WO2008044179A1

WO2008044179A1 - Biosensors and preparation thereof

Info

Publication number: WO2008044179A1
Application number: PCT/IB2007/054066
Authority: WO
Inventors: Menno W. J. Prins
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2006-10-12
Filing date: 2007-10-05
Publication date: 2008-04-17

Abstract

A low-temperature preparation method for a biosensor device (1) with a layer of reagent (5) on the sensor surface (3) is disclosed. During manufacturing biological interaction between the biosensor substrate and the reagent layer (5) material is reduced, e.g. by cooling the biosensor substrate (2) and depositing the reagent layer (5)on the cooled biosensor substrate (2). Alternatively, the biological interaction between the biosensor substrate (2) and the reagent layer (5) material may also be reduced by providing a separation layer. Furthermore, the method comprises, after depositing of the reagent layer, at least maintaining the biological interaction until the reagent layer is substantially mechanically stable. The method reduces or even avoids interaction between reagent and biologically or biochemically active moieties (4) present on the substrate surface (3). The latter nevertheless allows to obtain a biosensor device (1) with high reactivity of the reagent layer (5) during operation of the biosensor. Biosensors (1) thus obtained allow for fast assays, low reagent use, multiplexing, and small sample volume.

Description

Biosensors and preparation thereof

FIELD OF THE INVENTION

The present invention relates to the field of biosensors. More particularly, the present invention relates to methods and systems for obtaining biosensors suitable for detecting analytes, e.g. for qualitative or quantitative detection of biological, chemical or bio- chemical particles.

BACKGROUND OF THE INVENTION

Biosensors typically are devices that allow qualitatively or quantitatively detection of target molecules, also called "analytes", such as e.g. proteins, viruses, bacteria, cell components, cell membranes, spores, DNA, RNA, etc. in a liquid, such as for example blood, serum, plasma, saliva, tissue extract, interstitial fluid, cell-culture extract, food or feed extract, drinking water,.... In almost all cases, a biosensor uses a surface that comprises specific recognition elements for capturing the analyte. Therefore, the surface of the sensor device may be modified by attaching specific molecules to it, which are suitable to bind the target molecules which are present in the liquid.

One of the measuring principles is the counting of labelled molecules attached at predetermined sites on the biosensor. For example, the molecules may be labelled with magnetic particles or beads and these magnetic particles or beads can be detected with a magnetic sensor. Alternatively, the amount of analyte may be detected by fluorescence. In this case the analyte itself may carry a fluorescent label, or alternatively an additional incubation with a fluorescent labelled second recognition element may be performed. In most biosensors, a sensor chip is provided with a dry reagent and a detector surface covered with a biologically-active surface coating. The reagent may e.g. comprise labels coupled to biologically-active moieties, e.g. an anti-drug antibody. When the test fluid arrives, the dry reagent dissolves and mixes into the fluid. Thereafter the fluid is transported towards the sensor surface and wets the sensor surface. The labels as well as the sensor are exposed to the drug molecules. This influences the binding of the labels to the sensor surface, which is detected. Electrochemical biosensors are known, e.g. from US 20050016844 Al, in the form of test strips wherein a dry dissolvable layer is provided near or on the electrodes. The layer generally comprises chemical components for reacting with the analyte or target molecule to produce an electrochemical signal that represents the presence of the analyte in the sample fluid, such as one or more enzymes, co-enzymes, co-factors, buffer salts, and adjuvants to enhance the reagent properties or characteristics such as de- and rehydration. The latter may be used for detecting analytes present in relatively high concentrations in the sample. In such electrochemical biosensors, a chemical reagent layer is deposited directly over the electrodes using a standard deposition technique.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method of manufacturing of accurate and sensitive biosensors. The above objective is accomplished by a method and device according to the present invention. The present invention relates to a method of manufacturing a biosensor device, the biosensor device comprising a biosensor substrate and at least one reagent layer, the method comprising preparing the biosensor substrate and/or a material for the at least one reagent layer for reducing biological interaction between at least one reagent layer and the biosensor substrate, depositing the at least one reagent layer on the biosensor substrate to achieve a reduced biological interaction between the at least one reagent layer and the biosensor substrate, and after the depositing, - at least maintaining the reduced biological interaction between the at least one reagent layer and the biosensor substrate until the at least one reagent layer is mechanically stable such as flow resistant, set, solidified or dried.

It is an advantage of such embodiments that control of the binding to or mixing with the underlying material of species from the at least one reagent layer is obtained. It is an advantage that such binding or mixing can be prohibited during preparation of the biosensor. It is also an advantage that such binding or mixing can be postponed till the use of the biosensor. It is an advantage of some embodiments according to the present invention that the label-to-surface binding is formed freshly during the assay with the fluid to be tested, and not already during the fabrication process. It is an advantage of embodiments according to the present invention that accurate and sensitive bio-sensors can be manufactured. Reducing interaction thereby may comprise reducing the strength of the interaction and/or may comprise reducing the duration of the interaction.

Maintaining the reduced biological interaction until the at least one reagent layer is mechanically stable may be at least maintaining the reduced biological interaction between the at least one reagent layer and the biosensor substrate until said at least one reagent layer is flow resistant, set, solidified or substantially dried. Preparing the biosensor substrate may comprise cooling the biosensor substrate to obtain a cooled biosensor substrate, and depositing may comprise depositing the at least one reagent layer on the cooled biosensor substrate. It is an advantage of such embodiments that cooling can be easily applied.

Cooling the biosensor substrate may comprise cooling the biosensor substrate below a freezing temperature of components of the at least one reagent layer deposited. It is an advantage of such embodiments that the method is applicable for a variety of different reagent layers, e.g. without the need for additional chemistry.

Preparing the biosensor substrate may comprise depositing a separation layer on the biosensor substrate prior to depositing the at least one reagent layer. The method further may comprise preventing dissolution of the separation layer during depositing of the at least one reagent layer. At least maintaining the reduced biological interaction may comprise active drying the at least one reagent layer. It is an advantage of such embodiments that these allow obtaining biosensors that may be stored without influence on their efficiency. In other words, in such embodiments, good conditions may be generated for, after drying, further processing and/or storing the biosensors at higher temperatures than during the cooling. The drying may be performed during the cooling of the substrate or while the substrate is still cool.

Active drying the at least one reagent layer may comprise providing a low ambient pressure. It is an advantage of such embodiments that the efficiency of the drying process may be increased compared to not using a low ambient pressure.

Drying the at least one reagent layer may comprise inducing sublimation of salt present in the at least one reagent layer. It is an advantage of such embodiments that biosensors may be obtained with a reagent layer comprising a low salt concentration. It is an advantage of such embodiments that biosensors may be obtained with a nanoporous or microporous reagent layer. The latter may result in a biosensor with potential for fast assays and high reactivity during the operation of the biosensor. At least maintaining the reduced biological interaction may comprise cooling the biosensor substrate after depositing the at least one reagent layer.

Preparing the material for the at least one reagent layer may comprise cooling the material for the at least one reagent layer. At least maintaining the reduced biological interaction may comprise cooling the at least one reagent layer.

The biosensor substrate may comprise a plurality of different sensor surfaces, wherein depositing at least one reagent layer may comprise depositing different reagent layers on the different sensor surfaces on the biosensor substrate. It is an advantage of such embodiments that biosensors may be manufactured that allow for multiplexing.

Depositing at least one reagent layer on the prepared biosensor substrate may comprise depositing a plurality of reagent layers on the prepared biosensor substrate. It is an advantage of such embodiments that layers providing different functions may be applied to the biosensor substrate.

The method may comprise, prior to depositing at least one reagent layer, providing a biosensor substrate having biologically or biochemically active moieties. It is an advantage of some embodiments according to the present invention that the method of manufacturing biosensors is compatible with other steps of processing the biosensor such as for example providing biologically or biochemically active moieties on the biosensor substrate prior to providing at least one reagent layer.

It is an advantage of the some embodiments according to the present invention that the method of manufacturing biosensors is compatible with a large number of reagent layer deposition techniques. The method may be a method for manufacturing a biosensor device adapted for label-based detection or assisting therein.

The present invention also relates to a method of manufacturing a detection system, the detection system comprising a biosensor device and a detector adapted for detecting a sensing signal from a sample in contact with the biosensor device, the biosensor device having a substrate, the method comprising preparing the substrate and/or a material for at least one reagent layer for reducing biological interaction between the at least one reagent layer and the biosensor substrate, depositing the at least one reagent layer on the biosensor substrate to achieve a reduced biological interaction between the at least one reagent layer and the biosensor substrate, and after the depositing, at least maintaining the reduced biological interaction between the at least one reagent layer and the biosensor substrate till the at least one reagent layer is mechanically stable such as flow resistant, set, solidified or substantially dried.

A biosensor comprising: a biosensor substrate, a detection surface on the biosensor substrate comprising biologically or biochemically active moieties and at least one reagent layer deposited on the biosensor substrate, there being no interaction between the active moieties and the at least one reagent layer. The at least one reagent layer may be deposited in direct contact with the biologically or biochemically active moieties or it may be in indirect contact, other material, e.g. solid material, being in between. Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate and not merely as explicitly set out in the claims.

The teachings of the present invention permit the design of improved methods and apparatus for biosensing, the biosensors and biosensing method being accurate and efficient and sensitive.

The above and other characteristics, features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention. This description is given for the sake of example only, without limiting the scope of the invention. The reference figures quoted below refer to the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic representation of a first exemplary biosensor that may be obtained using a method of manufacturing according to embodiments of the present invention.

Fig. 2 is a schematic representation of a second exemplary biosensor that may be obtained using a method of manufacturing according to embodiments of the present invention. Fig. 3 is a flow diagram of a method for manufacturing a biosensor according to a first embodiment of the present invention.

Fig. 4 is a schematic representation of a detection system comprising a biosensor that may be obtained using a method of manufacturing according to embodiments of the present invention. In the different figures, the same reference signs refer to the same or analogous elements.

DETAILED DESCRIPTION OF THE EMBODIMENTS The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Moreover, the terms top, bottom, over, under and the like in the description and the claims are used for descriptive purposes and not necessarily for describing relative positions. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other orientations than described or illustrated herein.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention and is not necessarily referring to the same embodiment each time, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments. Grouping of features of the invention together in a single embodiment, figure, or description thereof is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination. In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. The following terms or definitions are provided solely to aid in the understanding of the invention. In the present application, with the term "nanoporous material" there is meant porous material with pores having a pore diameter between 0.5 nm and 500 nm. With the term "microporous material" there is meant porous material with a pore diameter in the range between 0.5 micrometer and 500 micrometer. The term "probe" is used to indicate components, parts or particles that are capable of selectively binding to a potential target molecule. Probes may include biologically-active moieties such as whole antibodies, antibody fragments such as Fab' fragments, single chain Fv, single variable domains, VHH, heavy chain antibodies, peptides, epitopes, membrane receptors or any type of receptor or a portion thereof, substrate-trapping enzyme mutants, whole antigenic molecules (haptens) or antigenic fragments, oligopeptides, oligonucleotides, mimitopes, nucleic acids and/or mixtures thereof. Antibodies can be raised to non-proteinaceous compounds as well as to proteins or peptides. Probes are typically members of immunoreactive or affinity reactive members of binding-pairs. The nature of the probe will be determined by the nature of the target to be detected. Most commonly, the probe is developed based on a specific interaction with the target such as, but not limited to, antigen- antibody binding, complementary nucleotide sequences, carbohydrate-lectin, complementary peptide sequences, ligand-receptor, coenzyme-enzyme, enzyme inhibitors-enzyme, etc. Probes also include "capture probes" for immobilizing targets and/or labeled targets on the detection surface via recognition or binding events. Probes and capture probes may be labeled.

The term "sample", as used herein, relates to a composition which may comprise at least one analyte of interest. The sample is preferably a fluid e.g. an aqueous composition also referred to as "sample fluid".

The term "analyte", as used herein, refers to a substance whose presence, absence, or concentration is to be determined using biosensors obtained according to the present invention. Typical analytes may include, but are not limited to small organic molecules, metabolites such as glucose or ethanol, proteins, peptides, nucleic acid segments, molecules such as small molecule pharmaceuticals, antibiotics or drugs, molecules with a regulatory effect in enzymatic processes such as promoters, activators, inhibitors, or cofactors, viruses, bacteria, cells, cell components, cell membranes, spores, DNA, RNA, micro-organisms and fragments and products thereof, or any substance for which attachment sites, binding members or receptors (such as antibodies) can be developed. Presence, absence or concentration of the analyte may be determined directly by assessing the presence, absence or concentration of the analyte itself, or may alternatively be determined indirectly by assessing the presence, absence or concentration of a target or target molecule.

The term "target" or "target molecule", as used herein, refers to a substance whose presence, absence, or concentration is actually be determined using biosensors obtained according to the present invention. The term "target molecule" should be construed broadly and can be, for example, an individual molecule, can be a cluster of molecules, can be a complex of molecules, can be a molecule embedded in other material such as a substrate, etc. The target and analyte may be identical, or the target may be indicative of the presence or absence of the analyte. In particular, targets such as proteins or DNA may be a distinctive component or product of analytes such as viruses, bacteria, or other organisms, and therefore indicative of their presence. Where detection involves an enzymatic assay, the target may be a product of an enzymatic conversion of a substrate by an enzyme, and may therefore be indicative of the amount of substrate or the activity of the enzyme. Target molecules may also be polymers, metal ions, and low molecular weight organic species such as toxins, illicit drugs, and explosives, the invention clearly not limited thereto. During the detection assay, the target may become labelled to emanate a detectable signal. The target may also become immobilized on the detection surface as part of a biologically-active coating.

The term "label", as used herein, refers to a molecule or material capable of generating or assisting in generating a detectable signal. Suitable labels for use in the different detection systems and methods of the present invention are numerous and extensively described in the art. These may be optical labels, radioactive labels, magnetic labels, etc. Labels can be direct labels, which can directly be detected by a sensor. Alternatively, labels can be indirect labels, which become detectable after a subsequent development process. Typically, the label used in the methods of the present invention is a target-specific label, i.e. capable of binding specifically to the target. Nevertheless it is also envisaged that where the target is present in a purified form, it is sufficient that the label binds to the target.

The method of manufacturing a biosensor device as described in the present invention will be especially suited for manufacturing a biosensor device with a reagent layer on top of the detection surface. By way of illustration, the present invention not being limited thereto, an exemplary biosensor obtainable using methods of the present invention is shown in Fig. 1 and Fig. 2. The biosensor device 1 preferably comprises a biosensor substrate 2 having a surface 3. The biosensor substrate 2 preferably comprises on the surface biologically or biochemically active moieties 4, such as e.g. antibodies or target analogues. In biosensors, as obtained using the method of manufacturing according to the present invention, typically the biologically or biochemically active moieties 4 are covered with a reagent layer 5. Such a reagent layer 5 may comprise a dissolvable matrix 7 and reagent particles 6, such as e.g. detection labels. Typically the detection labels may be provided with biologically or biochemically active moiety 61. Typically, when the biosensor device 1 is exposed to a fluid sample, the dissolvable matrix 7 and the reagent particles 6 will dissolve in the fluid sample and interact. Additional optional layers, such as e.g. separation layer 8 and/or calibration layer 9 may be provided. Such layers 8, 9 may e.g. comprise a calibration reagent, wherein a well-controlled amount of target molecules is present. For example layer 8 also may function as separation layer assisting in avoiding that reagent particles 6 bind to moieties 4 during the biosensor preparation process. The latter is illustrated in the exemplary biosensor shown in Fig. 2.

In a first aspect, the present invention relates to a method of manufacturing a biosensor device 1, whereby a reagent layer 5 is deposited on top of biologically or biochemically active moieties 4 on the biosensor substrate surface 3 under predetermined conditions in order to reduce or avoid interaction between the reagent layer and the biologically or biochemically active moieties during preparation and manufacturing of the biosensor device 1. The method of manufacturing a biosensor device according to the present aspect comprises preparing a biosensor substrate and/or a material for the at least one reagent layer for reducing biological interaction between at least one reagent layer and the biosensor substrate and depositing the at least one reagent layer on the biosensor substrate to achieve a reduced biological interaction between the at least one reagent layer and the biosensor substrate. Reducing interaction thereby may comprise both reducing the interaction strength and/or the duration of the interaction between the at least one reagent layer and the biosensor substrate. The method furthermore comprises, after the depositing of the reagent layer, at least maintaining the reduced biological interaction between the at least one reagent layer and the biosensor substrate till the at least one reagent layer is substantially mechanically stable. Mechanically stable may for example mean that the reagent layer is flow resistant, set, solidified or substantially dried. During deposition, the at least one reagent layer may be in fluid phase, or e.g. when deposition occurs whereby cooling below the freezing point of the reagent is performed, in a solid phase. With substantially mechanically stable, e.g. resistant, set, solidified or substantially dried, there is meant that the components of the at least one reagent layer are not mobile enough to migrate towards components of the biosensor substrate. In other words, during manufacturing there is reduced, preferably minimal, biological interaction between the reagent layer and the underlying surface. Preparing the biosensor substrate and/or a material for the at least one reagent layer may include taking measures for lowering the reaction rate both prior and during the deposition step. Reduction of biological interaction may for example be obtained by cooling at least one of the biosensor substrate or the material for the reagent layer or by providing a separation layer, also referred to as buffer layer, for being positioned between the biosensor substrate and the reagent layer. At least maintaining the reduced interaction may include taking measures to prepare the biosensor for storage. At least maintaining the reduced interaction preferably may comprise at least one of cooling the biosensor substrate and/or the at least one reagent layer or active drying of the reagent layer. The present invention will be further illustrated using particular embodiments, the present invention not being limited thereto.

In a first particular embodiment according to the present invention, a method as described above is disclosed, wherein preparing the biosensor substrate and/or a material for the at least one reagent layer 5 and the biosensor substrate 2comprises cooling of the biosensor substrate 2. The biosensor substrate 2 thereby already comprises the biologically or biochemically active moieties 4. Depositing the at least one reagent layer 5 and depositing at least one reagent layer 5 on top of the prepared, e.g. cooled, biosensor substrate 2. During the depositing of the at least one reagent layer 5, cooling of the biosensor substrate may be performed, or cooling may be performed prior to the provision of the reagent layer 5, such that during provision of the reagent layer 5, the biosensor substrate 2 may still be in a cooled condition. At least maintaining the reduced biological interaction between the at least one reagent layer 5 and the biosensor substrate 2 until the at least one reagent layer 5 is substantially mechanically stable, e.g. flow resistant, set, solidified or dried, may comprise for example cooling, e.g. further cooling, the substrate 2 while the at least one reagent layer 5 is passively dried, i.e. without applying additional drying means, or actively drying the at least one reagent layer 5 such that interactions are reduced. In a dried layer, typically the reaction may be terminated. In the present embodiment of the invention, the biosensor substrate 2 preferably is cooled such that the temperature of the biosensor substrate 2 during provision of the reagent layer 5 is such that the deposited reagents have a limited binding affinity to the underlying species. Preferably the temperature during deposition is substantially lower than the sensor temperature during assay operation and use of the biosensor system and/or the drying time is much shorter than the incubation time of the sensor surface during use of the biosensor system. Preferably the temperature of the surface during deposition is at least 5°C lower, more preferably 10⁰C lower than the temperature during assay operation and use of the biosensor system. Note that the temperature of the sensor surface during biosensor use depends on the application. In some applications temperature control is not very important and the biosensor surface is close to ambient temperature during use. In more advanced applications, the system is designed to allow temperature control of the biosensor for improved assay control. For example, assays can be performed at a temperature for optimal speed and specificity. An exemplary description of a method of manufacturing according to the first aspect will further be given with reference to Fig. 3, the present invention not being limited thereto. Fig. 3 shows standard and optional steps of an example method 200 of manufacturing according to the first embodiment of the first aspect of the present invention. In a first step, the method 200 according to the present invention may comprise obtaining 202 a biosensor substrate 2. Obtaining 202 a biosensor substrate 2 may comprise obtaining a pre-made biosensor substrate 2 whereon biologically or biochemically active moieties 4 are already provided, or it may comprise obtaining a biosensor substrate 2 and coating the surface 3 thereof with biologically or biochemically active moieties 4. The biosensor substrate 2 may be made of any suitable substrate material, such as e.g. but not limited to a flexible organic material, e.g. a polymeric material such as polyester, especially high temperature polyester materials, polyethylene naphthalate (PEN), and polyimide, or mixtures of two or more of these. Another particularly preferred chip carrier material is an inorganic material, for example a semiconductor material such as e.g. silicium, or a glass- type material such as e.g. glass or quartz. The biosensor substrate 2 also may be constituted by the solid surface of a detection surface of the detector used. The detector used may for example be an optical detector or a magnetical detector, the invention not being limited thereto. Biologically or biochemically active moieties 4 may for example refer to capture probes and/or target molecules that are attached to the biosensor substrate 2 and that are capable of binding, or that are reactive with, a target or a labelled probe, respectively, when in appropriate conditions. The capture probes and/or target molecules of the biologically- active layer may be immobilized on the surface 3 by any method known in the art. These biologically-active moieties 4 may be attached to the detection surface in a site-specific manner meaning that specific sites on these moieties are involved in the coupling, e.g. through a protein-resistant layer on the substrate 2. The biosensor substrate 2 may have a porous surface in order to enhance the surface-over- volume ratio.

In a second step, the method 200 according to the present aspect comprises cooling 204 the biosensor substrate 2. In other words, the biosensor substrate 2 with the biologically or biochemically active moieties 4 are cooled such that a reagent layer 5 to be deposited will have a very limited binding affinity to the cooled biologically or biochemically active moieties 4. A low biosensor substrate temperature may decrease the reaction, diffusion and/or dissolution rates of applied reagents and biolocically or biochemically active moieties 4. Particularly biological binding rates may depend very strongly on temperature. Preferably the temperature during deposition is substantially lower than the sensor temperature during assay operation and use of the biosensor system and/or the drying time is much shorter than than the incubation time of the sensor surface during use of the biosensor system. Preferably the temperature of the surface during deposition is at least 5°C lower, preferably at least 10⁰C lower than the surface temperature during assay operation and use of the biosensor system. Such cooling may be performed in any suitable way, such as using a heat sink, using fan cooling, using forced fluid flow, using electrical cooling such as e.g. using a Peltier element, a refrigerator element, etc. In a particular embodiment, the cooling may be such that a temperature below the freezing point of the reagent material is obtained, such that the material deposited on the substrate is solid. A third step of the method comprises depositing 206 at least one reagent layer

5 on the cooled biosensor substrate 2. The latter may be performed in any suitable way, such as e.g. using micro-deposition techniques. Such techniques may comprise inkjet printing, tampon printing, microcontact printing, screen printing, stamp printing, etc. It is to be noticed that components of the printing tool used, e.g. the inkjet head, stamp, tampon, etc. may be at a temperature higher than the temperature of the cooled biosensor substrate 2. The latter may be advantageous for obtaining a high reliability of operation of the deposition tool and/or a high quality of the deposited reagent layer 5. As indicated above, deposition on the cooled biosensor substrate may be performed simultaneously with the cooling or after cooling has been performed but whereby the biosensor substrate 2 still is cooled substantially, such that reagents to be deposited will have a very limited binding affinity to the cooled species on the biosensor substrate 2. In other words, deposition may be performed simultaneously with an active cooling step as described in step 204 or after such a cooling step while the effect of the cooling still is present. The at least one reagent layer 5 preferably is a dissolvable reagent layer, i.e. a reagent layer 5 adapted for dissolving once in contact with the sample fluid. The reagent layer 5 may be assisting in label-based analyte detection. The at least one reagent layer 5 of the sensor chip 1 may comprise reagents of chemical or biochemical nature for reacting with the target to produce a detectable signal that represents the presence of the analyte in the sample. The term "reagent", as used herein comprises a chemical, biological or biochemical component for reacting with the analyte and/or the target thus assisting in producing a detectable signal that represents the presence of the analyte in the sample. Suitable reagents for use in different detection systems and methods include a variety of active components selected to determine the presence and/or concentration of various analytes. There are numerous chemistries available for use with each of various targets. They are selected with respect to the target to be assessed. The reagent may contain for example an enzyme, a co-enzyme, an enzyme inhibitor, an enzyme substrate, a co-factor such as ATP, NADH, etc. to facilitate enzymatic conversions, a vitamin, a mineral, the invention clearly not limited thereto. For example, in one preferred embodiment, the at least one reagent layer can include one or more enzymes, co-enzymes, and co-factors, which can be selected to determine the presence of metabolites or small molecules in a sample. Furthermore, the at least one reagent layer may further contain labels, buffer salts, detergents, sugars, etc.

Depositing 206 at least one reagent layer 5 on the cooled biosensor substrate 2 may be depositing a reagent layer 5 that is sufficiently thin and/or porous such that, in operation of the biosensor, the sample fluid will hydrate or dissolve the thin reagent layer 5 rapidly. Preferably, a reagent layer 5 may be deposited having a thickness in a range having a lower limit of 0.1 μm, preferably lμm, and an upper limit of 150μm, preferably 50μm, still more preferably 15μm. The thickness referred to in the present application may be the average thickness of the layer. The reagent layer 5 may be a substantially uniform layer, or it may comprise an island or stripe structure. The thickness then refers to the average thickness of the islands or stripes. It is an advantage that a thin layer may be deposited leading to biosensors with a larger capture efficiency than for a thick layer. A thick reagent layer will take more time for the sample fluid to hydrate or dissolve, and more time will be needed for the reagent to approach the detection surface. This can delay the time to determine the analyte concentration and introduce errors into the determination. In a further step, the method 200 comprises at least maintaining the reduced biological interaction between the at least one reagent layer and the biosensor substrate till the at least one reagent layer is substantially mechanically stable. Substantially mechanically stable thereby may be that the at least one reagent layer is flow resistant, set, solidified or substantially dried. At least maintaining the reduced biological interaction may comprise active drying 208 of the reagent layer 5 on the biosensor substrate 2. Drying of the reagent layer 5 on the biosensor substrate 2 preferably may be performed by application of a low ambient vapor pressure, although the latter is not obligatory. Drying may comprise both drying a reagent layer from its fluid phase as well as drying a reagent layer from its solid phase. It may comprise reducing the amount of aqueous components present in the reagent layer. During drying, cooling the biosensor substrate 2 or operating on a cooled biosensor substrate 2 may be preferred, in order to further prevent interaction of non-dried parts of the reagent layer 5. As soon the reagent layer is sufficiently dry that no further reaction occurs between the reagent material and underlying materials, further drying, e.g. to obtain improved storage properties, may be performed using heating of the substrate. At least maintaining the reduced biological interaction alternatively or in addition to active drying may comprise cooling the biosensor till the at least one reagent layer is substantially mechanically stable, e.g. dried, such that further interaction of non-dried parts of the reagent layer is prevented. Such cooling may be performed in a similar way as described above. In another optional step, the method 200 comprises further processing 210 of the biosensor device 2. The latter may comprise providing additional layers, providing additional treatments to the biosensor device 1, sealing of the biosensor device 1, packaging of the biosensor device 1, etc. For example, a layer comprising calibration materials may be additionally provided, a cover layer that acts as a protection and lift-off layer against contaminations e.g. organic contaminants excreted by surrounding cartridge materials during processing or storage, may be provided, etc. After these further processing steps, the biosensor device also may be stored, indicated by storing step 212. The further processing as well as the storing may be performed at substantially higher temperatures than when the biosensor device is cooled, without automatically lowering the sensitivity or accuracy of the device due to preliminary interaction of reagent material with the biologically or biochemically active moieties. Similarly to the processing and storing, also the use of the biosensor device at higher temperatures than when the biosensor device is cooled for depositing the reagent layer may be performed without automatically lowering the sensitivity or accuracy of the device. An alternative embodiment relates to a method as described in the first embodiment, comprising the same features and advantages as described above, but wherein the initial cooling prior and/or during the deposition, i.e. for the preparing of the biosensor substrate, may comprise cooling of the material of the reagent layer. Alternatively or in addition thereto, further cooling after deposition, i.e. for at least maintaining the reduced biological interaction, may comprise cooling of the reagent layer. Cooling of the reagent layer or its material may be performed alternatively to or in combination with cooling of the biosensor substrate. As, due to the large thermal mass of the substrate, a cooled reagent material, e.g. in fluid phase, will very rapidly assume the temperature of the substrate once deposited, at least maintaining the reduced biological interaction needs to be very efficient in this alternative embodiment. Such at least maintaining the reduced biological interaction may then e.g. be obtained by rapid drying, in order to reduce or minimise the interactions.

In an exemplary embodiment, a method as described in any of the other embodiments is provided, comprising the same features and advantages as described above, but wherein the ambient provided during depositing and/or drying of the at least one reagent layer has a very low humidity. The latter has the advantage that the drying proceeds rapidly. It also has the advantage that water condensation and ice deposition from the environment onto the sensor substrate are reduced during the processing, e.g. during the deposition of the reagent layer. An inert gas can be used as a carrier. With very low humidity there is meant a relative humidity less than 30%, more preferably a relative humidity less than 10% even more preferably a relative humidity less than 3%

In one particular embodiment, a method as described in any of the other embodiments is provided, comprising the same features and advantages as described above, but wherein a biosensor with a porous reagent layer is manufactured. The latter is obtained by depositing a reagent layer comprising material that sublimes during drying and by drying the reagent layer, e.g. sublimation of water and/or of a salt such as ammoniumcarbonate. The reagent layer thus obtained furthermore may be nano- and/or microporous. The latter is advantageous as it assists in improved dissolving of the reagent layer components. In a further particular embodiment, the present invention relates to a manufacturing method according to any of the other embodiments according to the present invention, whereby in between the biologically or biochemically active moieties on the biosensor substrate an additional separation layer is applied. An example of a resulting biosensor chip is shown in Fig. 2. Such a separation layer preferably may be a layer that, in use of the biosensor chip, is dissolvable. During manufacturing, the separation layer may act as a penetration barrier, assisting in avoiding that reagent particles bind to biologically or biochemically moieties. Penetration can be suppressed by using a condition wherein the deposited reagent dries in a time that is lower than the time needed to redissolve the separation layer. At least maintaining the reduced biological interaction therefore may comprise active drying, preferably fast drying, of the reagent layer. Provision of a separation layer also can be performed as an alternative to the cooling prior and/or during deposition of the reagent layer on the biosensor substrate, i.e. as an alternative form of preparing the biosensor substrate and/or material. Providing a separation layer thereby may assist in lowering the reaction rate between the deposited reagent material and the biosensor substrate. Typically a separation layer can contain organic material, e.g. a carbohydrate material such as a sugar. Such separation layer thus may be a buffer layer that does not contain biologically- active species to further suppress that labels bind to biologically-active species during the fabrication process.

An alternative embodiment relates to a method as described in any of the other embodiments, comprising the same features and advantages as described above, but wherein preparing the biosensor substrate and/or a material for the at least one reagent layer comprises adding an interaction limiting material to the to-be deposited reagent material which biologically or chemically suppresses the biological active of the reagent, in addition to or as alternative for depositing during cooling or for depositing using a separation layer between the biosensor substrate, and biological or biochemical moieties thereon, and components of the reagent material. Optionally, the additive can be a sublimable material that disappears during the drying process, e.g. a sublimable salt such as ammoniumcarbonate. The interaction limiting material may limit the migration of components of the reagent layer.

In still another embodiment, the present invention relates to a manufacturing method according to any of the other embodiments of the present invention, whereby more than one reagent layer 5 is deposited on the biosensor substrate 2. The latter may be advantageous to improve assay performance or to perform different types of assays simultaneously. The more than one layers can be deposited on top of each other and/or besides each other. By depositing the different reagent layers 5 at a cooled biosensor substrate 2, possible reactions or mixing, e.g. by diffusion or by spread out of fluids, of the different reagent layers 5 is reduced, resulting in more accurate and more efficient biosensors for multiplexing.

The biosensor device as described above, typically also referred to as biosensor chip, may be a fixed part of a biosensing detection system or it may be disposable, such as a disposable cartridge that is fitted into a biosensing detection system.

It is an advantage of embodiments of the present invention that accurate and sensitive biosensor devices are obtained that allow ultrahigh speed of the test.

It is an advantage of embodiments of the present invention that biosensors allowing fast dissolution and mixing of the reagent with the sample fluid are obtained. It is also an advantage of embodiments of the present invention that biosensors allowing fast transport of reagent to the detection region are obtained. It is furthermore an advantage of embodiments of the present invention that biosensors obtained using a method of manufacturing according to the present invention allow a high concentration of mobile reagent at the sensor surface during use.

It is an advantage of embodiments of the present invention that the label-to- surface bindings are formed freshly during the assay with the test fluid, and not already duing the manufacturing process. It thus is an advantage of embodiments of the present invention that the species are not allowed to bind to and/or mix with the underlying biologically or biochemically active moieties or that the amount of binding or mixing is substantially restricted.

It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. For example, whereas the present invention is described by way of a method for manufacturing a biosensor device, the present invention also relates to a method of manufacturing a detection system with a biosensor chip, made using a method according to the present invention. An example of a detection system obtainable using such a method is shown in Fig. 4. A detection system 300 is shown comprising a detector 302, a biosensing device 1 and a sample chamber 304 for holding the sample. When sample, e.g. sample fluid, is contacted with the biosensing device 1, the at least one reagent layer 5 is dissolved resulting in a detectable sensing signal if analyte is present in the sample fluid. The detectable signal is detected by the detector 302 and the obtained measurement results are obtained through a detector read-out 306. Other optional components such as fluid inlet, fluid outlet, label activation means, etc. may be present in the detection system 300, as known in the art.

Claims

CLAIMS:

1. A method (200) of manufacturing a biosensor device (1), the biosensor device

(1) comprising a biosensor substrate (2) and at least one reagent layer (5), the method comprising : preparing the biosensor substrate (2) and/or a material for the at least one reagent layer (5) for reducing biological interaction between at least one reagent layer (5) and the biosensor substrate (2), depositing the at least one reagent layer (5) on the biosensor substrate (2) to achieve a reduced biological interaction between the at least one reagent layer (5) and the biosensor substrate (2), and after said depositing: at least maintaining the reduced biological interaction between the at least one reagent layer (5) and the biosensor substrate (2) until said at least one reagent layer (5) is substantially mechanically stable.

2. A method (200) according to claim 1, wherein at least maintaining the reduced biological interaction is at least maintaining the reduced biological interaction between the at least one reagent layer (5) and the biosensor substrate (2) until said at least one reagent layer (5) is substantially dried.

3. A method (200) according to any of claims 1 to 2, wherein preparing the biosensor substrate (2) comprises cooling (204) the biosensor substrate (2) to obtain a cooled biosensor substrate (2), and wherein depositing comprises depositing (206) the at least one reagent layer (5) on the cooled biosensor substrate (2).

4. A method (200) according to claim 3, wherein cooling (204) the biosensor substrate (2) comprises cooling the biosensor substrate (2) below a freezing temperature of components of the at least one reagent layer (5) deposited.

5. A method (200) according to any of the previous claims, wherein preparing the biosensor substrate (2) comprises depositing a separation layer on the biosensor substrate (2) prior to depositing the at least one reagent layer (5) further comprising preventing dissolution of the separation layer during depositing of the at least one reagent layer (5).

6. A method (200) according to any of the previous claims, wherein the at least maintaining the reduced biological interaction comprises active drying (208) the at least one reagent layer (5).

7. A method (200) according to claim 6, wherein active drying (208) the at least one reagent layer (5) comprises providing a low ambient pressure.

8. A method (200) according to any of claims 6 to 7, wherein drying (208) the at least one reagent layer (5) comprises inducing sublimation of salt present in the at least one reagent layer (5).

9. A method (200) according to any previous claim, wherein at least maintaining the reduced biological interaction comprises cooling the biosensor substrate (2) after depositing said at least one reagent layer (5).

10. A method (200) according to any previous claim, wherein preparing the material for the at least one reagent layer (5) comprises cooling the material for the at least one reagent layer (5).

11. A method (200) according to any previous claim, wherein at least maintaining the reduced biological interaction comprises cooling the at least one reagent layer (5).

12. A method (200) according to any of the previous claims, the biosensor substrate (2) comprising a plurality of different sensor surfaces (3), wherein depositing (206) at least one reagent layer (5) comprises depositing (206) different reagent layers (5) on the different sensor surfaces (5) on the biosensor substrate (2).

13. A method (200) according to any of the previous claims, wherein depositing

(206) at least one reagent layer (5) on the prepared biosensor substrate (2) comprises depositing (206) a plurality of reagent layers (5) on the prepared biosensor substrate (2).

14. A method (200) according to any of the previous claims, the method comprising, prior to depositing (206) at least one reagent layer (5), providing a biosensor substrate (2) having biologically or biochemically active moieties (4).

15. A method (200) according to any of the previous claims, wherein the biosensor device is adapted for label-based detection.

16. A method of manufacturing a detection system (300), the detection system (300) comprising a biosensor device (1) and a detector (302) adapted for detecting a sensing signal from a sample in contact with the biosensor device (1), the biosensor device having a substrate, the method comprising preparing the substrate and/or a material for at least one reagent layer (5) for reducing biological interaction between the at least one reagent layer (5) and the biosensor substrate (2), depositing the at least one reagent layer (5) on the biosensor substrate (2) to achieve a reduced biological interaction between the at least one reagent layer (5) and the biosensor substrate (2), and after said depositing, at least maintaining the reduced biological interaction between the at least one reagent layer (5) and the biosensor substrate (2) until said at least one reagent layer (5) is substantially mechanically stable.

17. A biosensor comprising: a biosensor substrate (2), a detection surface on the biosensor substrate comprising biologically or biochemically active moieties and at least one reagent layer (5) deposited on the biosensor substrate, there being no interaction between the active moieties and the at least one reagent layer (5).