Sensor system for detecting analytes in tear fluid
The subject matter of the invention is a sensor system for detecting endogenous or exogenous analytes in tear fluid.
Soft contact lenses usually consist of hydrogels. These hydrogels are permeable to a number of substances contained in lachrymal fluid, such as salts, sugars, medicament constituents, proteins and other biomolecules, and may therefore serve as carriers for biosensors based on specific detection systems. With the assistance of a detection system immobilised in the lens material, individual substances (analytes) may thus be specifically optically detected. In this system, the spectroscopic characteristics of the detection system usually undergo a change that can be detected by appropriate measuring instruments.
For example, the process of forming a bond or the release of a bond between a receptor and a ligand may be detected by means of the so-called Fluorescence Resonance Energy Transfer (FRET) by the change of fluorescence spectra of suitable dyes.
In this, a sensor molecule which specifically binds the analyte is labelled by a fluorescence marker (donor): the sensor molecule is bound to an analyte-like molecule, which likewise has fluorescence labelling (acceptor). In the absence of the analyte, the fluorescence of the donor is suppressed by (non-radiative) energy transfer to the acceptor (FRET). In the presence of analyte molecules, if the acceptor is forced away from the donor by these, the FRET process does not come into operation, whereupon the donor fluorescence is increased. This change in the intensity of fluorescence is proportional to the concentration of analyte and may be measured by a measuring instrument (fluorescence spectrometer). Numerous scientific reports and patents have already been published on this subject (WO 01/13783 A1 ). Here, special attention was paid to the determination of concentrations of medically relevant analytes, for example glucose, which is of great interest in respect of blood-sugar measurement of diabetes patients (Schultz et al.). If such a detection system is to be used for measuring medically relevant analytes in lachrymal fluid, it must be inserted into the hydrogel of a contact lens in an appropriate manner without any adverse affect on its functionality.
Previous attempts to insert the detection system directly into the hydrogel matrix as a solution had the following undesired effects: a) The sensitivity of the optical detection system is greatly reduced in the hydrogel structure. b) The kinetics of the detection system is slowed down considerably.
These characteristics are caused on the one hand by the restricted mobility of the sensor molecules in the hydrogel, and on the other hand by the inhibited diffusion of the analyte. c) In addition, many relevant sensor molecules (for example for glucose detection) are not sufficiently stably enclosed in the hydrogel, and can slowly diffuse out. Since the possibility cannot be excluded that the sensor and marker molecules are toxic or at least harmful to health, this should be avoided.
The problem is therefore to overcome the above-mentioned disadvantages and to develop a detection system, which may be integrated into a soft contact lens and allows endogenous or exogenous substances in lachrymal fluid to be simply and reliably detected.
This problem is solved by a sensor system, which allows the detection of endogenous or exogenous substances in lachrymal fluid by means of a substance-specific detection system arranged preferably annularly or in another shape in a contact lens.
In a first variant, the detection system may be contained in annularly designed cavities or microchambers embedded into the hydrogel layer of a contact lens, the cavities or microchambers being sealed by a semi-permeable membrane.
In another variant, the detection system may be contained in annularly designed microchambers, which are formed from semi-permeable films and are embedded into the hydrogel layer of a contact lens. Finally, the detection system may also be contained in a spirally designed microtube, which has a semi-permeable wall and is embedded into the hydrogel layer of a contact lens.
The essential advantages of these ideas may be seen from the fluorescent sensor molecules, which are enclosed in the microchambers or in the microtube, but are freely mobile in the solution. They are prevented from escaping from the chambers by the semi-permeable walls, whereby the diffusion selectivity (cut-off) of these walls is set such that the analyte molecules can simultaneously diffuse freely into the chambers. Owing to the free mobility of the sensor molecules in the microchambers and microtubes, they retain their full sensitivity and reaction kinetics. The wall of the microchambers or of the microtube is chosen so that it is permeable to molecules up to a diameter of 5000 Daltons.
A further advantage of the above-mentioned microchamber concept is the possibility of including various sensor molecules in the detection system at a distance from each other. The different designs of the object according to the invention are illustrated in detail as follows:
Structures for enclosing the sensor solution 1 Microstructured hydrogel
Since the lens-specific hydrogel has a certain permeability for the sensor and marker molecules, according to the invention another hydrogel or a more strongly crosslinked form of the lens material is used for encapsulation of the sensor solution, so as to ensure safer enclosure of the sensor molecules with simultaneous unhindered diffusion of the analyte. The geometry of the finished object is selected such that it can be integrated into the lens without problems (fig. 1 ). So as not to impair vision through the centre of the lens, a ring-shaped geometry of the microstructure is offered in particular ("sensor ring"), though other shapes may similarly be conceived.
1.1 Hydrogel with microchambers
In this embodiment, recesses are inserted into the hydrogel surface (fig. 2). This may take place, for example, by stamping when the hydrogel is crosslinked or by subsequently cutting out with a laser. The dimensions and spacing of the microchambers are chosen such that their density and mechanical strength are assured whilst simultaneously providing flexibility of the microstructure. The chambers may have a round or square shape. The microchambers filled with the sensor solution are covered with a thin semi-permeable film (for example dialysis film) or with a thin hydrogel layer. The strength of the whole system is less than the strength of the lens. This dimension applies to all further sensor rings.
The hydrogel and the crosslinking thereof are adapted such that the sensor molecules remain firmly enclosed in the microchambers and the analyte can diffuse in.
1.2 Microchambers in the hydrogel with a coated surface
The microchambers may be formed directly in the lens material. However, the selective permeability of the chamber walls must be guaranteed. This can be assured by a coating of the chamber walls (fig. 3). The selective permeability of the coating enables free diffusion of the analytes to take place and blocks diffusion of the detection system. The filled microchambers are covered with a semi-permeable film (dialysis film) or with a similarly coated hydrogel layer (fig. 3).
1.3 Drops of the detection solution in the hydrogel
In order to avoid micro-structuring, microscopic drops of the detection system may be injected into the still liquid hydrogel (fig. 4). After rapid polymerisation, the drops remain in the hydrogel. As in 1.1 , the hydrogel and its crosslinking are chosen so as to be suitable for the said prerequisites.
2. Structured dialysis films 2.1 Multi-layered film with microchambers
The microchambers may be formed directly in semi-permeable membranes (dialysis films). An example is shown in fig. 5. This structure consists of three layers/films: one thicker layer, in which the microchambers are shaped, and two thin coverings. If all films have the same properties (selective permeability, cutoff), optimum functioning may be expected; however, selective permeability is only necessary for a covering film. The dimensions and pattern of the microchambers may be chosen as in section 1. 2.2 Structured film with microchambers
Thin films can be easily structured, whereupon it is possible to create the microchambers. Fig. 6 shows an example. The microchambers formed by stamping and filled with sensor solution are sealed by a covering film. Permeability and cut-off of the films are chosen in accordance with the relevant detection method.
2.3 Structured film with double-sided filling
This solution is similar to the above embodiment (2.2). The difference is that here the intermediate areas on the reverse side are likewise filled (fig. 7). A further film is necessary to cover these areas. Because of the structural areas that are used on both sides, this system offers a larger sensor area.
2.4 Structured film with capillary chambers
Compared with the round or polygonal microchambers (see above), microcapillaries may have an advantage, since filling of the microstructures with sensor solution is simplified in this case by capillary forces. The capillary chambers may be arranged radially or in concentric circles (fig. 8). First of all, a wavy structure is formed from a film, which is sealed on both sides by covering films. The capillary chambers are then filled with the sensor solution by capillary forces.
2.5 Structured film with microrings
This embodiment is similar to that above (2.4), but here the concentric rings are not capillaries, but are flat ring chambers (fig. 9). After filling the ring chambers, a covering film is used to seal in the sensor solution.
2.6 Dialysis tubing
A simple solution is offered by the usage of a dialysis tube of an appropriate dimension (for example a hollow fibre) (fig. 10). After filling the microtube with the sensor solution, it is coiled up. A snake-like structure is also conceivable. Subsequently, hydrogel may be used to fix the stricture (see production methods below). General production technology
The illustrated designs of the microstructured contact lens can be produced efficiently by mass production. The parallel production process comes to the fore here. Parallel shaping and filling of hundreds of sensor rings may be carried out for the illustrated concepts (with the exception of the dialysis tube) (figs. 11 and
12).
Microstructuring of the sensor rings is carried out on gel layers or films having a large area (fig. 11 ). Depending on the implementation and materials, various operational methods may be used:
- pouring hydrogels into microstructured molds
- punching out films
- stamping films
- injection processes ' laser ablation laser welding
- lithography
All these methods enable rapid, reproducible and precise microstructuring to take place.
The following different materials may be used as the basic material for the microchambers and for the coating:
- various types of hydrogels with different degrees of crosslinking
- thin protective film dialysis membranes hollow-fibre membranes
- selectively permeable coatings of the lens material
These materials may be produced in the desired dimensions and processed by the above methods. Any combination of the above-listed materials and operations may likewise be advantageous. Filling of the microchambers in the large-area parallel operation is shown in fig. 12. The covering film is pressed by a roller onto the edge of the filled microchambers, whereby the loose film pushes the sensor solution further ahead. At the point where the roller presses the covering film onto the edges of the microchambers, the covering film and the chamber holders are joined together using a suitable method (for example laser welding of dialysis films or crosslinking with UV light in the case of hydrogels).
Production and sealing of the microchambers with dialysis film is illustrated in the example of the structured film (fig. 13). Here, laser welding is chosen as the operational method. The system should be made up of a film which is non- transparent (absorbing) to the laser and from a transparent film. The laser beam is focussed onto the level of the contact area of the two films. The surface of the absorbing film is warmed up locally and welded to the transparent film.
Production methods according to the invention
1. Hydrogel ring with microchambers
The microchambers in the hydrogel may be shaped in different ways, whereby the hydrogel used for the sensor ring is not identical to that of the lens material. Figs. 14 and 15 illustrate two examples. In fig. 14, the microstructure in the hydrogel is formed by a stamp. After polymerisation, the desired structure is obtained. The filled microchambers are sealed by a thin covering film (dialysis film) or by a thin hydrogel layer. At first, the hydrogel layer should be only slightly polymerised, so that handling is possible. Complete polymerisation should be carried out after covering the microchambers. In this operating step, the edges of the microchambers and the covering layer are joined together.
The microchambers in the polymerised hydrogel may likewise be shaped by laser ablation (fig. 15). The remaining steps of this production process are identical to the above method.
2. Microchambers with a coated surface The lens-specific hydrogel is not suitable for incorporation of the sensor solution, as was explained above. However, after appropriate coating of the microchamber walls, the desired selective permeability may be attained. In this concept, the contact lens consists of two layers. In one layer, the microchambers are shaped in the course of the molding process (fig. 16). After coating the chamber walls, the chambers are filled up. The chambers are sealed by the second lens part which is similarly coated. The second lens part is only slightly polymerised prior to covering, as described above, and, after covering, polymerisation is concluded in order to seal the chambers safely.
If the geometry of the contact lens allows it, the second lens part can be replaced by a dialysis film. In this case, the microchambers should similarly be arranged in annular manner around the edge of the lens, in order to maintain compete quality of vision.
3. Drops of sensor solution in the hydrogel
The production process of this embodiment is shown in fig. 17. Prior to polymerisation, the sensor solution is injected into the hydrogel by an injection system. After rapid crosslinking, the microdrops remain in the hydrogel at the desired depth.
It is similarly conceivable for the microdrops to be injected directly into the lens material. In this embodiment, a further crosslinker is mixed into the sensor solution, and leads to further polymerisation at the walls of the cavities, thereby ensuring the desired selective permeability.
4. Multi-layered film
The production process for the sensor ring consisting of a multi-layered film is illustrated in fig. 18. First of all, the thick middle layer is worked. The chamber structure is shaped by stamping or by laser. The middle layer is then welded together with the lower film, as demonstrated in fig. 13. After filling the microchambers, an appropriate welding~procedure takes place, in order to seal the chambers. 5. Structured film with microchambers
Structuring of a thin film may be carried out in two different ways (figs. 19 and 20). In one method, a stamp and a mold are used (fig. 19). As usual, the recesses are filled with the sensor solution and sealed. In the other method (fig. 20), only a stamp is used, which presses the film into the viscous, lens-specific hydrogel. The subsequent polymerisation ensures the structure. After filling, first of all a covering film is applied and finally the upper lens part.
It is likewise conceivable for the second lens part to be unnecessary, if the covering film does not have sharp edges and/or the full lens surface is covered and the otherwise customary characteristics of the contact lens are not changed (for example oxygen permeability, comfort, etc.).
6. Structured film with double-sided filling
Microstructuring of the dialysis film, as well as filling and sealing, are carried out in a similar manner to that above (5) (fig. 21 ). Subsequently, the ring is turned over and the intermediate spaces are similarly filled. Covering is carried out as usual with a further film.
7. Structured film with capillary chambers
In this embodiment, first of all the microstructure is stamped into the film with stamps and molds (fig. 22). Subsequently, covering films are welded to the top and bottom of the structured middle film, whereby both ends of the capillary chamber should remain open. The capillaries are filled by capillary forces. The capillary chambers are sealed by the pressing and welding operations.
8. Structured film with microrings
In this embodiment, the processing steps for the film (structuring, filling, sealing) may be carried out in a similar manner to the above (fig. 23).
9. Sensor ring consisting of dialysis tubing or hollow fibre membrane
Fig. 24 shows a schematic illustration of the production steps for the sensor ring. First of all, the tube is filled with the sensor solution. Then, the tube is wound onto a holder, the base of which is coated with hydrogel that has not yet been polymerised. The tube spool is coated from the top with a further layer of hydrogel. The hydrogel holds the spool in the desired shape after polymerisation.
Reflecting layer on the ring structure
Small optical signals are measured by the sensor ring. To increase the intensity of signal, a reflecting layer is applied to the rear surface of the ring.
Quality control
The finished sensor rings are checked for the following qualities prior to integration into the contact lens:
- filling process is free of air-bubbles
- dimensions
- leakage of the sensor solution (satisfactory seal)
- concentration and sensitivity of the sensor solution
In order to check these parameters, the following testing methods may be used:
- light diffusion or microscopic image analysis for air-bubble-free filling
- microscopic image analysis to check the dimensions and to check for leakage
- absorption measurement to test the marker concentrations
- fluorescence measurement to check the sensitivity
Integration of the sensor ring into the contact lens
The sensor rings may be integrated into the contact lens in various ways. One possibility is a three-layered contact lens, as has already been used for coloured contact lenses (EP 0 369 942 A1). The contact lens consists of a lower layer, middle film layer with the sensor ring and an upper layer (fig. 25 left).
The sensor rings can be stamped out of a film of large area, placed into the lens mold and hydrogel poured around them (fig. 25 right). In this case, knubs should be inserted on the film or in the mold, in order to keep the ring at a defined distance from the surface of the contact lens.