxides, silicon nitrides, III-V materials, such as, e.g., GaAs, AIAs, etc. Further examples of suitable

materials include organic materials, such as, but not limited to, SU-8, polymethylmethacrylate (PMMA),

polycarbonate (PC), polystyrene (PS), TOPAS (R) (cyclic olefin copolymer) , organically modified ceramics

(ORMOCER(R)) . The material may be optically transparent or reflective at the used wavelengths of light or other electromagnetic radiation. In one embodiment, the microstructure area comprises a plurality of pillars having a height between 0.1 pm and 500 pm and a distance between each pillar between 0.1 pm and 500 pm. The microstructure may also be applied to a curved surface. However, irrespective of whether the surface is curved or not, the shape of the pillars forming the

microstructure area may be designed such that the

microstructure area also improves or even optimizes the extraction of the light from a deposited sol-gel based matrix material during use as an optical sensor. For example, when the microstructure is a multitude of pillars that have a truncated-conical shape, the light emitted from a deposited sensor material may be directed to the optical sensing element through reflections on the inner surfaces of the pillar.

In one embodiment, the layer of the sol-gel based matrix has a thickness smaller than the height of the microstructure or the height of the pillars. Thus, the microstructure or the pillars of the microstructure penetrates the layer of the sol-gel based matrix, thereby providing stability to the sol- gel based matrix. Thus, the attachment of the sol-gel based matrix to the microstructure is improved.

In another embodiment, the composite comprises one or more sol- gel based matrixes comprising luminescent lanthanide entities. If the composites of the invention comprise different luminescent lanthanide entities, it will be possible to monitor one or more analytes and/or

concentrations thereof simultaneously. The use of the composites of the invention reduces the amount of space required for the monitoring.

In yet another embodiment, the invention relates to an array of sol-gel based matrixes attached to different areas on the microstructure area. In yet another embodiment, the

plurality of sol-gel based matrixes includes at least two sol-gel based matrixes having different luminescent

lanthanide entities.

A plurality of separate platform areas, e.g. a plurality of sol-gel based matrixes on respective sol-gel based matrixes areas, may be provided. Particularly, the plurality of sol- gel based matrixes may include at least two of sol-gel based matrixes having different thickness of the respective layer of the sol-gel based matrixes. Hence, different properties, e.g. sensitivity, may be provided. Further, different layers of sol-gel based matrixes may be obtained by providing variations in height/spacing profile of the microstructure.

This is particularly suitable for providing of sol-gel based matrixes on an inside surface of a container, e.g. a container for accommodating a fluid, e.g. a bottle, a tube, a flask, a bag, a microtiter plate, and/or the like. The surface may be planar or have a curvature in one or more directions. The deposited sol-gel based matrixes may thus be used to sense e.g. analytes or other properties of a medium (e.g. a fluid) in contact with the surface, e.g. a medium inside a container or laboratory consumable. Particularly, the sol-gel based matrixes may be read by detecting light emitted from the of sol-gel based matrixes responsive to the detected property. The light emission may be detected through the wall of a container by a detector placed outside the container or laboratory consumable.

In yet another embodiment, the platform of the composite is an inner surface of a container or conduit for transporting a fluid. In yet another embodiment, the container comprises an opening, cylindrical or tapered sides, and is closed opposite to the opening. In yet another embodiment, the platform is an inner surface of a disposable container for transporting a fluid. The composite may amongst other without being limited be deposited in and/or constitute a part of open or closed containers, or laboratory vessels, dedicated sensing equipment and laboratory consumables to act as a build-in sensor for analytes such as pH, dissolved oxygen (DO), conductivity, etc.

The composite may be deposited and constitute a part of open or closed containers, or laboratory vessels to yield a sensor spot, which may be circular or take any other form. The amount deposited may be 1 ul, 10 ul, 100 ul or even more. Any number of sensor spots can be deposited in a piece of equipment, consumable or vessel. The size of the spot may be 100 pm, 1 mm², 10 mm², 100 mm², 1 cm², 10 cm², 100 cm² or even more.

The container or laboratory consumable may be made of glass, polystyrene, polycarbonate or any polymer or composite material transparent to light, preferable green and red light (400 nm to 800 nm) .

In one aspect, the invention relates to the use of a composite according to the invention for monitoring of a bioculture. The environment and development of the

bioculture may be followed, periodically or continuously, e.g. by detecting light emitted from the composite or sensor. In another application of the invention, the composite is used for monitoring a body liquid, such as blood, saliva, serum, etc. for the concentration of a target analyte, such as glucose, carbonate, etc.

Thus, the sol-gel based matrixes and composites of the invention may be used as integrated sensors or probes, thereby reducing the risk of contamination as these can be read from the outside of the container and/or laboratory vessels .

Methods for deposition of matrix material, such as a sol-gel based matrix, on a homogeneous layer in a well-defined region of a surface are well known in the art, Quere D 2008, Annu. Rev. Mater. Res. 38 71-99. A drop of liquid material that is deposited on the microstructured area will spread, guided by the structures of the pillars, to homogeneously fill the volume between the pillars. In other embodiment of the invention, the matrix material is applied on the substrate in a thin film in a continuous layer covering high as well as lower areas of the microstructure.

Generally, a sol-gel process, also known as chemical solution deposition, is a wet-chemical technique suitable for the fabrication of materials, e.g. a metal oxide, or glass, starting from a chemical solution acting as a precursor for an integrated network, or gel, of discrete particles or network polymers. The process typically includes the removal of liquid after deposition of the precursor on the surface, e.g. by sedimentation and removal of the remaining solvent, by drying, and/or the like.

Afterwards, a thermal treatment, or firing process, may be employed .

Microstructuring of e.g. the inside of blow-molded plastic containers may be performed using step-and-stamp imprint lithography (Haatainen T and Ahopelto J 2003 Phys . Scr. 67 357), and for plastic components produced by injection molding, microstructures can be integrated directly in the mold (Utko P, Persson F, Kristensen A and Larsen N B 2011 Lab Chip 11 303-8) . Both of these fabrication methods are suited for large-scale industrial production. Spreading of the liquid is governed by the geometry of the microstructures and the thickness of the deposited film is determined by the height of the pillars and is thus

independent of the volume of the deposited drop. This enables easy and reproducible deposits of spots of the sol- gel based matrix of precise thickness to be made surfaces, such as metallic and plastic surfaces.

Spreading of the sol-gel based matrix enables direct, controlled deposition of spots of the sensor material inside containers, and it simplifies the fabrication of optical sensors in disposable lab ware.

The deposited layer generally remains fixed as an integral layer covering and attached to at least a portion of the deposition area. The fixation of the deposited matrix may be performed by a variety of techniques, e.g. by curing, hardening the deposited liquid, by evaporation of a solvent, by a sedimentation process, by covering the deposited matrix by a sealing layer, e.g. a foil, membrane etc. and/or a combination of the above. For example, the deposited matrix may be fixed on the surface by solvent evaporation, by cross-linking due light exposure, exposure by other forms of electromagnetic radiation, and/or by thermal treatment, and/or by any other suitable curing process. Materials which remain liquid after deposition on the microstructures are also a possibility; such materials may be fixed by

depositing a cover layer, e.g. a membrane, on top of the deposited matrix. Hence, the process results in a composite layered product in which the microstructured area and a layer of deposited matrix are efficiently bonded to each other .

In one embodiment of the invention, at least a part of the matrix material is immobilised resulting in an immobilised layer of sol-gel based matrix attached to the surface of the microstructure area. In another embodiment of the invention, the immobilised layer of sol-gel based matrix has a

thickness smaller than the height of the microstructure. In yet another embodiment of the invention, the microstructured area is prepared by a process chosen from 3D printing, injection molding, hot embossing, laser microstructuring, micromachining, chemical etching, photoresist layer

structuring .

The system according to the present invention is

schematically depicted in Fig. 4, which shows luminescent lanthanide entities 1 immobilized in a matrix material 2. A target analyte is not shown. The matrix material is

deposited or adhered to a substrate 3. The substrate is transparent for the wave lengths used for excitation and emission. After a not shown excitation light has been directed to the luminescent lanthanide entities immobilised in the matrix material, an emission signal is transduced and measured by the luminescence detector 4. The signal is subsequently directed to the computer 5, for processing. The processing i.e. compares the measured excitation light to a standard curve similar to the one shown in Fig. 3 for conversion of the intensity of the emitted light to a concentration of the target analyte.

In Fig. 5a-5c three embodiments of immobilizing the

luminescent lanthanide entity are shown. In Fig. 5a the luminescent lanthanide entities are directly deposited on a matrix material in the form of a flat substrate. In an embodiment, the matrix material is a polymer of glass having the ability of attaching the linker part of the lanthanide entity to the surface of the matrix material. In the event a silane linker is used, it can be directly attached to a glass substrate. A polymer matrix material may require pretreatment of the surface therefore for a silane linker to be able to be covalently attached.

In Fig. 5b, the luminescent lanthanide entity is immobilized to a matrix material, such as a sol-gel, which subsequently is deposition as a thin film on a flat substrate. As the sol-gel is porous material it allows relatively fast

diffusion of the target analyte from the environment, through the sol-gel to the lanthanide chromophore.

Fig. 5c discloses a deposition of the matrix material, such as a sol-gel, having attached the luminescent lanthanide entity to a micro structured surface of a substrate. The microstructures are parallel pillars and the matrix material is deposited as a thin layer following the microstructured surface so that a continuous layer of the matrix material is obtained.

Fig. 5d discloses a deposition of a matrix material on a substrate having a microstructure, i.e. a stepped structure. The luminescent lanthanide entity is attached to the matrix material by chemical linkage to the polymers making up the matrix structure to protrude into the external medium as shown on the drawing or embedded in the matrix structure. The latter embodiment requires the analyte to diffuse into the matrix material before it can be sensed by the

lanthanide chromophore. The matrix material is deposited as a thin layer following the stepped surface so that a continuous layer of the matrix material is obtained.

Fig. 6 shows the (1) absence, (2) presence in the chemical structure, and (3) presence in the matrix material of the antenna dye. For some embodiments of the present invention the antenna dye may, as shown in embodiment (1) be dispensed with because the intensity is sufficient for measuring the concentration of the target analyte. In a case a higher intensity of the emitted signal is desired, an antenna dye may be present in the same chemical structure as the lanthanide chromophore, as shown in embodiment (2) . The distance between the lanthanide chromophore and the antenna dye in the chemical structure should not extend 10 nm because the energy transfer from the antenna dye to the lanthanide chromophore may be impeded. In a preferred aspect that distance between the lanthanide chromophore and the antenna dye in the chemical structure is less than 5 nm, such as less than 1 nm for enabling efficient transfer of energy. In embodiment (3), the antenna dye is attached to the matrix material, e.g. by a covalent bonding. The distance between the antenna dye and the lanthanide

chromophore may be adjusted by the concentration of antenna dye and the lanthanide chromophore. Thus, a higher intensity of the emitted signal may be obtained by increasing the concentration of the antenna dye.

Examples

Example 1

Preparation of the sol-gel

The sol-gel is prepared by mixing a first sol-gel component and a second sol-gel component. The first sol-gel component is prepared from polymerization of ethyltriethoxysilane (ETEOS) under acidic conditions. ETEOS is hydrolysed under acidic conditions, which initiates a polymeric condensation reaction upon formation of a polymer silicon oxide network.

Procedure for preparation of first sol-gel component: 5 ml ETEOS (0.02 mol) is dissolved in 8 ml absolute ethanol (0.14 mol) upon stirring. Hereafter, 1.6 ml of 0.1 M HC1 solution (0.16 mmol) is added dropwise. This mixture is then left on a stirring table for a minimum of 7 days to allow the polymerization process to proceed.

The second sol-gel component is prepared from polymerization of 3- ( glycidoxy) propyltrimethoxysilane (GPTMS) using a Lewis acid as initiator. In this procedure we use boron

trifluoride diethyletherate as the Lewis acid. The Lewis acid attacks the epoxy ring that allows for ring opening of the epoxy ring upon formation of a secondary carbocation. This intermediate carbocation can then react with another GPTMS molecule, initiating a polymerization reaction. Due to the acidic environment a polymerization of the silicon network equivalent to that described for the first sol-gel component will proceed alongside.

Procedure for preparation of second sol-gel component: 6 ml of GPTMS (0.027 mol) is mixed with 11 ml of absolute ethanol (0.19 mol) upon stirring. Then 0.75 ml of cold borontri- fluoride diethyletherat (BF3 · 0 (CH2CH3) 2, 5.8 mmol) is added dropwise. The mixture is left with stirring for 30 min in a sealed container until the temperature of the mixture has dropped to room temperature. After 30 min 2 ml of MilliQ water (0.11 mol) is added to the solution. The resulting mixture was left with stirring for 4 h.

Example 2

Preparation of the luminescent lanthanide entity: The

Ln . DOTA-diamide phenylboronic acid in which Ln is Eu, was chosen as the lanthanide chromophore.

The lanthanide chromophore was activated by linking to a trialkoxysilane group that can mix into the silicon network of the first sol-gel component. Similar activation was prepared for the following chromophores :

The first and second sol-gel components are prepared and mixed as described above, with the exception that the silane functionalized lanthanide chromophore is mixed into the either the first or the second sol-gel component after 1 h of polymerization.

The first and second sol-gel components are left for polymerization. The two sol-gel components are then mixed in the described 1:1 molar ratio and left at a stirring table for no less than 3 days to allow the networks to mix. The lanthanide chromophore should be added in an amount so that a final concentration of 0.1 mM of chromophore is obtained in the final sol-gel mixture. The resulting mixture is then deposited on a glass platform and cured at 140°C for 2 hours .

Example 3

The sol-gels for compounds 2-4 were illuminated with a pulsing lamp having a wave length of 337 nm and the emission intensity was measured 0.2 to 1 ms after the pulse at 620 nm. Table 1 show the result:

Table 1 :

The data show that a reasonable emission is obtained.

Example 4

In a second experiment, the antenna dye 1 was used. The activated antenna dye was mixed with the activated

lanthanide chromophore into the first sol-gel component as described above.

As the antenna dye is excited at a slightly lower wave length the mixed sol-gels were illuminated at 400 nm and the emission was obtained at 590nm. The results are shown in table 2 below:

Table 2 :

The results show that the antenna dye increases the emission considerably. Thus, when lanthanide chromophore 2 is combined with the antenna dye 1, the emission increases 423 folds, for the combination of lanthanide chromophore 3 and the antenna dye 1, the increase in emission is 3 folds, and for the combination of lanthanide chromophore 4 and the antenna dye 1, the increase in emission is 78 folds. Example 5

An experiment was performed in which the sol-gel components were layered instead of being mixed. The results are shown in table 3:

Table 3 :

Generally, the experiment show that a mixture of the antenna dye and the lanthanide chromophore provides a high-intensive response, whereas the layering of the sol-gel components results in a low emission. The close proximity of the antenna dye and the lanthanide chromophore appears to be essential for a high emission to be obtained.

Example 6

The 4+1 mixture was subjected to carbonate buffer and a mixture of the target analyte glucose and carbonate buffer by dipping the glass platforms comprising the sol-gels in a solution of 0.5 M NaC0₃ and 0.5 M NaC03 plus 0.5 M D- glucose, respectively. The results are shown in table 4 below :

Table 4 :

Table 4 shows that a distinct and readily recongnisible signal is detected in the carbonate buffer, indicating that the lanthanide chromophore 4 is suitable as a lanthanide chromophore in a sensor. When the glass platform is dipped in a combination of carbonate solution and glucose the intensity of the emission decreases a as expected from the recorded standard curve in Fig. 3., indicating that the lanthanide chromophore is influenced by glucose.

Example 7

The sensor 4+1 was subjected to a various glucose and fructose concentrations. The antenna dye was exited at 400nm and the intensity of the emission was measured in the visible spectrum. The result is shown in Fig. 1.

It is observed that emission is most pronounced at 616 nm. A minor top is present at about 590 nm. Most importantly, it is observed that the emission intensity depends on the glucose concentration. Thus, the highest emission intensity is obtained for low glucose concentrations. The emission intensity gradually decreases with increasing glucose concentrations, indicating that the sensor can be used for measuring the glucose concentration.

A similar experiment was prepared using fructose as target analyte. The result is shown in Fig. 2. It is noted that emission occurs at the same wave lengths as for glucose. However, the intensity of the emission does not vary

substantially in response to varying fructose

concentrations. In other words, the sensor is specific for glucose and can be used for selectively measuring of the glucose concentration in a mixture also comprising fructose.

In fig. 3 the glucose response is measured for two different wave lengths, i.e. 590 nm and 616 nm for glucose

concentrations between 0 mM and 11.5 mM. For both

wavelengths of the glucose response, the concentration dependency is pronounced and can be used to accurately measure the glucose concentration within reasonable error margins based on the intensity of the emitted light.

Example 8

The following luminescent lanthanide chromophore was prepared :

Note that the cores structure is a heptadentate . The lanthanide chromophore was activated as described above by linking to a trialkoxysilane group that can mix into the silicon network of the first sol-gel component. The linker may be positioned in any of the positions indicated below.

The first and second sol-gel components are prepared and mixed as described above in example 1, with the exception that the silane functionalized lanthanide chromophore is mixed into the either the first or the second sol-gel component after 1 h of polymerization. The first and second sol-gel components are left for polymerization. The two sol-gel components are then mixed in the described 1:1 molar ratio and left at a stirring table for no less than 3 days to allow the networks to mix. The lanthanide chromophore is added in an amount so that a final concentration of 0.1 mM of chromophore is obtained in the final sol-gel mixture. The resulting mixture is then deposited on a glass platform and cured at 140°C for 2 hours .

The response is monitored following direct excitation of the lanthanide ion (here europium ( II I ) ) at 392 nm. In Fig. 7 the emission data is shown for incremental additions of NaHC03 (0.1M PBS - H₂0, pH 7.4).

The intensity change of the Eu(III) emission in the band at 617 nm with incremental addition of NaHC03 ( 0.1M HEPES buffer - H20, pH 7.4) is reported in Fig. 8. The Intensity change of Eu(III) emission in the band at 580 nm with incremental addition of NaHC03 ( 0.1M HEPES buffer - H20, pH 7.4) is shown in Fig. 9. The change in the ratio of emission intensity observed in the 615 nm band to that of the

intensity observed in the 580 nm band of Eu(III) emission in the band at 617 nm with incremental addition of NaHC03 ( 0.1M HEPES buffer - H20, pH 7.4) is shown in Fig. 10.

The data shows that an accurate measurement of the carbonate concentration can be obtained by measuring the intensities at two different wavelengths.

Example 9

To show that the approach is also applicable for other lanthanide complexes than europium, the complex Eu. 1 below was synthesized for europium ( III ) as well as terbium(III) and samarium ( I I I ) ions (Ln = Eu, Tb and Sm) . The bicarbonate responsiveness was measured for the complexes synthesized. The equilibrium for the synthesized complexes is shown below for the reaction with the bicarbonate ion.

The response of the complex with europiu ( III ) is shown in fig. 11, the response for complex with terbium(III) is shown in fig. 12, and the response for the complex with samarium ( I I I ) is shown in Fig. 13. The arrows indicate the tendency of the intensity and the concentration. Thus, an arrow pointing upwards indicates that the intensity increases with increasing concentration of the bicarbonate ion and a downward pointing arrow indicates that the intensity decreases when the concentration of the bicarbonate increases.

The results shown in Fig, 11 to Fig. 13 show that the concept of changing intensity for in dependency of the concentration of an analyte applies to a wide range of lanthanide complexes.

Example 10

To expand on the scope of the methodology, two additional europium ( I I I ) complexes were synthesized in addition to the europium ( I I I ) complex of example 9.

EU.2:(H₂0)₂ EU.3:(H₂0)₂

The responsiveness to change in bicarbonate concentration for the complexes was measured and the data is reported in Fig. 14 to Fig. 16.

The response to changes in the bicarbonate concentration was monitored as a change in spectrum shape, a change in the relative intensities, and a change in luminescence lifetime.

The data shows that the design is general, and that various responsive complexes for bicarbonate can be made using a generic heptadentate lanthanide complex. It is assumed that the specificity for the analyte can be modified by changing the design of the lanthanide complex. Thus, it is possible to the tailor lanthanide complex to be specific for a single or a few analytes and non-responsive for others.

Example 11 The matrix material immobilizing the luminescent lanthanide was varied in this experiment. Thus, in two first variants, the Eu. 1 was functionalized to covalently attach to a polyvinyl alcohol and a polystyrene using points of attachments on the cyclen nitrogen. In the third variant, the linker to the polysilan matrix material is attached to one of the coordinating arms:

Example 12

Antenna dies for enhancing the lanthanide luminescence.

The optical signal from the lanthanide complex may favorably be enhanced using an antenna chromophore. In a composite polymer lanthanide complexes and various sensitizer dyes was included and the enhancement factor determined.

The following antenna dyes were tested:

Compound 4 was attached to a silane matrix material, while compounds 5 and 6 was tested in a PVA matrix.

The results are determined as an enhancement factor for each antenna dye on a flat glass surface. The enhancement factor is calculated by dividing the signal obtained with the antenna dye together with compound Eu. 1 with the signal obtained from a matrix material with the lanthanide complex but without the antenna dye. The enhancement factors were determined from a series of measurements and the result is presented table below:

Table 5 :

We believe these data show that any aromatic dye with absorption above 300 nm can be used as an antenna dye when creating and optical sensor.

Example 13

Lactic acid responsive dyes.

The responsiveness of the lanthanide complexes to various lactic acid concentration was measured. The same compounds used in example 10 were used for the experiment:

The results of the measurements are shown in Fig. 17 to 19. While the response was not in the physiologically relevant concentration range for lactic acid, the complexes are clearly able to sense changes in the lactic acid concentration. Thus, the data show that the protected lanthanide complexes show a luminescent response that is proportional to the lactic acid concentration.

Example 14

Optodes on flat and structured surfaces for enhanced response

To document that the surface structure changes the intensity of the signal, the lanthanide entity and the antenna dye Eu.l + 4 (Example 12) were deposited on different structured surfaces. The structured surfaces were prepared on either polycarbonate or glass and the microstructures had dimension of 5-50 micrometer.

Structured surfaces:

A - Polycarbonate pillars - diameter 30 pm0, 50 pm pitch (Figure 5c)

B - Furrows on glass - width 6 pm, 7 pm pitch (Figure 5c)

C - Glass pillars - diameter 6 pm0, 7 pm pitch (Figure 5c)

D - Holes in glass - 50 pm pitch (Figure 5c)

E - Holes in glass - 40 pm pitch (Figure 5c)

F - Holes in glass - 30 pm pitch (Figure 5c)

G - Indents in glass - 30 pm pitch (Figure 5d, however, with smooth corners)

H - Ridges on glass - 7 pm pitch (Figure 5d - sharp corners as drawn)

I - Rough glass surface

The enhancement factor of the structures was determined by dividing the signal obtained on the structured surface to the signal determined on a flat glass substrate. The data is shown in the table below:

Table 6:

Area A B C D E F G H I

Enhancement 1,71 7,30 3,60 2,79 4, 97 2, 60 12,1 2,22 0, 87 factor

The structured surfaces with defined microstructures was able to enhance the sensor signal by an additional factor of up to 12. Thus, the total enhancement factor (antenna dye and microstructured surface) of the invention is up to 60 for responsive dyes based europium ( III ) ions and 3600 for responsive dyes based on terbium(III) ions.

Claims

1. A sensor for emitting luminescence proportional to the concentration of a target analyte, comprising

2. A sensor according to claim 1, wherein luminescent lanthanide entity comprises a luminescent lanthanide chromophore capable of interacting with a target analyte and a linker for immobilization to the matrix material.

3. A sensor according to claims 1 or 2, wherein the luminescent lanthanide entity has the general formula:

L² _xLn¹L¹ wherein

Ln¹ is a lanthanide ion;

L¹ is a polydentate ligand bonded to Ln¹;

each L² is the same or different and is a ligand bonded to Ln¹; and

x is 0 or an integer of from 1 to 8.

4. A sensor according to claims 1, 2 or 3, wherein the lanthanide chromophore has the following composition:

wherein

Ln is a lanthanide ion;

each L⁴ is the same or different and is a monodentate ligand bonded to Ln;

p is zero or an integer of from 1 to 4 ;

each L⁶ is the same or different and is optionally substituted methylene optionally substituted with a linker ;

R^a, R^b, R^c, R^d, R^e, R^f, and R^g are independently selected from H or the groups of formulae (Ila), (lib) and

( H e ) :

wherein

X¹ is 0, which 0 is also coordinated to Ln;

wherein

X² is 0, which 0 is optionally coordinated to Ln; R^h and R¹ are the same or different and are selected from H, unsubstituted or substituted Ci-₆ alkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl and a linker; and

v is 0 or 1 ;

Optional linker

X' LyA

X' (lie)

wherein

X¹ is 0, which 0 is also coordinated to Ln;

X³ is C or N, which N is optionally coordinated to Ln; and

X⁴ is C(=0) or P (=0) (0R^X) wherein R^x is H, unsubstituted or substituted Ci-₆ alkyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; j is 0 or 1; and

k is 0 or 1 ;

or a salt or solvate thereof.

5. A sensor according to any one of the preceding claims, wherein the linker of the luminescent lanthanide entity is covalently attached to the matrix material .

6. A sensor according to any one of the preceding claims, wherein the linker of the luminescent lanthanide entity is a silane linker.

7. A sensor according to anyone of the preceding claims, wherein the linker has the general formula:

M-R3-NH-C (=0) -X-R3-Q wherein

R3 independently represents a group of the formula -R4- (X-R4)n-, wherein R4 independently is selected from straight or branched C2-C6 alkylene, C₂-C₂₄-haloalkylene,

X is a hetero atom or a group selected among 0, S, NH, n is an integer of 0-12,

M is matrix material, and Q independently is the lanthanide chromophore and/or the antenna dye.

8. A sensor according to any of the preceding claims, wherein the matrix material is a sol-gel.

9. A sensor according to any one of the preceding claims, wherein the sensor further comprises a substrate to which the matrix material is adhered.

10. A sensor according to any one of the preceding claims, wherein the substrate is transparent for the excitation light as well as the emitted light.

11. A sensor according to any one of the preceding claims, wherein the matrix material is deposited as a thin film on the substrate.

12. A sensor according to any one of the preceding claims, wherein the matrix material is mesoporous, allowing for less impeded diffusion of the target analyte.

13. A sensor according to any one of the preceding claims, wherein the surface of the substrate has a predetermined microstructure comprising valleys and ridges.

14. A sensor according to any one of the preceding claims, wherein the predetermined microstructure comprises a pillared structure.

15. A sensor according to any one of the preceding claims, wherein the predetermined microstructure comprises a stepped structure .

16. A sensor according to any one of the preceding claims, wherein an antenna dye is present in the vicinity of the luminescent lanthanide entity.

17. A sensor according to any one of the preceding claims, wherein the antenna die is chemically integrated in the luminescent lanthanide entity.

18. A sensor according to anyone of the preceding claims, wherein the antenna dye is an polyaromatic dye with a absorption above 300nm.

19. A sensor according to anyone of the preceding claims, wherein the antenna dye is an acridone chromophore.

20. A sensor according to any of the preceding claims, wherein the triplet excited state of the antenna dye has a triplet energy of more than 20.000 cnr¹.

21. A sensor according to anyone of the preceding claims, wherein the structure of the antenna dye is a heteroaromatic ring system comprising at least one heteroaromatic ring fused to at least one phenyl ring, said heteroaromatic ring comprising 0, S, or N as the ring atom.

22. A system for measuring a concentration of a target analyte, comprising

- an excitation light source,

- a sensor according to any one of the claims 1 to 21, producing a luminescence emission in response to the excitation light,

23. A composite comprising a layer of matrix material immobilizing a luminescent lanthanide entity and a substrate comprising a microstructure area, wherein the matrix material is attached to the microstructure area.

24. The composite according to claims 23, wherein the microstructure area comprises a plurality of pillars

furrows, ridges, holes, and/or indents having a height or depth between 100 nm and 500 pm and a distance between each of between 100 nm and 500 pm.

25. The composite according to claims 23 or 24, wherein the distance between each pillar, furrow, ridge, hole, and/or indent is between 5 and 40 pm, the height or depth of the pillars, furrows, ridges, holes, and/or indents is between 10 and 40 pm and the width of each pillar, furrow, ridge, hole, and/or indent is between 5 and 40 pm.

26. The composite according to any one of the claims 23 to 25, wherein the layer of the matrix material has a thickness smaller than the height of the microstructure.

27. The composite according to any one of the claims 23 to

26, comprising an array of matrix materials attached to different areas on the microstructure area.

28. The composite according to any one of the claims 23 to

27, wherein the composite further comprises one or more antenna dyes.

29. The composite according to claims 23 to 28, wherein the matrix material includes at least two matrix materials having different luminescent lanthanide entities.

30. The composite according to anyone of the claims 23 to 29, wherein the substrate is an inner surface of a container or conduit for transporting a fluid.

31. The composite according to claim 30, wherein the container comprises an opening and cylindrical or tapered sides, and is closed opposite to the opening.

32. The composite according to claims 23 to 31, wherein the substrate is an inner surface of a disposable container for transporting a fluid.

33. Use of a composite as defined in claims 22 to 32 for monitoring of a bioculture or a body fluid.