JP2004531703A - Method for producing optical (bio) chemical sensor device - Google Patents

Abstract

The present invention relates to a method of making a miniature optical chemistry or biochemical sensor device (such as a bulk optode for ion sensing), said device comprising a substrate material having a planar portion, wherein said planar portion is exposed to optical phenomena, such as evanescent waves. A multi-analytical array of (bio) chemical sensor dots arranged on the plane part at spatially separated predetermined positions, corresponding to a transducer based on surface plasmon resonance. Wherein the sensor dot comprises (i) a polymer matrix and (ii) one or more (bio) chemical recognition moieties, the method comprising: (a) providing a substrate material having planar portions; C) providing one or more spotting fluids; and (c) providing the one or more spotting fluids on a planar portion of the substrate material. It was deposited by pin printer a deposition coating mechanism (arrayer), comprising thereby consolidating the spotting fluid.

Description

【Technical field】
[0001]
The present invention relates to the production of optical (bio) chemical sensor devices useful for monitoring a large number of different compounds simultaneously. Other possible applications include high-throughput screening of combinatorial libraries, monitoring of food quality and detection of biological components. More particularly, the present invention relates to a method of making an optical (bio) chemical sensor device comprising a plurality of polymer-type (bio) chemical sensor dots.
[Background Art]
[0002]
Trends in the field of chemical and biochemical sensors [R. Kellner, M. Otto, M. Wildmer, Analytical Chemistry: The Approved Text to the FECS Curriculum Analytical Chemistry, Wiley-VCH 1998, p 359-360 and 375ff] Along with the screening of new compounds for development, the development of new approaches to implementing classical analytical methods, for example, to meet the increasing demand for high-throughput analysis of environmental and clinical samples. In particular, the miniaturization of chemical and biochemical sensing technologies has been of great interest, and the processes further supported by the development of new approaches for chemometric data processing and neural networks will require discrete results. Enables access to information embedded in response patterns that exceed the total.
[0003]
Chemical and biochemical sensors [ie (bio) chemical sensors] are devices that can continuously recognize the concentration of a chemical component in a liquid or gas and convert this information into electrical or optical signals in real time. [R. Kellner, M. Otto, M. Wildmer, Analytical Chemistry: The Approved Text to the FECS Curriculum Analytical Chemistry, Wiley-VCH 1998]. In this context, a chemically sensitive layer is associated with a so-called transducer, which converts (bio) chemical information into electrical or optical signals, which are converted into data Recorded by the evaluation unit. The chemically detectable layer can be a surface directly modified with the recognition system or a surface coated with a thin film doped with the recognition system; for example, a chemically detectable polymer film can be attached to the electrode to Either their potential difference to the sample solution can be measured as a function of concentration (or activity); or they can be coated on an optical transducer, such as an optical fiber, to determine the optical change (such as absorbance or refractive index) as a function of analyte concentration. Can be measured.
[0004]
Bulk optode films as described in EP 0 358 991 are examples of chemical sensing layers for optical ion sensing. Chemicals that selectively interact with specific ions are called ionophores and are conventionally used in potentiometric membrane electrodes to increase or control their selectivity. The combination of other factors, particularly lipophilic ions ("counter ions") and ionophores with pH-sensitive dyes ("chromoionophores"), produces specific ions at a given pH with reversible and reproducible color changes. To provide a membrane material responsive to the Unlike those electrochemical analogues where a reference electrode is a requirement for the measurement, optical sensors based on such membranes ("opt (r) odes)" have no reference And is less susceptible to electrical interference, making it much easier to integrate the sensor into miniaturized systems. Further, because more than one parameter can be derived from the sensor, optical methods can be combined with chemometric techniques, pattern recognition, etc .: For example, electrochemical sensors generally have one value (eg, potential or current). ), Data on both absorbance and refractive index, temporal information or spectral shape can be measured.
[0005]
To combine optical sensing using chemically responsive polymers with the concept of a sensing array, individual small dots are arranged on a substrate in such a way that the signals from each individual sensing element can be distinguished from each other. There is a need. One approach is to configure the optical fibers as bundles or arrays as demonstrated by Dickinson et al. [Nature 1996, 382, 697-700; Johnson et al., Anal. Chem. 1997, 69, 4641-4648]. A representative example performed to demonstrate the benefits of multi-sensing using optical changes of polymer probes is the work of Walt's group (eg, Anal. Chem 70 1998 1242-1248). Here, microsphere sensors are randomly incorporated into thousands of micrometer-scale wells. These are etched from the surface of the optical fiber with hydrofluoric acid using different etching rates of the fiber core and the clad. Alternatively, sophisticated site-selective photopolymerization has been adopted by the group for such fiber bundle phases (Mikrochim. Acta 131, 99-105, reviewed by Steelers and Walt). (1999)). Such studies strongly confirm the potential of sensing technologies based on miniaturized polymer arrays. However, because of the individual modifications and the subsequent bundling of the fibers, it is clear that this approach cannot be implemented for mass production of sensor layers or for production of sensor layers with a variety of different sensor systems. On the other hand, for many viable devices, it will not be necessary to scale individual sensor elements down to the size (several micrometers) realized in such studies. For example, 36 dots of 120 μm diameter can still be easily arranged in a 1 cm × 1 cm area with a 30 μm dot-to-dot distance.
[0006]
An ideal sensor device should comprise a plurality of different, independently spatially separated polymer sensor areas (these areas, referred to in this context as sensing dots, are roughly circular in shape and micro-area size (Having a diameter of 10 to 1000 μm). Imaging techniques, such as cameras or linear or two-dimensional CCD arrays, and appropriate software can then be used to differentiate the response between the different sensing dots from each other. These sensor devices may exhibit greater flexibility and reproducibility, and it may be easier to increase the number of sensing regions. The potential of an array of optical sensing areas for analysis via imaging has recently been demonstrated by Rakow et al. In a colorimetric sensor for odor detection [Rakow et al., Nature 406 (2000) 701-713].
[0007]
In principle, there are a number of different optical sensing schemes that can be imaged and thus used in an array of polymer-based sensing dots. In the simplest case, a transparent carrier plate can be modified with sensing dots. If appropriate gaskets and cover plates are used, flow channels can be formed, which allow, for example, the flow of groundwater samples above the dots. A light source can be mounted on the top of the cover plate and illuminated through both plates and the sample, and then an imaging detector (eg, camera) below the lower plate contains an image containing all information about the color response of the individual dots Can be recorded. Of course, it would be possible to devise a similar system that utilizes the total internal reflection or diffusion of the sensing dots on a suitable non-transparent surface. Fluorescence monitoring in a similar system would also be possible.
[0008]
A more sophisticated optical sensing method is the phenomenon of evanescent wave, an attenuating standing wave, which occurs on the surface between two phases with different refractive indexes due to the total reflection of light in an optically dense medium. Is used. Such waves may occur directly at the total internal reflection surface in a suitable structure, such as a diffraction grating, or in a thin film of a suitable metal (typically gold or silver) on a relevant suitable optical structure. Occur through the excitation of so-called surface plasmons (collective electron oscillations). Combining with an array of polymer sensing dots as described in WO 00/46589 (Vir A / S) since such a device allows the measurement of optical properties very close to the transducer surface You can also. In addition, evanescent waves can be used to excite fluorescence rather than monitor absorption, thereby enabling fluorimetric sensing.
[0009]
Suitable optical transducers allow the detection of changes in the optical properties of the polymer dots, such as, in particular, changes in absorption, refractive index or fluorescence (so that the chemical response of the polymer dots can be monitored). , Optical waveguides, surface plasmon resonance films, reflective grating couplers, optical waveguides, Mach-Zehnder interferometers or Hartmann interferometers.
[0010]
However, there has been no automated method for producing arrays of spatially separated optical sensing (bio) chemical sensor dots for optical sensing.
[0011]
Several techniques are available for automated spraying of fluid droplets. For example, ink jet technology, known from printing technology, is characterized in that a fluid is deposited from a capillary. The release of fluid from the capillary can be achieved by different approaches. In the drop-on-demand approach, the application of a voltage by a piezo actuator causes a ceramic collar around the capillary to generate sound waves in the capillary, and the resulting deformation of the capillary controls the liquid column. The ejected portion is released as a droplet. In the continuous approach, the fluid is released under pressure, resulting in a fluid flow, and the application of a voltage by the piezoelectric actuator causes the droplet to be generated and released again. Continuous ink jet technology is widely used in the food and pharmaceutical industry for product labeling. Ink-jet technology has also been used by Newman et al. In the preparation of membranes for amperometric biosensors [Newman et al. Analytical Chemistry 1995, 67, 4594-4599], where droplets are generated. Fused to form a film. Other related technologies are micro and nano dispensing devices such as micro pipettes and the like.
[0012]
None of these techniques are suitable for the deposition of fluids with low surface tension and high viscosity, and the application of these fluids often results in the formation of gas bubbles in the dispensing device. . Heating the printhead can reduce the viscosity of the fluid, but can also cause evaporation of volatile solvents, which can cause clogging of the printhead. Further, viscoelasticity is known to cause significant performance problems in such printers. Non-Newtonian behavior under high shear forces in the nozzle can result in unstable droplet formation or droplet satellite formation.
[0013]
From the brief overview above, it is clear that inkjet technology is not a suitable choice for the deposition of polymers or polymer precursor fluids. The physicochemical properties of these fluids will prevent not only the deposition process but also the suction through the pump into the dispensing mechanism.
[0014]
Another technique for microarraying of fluids without sample aspiration, pumping and flushing is a pin printer or arrayer, such as the quill described in US Pat. No. 5,807,522. quill) · An open deposit device such as a printer. In pin printer technology, the fluid to be deposited is picked up from a small container, usually a hole in a microtiter plate. This reduces the amount of spotting fluid needed to fill the device, which is an important feature in the preparation of highly valued sensing membranes for (bio) chemical recognition factors. However, quill printers are not suitable for the deposition of high viscosity polymers and polymer precursors, as fluid reception is based on wicking into the quill and the problems described above can occur. Another disadvantage is that it is very difficult to reproduce the size of the deposited fluid dot, e.g. some commercial printers achieve a constant dot size in the pre-printing step Need that.
[0015]
Another example of an open deposit device is the pin-ring technique as described in WO 99/36760. Pin-ring technology has been developed for the production of reproducible microarrays of biological samples and is based on surface tension as the basic mechanism for holding and transporting fluids.
DISCLOSURE OF THE INVENTION
[0016]
The present invention relates to a method for producing an optical (bio) chemical sensor device as described in claim 1.
[0017]
The invention also relates to an optical (bio) chemical sensor device obtainable by the above method and a method of using said device.
BEST MODE FOR CARRYING OUT THE INVENTION
[0018]
The present invention relates to a method for making an optical (bio) chemical sensor device. The (bio) chemical sensor comprises well-spaced (bio) chemical sensor dots arranged on a substrate material. More specifically, the (bio) chemical sensor device comprises a substrate material having a planar portion, which corresponds to a transducer based on optical phenomena, on which the planar A plurality of (bio) chemical sensor dots are arranged at predetermined spatially separated positions, and the sensor dots are composed of (i) a polymer matrix and (ii) one or more kinds of (bio) chemical recognition. Has parts.
[0019]
The term "plurality" in the context of the phrase "plurality (bio) chemical sensor dots" is synonymous with the term "array" frequently used in technical literature.
[0020]
The term "(bio) chemical" has the same meaning as "biochemistry and chemistry" and is intended to encompass reactions and reagents in the biochemistry as well as in the chemistry field. You.
[0021]
The term “recognition moieties” refers to chemical groups that interact with the analyte, as well as cascades, to directly or indirectly alter the optical properties of the associated polymer matrix. It is also intended to include chemical groups that participate in a change in property. The term "recognition system" embraces systems comprising one or more recognition moieties that are responsible (eg, via a cascade reaction) for altering the optical properties of the associated polymer matrix.
[0022]
The sensor device comprises a "substrate material" having a planar portion. The term "substrate material" is intended to mean a base material that may optionally be coated with one or more layers of a surface layer material (see below). It should be understood that the planar portion of the substrate material should correspond to a transducer based on optical phenomena.
[0023]
The dimensions of the planar portion of the substrate material are generally 1-50 mm wide and 2-100 mm long, more specifically 2-25 mm wide and 5-50 mm long, for example 4-8 mm wide and 8-16 mm long.
[0024]
The substrate material includes a base material and optionally a surface layer material corresponding to a planar portion of the base material. In some embodiments of the present invention, it is possible to utilize a substrate material in which the base material and the surface layer material are the same. The substrate may, of course, include multiple layers of surface layer material.
[0025]
An important requirement for the method to be applicable to the fabrication of multiple (bio) chemical sensor dots is that the deposited fluid remains localized in place and spread to wet the entire substrate material. That is not. The contact area of the sensor dot determines the size (diameter) of the dot.
[0026]
The base material constitutes an integral part of the (bio) chemical sensor device and is generally made of glass, silica, a dielectric inorganic material such as SiO_Two , PtO_x (Where x = 1 or 2), Al_TwoO_Three , TiO_Two , Ta_TwoO_Five , MgF_Two Or Si_ThreeN_Four Materials selected from plastics, such as acrylics, cycloolefin polymers (TOPAS ™), polycarbonates, polyetherimides (ULTEM ™), or silicon with hydrogen or deuterium terminated surfaces, especially glass And in plastic, in the form of a smooth plane.
[0027]
In this context, the term "dielectric" means a material that is a poor conductor of electricity and that sustains the force of an electric field therethrough.
[0028]
The base material is often coated with at least one surface layer material to control the optical performance of the transducer. Such surface layer materials are generally selected from metals (such as gold, silver, copper or platinum), silica and silicon, preferably gold, silver, copper and silicon.
[0029]
The surface layer material generally has a thickness of 10-500 nm, more specifically 20-80 nm, which is particularly relevant for surface plasmon resonance measurements.
[0030]
In one embodiment of the invention, the substrate material is a multilayer structure of one or more metals and a dielectric inorganic material as defined above, for example, a metal-dielectric or metal-dielectric- It may be a metal sandwich structure.
[0031]
In one embodiment of the invention, the substrate material is transparent, allowing the measurement of the bulk absorption of the (bio) chemical sensor dot.
[0032]
In another embodiment of the invention, the support surface is totally reflective, allowing reflection spectroscopy measurements of the optical properties of the (bio) chemical sensor dot.
[0033]
In another embodiment of the invention, the support surface is diffusely reflective to allow diffuse reflectance spectroscopy of the optical properties of the (bio) chemical sensor dot.
[0034]
The size, shape and adhesion of the (bio) chemical sensor dots are such that the modification of the surface of the substrate material reduces or increases the ability of the spotting fluid to wet or chemically bind to the surface. Can be further controlled by
[0035]
In one embodiment of the invention, the plane of the substrate material is chemically modified by treatment with a bifunctional reagent as described below.
X-Z-Y
[Wherein X is -OR ', asymmetric or symmetric disulfide (-SSR'Y', -SSRY), sulfide (-SR'Y ', -SRY), diselenide (-SeSeR'Y', -SeSeRY ), Selenide (-SeR'Y ', -SeRY), thiol (-SH), selenol (-SeH), -N = C, -NO_Two , Trivalent phosphorus compound group, -NCS, -OC (S) SH, thiocarbamate, phosphine, thioacid (-COSH), dithioacid (-CSSH), -Si (OR / R / H)_Three And halogen,
The substituents R and R 'are each independently an optionally substituted C_1-30 Alkyl, optionally substituted C_2-30 Alkenyl, optionally substituted C_2-30 Selected from alkynyl and optionally substituted aryl;
Y and Y 'are selected from hydroxyl, carboxyl, amino, formyl, hydrazine, carbonyl, epoxy, vinyl, allyl, acrylic, epoxy and methacryl;
Z is a linker (biradical) between the bifunctional groups, and generally is an optionally substituted C_1-12 Alkylene, optionally substituted C_2-12 Alkenylene and optionally substituted C_2-30 Represents alkynylene (heteroatoms such as N, S, O and Si may be interposed therein). ]
[0036]
In the present context, "C_1-30 The term "alkyl" means a linear, cyclic or branched hydrocarbon group having 1 to 30 carbon atoms, such as methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, tert-butyl, isobutyl, cyclobutyl. , Pentyl, cyclopentyl, hexyl, cyclohexyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and the like._1-6 The term "alkyl" means a straight, cyclic or branched hydrocarbon group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, isobutyl, pentyl, cyclopentyl, Hexyl, cyclohexyl and the like, especially methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl and cyclohexyl.
[0037]
Similarly, "C_2-30 The term "alkenyl" is intended to mean a straight, cyclic or branched hydrocarbon group having from 2 to 30 carbon atoms and one or more unsaturated bonds, as well as "C"._2-6 The term "alkenyl" is intended to mean a hydrocarbon group having 2 to 6 carbon atoms, linear, cyclic or branched, and one or more unsaturated bonds. Examples of alkenyl groups are vinyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, heptadecenyl. Examples of alkadienyl groups are butadienyl, pentadienyl, hexadienyl, heptadienyl, heptadecadienyl. Examples of alkatrienyl groups are hexatrienyl, heptatrienyl, octatrienyl and heptadecatrienyl.
[0038]
Similarly, "C_2-30 The term "alkynyl" is intended to mean a straight-chain or branched hydrocarbon radical having 2 to 30 carbon atoms and containing a triple bond. Examples are ethynyl, propynyl, butynyl, octynyl and dodecainyl.
[0039]
In the context of the terms "alkyl", "alkenyl" and "alkynyl", the term "optionally substituted" means that the group in question is hydroxyl, C_1-6 Alkoxy, carboxyl, C_1-6 Alkoxycarbonyl, C_1-6 Alkylcarbonyl, formyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono and di (C_1-6 Alkyl) amino, carbamoyl, mono and di (C_1-6 Alkyl) aminocarbonyl, amino-C_1-6 Alkyl-aminocarbonyl, mono and di (C_1-6 Alkyl) amino-C_1-6 Alkyl-aminocarbonyl, C_1-6 Means one or more times, preferably 1 to 3 times, substituted by a group selected from alkylcarbonylamino, cyano, carbamide, halogen, wherein aryl and heteroaryl are_1-4 Alkyl, C_1-4 It may be substituted 1 to 5 times, preferably 1 to 3 times, by alkoxy, nitro, cyano, amino or halogen. Particularly preferred examples are hydroxyl, C_1-6 Alkoxy, carboxyl, aryl, heteroaryl, amino, mono and di (C_1-6 Alkyl) amino and halogen wherein aryl and heteroaryl are C_1-4 Alkyl, C_1-4 It may be substituted 1-3 times by alkoxy, nitro, cyano, amino or halogen (such as fluoro, chloro, bromo and iodo).
[0040]
The term "aryl" in this context means a wholly or partially aromatic carbocyclic ring or ring system, such as phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracyl, There are Nantrasil, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl, of which phenyl is a preferred example.
[0041]
The term “optionally substituted” in the context of the term “aryl” refers to the group in question_1-4 Alkyl, C_1-4 It means that it may be substituted 1 to 5 times, preferably 1 to 3 times by alkoxy, nitro, cyano, amino or halogen.
[0042]
The term "heteroaryl" refers to a wholly or partially aromatic, wherein one or more carbon atoms are replaced by a heteroatom, for example a nitrogen (= N- or -NH), sulfur and / or oxygen atom. Carbocyclic ring or ring system. Examples of such heteroaryl groups include oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazoyl, pyrazoyl, pyridinyl, pyrazinyl, pyridazinyl, piperazinyl, cumaryl, furyl, quinolyl, benzothiazoyl, benzotriazolyl, benzodiazolyl, benzoyl. Oxazolyl, phthalazinyl, phthalanyl, triazolyl, tetraazolyl, isoquinolyl, acridinyl, carbazolyl, dibenzoazepinyl, indolyl, benzopyrazolyl, and phenoxazonyl.
[0043]
The functional group Y is often chosen to interact with the polymer or polymer precursor. In some embodiments, Y and Y 'are the same.
[0044]
Chemical modification of the surface can change the ability of the spotting fluid to wet the substrate material, and thus adjust the material to produce distinct sensor dots for a particular composition of the spotting fluid.
[0045]
In one embodiment of the present invention, Y represents an amino group capable of reacting with polyvinyl chloride to produce a polyvinyl chloride dot covalently bonded to the surface material.
[0046]
In one embodiment of the present invention, the surface coated with gold or silver is treated with allyl thiol or methacryloyl thiol to create a surface that can react with a polymer precursor such as methacrylate or acrylate during polymerization and shared with the substrate material. Multiple linked methacrylate or acrylate sensor dots can result.
[0047]
In yet another embodiment of the present invention, treating a glass or silicon oxide surface with allyl silane or methacryloyl silane creates a surface that can react during polymerization with a polymer precursor such as methacrylate or acrylate to provide a substrate material with Multiple methacrylate or acrylate sensor dots can be covalently linked.
[0048]
In yet another embodiment of the present invention, a gold or silver coated surface is treated with a hydroxyl terminated aliphatic thiol such as 11-mercaptoundecanol to provide dodecyl methacrylate and 1,6-hexanediol dimethacrylate. Creates a surface that allows for stable droplet deposition of a spotting fluid containing.
[0049]
In an alternative embodiment of the invention, the wetting of the substrate material is controlled by a microstructure such as a small well on the surface of the substrate material. Spotting fluid is deposited in the holes and allowed to spread therein. The diameter and depth of the hole determine the size and height of the resulting sensor dot. In one preferred embodiment of the invention, the diameter of the holes is between 50 and 1000 μm and the depth of the holes is between 1 and 50 μm.
[0050]
The substrate material has a planar portion. It should be understood that not all of the substrate material need correspond to a plane or planes in the device. The substrate material may have other portions with diffraction gratings, rims extending above a plane, mounting holes, and the like. What is important in the context of the present invention is that the substrate material has at least one planar part, on which a plurality of (bio) chemical sensor dots are provided.
[0051]
This plane portion corresponds to a “converter based on an optical phenomenon”. The term "optical phenomena" refers to refraction, reflection, diffuse reflectance, attenuated reflectance, transmission, spectral change, color change, absorption, critical angles of reflection, surface plasmon resonance, etc., fluorescence and fluorescence quenching. And preferably transmission, fluorescence and surface plasmon resonance, especially surface plasmon resonance. These phenomena include spectroscopy, spectrophotometry, photometry, SPR technology, Total Internal Reflection Fluorescence (TIRF) sensing, Grating Coupler sensing (GCS), resonant mirror sensing, and reflection interference spectroscopy. It is the basis for a number of transducer technologies such as Reflectometric Intererence Spectroscopy (RIFS), integrated optical devices (waveguides), integrated optical interferometers, critical angle refractometers, and the like.
[0052]
The term "change in optical properties" and similar terms are used to allow the detection of changes in the optical properties of a (bio) chemical sensor dot, such as, in particular, changes in absorption, refractive index or fluorescence (by which the polymer dot It is intended to cover changes in the optical phenomena described above, which allows the chemical response to be monitored).
[0053]
Although individual (bio) chemical sensor dots can have the same chemical composition, in general, the sensor dots are not all identical. Thus, devices made according to the present invention generally comprise at least 5, for example at least 15 different sensor dots. This means that the polymer matrices of the different sensor dots are generally the same, but the one or more (bio) chemical recognition moieties are different, thereby allowing multiple analytes to be identified on the same device. I can say that. In a preferred embodiment, each of the spatially separated (bio) chemical sensor dots comprises a different (bio) chemical recognition moiety.
[0054]
In an alternative embodiment, the composition of some sensor dots may be the same to image the distribution of the analyte in the heterologous sample.
[0055]
The (bio) chemical sensor device formed by the method according to the invention comprises a plurality of (bio) chemical sensor dots at predetermined spatially separated positions in the xy plane of the planar part of the substrate material. For practical purposes, there is an equal distance between the sensor dots in the x direction and an equal distance between the sensor dots in the y direction (where the distance between the dots in the x and y directions is the same). Or different) may be desirable in many cases. In a preferred embodiment of the invention, the distance between the centers of the (bio) chemical sensor dots in the x and y directions is independently in the range of 1.1 to 10 times the diameter of the (bio) chemical sensor dot.
[0056]
To provide multiple (bio) chemical sensor dots, the method comprises:
(a) providing a substrate material having a planar portion;
(b) one or more spotting fluids, each of which is
(i) a polymer and / or a polymer precursor; and
(ii) one or more (bio) chemical recognition moieties;
Providing at least one of the following:
(c) depositing the one or more spotting fluids simultaneously or sequentially at predetermined spatially spaced locations on the planar portion of the substrate material by a “pin ring” deposition mechanism, and solidifying the spotting fluids; Tying.
[0057]
The term "consolidation" includes, for example, polymerization, polycondensation, crosslinking, sol-gel processes, evaporation of solvents, by exposure to heat, irradiation with ultraviolet light, irradiation with visible light or by electron-induced excitation. Is intended.
[0058]
The method comprises spotting a fluid containing one or more polymers and / or polymer precursors (hereinafter "spotting fluid") onto a planar portion of a substrate material by a "pin ring" deposition method. including. Next, a plurality of spatially separated sensor dots are created by consolidating the spotting fluid on the support surface, either by evaporation of a solvent, polymerization of a polymer precursor, or a combination thereof.
[0059]
The “pin ring” deposition method applied to the present invention was originally introduced by Genetic MicroSystems ™ (WO 99/36760) as a method of making biological materials, especially microarrays, where The biological material is adhesively or covalently bonded to the dimensional surface. The present invention has newly found that the "pin ring" deposition system can be advantageously used for the production of a plurality of (bio) chemical sensor polymer dots, wherein a spatially separated sensor dot is used. The (bio) chemical recognition moiety is arranged in a three-dimensional matrix on or on the surface.
[0060]
The "pin ring" deposition method is based on surface tension as the basic mechanism for holding and transferring fluid. The key mechanism element consists of a circular open "ring" held in place by a vertical rod extending perpendicular to the ring and oriented parallel to the substrate. A vertical pin is located at the center of the ring. Both the ring rod and the pin are mounted to control the device so that each part can move separately in the z-axis while keeping both the ring rod and the pin in a fixed relationship to each other in the xy plane. Have been. As the ring is immersed in the spotting fluid and lifted, it collects an aliquot of the sample, which is held in the center of the ring by surface tension. Next, the pin ring mechanism is positioned at any desired position in the xy plane. When it is desired to create a dot on the substrate material, the pin is driven down through the ring. As the pin passes through the ring, a portion of the spotting fluid is transferred from the inner ring meniscus to the bottom of the pin, forming a new dripping drop on the lower surface of the pin. The pin continues to move downward until the fluid on the pin contacts the substrate material. Next, when the pin is lifted, the combined force of gravity and surface tension causes the spotting fluid to be deposited as dots on the substrate material.
[0061]
No shock or mechanical contact is required between the pin and the substrate to transfer the fluid.
[0062]
Movement of the pin through the inner meniscus of the ring does not destroy the meniscus until a sufficient portion of the fluid has been removed to pass some minimum threshold. Given a given amount of fluid on the pins in the ring, the pin drive process can be repeated many times, thus creating a large number of similar dots with a single moving pin and ring assembly Can be.
[0063]
The amount of this deposit fluid depends on the dimensions of the pin and is approximately equal to the volume of a hemisphere with a radius equal to the radius of the pin, which is in the range of 50 to 500 μm with currently available hardware. This is generally desirable for the embodiments described herein.
[0064]
Characteristic to the invention are that at least one of the spotting fluids comprises a polymer and / or a polymer precursor, and that at least one of the spotting fluids corresponds to one or more (bio) chemical recognition moieties. It is to include.
[0065]
In one embodiment, only one spotting fluid is used, which contains not only the polymer and / or polymer precursor, but also elements corresponding to one or more (bio) chemical recognition moieties.
[0066]
In a preferred embodiment, at least two spotting fluids are used; the first spotting fluid comprises a polymer and / or a polymer precursor, and the second spotting fluid comprises one or more (bio) chemical recognition moieties. Including elements corresponding to In a preferred embodiment, the first spotting fluid is deposited before the second spotting fluid.
[0067]
Examples of suitable polymers include polyacrylates, such as poly (methylpropenoate) or poly (2-methylpropenoate), polyanilines, poly (butadiene), polyethylene, ethylene-vinyl acetate copolymer, poly (vinyl acetate). Methacrylates such as poly (methyl methacrylate), poly (octyl methacrylate), poly (decyl methacrylate) or poly (isodecyl methacrylate), polystyrenes such as polystyrene, poly (4-tert-butylstyrene) or poly (4-methoxy) Styrene), polypyrroles, polythiophenes, polyurethanes, such as Tecoflex® EG 80A, poly (vinyl acetate), poly (vinyl alcohol), poly (vinyl chloride), epoxy novolak resins, such as shell ( Shell 5U 8, and copolymers or terpolymers of the polymer from, for example, ethylene-vinyl acetate copolymer, a plastic resin containing. Specific examples include poly (decyl methacrylate), poly (isodecyl methacrylate), Tekoflex® EG 80A, and poly (vinyl chloride).
[0068]
In the present context, the term "polymer precursor" refers to crosslinkers as well as monomers, dimers, oligomers, prepolymers, which form macromolecular, polymeric structures by polymerization, polycondensation and crosslinking.
[0069]
Examples of plastic monomers include monomeric acrylates such as acrylic acid, n-butyl acrylate, isodecyl acrylate, acrylamide, hexanediol diacrylate, cyclohexanediol diacrylate, N, N'-methylenebisacrylamide or tripropylene glycol diacrylate , Monomeric methacrylates such as methacrylic acid, methyl methacrylate, ethyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, nonyl methacrylate, decyl methacrylate, dodecyl methacrylate, hydroxyethyl methacrylate, glycidyl methacrylate, trifluoroethyl methacrylate, ethylene glycol diacrylate Methacrylate, triethylene It is recalled dimethacrylate or 1,6-hexanediol dimethacrylate. Particular examples of plastic monomers include n-butyl acrylate, isodecyl acrylate, decyl methacrylate, and 1,6-hexanediol dimethacrylate.
[0070]
Another class of monomeric units included by the present invention are metal or metalloid compounds such as organosilanes and the like, for example, tetramethoxysilane, 3-aminopropyltrimethoxysilane and tetramethyl orthosilicate, which are Hydrolysis of the alkyl-O-Si bond produces silanols (SiOH groups), which form sol-gels (-Si-O-Si-) by polycondensation. In this example, a sol-gel ("polymer" precursor) is used in combination with alcohol, water and acid as solvents.
[0071]
Examples of oligomers include aliphatic urethane diacrylate oligomers such as Ebecryl 230 (molecular weight 5000) and Ebecryl 270 (molecular weight 1500) (from UCB chemicals), and in combination with a cross-linking agent such as glutaraldehyde. There are proteins, such as bovine serum albumin (BSA), that form a water-insoluble macromolecular polymer-type structure that can physically incorporate a biochemical recognition element.
[0072]
It will be apparent that the spotting fluid may further include one or more solvents. The solvent or solvent mixture is such that the polymer and / or polymer precursor remains dissolved or suspended in the solvent during the deposition step, and the spotting fluid is small on the substrate material. It should be selected so that caking of the spotting fluid does not occur until it is deposited as drops. Preferably, after deposition of the spotting fluid onto the substrate material, the solvent or solvent mixture evaporates spontaneously. However, in the case of some non-volatile solvents or solvent mixtures, heat or reduced pressure may be applied to ensure proper and rapid evaporation of the solvent or solvent mixture and subsequent solidification of the sensor dots. May be needed.
[0073]
Suitable solvents include ketones such as acetone, butanone, 4-methyl-2-pentanone, cyclopentanone or cyclohexanone, hydrocarbons such as n-hexane, n-pentane, benzene, toluene or xylene, esters such as ethyl acetate Propyl acetate, butyl acetate or diethyl sebacate, alcohols such as methanol, ethanol, glycerol, ethanolamine or phenol, acids such as formic acid or acetic acid, amides such as N, N-dimethylformamide, N, N-dimethylacetamide or N -Methylpyrrolidone, halogenated hydrocarbons such as dichloromethane, chloroform, tetrachloroethane or chlorobenzene, nitromethane, nitrobenzene, water and mixtures thereof.
[0074]
The spotting fluid may further include one or more plasticizers. Examples of suitable plasticizers include esters such as bis (1-butylpentyl) adipate, bis (1-butylpentyl) decane-1,10-diyl-diglutarate, bis (2-ethylhexyl) adipate, bis (2- Ethylhexyl) phthalate, bis (2-ethylhexyl) sebacate, dibutylphthalate, dibutylsebacate, 10-hydroxydecylbutyrate, tetraundecylbenzhydrol-3,3 ′, 4,4′-tetracarboxylate, tetraundecyl Benzophenone-3,3 ', 4,4'-tetracarboxylate, tris (2-ethylhexyl) trimellitate, dibutyltin dilaurate, dioctylphenyl phosphonate, isodecyldiphenyl phosphate, tributyl phosphate or tris (2-E Lehexyl) phosphate, ethers such as dibenzyl ether, benzyl 2-nitrophenyl ether, 2-cyanophenyloctyl ether, dodecyl 2-nitrophenyl ether, dodecyl [2- (trifluoromethyl) phenyl] ether, [12- (4 -Ethylphenyl) dodecyl] 2-nitrophenyl ether, 2-fluorophenyl 2-nitrophenyl ether, 2-nitrophenylphenyl ether, 2-nitrophenyloctyl ether, 2-nitrophenylpentyl ether or octyl [2- (trifluoro Methyl) phenyl] ether, alcohol such as 1-decanol, 1-dodecanol, 1-hexadecanol, 1-octadecanol, 5-phenyl-1-pentanol or 1-tetradecanol, Halogenated hydrocarbons, such as 1-chloronaphthalene or chloroparaffins, phosphine oxides, for example trioctylphosphine oxide, and mixtures thereof. Specific examples include bis (2-ethylhexyl) sebacate, dodecyl [2- (trifluoromethyl) phenyl] ether, and 2-nitrophenyloctyl ether.
[0075]
In one embodiment of the present invention, the plasticizer comprises the solvent.
[0076]
Particular examples of spotting fluids according to the present invention include poly (vinyl chloride) (PVC) and bis (2-ethylhexyl) dissolved in cyclohexanone at a ratio of 1: 1 to 1: 4, eg, approximately 1: 2. There is Sebacate (DOS). Cyclohexanone evaporates at a moderately slow rate, allowing the deposition of about 100-400 fluid droplets on a substrate material without clogging the deposition mechanism.
[0077]
When the spotting fluid contains a polymer precursor, polymerization (one type of consolidation) of the polymer precursor is required to obtain polymer dots. The polymerization / consolidation process is initiated after deposition of the spotting fluid droplet and by exposing the spotting fluid droplet to irradiation with heat, ultraviolet or visible light, or by electron-induced excitation. It preferably does not occur until it has or has occurred spontaneously. However, for some polymer precursors, unless the polymerization initiator is present, the polymerization process is not initiated or is undesirably slow. As such, the spotting fluid may further include a polymerization initiator. Examples of polymerization initiators are the radical initiators α, α-dimethoxy-α-phenylacetophenone which can be used further in combination with a photosensitizer such as benzophenone or benzoyl peroxide.
[0078]
Similarly, polycondensation of a polymer precursor, such as in the formation of a sol-gel, may require the presence of water and / or acid, which may be included in the spotting fluid. In some cases, polycondensation can be inhibited by exposing the deposited sol-gel precursor spotting fluid to water and / or acid vapor.
[0079]
The function of the polymer matrix is to provide a carrier for the (bio) chemical recognition system. In the present invention, a (bio) chemical recognition system is a complex that can include one or more elements that induce a change in a physical property of a polymer matrix, such as an optical property, when exposed to a specific analyte. About. This facilitates the detection of changes in the physical (optical) properties of the polymer matrix by the planar portions of the substrate material forming a suitable transducer, thereby allowing the detection and quantification of a particular analyte. It is understood that not all (bio) chemical recognition moieties need to interact directly with the analyte, and that their combination may cause a change in the physical properties of the polymer matrix (eg, in a cascade). Should be.
[0080]
Elements that correspond to (bio) chemical recognition moieties may be physically incorporated into the polymer network, covalently bonded to the polymer backbone, interact ionicly with charged groups on the polymer, or be incorporated into the polymer phase. It is retained either in the matrix of the sensor dots or near the interface, either by physical dissolution.
[0081]
In an alternative embodiment of the invention, one or more components of the (bio) chemical recognition system are immobilized directly on the surface of the sensor dot. This includes enzymes, antibodies, catalytic antibodies, proteins, nucleic acids and derivatives thereof, such as PNA (peptide nucleic acids) or LNA (locked nucleic acids), aptamers, receptors, or cells or tissue segments (organisms). Of particular importance for the chemical recognition part. However, these (bio) chemical recognition portions may be held in the matrix of the sensor dots or near the interface as described above. However, recognition moieties attached to the surface of such polymer matrices are only considered as part of the recognition system if there are direct chemical bonds to the remaining (embedded) elements of the recognition system. It should be understood that.
[0082]
In a preferred embodiment of the present invention, the sensor device made by the method comprises a plurality of optode films. The optode membrane is considered as a single thermodynamic homogeneous phase that responds reversibly to the activity of the analyte. Optode membranes consist of a polymer matrix that functions as a carrier for the chemical recognition moiety. The chemical recognition system may include ligands (ion carriers, ionophores, indicators, complexing agents) that are physically incorporated or chemically bound into the polymer matrix. The interaction of the analyte with the ligand generates an optical signal that allows the ligand itself or additional compounds (chromoionophores, fluoroionophores, indicator dyes) to form their optical properties by complexing with other ions. To change.
[0083]
In one embodiment of the present invention, the sensor device made by the method is a plurality of ion-selective optode membranes, wherein the chemical recognition system comprises, for example, an ion-selective electrically neutralizing ionophore and H⁺ It contains a lipophilic anionic site as well as a selectively electrically neutralized chromoionophore. By exchanging hydrogen ions for analyte cations, the membrane changes its color. This change in spectral properties is used for optical detection. Lipophilic anionic sites are added to ensure a certain amount of ions present in the polymer matrix.
[0084]
Examples of ionophores include ion-specific ionophores, such as the lithium-specific ionophore N, N'-diheptyl-N, N ', 5,5-tetramethyl-3,7-dioxanonanediamide or N, N, N ', N'-tetraisobutyl-cis-cyclohexane-1,2-dicarboxamide, a sodium-specific ionophore N, N', N "-trimethyl-4,4 ', 4" -propylidinetris (3- Oxabutylamide), 4-octadecanoyloxymethyl-N, N, N ', N'-tetracyclohexyl-1,2-phenylenedioxydiacetamide or 4-tert-butylcalix [4] arene-tetraethyltetraacetate Esters, potassium-specific ionophore valinomycin, 2-dodecyl-2-methyl-1,3-propanediyl- Bis [N- [5'-nitro (benzo-15-crown-5) -4'-yl] carbamate] or 4-tert-butyl-2,2,14,14-tetrahomo-2a, 14a-dioxacalix [4] Allen-tetraacetic acid tetra-tert-butyl ester, 4- [N- (1-adamantyl) carbamoylacetyl] -13- [N- (n-octadecyl) carbamoylacetyl] -1, which is an ammonium-specific ionophore , 7,10,16-Tetraoxa-4,13-diazacyclooctadecane, cesium-specific ionophore calix [6] arene-hexaethylhexaethyl ester, magnesium-specific ionophore N, N ″ -octamethylene- Bis (N'-heptyl-N'-methyl-methylmalonamide), N, N "-octamethylene-bis (N -Heptyl-N'-methyl-malonamide), N, N ', N "-tris [3- (heptylmethylamino) -3-oxopropionyl] -8,8'-iminodioctylamine, 7-[(1- Adamantylcarbamoyl) acetyl] -16-[(octadecylcarbamoyl) acetyl] -1,4,10,13-tetraoxa-7,16-diazacyclooctadecane, a calcium-specific ionophore (-)-(R, R) -N, N'-bis [11- (ethoxy-carbonyl) undecyl] -N, N'-4,5-tetramethyl-3,6-dioxaoctane-diamide, calcimycin, 10,19-bis- [ (Octadecylcarbamoyl) methoxyacetyl] -1,4,7,13,16-pentaoxa-10,19-diazacyclopheneicosane, N, N, N ', N'-tetracyclohexyl-oxybis (o-phenyleneoxy) diacetamide which is a barium-specific ionophore, o-xylylenebis (N, N-diisobutyldithiocarbamate) which is a heavy metal-specific ionophore (particularly copper) ), S, S'-methylenebis (N, N-diisobutyldithiocarbamate) (especially silver), O, O "-bis [2- (methylthio) ethyl] -tert-butylcalix [4] arene (especially silver), Methylenebis (2-thiobenzothiazole) (especially silver), 5-tetradecyl-1,4-dioxa-8,11-dithiacyclotetradecane (especially silver), 7-tetradecyl-6,9-dioxa-2,13- Dithiatetradecane (especially silver), tetrabutylthiuram disulfide (especially zinc), N-phenyl-iminodivinegar Acid N ', N'-dicyclohexyl-bis-amide (especially zinc), N, N, N', N'-tetrabutyl-3,6-dioxaoctanedi (thioamide), [1,1'-biscyclohexyl] -1,1'-2,2'-tetrol (especially cadmium), N, N-dioctadecyl-N ', N'-dipropyl-3,6-dioxaoctanediamide (especially lead), N, N, N ', N'-tetradodecyl-3,6-dioxaoctanedithioamide (particularly lead), tert-butylcalix [4] arene-tetrakis (N, N-dimethylthioacetamide) (particularly lead), tert-butylcalix [6] arene-ethyleneoxydiphenylphosphine (particularly lead), N, N, N ', N'-tetradodecyl-3,6-dioxaoctane-1-thio-8-oxadiamide (particularly lead) , 5,7,12,14-tetramethyldibenzotetraazaannulene (especially lead), 1,10-dibenzyl-1,10-diaza-18-crown-6 (especially lead), O-methyldihexylphosphine oxide O '-Hexyl-2-ethylphosphate (especially uranyl ion), an anion-selective ionophore such as tridodecylmethylammonium chloride, or the fluoride and chloride-specific ionophore chloro (2,3,7,8,12,13) , 17,18-octaethylporphyrinato) gallium (III), chloro (5,10,15,20-tetraphenylporphyrinato) gallium (III), hydroxo (5,10,15,20-tetrakis (o) -Pivalamidophenyl) porphyrinato) gallium (III), chloro (2,3,7,8,12,1) 3,17,18-octaethylporphyrinato) indium (III), chloro (5,10,15,20-tetraphenylporphyrinato) indium (III), hydroxo (5,10,15,20-tetrakis ( o-pivalamidophenyl) porphyrinato) indium (III), chloro (2,3,7,8,12,13,17,18-octaethylporphyrinato) thallium (III), chloro (5,10 , 15,20-Tetraphenylporphyrinato) thallium (III), [N, N- [4,5-bis (dodecyloxy) -1,2-phenylenebis [nitrilomethylidine (2-hydroxy-1,3 -Phenylene)] acetamido] -N, N ', O, O'] dioxouranium, 4,5-dimethyl-3,6-dioctyloxy-1,2-phen Lenbis (mercury trifluoroacetate), 3,6-didodecyloxy-4,5-dimethyl-1,2-phenylenebis (mercury chloride), [9] mercuracarborand-3, ruthenium (II) (2,3,7,8,12,13,17,18-octaethylporphyrin) which is selected from the group consisting of carbonyl, trioctyltin chloride and tricyclohexyltin chloride; other ionophores include triiodide Heptakis, a specific ionophore, 2,4,6,8-tetraphenyl-2,4,6,8-tetraazabicyclo [3.3.0] octane, a nitrite-specific ionophore cyanoaquacobilininate ( 2-phenylethyl ester), heptapropyl dicyanocobilinate, aquo-cyano-cobinamide, carbonic acid and sulfide Specific ionophores 3,12-bis (trifluoroacetobenzoyl) cholic acid, trifluoroacetyl-p-butylbenzene, octadecyl 4-formylbenzoate, and sulfuric acid Specific ionophores dibecaine sulfate and α, α′-bis (N'-phenylthioureylene) -m-xylene. Specific examples include 4-tert-butylcalix [4] arene-tetraacetic acid tetraethyl ester, 2-dodecyl-2-methyl-1,3-propanediylbis [N- [5′-nitro (benzo-15- Crown-5) -4'-yl] carbamate], 4- [N- (1-adamantyl) carbamoylacetyl] -13- [N- (n-octadecyl) carbamoylacetyl] -1,7,10,16- Tetraoxa-4,13-diazacyclooctadecane, (-)-(R, R) -N, N'-bis [11- (ethoxy-carbonyl) undecyl] -N, N'-4,5-tetramethyl- 3,6-dioxaoctane-diamide, tridodecylmethylammonium chloride, hydroxo (5,10,15,20-tetrakis (o-pivalamidophenyl) porphyrina G) Indium (III), and cyanoaquacobilinic acid heptakis (2-phenylethyl ester).
[0085]
Examples of chromoionophores include 9- (diethylamino) -5- (octadecanoylimino) -5H-benzo [a] phenoxazine, 9-dimethylamino-5- [4- (16-butyl-2,14-dioxo). -3,15-dioxaeicosyl) phenylimino] benzo [a] phenoxazine, 9- (diethylamino) -5-[(2-octadecyl) imino] benzo [a] phenoxazine, 5-octadecanoyloxy- 2- (4-nitrophenylazo) phenol, 9- (diethylamino) -5- (naphthoylimino) -5H-benzo [a] phenoxazine, 4 ′, 5′-dibromofluorescein octadecyl ester, fluorescein octadecyl ester , 4- (octadecylamino) azobenzene, and N-2,4-dinitro-6 (Octadecyloxy) phenyl-2 ', 4'-dinitro - those selected from the group consisting of (trifluoromethyl) phenylamine. Specific examples include 9- (diethylamino) -5- (octadecanoylimino) -5H-benzo [a] phenoxazine and 9-dimethylamino-5- [4- (16-butyl-2,14- Dioxo-3,15-dioxaeicosyl) phenylimino] benzo [a] phenoxazine.
[0086]
Examples of complex lipophilic inorganic ions include tetrakis [3,5-bis (trifluoromethyl) phenyl] borate, tetrakis (4-chlorophenyl) borate, tetrakis (4-fluorophenyl) borate, and Some are selected from the group consisting of tetradodecyl ammonium. Specific examples include tetrakis [3,5-bis (trifluoromethyl) phenyl] borate, and tetradodecylammonium.
[0087]
In one embodiment of the present invention, the chemical recognition system comprises one ionophore, one chromophore, and one complex lipophilic inorganic ion.
[0088]
In another embodiment of the invention, the biochemical recognition system comprises an enzyme or enzymes and a coloring reagent, for example, when an analyte (eg, glucose) is bound to an enzyme (eg, glucose oxidase), the enzyme Oxidize the analyte in the presence of The recognition system may include an element that can interact with the reaction product of this oxidation, gluconic acid or hydrogen peroxide, to facilitate optical detection (eg, the former can protonate a pH indicator or the latter can be a dye). May be oxidized).
[0089]
In one embodiment of the invention, the optical phenomenon is surface plasmon resonance, the substrate material is made from a plastic base material and a metal surface layer material, and the sensor dots are made from polyvinyl chloride or cross-linked acrylate with a plasticizer. . In particular, the metal is gold and the base material is polyetherimide.
[0090]
In an alternative embodiment of the invention, the chemical recognition is provided by the polymer structure itself. Polymer-type materials that respond reversibly to the activity of the analyte and are characterized by having a polymer-type structure that includes cavities (voids) are referred to as molecular imprinted polymers. The preparation of molecularly imprinted polymers involves polymerizing a polymer precursor, for example, an acrylate, in the presence of a template molecule, often the analyte itself. The removal of template molecules from the polymer matrix then creates cavities within the polymer matrix that constitute binding sites specific for the analyte. Binding of the analyte to the cavity results in a change in the optical / physical properties of the polymer matrix. In this particular embodiment of the invention, the template molecule ("negative" but "recognition moiety" in the present application) is included in the spotting fluid and then chemically washed by washing the solidified sensor dots. / System is formed.
[0091]
As indicated above, the introduction of the (bio) chemical recognition system into the polymer matrix of the sensor dots can be achieved in different ways, including one or more sequential steps. These steps may include one or more cleaning steps, one or more “pin ring” deposition steps, and one or more consolidation steps, eg, exposure to heat, reduced pressure, or irradiation from a different source. May be included.
[0092]
In one embodiment of the invention, the components of the (bio) chemical recognition system are contained in the same spotting fluid as the polymer and / or the polymer precursor.
[0093]
In one embodiment, two or more spotting fluids are sequentially deposited at each predetermined location in the plane and the spotting fluid is consolidated after the last deposition of the spotting fluid.
[0094]
In another embodiment, two or more spotting fluids are sequentially deposited at each predetermined location on the plane and the spotting fluid is consolidated after each deposition of the spotting fluid.
[0095]
In another embodiment of the invention, one or more fluids containing one or more components of a (bio) chemical recognition system are deposited in a "pin ring" deposition on a preformed sensor dot. By superimposition by a method, a chemical or biological recognition system can be introduced into (or on) the polymer matrix of the sensor dot. In this embodiment, the fluid containing the (bio) chemical recognition element may further comprise a solvent and / or a plasticizer.
[0096]
This approach may not only be advantageous for the introduction of recognition elements that can be damaged when exposed to polymerization conditions, but may also represent an economical method of producing (bio) chemical sensor devices with different patterns.
[0097]
In one embodiment of the present invention, the introduction of the chemical recognition system into the polymer matrix of the sensor dot is performed in the following manner. A first spotting fluid comprising a polymer and / or a polymer precursor and a plasticizer is deposited on a substrate material. This spotting fluid is consolidated. The plasticizer is again removed by washing the consolidated sensor dots with a suitable solvent. On top of the consolidated polymer matrix is deposited a second spotting fluid comprising a (bio) chemical recognition system in the form of an element corresponding to one or more (bio) chemical recognition moieties and a plasticizer. The plastic matrix is again plasticized. Next, the combination is consolidated.
[0098]
In one embodiment of the present invention, the droplets of the unconsolidated sensor dots as well as the consolidated sensor dots are added to the droplets of a solvent, a plasticizer, a polymerization initiator and a (bio) chemical recognition element. The (bio) chemical sensor dots can be constructed on a support surface by the sequential superposition of one or more fluids containing one or more selected elements.
[0099]
In an even more preferred embodiment, one or more of the sensor dots have an unspecified change due to effects from temperature, degradation, analyte, bulk solution refractive index, swelling of the polymer matrix, ionic strength, or the sensor dot. Represents a reference sensor dot that includes a reference polymer matrix that responds to variations in the light source used in the transducer. The reference sensor dot may include all elements of the reference sensor dot except for one or more (bio) chemical recognition elements.
[0100]
(Bio) chemical sensor dots generally have a diameter of 1 to 1000 μm, more preferably 150 to 250 μm, and the height of the dots is 0.1 to 1000 μm, more preferably 1 to 5 μm. The number of fluid overlay steps usually controls the diameter and height of the sensor dots.
[0101]
The (bio) chemical sensor dots produced by the method of the present invention can be used for the parallel detection and quantification of two or more analytes contained in the same sample. Those skilled in the art will recognize the broad potential scope of the present invention.
【Example】
[0102]
Example 1
Improvement of commercially available "pin ring" arrayer
Adapt a commercially available "pin ring" arrayer (Affimetrix 417, formerly GMS 417 from Genetic Microsystems) for the deposition of spotting fluids containing polymers or polymer precursors but not biological or biochemical fluids That is, it is adapted for continuous use with an organic solvent such as ethanol for pin washing. The tubing (generally silicone) was replaced with more solvent resistant FEP (fluoroethylene-propylene) tubing. Similarly, remove the pump (AS Thomas) that sends the cleaning solution to the cleaning station and replace it with a "chemical resistant" version of the same model. Revoke the protective lock on the door to allow access to the pins for manual cleaning with tetrahydrofuran using a wash bottle. A flow restrictor (flow restrictor), rather than (or in addition to) a clamp, is attached to the cleaning solvent tubing to allow for increased control of the amount of solvent ejected from nozzles in the cleaning station. By using a protective foil to cover the inside of the transparent front door of the device, the device can be protected from damage if less washing solvent bounces off. The outlet of a vacuum pump that removes the washing liquid from the liquid tank by suction is connected to a laboratory ventilation system (hood) to prevent a large amount of solvent vapor from being introduced into the working environment. Introduce into the arrayer in the microtiter plate hole. Typical polystyrene plates are not resistant to many organic solvents, which is why polypropylene plates are chosen.
[0103]
Example 2
Fabrication of multiple miniaturized PVC dots on glass material
33 mg of poly (vinyl chloride) (high molecular weight) and 66 mg of plasticizer bis (2-ethylhexyl) sebacate (DOS) are dissolved in 800 μL of cyclohexanone, and 35 μL of the resulting spotting fluid is placed in a well of a 256-well polypropylene microtiter plate. Inject into A1. Using a GMS 417 arrayer with 125 μm pins, a demonstration array of PVC dots can be easily deposited on a substrate such as a commercially available glass or gold-coated glass microscope slide (FIG. 1). Other support surfaces may be provided in the device by using a custom metal adapter plate. Wash the pins with tetrahydrofuran to remove PVC-DOS residues. This may be done manually, or the use of a suitable solvent in the washing line and the reservoir in the correspondingly adapted equipment.
[0104]
Example 3
Fabrication of multiple miniaturized sodium selective (bio) chemical sensor dots
2.9 mg of 9- (diethylamino) -5- (octadecanoylimino) -5H-benzo [a] phenoxazine, 4.6 mg of sodium tetrakis [3,5-bis (trifluoromethyl) phenyl] borate, 4-mg 10.0 mg of tert-butylcalix [4] arenetetraacetic acid tetraethyl ester, 139.2 mg of bis (2-ethylhexyl) sebacate and 69.1 mg of poly (vinyl chloride) (high molecular weight) are dissolved in 2 ml of cyclohexanone. Inject 35 μL of the resulting spotting fluid into hole A1 of a 256-well polypropylene microtiter plate. Sodium selective (bio) chemical sensor dots based on plasticized PVC were made on gold-coated glass microscope slides using a GMS 417 arrayer with 125 μm pins. The functionality of the sensing dot, that is, the response to sodium as the target ion in the buffer solution, can be confirmed by fiber optic absorptiometry or surface plasmon resonance spectroscopy, respectively. The latter detects the refractive index change in the film which is related to the spectral / absorbance change by the Kramers-Kronig relation.
[0105]
Example 4
Fabrication of multiple sensor dots using spotting fluid containing methacrylate
160 mg of 1,6-hexanediol dimethacrylate, 100 mg of dodecyl methacrylate, 200 mg of bis (2-ethylhexyl) sebacate, and 1 mg of a radical initiator such as α, α-dimethoxy-α-phenylacetophenone, or benzophenone as a photosensitizer 35 μL of spotting fluid prepared from 2.5 mg of benzoyl peroxide along with 5 mg is injected into hole A1 of a 256-well polypropylene microtiter plate. Demonstration arrays of photopolymerized methacrylate sensor dots were fabricated on commercial microscope slides using a GMS 417 arrayer with 125 μm pins. After deposition of the spotting fluid, the pins were washed with a suitable solvent such as ethanol. After deposition, the spotting fluid droplets were photopolymerized by exposing the spotting fluid to UV light in an inert gas atmosphere, typically for 10-20 minutes. Such experiments convincingly demonstrate that a large number of methacrylate cocktail dots can be produced with high precision and can be photopolymerized immediately thereafter. The addition of (bio) chemical recognition elements (eg ionophores, chromoionophores, complex lipophilic inorganic ions) at a concentration of a few percent (w / w) to the spotting fluid results in an array of sensing dots without affecting the deposition process. Occurs. However, since many of these elements are photobleachable, replasticizing can be selected as an alternative route to introducing sensing elements. To this end, plasticized dots that do not contain recognition elements are deposited and then treated with tetrahydrofuran to remove the plasticizer from the material. Thereafter, a droplet of fluid containing (bio) chemical recognition elements (eg ionophores, chromoionophores, complex lipophilic inorganic ions) in bis (2-ethylhexyl) sebacate can be deposited directly on top of the polymer dots. . After sufficient time, the plasticizer and the sensing element therewith are incorporated into the polymer matrix, resulting in a functional ion-selective (bio) chemical sensor dot.
[0106]
Example 5
Superposition of polymer dots
5. I
A spotting fluid similar to the spotting fluid of Example 4 was deposited on a glass microscope slide such that four arrays A, B, C, D of 3 × 3 dots were obtained. Next, these dots were photopolymerized by UV irradiation in an inert gas. An image of the sensor dots was taken using an Affimetrix 418 fluorescence scanner (FIG. 2) to confirm that the overlay was successful (overlay failed in Array B due to experimental errors, where two individual arrays failed). Is displaced).
[0107]
5. II
In addition, a spotting fluid similar to the spotting fluid of Example 2 was deposited on a 20 × 20 array using features in the calibration software of the arrayer for the alignment test. This deposition was repeated so that the dot positions were shifted. The scanner image of FIG. 3 shows two arrays, showing the area where they overlap. Slight differences in the appearance of the array are the most likely causes caused by the use of different surfaces of the microscope slide.
[Brief description of the drawings]
[0108]
FIG. 1 is a photographic image of a plurality of sensor dots obtained by “pin ring” deposition of a spotting fluid containing PVC / DOS in cyclohexanone (Example 2). The dot diameter is about 200 μm.
FIG. 2 shows an array of spotting fluid droplets (AD) of photopolymerized methacrylate obtained by deposition of a spotting fluid containing methacrylate (Example 4) and subsequent polymerization of a polymer precursor on a support surface. It is a fluorescence image. A second set of arrays is superimposed directly on top of A and C and photopolymerized (Example 5.1).
FIG. 3 is a partially overlaid PVC-DOS dot array (Example 5.II) (image obtained with a fluorescence scanner) generated by a “pin ring” calibration function. The outer white frame surrounds the second array, and the inner white frame surrounds the dot overlap area.

Claims (24)

A method of making an optical (bio) chemical sensor device, said device comprising a substrate material having a planar part, said plane corresponding to a transducer based on optical phenomena, said planar part comprising: A plurality of (bio) chemical sensor dots arranged at predetermined positions spatially separated from each other are arranged, and the sensor dots are
(i) a polymer matrix, and
(ii) one or more (bio) chemical recognition moieties,
With
The method is
(a) providing a substrate material having a planar portion;
(b) one or more spotting fluids, each of which is
(i) a polymer and / or a polymer precursor; and
(ii) one or more (bio) chemical recognition moieties;
Providing at least one of the following:
(c) depositing the one or more spotting fluids simultaneously or sequentially at predetermined spatially spaced locations on the planar portion of the substrate material by a “pin ring” deposition mechanism, and solidifying the spotting fluids; A method comprising tying. The method of claim 1, wherein the optical phenomenon is selected from transmission, fluorescence and surface plasmon resonance. 3. The method according to claim 2, wherein the optical phenomenon is surface plasmon resonance. A method according to any preceding claim, wherein the substrate material comprises a base material selected from glass, silica, dielectric inorganic materials, plastics and silicon with hydrogen or deuterium terminated surfaces. The method according to any one of the preceding claims, wherein the substrate material comprises a planar portion comprising at least one surface layer material selected from metal and silicon. The method according to claim 5, wherein the surface layer material has a thickness of 10 to 500 nm. The plane of the substrate material has the following bifunctional reagent:
X-Z-Y
[Wherein X is -OR ', asymmetric or symmetric disulfide (-SSR'Y', -SSRY), sulfide (-SR'Y ', -SRY), diselenide (-SeSeR'Y', -SeSeRY ), selenide (-SeR'Y ', - SeR'Y') , thiol (-SH), selenol (-SeH), - N = C , -NO 2, trivalent phosphorous compounds, -NCS, -OC ( S) selected from SH, thiocarbamates, phosphines, thioacids (-COSH), dithioic acids (-CSSH), -Si (OR / R / H) ₃ and halogens;
The substituents R and R 'are each independently an optionally substituted _C1-30 alkyl, an optionally substituted _C2-30 alkenyl, an optionally substituted _C2-30 Selected from alkynyl and optionally substituted aryl;
Y and Y ′ are selected from hydroxyl, carboxyl, amino, formyl, hydrazine, carbonyl, epoxy, vinyl, allyl, acrylic, epoxy and methacrylic;
Z is a linker (biradical) between the two functional groups. ]
The method according to any one of the preceding claims, wherein the method is chemically modified by treatment with At least one of the one or more spotting fluids is a polyacrylate, a polyaniline, a poly (butadiene), a polyethylene, an ethylene / vinyl acetate copolymer, a polymethacrylate, a polystyrene, a polypyrrole, a polythiophene, Any of the preceding claims comprising a polymer selected from polyurethanes, poly (vinyl acetate), poly (vinyl alcohol), poly (vinyl chloride), epoxy novolak resins and copolymers or terpolymers of the above polymers. The method according to any one of the preceding claims. The method of any one of the preceding claims, wherein at least one of the one or more spotting fluids comprises a polymer precursor selected from monomeric acrylates, monomeric methacrylates, oligomers and crosslinkers. 10. The method of any one of claims 8 and 9, wherein at least one of the one or more spotting fluids comprises a plasticizer. The method according to any one of claims 8 to 10, wherein the spotting fluid comprises a polymerization initiator. The method according to any of the preceding claims, wherein the (biological) chemical recognition moiety is selected from ionophores, chromoionophores and complex lipophilic inorganic ions. The method according to any of the preceding claims, wherein the spotting fluid is consolidated by exposure to heat, irradiation with ultraviolet light, irradiation with visible light or by electron-induced excitation. A method according to any one of the preceding claims, wherein two or more spotting fluids are sequentially deposited at each predetermined location on the plane, and the spotting fluid is consolidated after the last deposition of the spotting fluid. the method of. 14. The method of claim 1, wherein two or more spotting fluids are sequentially deposited at each predetermined location on the plane, and the spotting fluid is consolidated after each deposition of the spotting fluid. The method described in. The method according to any of the preceding claims, wherein each of the (bio) chemical sensor dots comprises a different (bio) chemical recognition moiety. 17. The method according to claim 16, wherein the sensor device comprises at least five different sensor dots. The method according to any one of the preceding claims, wherein the optical phenomenon is surface plasmon resonance, the substrate material is made from a plastic base material and a metal surface layer material, and the sensor dots are made from crosslinked acrylate or polyvinyl chloride containing a plasticizer. The described method. 19. The method according to claim 18, wherein said metal is gold and said base material is polyetherimide. (Bio) chemical sensor device obtainable by the method according to any one of the preceding claims. 21. The (bio) chemical sensor device of claim 20, wherein each of the (bio) chemical sensor dots comprises a different (bio) chemical recognition moiety. 22. The method according to claim 21, wherein the sensor device comprises at least five different sensor dots. A method for monitoring and / or characterizing two or more analytes using an optical (bio) chemical sensor device according to any one of claims 20 to 22. 24. The method of claim 23, utilizing surface plasmon resonance in combination with an optical (bio) chemical sensor device.