DE102013210794B3 - Sensor with photocrosslinked hydrogel - Google Patents

Abstract

The present invention relates to a sensor comprising - a substrate; - a polymer optical functional layer; A layer of a photocrosslinked hydrogel polymer which has covalently bound analyte-specific recognition groups and is crosslinked via photoreactive groups; wherein the hydrogel polymer is covalently connected to the polymer of the optical functional layer via photo-reactive groups and the optical functional layer can indicate a change in the refractive index in the layer of the hydrogel polymer via a change in one of its optical properties.

Description

The present invention relates to optical sensors, preferably biochemical or bio-sensors, in which specific analytical detection layers based on immobilized photocrosslinked hydrogels are an integral part of the optical sensor and permit marker-free detection, and a method for their preparation.

Sensors based on microstructured polymer layers such. "Distributed feedback" lasers (also referred to as DFB lasers) or ring resonators offer cost-effective, miniaturizable and easily scalable opportunities for implementing sensors (eg, biosensors) that are technically established and lightweight to be implemented methods such as nanoimprinting, printing or spin coating. In most cases, the use as a biosensor requires the immobilization of biological or biochemical recognition units on the actual sensor surface. There is a risk that the recognition units denature when it comes to sensitive biomolecules.

The physical basis of such sensors is often the use of evanescent fields that respond to the change in local refractive index near the sensor surface. If the refractive index of the surrounding medium is smaller than the refractive index of the bound analytes, the intensity of the sensor signal is proportional to the number of bound analytes in the area of the evanescent field. Immobilization of analyte-specific recognition groups via adsorption restricts the maximum possible number of analytes that can be bound to a monolayer specifically, ie through a defined interaction between recognition group and analyte. This also sets an upper limit for the signal intensity.

The immobilization of proteins in photocrosslinked hydrogels is described in Langmuir 2011, 27, p. 6116-6123, Thomas Brandstetter et al. and in Analytica Chimica Acta 671 (2010), p. 92-98, Jürgen Rühe et al. described. The proportion of crosslinking (i.e., crosslinking points) repeating units in the hydrogel polymer is quite high at 5% and 1%, respectively. Accordingly, the degrees of swelling are quite low. The immobilized protein levels do not exceed those of a monolayer. These sensors work with fluorescent markers.

In view of the above, it is an object of the present invention to provide a sensor on which the largest possible number of recognition groups can be immobilized as gently as possible. The sensor should preferably be inexpensive, miniaturizable and easy to produce in large quantities. The sensor should have a good signal-to-noise ratio and a low detection limit and enable marker-free detection.

• – ein Substrat;
• – eine polymere optische Funktionsschicht;
• – eine Schicht eines photovernetzten Hydrogel-Polymers, das
• – kovalent gebundene Analyt-spezifische Erkennungsgruppen aufweist, und
• – über photoreaktive Gruppen vernetzt ist;

wobei das Hydrogel-Polymer über photoreaktive Gruppen kovalent mit dem Polymer der optischen Funktionsschicht verbunden ist und
die optische Funktionsschicht eine Brechzahländerung in der Schicht des Hydrogel-Polymers über eine Veränderung einer ihrer optischen Eigenschaften anzeigen kann.According to the present invention, the object is achieved by a sensor comprising

• A substrate;
• A polymeric optical functional layer;
• A layer of a photocrosslinked hydrogel polymer, the
• - has covalently bound analyte-specific recognition groups, and
• Is crosslinked via photoreactive groups;

wherein the hydrogel polymer is covalently connected via photoreactive groups with the polymer of the optical functional layer, and
the optical functional layer may indicate a refractive index change in the hydrogel polymer layer via a change in one of its optical properties.

In the sensor according to the invention, the analyte-specific detection layer based on the immobilized hydrogel is an integral part of the sensor. With the sensor according to the invention a marker-free detection and detection in real time are possible.

As explained above, the optical functional layer of the sensor according to the invention is one which exhibits a refractive index change in the layer of the hydrogel polymer (eg by immobilization of analyte molecules due to interaction with the analyte-specific recognition groups) via a change show one of their optical properties (sensory). Within the scope of the present invention, changing optical properties of the optical functional layer may be, for example, a change in the wavelength of the emitted laser light, a change in the resonant frequency or a change in the optical path length.

Optical functional layers that can indicate a refractive index change of an adjacent medium by altering one of its optical properties are known in the art. These are usually microstructured layers, the desired optical detection being achieved on account of the defined microstructuring (for example ring resonators, lattice structures (for example surface gratings or volume gratings) or two-arm waveguide arrangements).

The optical functional layer of the sensor according to the invention contains at least one polymer. As explained in more detail below, the polymer of the optical functional layer is covalently bonded to the hydrogel polymer.

The optical functional layer is preferably a microstructured optical functional layer, i. H. a layer with optical structures or with structural elements in the micrometer range. Hereinafter, these optical structures or structural elements in the micrometer range are also referred to as microstructured optical elements. Exemplary optical structures or microstructured optical elements include periodic grating structures (eg, periodic surface relief gratings or volume gratings), ring resonators, or even two-arm waveguide arrangements. As mentioned above, such microstructured optical functional layers and methods for their preparation are known per se.

Preferably, the microstructured optical elements are at least partially or completely formed by the polymer present in the optical functional layer.

In a preferred embodiment, the optical functional layer is a DFB laser, a ring resonator-containing layer or a layer having a Mach-Zehnder interferometer structure.

The optical functional layer contains at least one polymer. Due to the covalent bonds between the hydrogel polymer and the polymer of the optical functional layer immobilization of the hydrogel takes place. As will be described in more detail below, this covalent bonding of the hydrogel to the polymer of the optical functional layer takes place via photoreactive groups. These photoreactive groups can react both with the polymer chains of the optical functional layer (photoreactive groups which ensure the covalent attachment of the hydrogel to the optical functional layer) as well as with adjacent polymer chains in the hydrogel (photoreactive groups which cause the three-dimensional cross-linking of the hydrogel). The photoreactive groups for crosslinking in the hydrogel and the photoreactive groups for attachment to the optical functional layer may be the same or different, preferably they are the same.

In a preferred embodiment, the optical functional layer is a DFB (Distributed Feedback) laser. As is generally known to the person skilled in the art, DFB lasers are lasers in which the active material is periodically structured. The structures of changing refractive index form a resonator structure. This can be realized by a surface relief grid (variation of the surface topology) or by a volume grid (variation of the refractive index). The resulting interference causes the optical feedback of the laser. Alternatively, it is also possible for the optical functional layer containing the laser-capable material to simultaneously also embody the resonator structure.

A change in the refractive index in the hydrogel polymer layer (for example, by immobilization of analyte molecules) causes a sensorially usable wavelength shift of the emitted laser light.

In the case of the DFB laser, the optical functional layer contains a laser-capable material in combination with a resonator structure (grating), i. H. a material emitting laser light when excited optically. Such materials are generally known to the person skilled in the art. By way of example, fluorescent dyes, fluorescent polymers, semiconductive fluorescent polymers or mixtures thereof may be mentioned.

The fluorescent dyes are usually present in the optical functional layer in an amount of 0.5 to 20% by weight. These fluorescent dyes may be embedded in the polymer of the optical functional layer. The polymer of the optical functional layer then functions not only as a microstructured optical element (eg in the form of or as part of a periodic surface relief or volume grating of the DFB laser) but also as a matrix for the laser-capable component.

When using laser-capable fluorescent polymers, the optical functional layer predominantly, z. At least 70% by weight, more preferably at least 85% by weight or even consist entirely of the fluorescent laser-capable polymer. In this case, the polymer of the optical functional layer functions not only as a microstructured optical element (eg in the form of or as part of a periodic surface relief grating of the DFB laser) but also as an active laser-capable component. Advantages also bring mixtures of two dyes or blends of two fluorescent polymer.

Laser-compatible materials are generally known to the person skilled in the art. By way of example, in this context, laser dyes, pyromethanes, 4- (dicyanomethylene) -2-methyl-6- (4-dimethylaminostyryl) -4H-pyran (DCM), perylene compounds such. B. perylenediimides, poly (p-phenylene-vinylene) (abbreviation: PPV), poly [(9,9-di-n-octylfluorenyl-2,7-diyl) -alt- (benzo [2,1,3] thiadiazole -4,8-diyl)] (abbreviation: F8BT), poly [2-methoxy-5- (2-ethylhexyloxy) -1,4-phenylenevinylene] (abbreviation: MEH-PPV), or mixtures thereof (such as e.g. F8BT / MEH-PPV).

As already mentioned above, in the case of the laser-capable optical functional layer, the specific interaction of the analyte molecules with the photocrosslinked hydrogel layer causes a change in the refractive index of this hydrogel layer, which leads to a shift in the wavelength of the laser light emitted by the optical functional layer. The DFB laser itself is the marker-free sensor.

If the sensor according to the invention contains a DFB laser, it can be produced by methods which are known in principle to the person skilled in the art.

As mentioned above, in a preferred embodiment, the optical functional layer may also be a layer containing a ring resonator. In order to supply light to the ring resonator, the optical functional layer still contains a waveguide. As is known to those skilled in the art, the ring resonator and waveguides differ from the surrounding material by the refractive index, so that the light is conducted in these optical elements (i.e., ring resonator and waveguide). Such optical ring resonators and methods for their preparation are known in the art. The ring resonator is preferably formed by the polymer present in the optical functional layer. As a result, the formation of covalent bonds between the polymer forming the ring resonator and the hydrogel polymer is also possible in this embodiment.

Suitable polymers for the formation of a ring resonator are known in principle to the person skilled in the art. By way of example, the following polymers may be mentioned: polyacrylates, polymethacrylates, polycarbonates, polyurethanes, or mixtures of these polymers.

The frequency of the ring resonator is detuned (i.e., altered) by the interaction of the analyte with the hydrogel layer via the change in refractive index.

As mentioned above, in a preferred embodiment, the optical functional layer may be a layer having a Mach-Zehnder interferometer structure. As is known to those skilled in the art, in a Mach-Zehnder interferometer the incident light is split by a beam splitter into two branches. So there is a two-arm waveguide structure. After passing through the two routes whose optical path length may be different, the two beams (reference and measuring beam) are superimposed again.

Preferably, the two arms e of the Mach-Zehnder interferometer structure are formed from the polymer of the optical functional layer. Suitable polymers for the formation of such waveguide structures in a Mach-Zehnder interferometer structure are basically known to the person skilled in the art. By way of example, the following polymers may be mentioned: polyacrylates, polymethacrylates, polycarbonates, polyurethanes, or mixtures of these polymers.

Preferably, the hydrogel polymer has both a common interface with the first branch (which, for example, acts as a waveguide for the reference beam) and a common interface with the second branch (which, for example, acts as a waveguide for the measurement beam). Furthermore, it is preferred that the layer of the hydrogel polymer is structured such that liquid diffusion from the region of the hydrogel polymer which at least partially covers the first branch to the region of the hydrogel polymer which at least partially covers the second branch , is prevented. This can be achieved, for example, by dividing the hydrogel polymer into two spatially separated regions, namely a first hydrogel polymer which at least partially covers the first branch, and a second hydrogel polymer which at least partially covers the second branch , The sensor is then preferably operated, for example, such that one of these two polymer regions of the hydrogel layer (eg. B. the one which covers the first branch or arm of the optical functional layer at least partially) comes into contact only with the solution without analyte, while the other polymer region of the hydrogel layer (ie the one who the second branch or arm of the optical functional layer at least partially covering) is contacted with the solution containing the analyte. The refractive index change of the measuring beam waveguide changes the optical path length of the second arm or branch of the Mach-Zehnder interferometer. This causes a sensorially usable phase shift.

The polymer-containing optical functional layer is present on a substrate. Substrates suitable for this purpose are generally known to the person skilled in the art. The substrate may be made of an inorganic material (eg, glass, silicon, silicon nitride, etc.) and / or plastic. The substrate may be single-layer or multi-layered. Suitable substrates include microfluidic chips or so-called biodisposables.

• – kovalent gebundene Analyt-spezifische Erkennungsgruppen aufweist, und
• – über photoreaktive Gruppen vernetzt ist;

und wobei das Hydrogel-Polymer über photoreaktive Gruppen kovalent mit dem Polymer der optischen Funktionsschicht verbunden ist.As noted above, the sensor of the present invention includes a photocrosslinked hydrogel polymer layer

• - has covalently bound analyte-specific recognition groups, and
• Is crosslinked via photoreactive groups;

and wherein the hydrogel polymer is covalently linked to the polymer of the optical functional layer via photoreactive groups.

In the context of the present invention, it is possible for the layer of the photocrosslinked hydrogel polymer to be a continuous layer that extends over the entire surface of the optical functional layer. Alternatively, it is also possible that the hydrogel layer covers only a part of the optical functional layer. Furthermore, it is also possible within the scope of the present invention that the hydrogel layer is not continuous, but has two or more regions which are spatially separated from one another. It is important above all that the hydrogel polymer and the polymer of the optical functional layer have a common interface at which covalent bonds can be formed via the photoreactive groups of the hydrogel.

The photoreactive groups thus causes two things. On the one hand, adjacent polymer chains in the hydrogel are linked to one another (that is, crosslinking points are formed in the hydrogel via these photoreactive groups), and the hydrogel polymer is covalently linked to the polymer of the optical functional layer via the photoreactive groups.

The photoreactive groups that cause crosslinking in the hydrogel polymer and the photoreactive groups that cause covalent bonding to the optical functional layer may be the same or different. Preferably they are the same.

A hydrogel or hydrogel polymer is understood to mean a three-dimensional network which is no longer soluble in water but absorbs it and swells with it. The polymer chains of hydrogels are interconnected via crosslinking points and thus form a three-dimensionally crosslinked structure.

In the context of the present invention, the formation of crosslinking points via photoreactive groups takes place. An additional crosslinking by tying additional covalent bonds, z. Chem. Int. Ed. 2001, 40, pp. 2004-2021) is optionally possible if further monomer units having suitable reactivity in the polymer chain of the copolymer to be built in. In a preferred embodiment, however, the crosslinking required for the formation of the hydrogel takes place exclusively via a photochemical crosslinking. In a particularly preferred embodiment, the photochemical crosslinking via photoreactive groups, which can react after activation by visible light or UV radiation by insertion reactions with organic radicals in the immediate vicinity. Examples of such particularly preferred photoreactive groups are benzo or acetophenone groups, diazirines or azides.

Hydrogel polymers and processes for their preparation are known to those skilled in principle.

In the present invention, the hydrogel may also be a switchable hydrogel. Switchable hydrogels are understood by those skilled in the art to mean those hydrogels that are under the action of an external stimulus (ie, under the action of an external, changing parameter) Swell water intake or alternatively can collapse under liquid delivery. These transitions are preferably reversible. Such switchable hydrogels are generally known to the person skilled in the art.

As one skilled in the art knows, each polymer is made up of repeating units. Each polymer is effectively the sum of all its repeating units. If all repeating units of a polymer are identical, it is called a homopolymer. Different repeating units have a copolymer.

Like any polymer, the photocrosslinked hydrogel polymer of the present invention is thus composed of repeating units. As will be described in more detail below, the photocrosslinked hydrogel polymer preferably comprises (i) repeating units WE ^AS , each having an analyte-specific recognition group; (ii) repeating units WE ^V , which have each formed a crosslinking point in the hydrogel polymer via a photoreactive group, and (iii) repeating units WE ^OF , which are each covalently connected to the polymer of the optical functional layer via a photoreactive group.

The hydrogel polymer also contains such repeating units that form the actual structural base of the hydrogel (hereinafter also referred to as repeating units WE ^base ) and usually in a proportion of over 50 mole%, more preferably over 60 mole% or over 70 mole% in the hydrogel Polymer present. In the context of the present invention, the repeat units WE ^Basis can be selected from those which are customarily present in known hydrogel polymers.

• (1)
  
  wobei R₁ = H, Alkyl wie z. B. C_1-4-Alkyl, bevorzugt -CH₃, R₂, = H, Alkyl wie z. B. C_1-4-Alkyl, -(CH₂)_n-COOH und x = 1–50, bevorzugter 2–20 sind,
• (2)
  
  wobei R₁ = H, Alkyl wie z. B. C_1-4-Alkyl, bevorzugt -H, R₄, = H, Alkyl wie z. B. C_1-4-Alkyl, -(CH₂)_n-COOH und x = 1–50, bevorzugter 3–20 sind,
• (3)
  
  wobei R₂, = H, Alkyl wie z. B. C_1-4-Alkyl, -(CH₂)_n-COOH und x = 1–50, bevorzugter 2–20 sind,
• (4)
  
  wobei R₂, = H, Alkyl wie z. B. C_1-4-Alkyl, -(CH₂)_n-COOH und x = 1–50, bevorzugter 2–20 sind,
• (5)
  
  wobei R₁ = H, Alkyl wie z. B. C_1-4-Alkyl R₂, R₃, = H, Alkyl wie z. B. C_1-4-Alkyl, Allyl,
  
  sind,
• (6)
  
  wobei R₁ = H, Alkyl wie z. B. C_1-4-Alkyl, bevorzugt H und x = 1–6, bevorzugter 3–4 sind,
• (7)
  
  wobei R₁, R₂, R₃ = H, Alkyl wie z. B. C_1-4-Alkyl sind,
• (8)
  
  R₁ = H oder eine Verzweigung der Polysaccharidkette R₂ bis R₅ = H, Alkyl wie z. B. C_1-4-Alkyl, Allyl, -(CH₂)_n-COOH sind,
• (9) Es können auch andere Polysaccharidstrukturen, wie z, B. Carboxymethylcellulose, Hydroxyethylstärke etc. als Strukturelemente für das Hydrogel dienen.
• (10)
  
  wobei R₁ = H, Alkyl wie z. B. C_1-4-Alkyl, bevorzugt -CH₃ ist,
• (11)
  
  wobei R₁ = H, Alkyl wie z. B. C_1-4-Alkyl, bevorzugt -CH₃ ist, x, y unabhängig voneinander Werte zwischen 1 und 12 annehmen können und Z = SO₃, COO, oder PO₃ ist.

The following repeat units WE ^{base of} the hydrogel polymer can be mentioned by way of example:

• (1)
  
  where R ₁ = H, alkyl such as. B. C _1-4 alkyl, preferably -CH ₃ , R ₂ , = H, alkyl such as. C _1-4 alkyl, - (CH ₂ ) _n -COOH and x = 1-50, more preferably 2-20,
• (2)
  
  where R ₁ = H, alkyl such as. B. C _1-4 alkyl, preferably -H, R ₄ , = H, alkyl such as. C _1-4 alkyl, - (CH ₂ ) _n -COOH and x = 1-50, more preferably 3-20,
• (3)
  
  wherein R ₂ , = H, alkyl such as. C _1-4 alkyl, - (CH ₂ ) _n -COOH and x = 1-50, more preferably 2-20,
• (4)
  
  wherein R ₂ , = H, alkyl such as. C _1-4 alkyl, - (CH ₂ ) _n -COOH and x = 1-50, more preferably 2-20,
• (5)
  
  where R ₁ = H, alkyl such as. B. C _1-4 alkyl R ₂ , R ₃ , = H, alkyl such as. C _1-4 alkyl, allyl,
  
  are,
• (6)
  
  where R ₁ = H, alkyl such as. C _1-4 alkyl, preferably H and x = 1-6, more preferably 3-4,
• (7)
  
  wherein R ₁ , R ₂ , R ₃ = H, alkyl such as. C _1-4 alkyl,
• (8th)
  
  R ₁ = H or a branch of the polysaccharide chain R ₂ to R ₅ = H, alkyl such as. C _1-4 alkyl, allyl, - (CH ₂ ) _n -COOH,
• (9) Other polysaccharide structures such as, for example, carboxymethyl cellulose, hydroxyethyl starch, etc., may serve as structural elements for the hydrogel.
• (10)
  
  where R ₁ = H, alkyl such as. C _1-4 alkyl, preferably -CH ₃ ,
• (11)
  
  where R ₁ = H, alkyl such as. C _1-4 alkyl, preferably -CH ₃ , x, y can independently of one another assume values between 1 and 12 and Z = SO ₃ , COO, or PO ₃ .

The photocrosslinked hydrogel polymer contains covalently bound analyte-specific recognition groups, i. H. These analyte-specific recognition groups are linked via covalent bonds either directly to the polymer chain of the hydrogel polymer or to a side group of the hydrogel polymer chain.

• (i) Wiederholeinheiten WE^AS, die jeweils zumindest eine Analyt-spezifische Erkennungsgruppe aufweisen,
• (ii) Wiederholeinheiten WE^V, die jeweils über eine photoreaktive Gruppe einen Vernetzungspunkt in dem Hydrogel-Polymer ausgebildet haben, sowie
• (iii) Wiederholeinheiten WE^OF, die jeweils über eine photoreaktive Gruppe kovalent mit dem Polymer der optischen Funktionsschicht verbunden sind,

In a preferred embodiment, the photocrosslinked hydrogel polymer comprises

• (i) repeating units WE ^AS , which each have at least one analyte-specific recognition group,
• (ii) repeat units WE ^V each having a crosslinking point in the hydrogel polymer via a photoreactive group, and
• (iii) repeat units WE ^OF , which are each covalently connected to the polymer of the optical functional layer via a photoreactive group,

As will be explained in more detail below, the covalent bonding of the analyte-specific recognition groups in the hydrogel polymer can be realized by monomer compounds are already present in the polymerization of a photocrosslinkable copolymer (from which the photocrosslinked hydrogel is formed later by irradiation) Contain analyte-specific recognition group. Alternatively, it is also possible that in this polymerization initially a monomer compound having an organic functional group is present and only after polymerization, these organic functional groups with a compound containing the analyte-specific recognition group, are reacted.

Suitable monomer compounds from which the repeating units WE ^{AS are} derivable or obtainable are known to the person skilled in the art. These are those compounds which can be incorporated into a polymer chain as part of a polymerization, in particular compounds with an unsaturated carbon-carbon bond. By way of example, olefins, styrenes, vinyl compounds, acrylates or methacrylates may be mentioned in this connection. As already mentioned above, these monomer compounds may already contain an analyte-specific recognition group or, alternatively, they may initially contain a functional organic group which after polymerization (ie after incorporation of the monomer molecules into the polymer chain) with a compound containing the analyte-specific recognition group , is reacted. For the latter variant, in addition to the analyte-specific recognition group, these compounds in turn contain a suitable functional organic group that can react with the functional organic group of the monomer compound (ie, complementary pairs of functional groups are used). Examples of such complementary pairs of functional groups are active esters and amino groups, epoxides and hydroxyl groups, thiol and ene groups or alkyne and azido groups.

Depending on the type of analyte to be detected, it is in principle known to the person skilled in the art which specific recognition groups (that is to say groups which can undergo a specific interaction with the analyte molecules) can be used.

For example, the analyte-specific recognition group may be selected from antibodies, F _ab fragments of antibodies, enzymes, enzyme fragments, coenzymes, prosthetic groups, aptamers, single-stranded DNA, single RNA strands, or combinations thereof.

In a preferred embodiment, the sensor is a biosensor or biochemical sensor and thus a sensor that uses biological or biochemical components to select the analyte.

Depending on the application of the sensor, the proportion of repeat units WE ^AS in the hydrogel polymer can vary over a wide range. For example, the repeat units may be present WE ^AS mol% in the hydrogel polymer in an amount of up to 50 mole%, or up to 95, based on the total amount of all repeating units constituting the hydrogel polymer. However, for many applications it may be sufficient if the repeat WE ^AS in the hydrogel polymer in an amount of up to 10 mole%, or up to 5 mol% are present, based on the total amount of all repeating units, from which the hydrogel Polymer exists.

As will be described in more detail below, the repeating units WE ^V and WE ^{OF can} preferably be obtained by virtue of the fact that at least one monomer compound which contains a photoreactive group is already present in the preparation of a photocrosslinkable copolymer. This monomer compound is incorporated into the polymer chain during the polymerization. Thus, a photocrosslinkable copolymer comprising repeat units WE ^PR each having a photoreactive group is included. Irradiation of the photocrosslinkable copolymer either leads to a reaction (eg an insertion reaction) between the photoreactive group and an organic group or an organic radical of an adjacent polymer chain and thereby to form a crosslinking point or to covalently bond to it Polymer in the optical functional layer.

Suitable photoreactive groups which can form such crosslinking points in the hydrogel polymer by irradiation are known in principle to the person skilled in the art.

In a preferred embodiment, the photoreactive group is selected from those which can react by insertion with an organic radical.

Preferably, the photoreactive group is selected from a benzophenone group, an acetophenone group, a diazirine group or an azide group.

When a benzophenone group is used as a photoreactive group, it may, for example, react under irradiation with an HC group of an adjacent polymer chain according to the following reaction, thereby forming a crosslinking point: > C = O + HC (R ¹ R ² R ³ ) →> C (OH) -C (R ¹ R ² R ³ )

Suitable monomer compounds from which the repeat units WE ^V and WE ^{OF can be} derived or are available are known to those skilled in the art. These are those compounds which can be incorporated into a polymer chain as part of a polymerization, in particular compounds with an unsaturated carbon-carbon bond. By way of example, olefins, styrenes, acrylates or methacrylates may be mentioned in this connection. Preferably, these monomer compounds already contain a photoreactive group. Alternatively, however, it is also possible that these monomer compounds initially contain a functional organic group which, after polymerization (ie after incorporation of the monomer molecules into the polymer chain), is reacted with a compound containing the photoreactive group. For this purpose, in addition to the photoreactive group, these compounds in turn contain a suitable organic functional group capable of reacting with the functional organic group of the monomer compound (ie, complementary pairs of functional groups are used). Examples of such complementary pairs of functional groups are active esters and amino groups, epoxides and hydroxyl groups, thiol and ene groups or alkyne and azido groups.

The photocrosslinked hydrogel contains the repeating units WE ^V and WE ^OF in an amount which is sufficient to ensure, in addition to a three-dimensional crosslinking of the hydrogel, that photoreactive groups are still available for covalent bonding to the optical functional layer.

However, in the context of the present invention, it has also proven advantageous for the sensitivity of the sensor and for the signal-to-noise ratio if the proportion of photoreactive units and thus also the degree of crosslinking of the hydrogel are as low as possible.

In a preferred embodiment, therefore, the repeating units WE ^V and WE ^{OF are present} in the hydrogel polymer in an amount of less than 1.0 mol%, preferably less than 0.5 mol%, more preferably less than 0.3 mol%, based on the total amount of all repeat units that make up the hydrogel polymer. The ratio of the repeat units WE ^V and WE ^OF each other can be varied over a wide range. It is preferred that the sum of the repeat units WE ^V and WE ^{OF is present} in an amount of less than 1.0 mol%.

Preferably, the repeating units WE ^V and WE ^{OF are present} in the hydrogel polymer in an amount in the range of at least 0.01 mol% to less than 1.0 mol%, more preferably 0.03 mol% to less than 0.5 mol%, more preferably, from 0.05 mole% to less than 0.3 mole%, based on the total amount of all repeat units making up the hydrogel polymer.

The amount of repeating units WE ^V and WE ^OF in the hydrogel polymer can be determined from the starting amount of the photoreactive group monomer compounds and the degree of conversion of these monomer compounds during the polymerization. For example, in the polymerization of the photocrosslinkable copolymer, if the monomer compound having the photoreactive group is present in an amount of 0.2 mol% (based on the total amount of the monomer compounds) and the polymerization reaction proceeds quantitatively, the photocrosslinked hydrogel polymer contains the repeating units WE ^V and WE ^S in an amount of 0.2 mol%. The degree of conversion of the monomer compounds in the polymerization can be determined by analytical methods known to the person skilled in the art.

The photocrosslinked hydrogel preferably has a degree of swelling of> 4, more preferably> 5, more preferably> 8.

The degree of swelling indicates how much water a hydrogel absorbs at most compared to its dry weight. It is determined by the quotient of the layer thickness d in the swollen state and the dry layer thickness d ₀ . For the determination of the degree of swelling, distilled water is used.

In a preferred embodiment, the sensor according to the invention detects the analyte label-free (ie the analyte molecules do not need to carry markers (eg fluorescence markers, dye markers, conjugated enzymes, etc.)).

The sensor of the present invention can be used for marker-free detection of analytes (preferably as biosensor or biochemical sensor).

• – die Bereitstellung eines Substrats mit einer darauf aufgebrachten Polymer-haltigen optischen Funktionsschicht;
• – die Bereitstellung einer Schicht eines photovernetzbaren Copolymers auf der optischen Funktionsschicht, wobei das photovernetzbare Copolymer und das Polymer der optischen Funktionsschicht eine gemeinsame Grenzfläche aufweisen und das photovernetzbare Copolymer Wiederholeinheiten WE^PR umfasst, die jeweils zumindest eine photoreaktive Gruppe aufweisen, und
• – die Bestrahlung des photovernetzbaren Copolymers unter Ausbildung eines photovernetzten Hydrogel-Polymers und Ausbildung von kovalenten Bindungen zwischen dem photovernetzten Hydrogel-Polymer und dem Polymer der optischen Funktionsschicht.

The subject of the present invention is also a method for producing the sensor described above, comprising

• The provision of a substrate having a polymer-containing optical functional layer applied thereon;
• The provision of a layer of a photocrosslinkable copolymer on the optical functional layer, wherein the photocrosslinkable copolymer and the polymer of the optical functional layer have a common interface and the photocrosslinkable copolymer comprises repeating units WE ^PR , each having at least one photoreactive group, and
• - The irradiation of the photocrosslinkable copolymer to form a photocrosslinked hydrogel polymer and formation of covalent bonds between the photocrosslinked hydrogel polymer and the polymer of the optical functional layer.

With regard to the preferred properties of the substrate and of the polymer-containing optical functional layer, reference may be made to the above statements.

The optical functional layer, for. Example, in the form of a DFB laser or a ring resonator layer or a layer with a Mach-Zehnder interferometer structure can be obtained by methods known in the art.

The repeating units WE ^PR containing the photoreactive group are preferably obtained by virtue of the presence of at least one monomer compound containing a photoreactive group in the preparation of the photocrosslinkable copolymer.

Irradiation of the photocrosslinkable copolymer then results in a reaction (eg, an insertion reaction) between the photoreactive group and either an organic group or organic residue of an adjacent polymer chain in the photocrosslinkable copolymer (thereby forming a crosslink point in the hydrogel). or a polymer chain present in the optical functional layer. Depending on whether the photoreactive groups with adjacent polymer chains form crosslinking points in the hydrogel or a covalent bond to the optical functional layer, the repeat units WE ^{PR of} the photocrosslinkable copolymer are either in repeating units WE ^V (ie repeating units which have formed crosslinking points in the hydrogel) or in repeating units WE ^OF (ie

Repeat units with covalent bonding to the optical functional layer) of the photocrosslinked hydrogel polymer.

With regard to suitable photoreactive groups, reference may be made to the above statements in the description of the sensor.

The photochemically initiated crosslinking of the copolymer to form the hydrogel is effected by irradiation with light of suitable wavelength. Depending on the photoreactive group used, it is known to the person skilled in the art at which wavelength the irradiation is to take place. By way of example, reference may be made in this connection to the irradiation conditions described in Laschewsky et al., Soft Matter 2013, 9, p. 929-937.

Suitable monomer from which the repeating WE ^PR are available, known to the expert. These are those compounds which can be incorporated into a polymer chain as part of a polymerization, in particular compounds with an unsaturated carbon-carbon bond. By way of example, olefins, styrenes, acrylates or methacrylates may be mentioned in this connection. Preferably, these monomer compounds already contain a photoreactive group. Alternatively, however, it is also possible that these monomer compounds initially contain a functional organic group which, after polymerization (ie after incorporation of the monomer molecules into the polymer chain), is reacted with a compound containing the photoreactive group. Included for this these compounds in turn, in addition to the photoreactive group, are a suitable organic functional group capable of reacting with the functional organic group of the monomer compound (ie, complementary pairs of functional groups are used). Examples of such complementary pairs of functional groups are active esters and amino groups, epoxides and hydroxyl groups, thiol and ene groups or alkyne and azido groups.

In the context of the present invention, it is possible that the photocrosslinkable copolymer already comprises repeating units WE ^AS which in each case have at least one analyte-specific recognition group.

The repeating units WE ^PR and WE ^AS in the photocrosslinkable copolymer can be obtained by preparing the photocrosslinkable copolymer by a polymerization in which a monomer compound containing the photoreactive group and a monomer compound containing the analyte-specific recognition group are present ,

With regard to the monomer compound containing the photoreactive group, reference may be made to the above statements.

Suitable monomer compounds from which the repeat units WE ^{AS are} available are known to those skilled in the art. They are preferably those compounds which can be incorporated into a polymer chain as part of a polymerization, in particular compounds with an unsaturated carbon-carbon bond. By way of example, olefins, styrenes, acrylates or methacrylates may be mentioned in this connection. If these monomer compounds already contain an analyte-specific recognition group, a copolymer having covalently bound analyte-specific recognition groups to the polymer chains is obtained directly from the polymerization.

Alternatively, it is also possible that the monomer compounds do not yet contain an analyte-specific recognition group, but initially a functional organic group which, after the polymerization (ie after incorporation of the monomer molecules into the polymer chain), is reacted with a compound containing the analyte-specific recognition group. is reacted. These compounds, in turn, contain, in addition to the analyte-specific recognition group, a suitable organic functional group that can react with the functional organic group of the monomer compound (i.e., complementary pairs of functional groups are used). Examples of such complementary pairs of functional groups are active esters and amino groups, epoxides and hydroxyl groups, thiol and ene groups or alkyne and azido groups.

• – eine Polymerisation, in der eine Monomerverbindung, die die photoreaktive Gruppe enthält, sowie eine Monomerverbindung, die eine funktionelle organische Gruppe enthält, anwesend sind und anschließend
• – eine Reaktion der funktionellen organischen Gruppe mit einer Verbindung, die die Analyt-spezifische Erkennungsgruppe enthält,

hergestellt wird.The repeating units WE ^PR and WE ^AS can be obtained in the photocrosslinkable copolymer by passing the photocrosslinkable copolymer through

• A polymerization in which a monomer compound containing the photoreactive group and a monomer compound containing an organic functional group are present, and then
• A reaction of the functional organic group with a compound containing the analyte-specific recognition group,

will be produced.

Alternatively, it is also possible in the inventive method, that the repeating units WE ^AS are formed in the photo-crosslinked hydrogel polymer after irradiation of the photo-crosslinkable copolymer.

• – das photovernetzbare Copolymer durch eine Polymerisation hergestellt wird, in der eine Monomerverbindung, die die photoreaktive Gruppe enthält, sowie eine Monomerverbindung, die eine funktionelle organische Gruppe enthält, anwesend sind, und
• – nach der Bestrahlung des photovernetzbaren Copolymers eine Reaktion der funktionellen organischen Gruppe mit einer Verbindung, die die Analyt-spezifische Erkennungsgruppe enthält, stattfindet.

The repeat units WE ^AS can be obtained in the photocrosslinked hydrogel polymer, for example, by

• The photocrosslinkable copolymer is prepared by a polymerization in which a monomer compound containing the photoreactive group and a monomer compound containing an organic functional group are present, and
• After the irradiation of the photocrosslinkable copolymer, a reaction of the functional organic group with a compound containing the analyte-specific recognition group takes place.

With regard to suitable functional organic groups of the monomer compound and the compound containing the analyte-specific recognition group, reference may be made to the above statements.

As explained above in the description of the sensor, the hydrogel polymer still contains "base" repeating units, which make up the actual structural basis of the hydrogel polymer. Of course, the same applies to the photocrosslinkable copolymer from which the crosslinked hydrogel is obtained by irradiation. With regard to suitable base repeat units WE ^base for the crosslinkable copolymer, reference may therefore be made to the statements made above in the description of the sensor.

Suitable reaction conditions for the preparation of the photocrosslinkable copolymer from the monomer compounds described above are known to those skilled in principle. For example, the preparation of the crosslinkable copolymer can take place via a free-radical polymerization.

In a preferred embodiment, the photocrosslinkable copolymer has a number-average degree of polymerization P _n in the range of 500 to 50,000, more preferably 500 to 10,000.

The number-average degree of polymerization P _n . is calculated from the molecular weight over the division by the average molecular weight of the repeat units. The molecular weight determination is preferably carried out by gel permeation chromatography.

The provision of the photocrosslinkable copolymer on the optical functional layer can conventional coating methods such. As a spin coating (spin-coating), a dip coating, printing or combinations thereof.

The invention will be further described by the following examples.

Examples

Preparation of the photocrosslinkable copolymer

A copolymer of the base monomers MEO ₂ MA (2- (2-methoxyethoxy) ethyl methacrylate) and OEGMA 475 (oligo (ethylene glycol) methyl ether methacrylate) was prepared. Furthermore, monomers were incorporated which contained a biotin group as an analyte-specific recognition unit. For photocrosslinking, monomers having a benzophenone group were incorporated into the polymer.

The photocrosslinkable copolymer obtained after the polymerization has the following structure:

In addition to the basic repeating units (subscripts p and q), the copolymer also contains repeat units WE ^PR (index r) which contain a photoreactive benzophenone group and repeat units WE ^AS (index s) which contain a biotin group as the analyte-specific recognition group contain.

The proportion of photoreactive repeat units in the copolymer was 0.5 mol%. The number-average degree of polymerization P _n was 580.

The proportions of the respective repeat units were as follows: p: q: r: s = 87.8: 9.7: 0.5: 2

Preparation of a sensor containing a polymeric DFB laser using the photocrosslinkable copolymer described above

This example describes the advantageous embodiment with a polymeric DFB laser. This DFB laser is made up of a polymer surface relief grating (SRG) formed on a glass surface with a silicone die, which consists of a polymer with a low refractive index (MY-136 from MYPolymers Ltd., Israel, 1374@633 nm). , This has a sinusoidal profile with an amplitude of 30-35 nm and a period of 400-405 nm. In addition, there is a laser-capable layer (ie the laser medium or gain medium) made of F8BT (poly 9,9-dioctylfluorenyl-2,7-diyl) -alt- (1,4-benzo {2,1 ', 3} -thiadiazole)], American Dye Source, Inc., Quebec, Canada) and MEH-PPV (Poly [2-methoxy-5- (2-ethylhexyloxy) -1,4-phenylene-vinylenes], American Dye Source, Inc., Quebec, Canada) prepared by spin-coating (30 mg F8BT and 7 mg MEH-PPV in 2 mL toluene, 3000 rpm). On the optical functional layer, the photocrosslinkable copolymer described above is applied by spin coating (0.4 wt .-% solution in ethanol, 3000 U / min.). By irradiation (UVACUBE 100 (Hönle, Gräfelfing) with an iron-doped 100 W mercury vapor lamp with glass filter (cut-off 310 nm), distance to the light source 20 cm), the copolymer is on the one hand crosslinked three-dimensionally and also covalently bound to the polymers forming the optical functional layer , The resulting hydrogel layer has a layer thickness of 25 nm in the dry state. After rinsing with PBS buffer (Fluka, 0.01 mol·l ^-1 phosphate (NaCl 0.138 mol·l ^-1 ; KCl 0.0027 mol·l ^-1 ); pH 7.4 at 25 ° C) to dissolve loosely remove attached material, the sensor is ready for use.

To check the sensor response, successive increasing concentrations were incubated with avidin solutions. As the avidin concentration increases, there is an increasing shift in the laser wavelength. The sensor enables detection in real time. This is in 1 shown.

Claims (17)

A sensor comprising A substrate; A polymeric optical functional layer; A layer of a photocrosslinked hydrogel polymer, the - has covalently bound analyte-specific recognition groups, and Is crosslinked via photoreactive groups; in which the hydrogel polymer is covalently linked to the polymer of the optical functional layer via photoreactive groups and the optical functional layer may indicate a refractive index change in the hydrogel polymer layer via a change in one of its optical properties. The sensor of claim 1, wherein the optical functional layer is a microstructured layer, preferably one or more microstructured optical elements selected from a periodic grating, a ring resonator, or a multi-arm waveguide structure or combinations thereof or be included. The sensor of claim 2, wherein the microstructured optical functional layer comprises one or more microstructured optical elements and at least one of the microstructured optical elements is formed of the polymer of the optical functional layer. The sensor according to one of the preceding claims, wherein the optical functional layer is a DFB laser, a ring resonator-containing layer or a layer having a Mach-Zehnder interferometer structure. The sensor according to one of the preceding claims, comprising the photocrosslinked hydrogel polymer comprising: (i) repeating units WE ^AS , each having an analyte-specific recognition group, (ii) repeating units WE ^V , which have each formed a crosslinking point in the hydrogel polymer via a photoreactive group, and (iii) repeating units WE ^OF , which are each covalently connected to the polymer of the optical functional layer via a photoreactive group. The sensor of claim 5, wherein the repeat units WE ^V and WE ^{OF are present} in the hydrogel polymer in a proportion of less than 1.0 mole%. The sensor according to any one of the preceding claims, wherein the photocrosslinked hydrogel has a degree of swelling> 4. The sensor according to any one of the preceding claims, wherein the photoreactive group is selected from those capable of undergoing insertion with an organic radical. The sensor according to any one of the preceding claims, wherein the photoreactive group is a benzophenone group, an acetophenone group, a diazirine group or an azide group. The sensor of any of the preceding claims, wherein the analyte-specific recognition group is selected from antibodies, F _ab fragments of antibodies, enzymes, enzyme fragments, coenzymes, prosthetic groups, aptamers, single-stranded DNA, single RNA strands, or combinations thereof. The sensor according to any one of the preceding claims, wherein the photocrosslinked hydrogel polymer comprises repeat units WE ^base , which are preferably selected from: (1)

where R ₁ = H, alkyl such as. B. C _1-4 alkyl, preferably -CH ₃ , R ₂ , = H, alkyl such as. C _1-4 alkyl, - (CH ₂ ) _n -COOH and x = 1-50, more preferably 2-20, (2)

where R ₁ = H, alkyl such as. B. C _1-4 alkyl, preferably -H, R ₄ , = H, alkyl such as. C _1-4 alkyl, - (CH ₂ ) _n -COOH and x = 1-50, more preferably 3-20, (3)

wherein R ₂ , = H, alkyl such as. C _1-4 alkyl, - (CH ₂ ) _n -COOH and x = 1-50, more preferably 2-20, (4)

wherein R ₂ , = H, alkyl such as. C _1-4 alkyl, - (CH ₂ ) _n -COOH and x = 1-50, more preferably 2-20, (5)

where R ₁ = H, alkyl such as. B. C _1-4 alkyl R ₂ , R ₃ , = H, alkyl such as. C _1-4 alkyl, allyl,

are (6)

where R ₁ = H, alkyl such as. C _1-4 alkyl, preferably H and x = 1-6, more preferably 3-4, (7)

in which R ₁ , R ₂ , R ₃ = H, alkyl such as. B. C _1-4 alkyl, (8)

R ₁ = H or a branch of the polysaccharide chain R ₂ to R ₅ = H, alkyl such as. C _1-4 -alkyl, allyl, - (CH ₂ ) _n -COOH, (9) polysaccharide-based repeating units, (10)

where R ₁ = H, alkyl such as. C _1-4 alkyl, preferably -CH ₃ ; (11)

where R ₁ = H, alkyl such as. C _1-4 alkyl, preferably -CH ₃ , x, y can independently of one another assume values between 1 and 12 and Z = SO ₃ , COO, or PO ₃ or combinations of these repeating units. A method of making the sensor of any one of claims 1-11, comprising - providing a substrate having a polymer-containing optical functional layer disposed thereon; The provision of a layer of a photocrosslinkable copolymer on the optical functional layer, wherein the photocrosslinkable copolymer and the polymer of the optical functional layer have a common Having the interface and the photocrosslinkable copolymer repeating units WE ^PR each having at least one photoreactive group, and - the irradiation of the photocrosslinkable copolymer to form a photocrosslinked hydrogel polymer and formation of covalent bonds between the photocrosslinked hydrogel polymer and the polymer of the optical functional layer , The method according to claim 12, wherein the photocrosslinkable copolymer comprises repeating units WE ^AS each having at least one analyte-specific recognition group. The process according to claim 12 or 13, wherein the repeating units WE ^PR and WE ^{AS are obtained} in the photocrosslinkable copolymer by preparing the photocrosslinkable copolymer by a polymerization in which a monomer compound containing the photoreactive group and a monomer compound containing the Contains analyte-specific recognition group present; or wherein the repeating units WE ^PR and WE ^{AS are obtained} in the photocrosslinkable copolymer by the photocrosslinkable copolymer by - a polymerization in which a monomer compound containing the photoreactive group and a monomer compound containing an organic functional group are present, and then - preparing a reaction of the functional organic group with a compound containing the analyte-specific recognition group. The method of claim 12, wherein the repeat units WE ^{AS are formed} in the photocrosslinked hydrogel polymer after irradiation of the photocrosslinkable copolymer. The process according to claim 15, wherein the repeating units WE ^{AS are obtained} in the photocrosslinked hydrogel polymer by: preparing the photocrosslinkable copolymer by a polymerization in which a monomer compound containing the photoreactive group and a monomer compound having a functional organic group Group contains, are present, and - after the irradiation of the photocrosslinkable copolymer, a reaction of the functional organic group with a compound containing the analyte-specific recognition group takes place. The method according to any one of claims 12-16, wherein the photocrosslinkable copolymer has a number-average degree of polymerization P _n in the range of 500 to 50,000; and / or wherein the provision of the photocrosslinkable copolymer on the optical functional layer comprises spin-coating, dip-coating, printing or combinations thereof.

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