Patent Application
20120046201
The present application is a National Phase entry of PCT Application No. PCT/FI2010/050354, filed Apr. 30, 2010, which claims priority from Finnish Application Number 20095501, filed May 4, 2009, the disclosures of which are hereby incorporated by reference herein in their entirety.
This invention relates to a method for characterizing and/or determining a sample. This invention particularly relates to an array of non-specific interacting means for characterizing and/or determining a sample and the use of the array.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Binder arrays have been used in detection of biomolecular liquids. The basis of these binder arrays has been the production of a library of components on surfaces or production of e.g. ion sensitive/selective surfaces. When the array is allowed to be in contact with the sample, components from the sample may interact with the array creating a fingerprint that describes the contents of the sample. The array designs vary from specific arrays that detect no or only a few more species that the dimension of the array to non-specific arrays that detect higher number of species. Arrays based on chemical libraries and affinity molecules have been reviewed e.g. in Journal of Biomedicine and Biotechnology; 2003:5 (2003) 257-266. In the field of the Invention both electronic interaction based and luminescence/light interaction based detection schemes have been proposed. Whereas non-specific interactions are widely used in sensing odors and volatile compounds (artificial olfaction) only a few examples can be found in detection of compounds from liquid phase.
Specific arrays have widely been applied in proteomics and genomics. In WO/2001/055702 a portable sensor array is disclosed. The array uses microparticles that are in their preferred embodiment depicted in cavities. The publication also suggests various ways of detection scheme including fluorescence quenching and resonance energy transfer. The main idea is to use specific “receptors” as binders on these particles and the publication lists several examples of specific receptors. The publication also reviews the area of sensor array detection. An array may also be viewed as a multiplex of particles. In a flow fluorometry based detection scheme (Luminex Corporation, www.luminexcorp.com), small (2 to 3 micrometer) microparticles are coded for different categories by internal fluorescent colours. The particles are coated bioactively and the surface attracts both the sample and a fluorescent marker. When the intensity of the fluorescent marker and the internal color coding are read in a flow fluorometric device, quantities of binding for each category in this multiplexed assay become apparent. Similar approaches have been suggested also by others. (U.S. Pat. No. 5,891,738; WO/2003/042698).
In publication by Yuri Vlasov (IUPAC Technical report, Pure Appl. Chem., Vol. 77, No. 11, pp. 1965-1983, 2005) in their wording “non-specific” potentiometric (electronic) sensor arrays and the analysis methods for the results are reviewed. The function of these sensors bases on sensing of ionic currents and typically they operate on micromolar to millimolar concentration range of the ions. The article begins with an explanation of Nikolsky-Eisenman equation that describes the selectivity of a sensor—low selectivity is seen as a problem and is either avoided by better design of the sensing system, or when not possible mathematically using signal processing and statistical analysis. It is clear that these sensors may only detect ionic small compounds such as metal ions. The publication also lists several typical ions that such detectors are tuned for—these ions include ions of iron (Fe), copper (Cu), chlorine (Cl) and sodium (Na). The article also presents (refers to) an analysis of more complex liquids (e.g. wine) in combination with sophisticated signal analysis (Principal component analysis). The described method allowed separation of two different red wines from different areas from Italy and made of a different grape as well as separation of red wines from a white wine.
Non-specific interactions have also been observed between specific capture molecules and molecules that are not their specific counterparts. In WO/2004/048937 an array sensor based on fluorescently labeled oligonucleotides to analyze vapor phase components is presented. The main idea of the technology is to observe changes in the fluorescence properties of the labeled oligonucleotides due to different volatile compounds.
In another approach, the Raise-technology (www.graffinity.com) a well characterized chemical “fragment” library is transferred to an array. These library elements are serving as fragments in lead compound screening of drug discovery studies. Although weak in specificity, the technology aims to find specific interactions between the sample and the array. Detection of these interactions follows by surface plasmon resonance (SPR).
Chemical interactions between specially synthesized chemical compounds (residues) and proteins have been demonstrated by You, Miranda et. al (Nature Nanotechnology, Vol 2., 2007, pp 318-323). The technique bases on synthesis of different chemical residues on metallic nanoparticles. These particles are then allowed to interact with proteins. Depending on the structure of the protein these particles are bound from their residue non-covalently to the proteins. Detection follows by a fluorophore whose fluorescence is quenched by the metal particle in cases when the particles are not bound to sample proteins—when bound, quenching effect is removed and luminescence of the fluorophore is restored. The article reported that by the use of six differently synthesized residues on nanoparticles allowed reliable separation of seven different proteins by the use of the technique in combination with linear discriminant analysis (LDA). Detection sensitivity of the technology is in sub-micromolar range with the best values approaching nanomolar (4 nM for β-galactosidase). The principle also requires separation of the different liquid binders and reactions are thus performed in separate wells.
A feature and advantage of embodiments of the present invention is to provide a method for characterizing and/or determining a sample.
Another feature and advantage of embodiments of the present invention is to provide an array for characterizing and/or determining a sample.
A further feature and advantage of embodiments of the present invention is to provide use of the array for characterizing and/or determining a sample.
Embodiments of the invention relate to a method according to claim 1, an array according to claim 10, use of the array according to claim 16, use of at least one non-specific labelling reactant and an array of plurality of different interacting surfaces according to claim 17, an arrangement according to claim 18, a portable device according to claim 19, and a computer program product for characterizing and/or determining according to claim 20 for characterizing and/or determining a sample.
According to an embodiment of the invention an array of at least two of different interacting surfaces is employed for characterizing and/or determining a sample, wherein at least one of the surfaces comprises a non-specific interacting material. The non-specific interacting material advantageously non-specifically interacts with the sample, at least one labelling reactant, and/or combination of the sample and at least one labelling reactant. In the embodiment the sample and at least one labelling reactant is introduced to said interacting surfaces of said array, such as e.g. letting them in contact with said interacting surfaces of said array. The labelling reactant either as such or in combination with said sample is adapted to change at least one electromagnetically readable property of at least one interacting surface of said array. The electromagnetically readable property may be e.g. optically or radioactively readable property, such as luminance, absorption, emission, reflectivity, polarization, scattering, raman scattering, chemiluminance, radioactive emission, or local change (enhancement) of electromagnetic field. It is to be noted that also at least one further labelling reactant may be employed before obtaining a fingerprint of the sample as is discussed elsewhere in this document.
In order to obtain the fingerprint of the sample the electromagnetically readable property of at least one of said at least two different interacting surfaces of said array is detected at a predetermined time point. The characterizing and/or determining the sample is carried out by comparing the fingerprint of the sample with
According to an embodiment the sample and/or at least one labelling reactant is introduced to a dilution medium before introducing to the interacting surfaces of said array. Advantageously the dilution medium comprising said sample and/or at least one labelling reactant is introduced to the interacting surfaces of said array so that the sample and/or at least one labelling reactant are not brought directly in contact with the interacting surfaces but via said dilution medium.
According to an embodiment the sample and/or at least one labelling reactant is introduced into a liquid medium before introducing to the interacting surfaces of the array so that the sample and/or at least one labelling reactant are not brought directly in contact with the interacting surfaces but via said liquid medium. It should be noted that even it is disclosed that it is the liquid medium which is introduced to the interacting surfaces, the sample and/or at least one labelling reactant may exist as a gas or solid particles in the liquid.
According to an embodiment the sample may be introduced to the interacting surfaces of said array before any labelling reactant. According to another embodiment at least one labelling reactant may be introduced to the interacting surfaces of said array before the sample, whereafter the sample may interact with the labelling reactant and/or the interacting surface. In addition according to an embodiment the sample and at least one labelling reactant may be introduced to the interacting surfaces of said array together.
According to an embodiment the interacting surfaces are washed with at least one washing medium before detecting said electromagnetically readable property of said interacting surfaces. The interacting surfaces may be washed several times, such as e.g. after introducing first labelling reactant, after introducing the sample and/or after introducing any possible subsequent labelling reactant.
The labelling reactant used in the embodiment of the invention may comprise at least one of the following: luminophore label, radioactive label, and/or a label changing at least luminance, absorption, emission, reflectivity, scattering, raman scattering, chemiluminescence, radioactive emission, local electromagnetic field and/or polarization of at least one interacting surface of the array when introducing said labelling reactant to interact with said interacting surface.
According to an embodiment the luminophore may be selected from the group consisting of coumarins; rhodamines; cyanines; boron-dipyrromethenes; lanthanide compounds, preferably chelates and cryptates; porphyrins; metalloporphyrins; fluorescent proteins; fluorescent polymers; particulate labels, preferably quantum dots, luminescent crystals and luminescent polymer particles; and any combination thereof.
According to an embodiment an essential portion, i.e. at least 10%, preferably at least 30%, even more preferably at least 50% of the interacting surfaces of the array used in the embodiments are selected from groups of interacting surfaces selected from peptides, proteins, detergents, surfactants, carbohydrates and/or derivatives, and polymers. The polymer may be physically treated or chemically treated with an acid, base and/or solvent. For example the pattern of the interacting surface may be treated so its interacting surface area is increased, whereupon the interacting surface may interacting, such as bind, the sample and/or labelling reactants more powerfully and thereby increasing sensitivity.
According to an embodiment the array may comprise at least three interacting surfaces, where the two of which comprise similar type interacting surfaces. When the array comprises similar type interacting surfaces the sensitivity of the array may be enhanced remarkably, especially when the concentration of the sample and/or labelling reactant is minimal, because the probability that at least minor part of the sample and/or labelling reactant would interact with at least one interacting surface is increased.
According to an embodiment the array may comprise plurality of same type interacting surfaces, the interacting force, such as binding force of which is adapted to change gradually or continuously between the minimum and maximum interacting force values. This can be achieved e.g. by changing the thickness, density or concentration of the interacting surfaces. According to an embodiment the array may comprise plurality of interacting surfaces, the type of which is adapted to change gradually or continuously from one type to another type. The array having interacting surfaces with changing interacting property may be used for detecting more fine-grained fingerprints, since much smaller differences with interacting properties of different samples and/or labelling reactants can be detected.
According to an embodiment at least 1, preferably at least 2, even more preferably at least 3 of the interacting surfaces are selected from each of at least 2, preferably at least 3, of the groups of interacting surfaces selected from the peptides, proteins, detergents, surfactants, carbohydrates and/or derivatives, and polymers.
According to an embodiment the array may also comprise an additional surface, such as a gel-like surface, which essentially encapsulates the interacting surfaces. The additional surface is advantageously adapted to convey at least part of said sample, at least one labelling reactant, and/or combination of the sample and at least one labelling reactant through said additional surface in order to interact with said interacting surfaces of the array encapsulated by said additional surface. According to an embodiment the sample can be caught even from the air by the array having additional surface.
In this disclosure, the term array refers to a particular plurality of different interacting, such as binding surfaces. Accordingly not all interacting surface of the array, i.e. elements of the array, are identical. However, the array can, and in many preferred embodiments does, comprise, in addition to a plurality of different interacting surfaces, also a plurality of each different type of interacting surface.
In the context of the present application the term interacting surface or binding surface shall be understood to refer to any surface or surfaces that form a uniformly coated or modified electromagnetically (e.g. optically or radioactively) readable and identifiable area with the potential of interacting, such as binding analytes to be characterized and/or determined. Thus each interacting surfaces typically comprises an area of at least 200 nm (nanometer) in diameter and up to 100 mm2 (square millimetres). In many preferred embodiments the diameter is from 0.01 to 10 mm, in more preferred embodiments from 0.5 to 3 mm in diameter. If each interacting surface is that of a particle, the preferred diameter of the particle is from 200 nm to 20 μm (micrometer) and a more preferred diameter from 1 to 5 μm. It should be noted that the sample and/or labelling reactant advantageously interact somehow with the interacting sample, such as change the electromagnetically readable property of the interacting surface. According to an embodiment sample and/or labelling reactant may bind e.g. chemically to the interacting surface, whereupon the term binding surface may be used.
It is clear to the skilled in art that the surface area may in some cases be smaller or larger than described above.
The term analyte or analytes refers, in the context of this application, to any constituent of the sample to be characterized and/or determined, which constituent contributes to the fingerprint of the sample in any specific embodiment of the invention.
The term plurality shall be understood to mean more than one, preferably at least three, more preferably at least ten, even more preferably at least 30. It is clear for the person skilled in the art (PSA) that in some preferred embodiments the term plurality can even refer to more than 100, 300, or 1 000.
The term sample shall, in the context of this application, be understood to cover any sample to be characterized and/or determined. The sample may be in liquid medium or form, preferably as such, or transformable into liquid form by dissolving it in a dilution medium. The sample typically comprises at least 100 nl but not more than 10 ml. The sample size is preferably from 1 μl to 1 ml, more preferably from 3 μl to 300 μl and most preferably from 10 μl to 100 μl.
The term dilution medium refers, in the context of this application, to any liquid medium suitable for dilution of the sample in a particular embodiment of the invention. Typically the dilution medium is an aqueous buffer.
The term luminophore label refers to a label that comprises a luminophore, i.e. inorganic or organic luminescent matter.
The term luminophore refers to an atom, group or particulate, i.e. of a luminophore label, that manifests luminescence and detection of the label accordingly consists of measuring the luminescence of the luminophore. Luminophores comprise e.g. of fluorophores, phosphors, but also conjugated pi systems and transition metal complexes. Luminophores preferred in embodiments of the present invention can be selected from the group consisting of coumarins, rhodamines, cyanines, boron-dipyrromethenes, lanthanide compounds (such as chelates and cryptates), porphyrins, metalloporphyrins, fluorescent proteins, fluorescent polymers, particulate labels (such as quantum dots, luminescent crystals and luminescent polymer particles). Luminophore may also be understood as a complex that is formed of a luminescent molecule or particle as defined above and non-luminescent molecules or molecular complexes.
The term washing medium or liquid, be it a first or second washing liquid, refers according to an embodiment to any liquid applicable for washing the array in any desired step of the method of the present invention. A washing medium or liquids are used in many preferred embodiments of the present invention to enable and/or improve characterization and/or determination. Typically a washing liquid is an aqueous buffer.
The terms detect and detecting shall be understood, in the context of this invention, to refer to any applicable measurement of luminescence of interacting surfaces of the array. Measurement can be accomplished e.g. using a luminescence plate reader, a dedicated luminescence array reader, a flow luminometric device and/or an automated imaging device.
The term fingerprint refers, in the context of the present application, to the array of results obtained through detection, i.e. measurement, of the plurality of different interacting surfaces of the array measured in any embodiment of the invention. If an embodiment of the present invention involves also a plurality of identical interacting surfaces and more than one of these are detected, the fingerprint can comprise all the results of the identical interacting surfaces or alternatively only a representative value, e.g. average, median, mode of the measurements of identical interacting surfaces or any combination thereof [Bartz A. E. (1999), Basic Statistical Concepts. 4th ed., Upper Saddle River, N.J.: Prentice Hall.]. A fingerprint can further refer to a profile of measured luminescent intensities subjected to numerical processing with an appropriate algorithm and in many preferred alternatives measured luminescent intensities of the interacting surfaces of the array are subjected to numerical processing by an appropriate algorithm before comparison with fingerprints of corresponding arrays without a sample and/or known samples.
The term corresponding sample or corresponding samples refer to samples that are believed to be virtually identical, highly similar or at least reasonably similar to those characterized and/or determined. This belief of similarity can be due to e.g. origin, i.e. relating to the same or a corresponding process or product, or classification.
The term known samples refers to any samples which composition is known in detail or is fully characterized.
The terms non-specific interacting means or non-specific binder refers e.g. to a binder or binders that are, in the context of a specific embodiment of the present invention, not specific: the selectivity of the binding is not predetermined. Accordingly a binder, which in some other context is a specific binder, might, in the context of the present invention be a non-specific binder due to that binding in the context of the present invention is not specific. Preferably the non-specific binder or binders of the present invention are not specific binders in any context.
The terms specific interacting means or specific binder refers e.g. to a binder that is specific or binders that are specific. A specific binder comprises a molecular recognition element or elements that bind to an epitope of the analyte bound. In the context of this invention a specific binder only binds to similar epitopes, i.e. epitopes that are chemically and structurally similar. A specific binder does not bind to more than 10, preferably not more than 3, chemically and conformationally non-identical different epitopes. Most preferably a specific binder only binds to one specific epitope.
In the contest of this invention the term bind refers to binding wherein the binding constant is at least 103 M−1, preferably at least 105 M−1, more preferably at least 107 M−1 and most preferably at least 109 M−1.
The exemplary embodiments of the invention presented in this document are not to be interpreted to pose limitations to the applicability of the appended claims. The verb “to comprise” is used in this document as an open limitation that does not exclude the existence of also unrecited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated.
Next the invention will be described in greater detail with reference to exemplary embodiments in accordance with the accompanying drawings, in which:
An exemplary embodiment of the method of the invention for characterizing and/or determining a sample employing an array of a plurality of different binding surfaces comprises e.g. the following steps:
In some exemplary embodiments of the invention at least one labelling reactant e.g. with a luminophore label is employed and said labelling reactant binds in the conditions prevailing in the method non-specifically to an essential portion, i.e. at least 10%, preferably at least 30%, more preferably at least 50%, even more preferably at least 70% and most preferably at least 90%, of said plurality of different binding surfaces of the array in the absence of a sample. In some of these exemplary embodiments at least one further labelling reactant e.g. with a luminophore label may also be employed and said labelling reactant binds in the conditions prevailing in the method specifically to at least one binding surface of the plurality of different binding surfaces of the array.
In some preferred embodiments of the invention at least 3, preferably at least 5 and most preferably at least 10 different binding surfaces are comprised in the array.
Often the array according to the invention comprises a plurality of at least one type of binding surface of the different binding surfaces of the array. Typically the array comprises at least 3, preferably at least 5 and most preferably at least 10 of at least 1 preferably 3, even more preferably 10 and most preferably all different binding surfaces comprised in the array.
In exemplary embodiments of the invention an essential portion, i.e. at least 10%, preferably at least 30%, even more preferably at least 50% of the binding surfaces are selected from groups of binding surfaces selected from peptides, proteins, detergents, surfactants and polymers. Preferably at least one binding, surface is a polymer, optionally treated with an acid, base and/or solvent.
In exemplary embodiments of the invention at least 1, preferably at least 2, even more preferably at least 3 of the binding surfaces are selected from each of at least 2, preferably at least 3, of the groups of binding surfaces selected from the peptides, proteins, detergents, surfactants and polymers.
Preferably the luminophore or luminophores employed in the invention are selected from the group consisting of coumarins; rhodamines; cyanines; boron-dipyrromethenes; lanthanide compounds, preferably chelates and cryptates; porphyrins; metalloporphyrins; fluorescent proteins; fluorescent polymers; particulate labels, preferably quantum dots, luminescent crystals and luminescent polymer particles; and any combination thereof.
In an exemplary array of a plurality of different binding surfaces according to the invention an essential portion, i.e. at least 10%, preferably at least 30%, more preferably at least 50%, even more preferably at least 70% and most preferably at least 90%, of said plurality of different binding surfaces consist of non-specific binders.
In exemplary embodiments at least 3, preferably at least 5, more preferably at least 10 and most preferably at least 30 different binding surfaces are comprised in the array.
In exemplary arrays of the invention an essential portion, i.e. at least 10%, preferably at least 30%, even more preferably at least 50% of the binding surfaces are selected from groups of binding surfaces selected from peptides, proteins, detergents, surfactants and polymers. Preferably at least one binding surface is a polymer, optionally treated with an acid, base and/or solvent.
In exemplary embodiments of the array at least 1, preferably at least 2, even more preferably at least 3 of the binding surfaces are selected from each of at least 2, preferably at least 3, of the groups of binding surfaces selected from the peptides, proteins, detergents, surfactants and polymers.
The array of the invention is typically used for characterizing and/or determining a sample.
According to an exemplary embodiment at least one non-specific label and an array of plurality of different binding surfaces are used, wherein
One of the basic paradigms of measurement of biomolecular binding reactions has been the specificity of the binder molecules. The higher the specificity, the better the assay. Non-specific binding has without an exception been seen as a burden that limits the sensitivity of the measurement by introducing a background component of often unknown dimension and origin. At least some of the embodiments of the present invention are based on an inverted approach to this traditional specific detection scheme and it discloses how non-specific interaction, such as binding can be used in measurement and analysis e.g. of complex fluids or other samples.
An exemplary method bases on the use of a simple array of non-specific binders, non-specifically reacting luminescent labels and analysis of the sample specific fingerprint on the array. The advantage of using non-specifically reacting components is the fact that a single interacting means, such as binder can interact or bind several different species. The combination of the results from different non-specific interacting means or binders creates a sample specific fingerprint. With a well-designed array the fingerprint as such or the numerically processed fingerprint may be extremely specific and thus a relatively small array of only a few binders can be used in detection of several sample species whereas the dimension of the array would limit a specific array. A further advantage of the method is the use of direct observation of electromagnetically readable property, such as luminescence intensity as such. From literature it is known that with specific binders and optimal conditions, direct luminescence detection (time-resolved) may reveal concentrations as low as a few attomoles/liter. Even with reduced affinity of binding, the sensitivity of a direct luminescence based method can thus be remarkable. When the exemplary method of this invention is compared with traditional chromatography or mass-spectrometry measurements, it is simple and straightforward. As compared with different electrical sensors or known luminescent sensors, the method offers much higher sensitivity and selectivity due to a higher number of surface coating and modification options offering a larger application scale than the known array screening techniques. When compared with known array sensors based on luminescence and specific interactions the invention offers simplified production and a much wider application area for a single array.
Specific binding in this context is understood as a strong, predetermined and selected binding force between a binder molecule and a single or a limited set of target molecules. Such specific binding may be for example binding between an antibody and its' specific antigen.
Non-specific binding, on the contrary, is understood as a binding force of often-undetermined origin that can vary from weak to extremely strong but has little selectivity with the target molecules. Such binding may be for example binding of the sample molecules to the walls of the assay vessel (test-tube). Thus non-specific binders show affinity towards more molecule classes than specific binders, usually 10 or more non-analogous and different targets bind to non-specific binder whereas specific binder typically bind only a single species and its structural analogues.
Specific binding mechanisms are usually well characterized, whereas non-specific binding may take place for a vast number of reasons from simple electrostatic attractive reasons to complicated binding including conformational changes of the binding partners. Binders for a non-specific array include but are not limited to proteins such as albumins; aminoacids; peptides; detergents; surfactants; sugars, such as glucose; glycine; polymers, such as polyethylene glycol, polypropylene glycol, polypropylene, pylyethene, polystyrene and their variants; crafted polymers, functionalized polymers; polyelectrolytes; small molecules; nucleic acids; cells; organisms; tissue; organic and inorganic materials; metals; crystalline or amorphous materials; ionic and non-ionic compounds. Small molecules refer, in this context, to molecules with a molecular weight (MW) of less than 10 000, preferably less than 1 000. Referral to cells can refer to wild type cells or genetically modified cells. The cells can be animal, including human, cells, bacteria or viruses. Binders may also introduce chirality to the surface. These binders can also be used as a mixture and their derivatives. A non-specific binder can also be a chemically or structurally modified surface, a hydrophobic or hydrophilic surface or the surface may contain halide, sulphate, carboxy, amine, thiol, hydroxyl, ketone, ether, epoxide, aldehyde and organometallic groups. The surface may also be treated with e.g. acids, bases or solvents.
Contrary to non-specific binding reactions specific binding reactions are characterized e.g. either as receptor-ligand or ligand-fragment interactions or nucleic acid hybridization reactions. Specific binding reactions in this context are typically also all reactions that trigger or catalyze an event or events, e.g. protein interactions such as RNA DNA transcription, polymerization, cell signaling and chaperon assisted protein folding.
Because of the vast number of different non-specific interactions, manufacturing of different non-specific binding surfaces is straightforward. In this context “surface” can be that of a microtiter plate well, a spotting slide, a particle or any other surface that forms a uniformly coated or modified optically readable identifiable area of 200 nanometers in diameter to 100 mm2 but typically 0.01 mm to 10 mm and preferably 0.5 mm to 3 mm in diameter. In that the surface forms a particle the preferred diameter of the particle is from 200 nm to 10 micrometers. In some embodiments of the invention the diameter of the particle or surface may also be smaller than 200 nm. A surface may, for example, be modified by charge and nanostructure, or it may be coated by layers of different molecular species and mixtures thereof.
A particle array may be realized in several ways that can be found in literature. In a preferred embodiment of a non-specific particle array the array is read by a flow-fluorometric device (see e.g. www.luminexcorp.com referred to in more detail above). Several other options for reading such an array may also be found in prior art (e.g. WO/2001/055702).
The approach of the present invention offers practically an unlimited non-specific chemical diversity for the surfaces. Specific binders, on contrary, are difficult to produce—for example production of a new antibody is a complex process and requires either a) use of animals or b) large artificial binder library searches. Further production of small molecular binder species, e.g. for drug discovery, requires complex chemical syntheses and a-priori knowledge of binding properties of different molecular groups or residues.
Specific binders may also be used as non-specific binders in cases where the binding partners bind to them without the specific targeted interaction. In a preferred embodiment of the current invention the binding array may also contain specific binders that act both as non-specific and as specific binders but typically less than 50% of all binders and preferably less than 10% of the binders of the array.
According to an exemplary embodiment of the present invention, the detection of a sample takes place by using a matrix of carefully selected surface modifications including modifications to the carrier surface nanostructure and charge as well as deposition of molecular layers on the surface. In preferred embodiments of the invention the array is brought into contact with the sample and non-specific interactions of the array and the sample are revealed by a luminophore interacting non-specifically with the array, in particular, with changes (modulation) of intensity of the luminophore, when compared to a reference array, e.g. array of a corresponding sample, array obtained without sample and/or array of known samples. In some embodiments of the invention the modulation may also be observed in the luminescence lifetime, luminescence polarization or luminescence energy transfer. The modulation in luminescence may also be observed as a function of time. Accordingly the invention can also be used for kinetic characterization and/or determining of a sample. In such cases detection of a sample or parallel samples at different time points can be carried out from the same array or from parallel arrays respectively.
Luminophores or components of a mixture of luminophores can be selected from the group consisting of coumarin; rhodamines; cyanines; boron-dipyrromethenes; lanthanide compounds, such as chelates and cryptates; porphyrins; metalloporphyrins; fluorescent proteins; nanolabels, such as quantum dots and other luminescent nanoparticles; luminescent nanocrystals and luminescent polymers.
In some embodiments of the invention the luminophore may consist of a donor and acceptor luminophore or a donor and a quencher such that the acceptor or quencher molecule are brought into contact with the donor luminophore or separated from the donor luminophore by the non-specific reactions of the invention.
In some embodiments of the invention the luminophore may be substituted by molecules or particles of the sample interacting with the array in which embodiments these molecules or particles are distinguishable by e.g. their absorption, scattering, raman scattering, chemiluminescence, radioactive emission and local enhancement of electromagnetic field or via other electromagnetically readable properties discussed e.g. in this document.
In many preferred embodiments of the invention the non-specific array may also contain a reference luminophore that is either a) not participating in the non-specific binding reactions or b) is participating in the reaction in a known fashion. The signal from the reference luminophore may e.g. be used for normalization of the results.
The mechanisms of non-specific interaction, such as binding are numerous, often undetermined and have low specificity and selectivity. The selectivity and specificity of the array depend from the interpretation of the fingerprint of several spots. In a well designed array this fingerprint is unique for each sample. In preferred embodiments of the invention the fingerprint is compared to a reference fingerprint or fingerprints. Detection may be carried out e.g. by a luminophore or a mixture of luminophores that also are non-specific binders. In some embodiments of this invention the luminophores may also be specific binders or a mixture of specific and non-specific binders. The luminophore may be used in revealing the fingerprint of an unknown sample by comparing the fingerprint of the luminophore in presence and absence of the sample. In comparative studies the fingerprint of the array is compared in presence of samples A, B, C etc. The comparisons of the acquired fingerprints against a library or comparison sets are made by known methods from bioinformatics and data mining. In many embodiments of the present invention fingerprints processed using appropriate algorithms are compared rather than observing the fingerprints of samples as such. Fingerprints and fingerprints processed using appropriate algorithms of the sample can be compared with fingerprints or fingerprints processed, respectively, using appropriate algorithms of:
These algorithms [see e.g. www.dtreg.com and Romesburg C, Cluster Analysis for Researchers, Lulu Press 2004] may e.g. be based on:
In exemplary embodiments of the invention a non-specific luminophore may bind directly to the non-specific array surface. In this case the sample can either increase or decrease the binding of the luminophore onto the surface. In further embodiments the luminophore may also be bound to sample molecules in the solution and thus be prevented from binding to the non-specific surface or be linked to the surface by the sample molecules. Because of these different alternatives modulation of the luminescence of a specific binding surface can either result in increase or decrease of the luminescence but does not necessarily affect the luminescence of a each particular binding surface of the array at all.
In some preferred embodiments of the invention the sample is first diluted in a buffer before contacting the array. The buffer may contain components that enhance modulation of the non-specific binding. These components may act with the sample non-specifically or specifically. The components may contain standard features for the fingerprint that can be used in normalization and interpretation of the results. These components may also act with the array surfaces increasing the probability of the luminophore to be bound or displaced by the sample. The buffer may also contain luminophores for reference purposes. The buffer may also contain the luminophore or luminophores that are used to detect the interaction of the sample and the array. The buffer may also be completely inert and act only as dilutant for the sample. In a preferred embodiment of the invention the pH of the buffer is often <7, preferably <5 and typically between 3 and 4 to increase the probability of non-specific binding. The electrolyte concentration of the buffer is typically <100 mM and preferably <10 mM. In an exemplary embodiment of the invention the assay is performed as follows.
It is clear for a person skilled in the art that the presented protocol and the order of the addition of the reaction components may vary depending e.g. on the application, sample, used reaction, washing buffers and used analysis methods. In addition is should be noted that even the luminiphore label is disclosed above, also other label e.g. disclosed in this document can be used for interacting with the interacting surfaces of the array and thereby changing the electromagnetically readable property.
In some embodiments of the invention for detection of water based fluids the array is preferably selected such that it contains non-specific surfaces that are coated with at least one surface from each of the groups: proteins, detergents, polymers and at least one surface that is treated with acids, bases or solvents. In such an array the luminophore is selected such that it is preferably hydrophobic and fluorescent or shows delayed fluorescence or phosphorescence upon excitation with suitable wavelength light. The luminophore may also be a combination or a mixture of a fluorescent substance and a substance exhibiting delayed luminescence upon excitation with suitable wavelength light.
The advantages of the current invention to known methods utilizing non-specificity and array detection are:
In this example each of the surfaces has different type of the electromagnetically readable properties due to different interactions with the samples and/or label, whereupon a specific fingerprint can be determined for each case.
According to an embodiment the arrangement 300 may also comprise an emitting means 302 for emitting a certain electromagnetic radiation, which reflection, absorption, polarization or scattering, for example, is detected advantageously by the detecting means 301.
In addition, according to an embodiment, the emitting means 302 may be adapted to emit a triggering pulse 310 for triggering e.g. luminescence effect 311 of the luminophore labels interacted with the surfaces of the array. The arrangement advantageously comprises also timing means 303 for triggering the emitting means 302 to emit the radiation, but also triggering the detecting means 301 to detect the electromagnetically readable property, such as luminescence, at an appropriate time point (t1, t2, . . . , tn) so that the triggering pulse 310 does not disturb the detection of the luminescence radiation 311.
The arrangement may also comprise controlling means 304 for controlling the emitting means 302 as well as the detecting means 301. For example, according to an embodiment, electromagnetically readable property of each individual interacting surface can be detected separately and independently of other surfaces by the detecting means, and/or the emitting means may be adapted to emit e.g. electromagnetic radiation or triggering pulse individually for each individual interacting surface. The controlling means 304 and timing means 303 may be adapted to control the emitting means and detecting means so that the electromagnetically readable property of each interacting surface can be read separately and independently of each other.
The arrangement 300 enables also the determination of kinematic of the emission, such as luminescence lifetime, which may be additional parameter when determining the fingerprint of the sample, since the lifetime may differ for different label reactants or samples.
It should be noted that the locations of the interface surfaces may differ from the locations of the exemplary arrays 200-250 disclosed in
According to an embodiment the detecting means 301 may be adapted to detect radioactive radiation emitted by the interacting surfaces, especially if a radioactive label is used. However, it is to be noted that also detecting means 301 capable of detecting radiation of another kind can be applied as well.
In addition the arrangement advantageously also comprises comparing means 305 for comparing said fingerprint of said sample with at least one of the following information advantageously stored in memory means 306
i) at least one fingerprint of at least one corresponding sample,
ii) at least one fingerprint of an array obtained without a sample, and/or
iii) at least one fingerprint of known samples,
in order to characterize and/or determine said sample.
Moreover the arrangement may also comprise receiving means 307 for receiving the array 200-240 and washing 308 means for washing the interacting surfaces of the array with at least one washing medium before detecting said electromagnetically readable property of said interacting surfaces. Furthermore the arrangement may also comprise user interface means 309, such as a keyboard, display or other controlling means for inputting commands and outputting results, for example.
4 wines (marked ‘A’, ‘B’, ‘C’, ‘D’) were chosen from 2 different grapes. Wines 1 and 2 were of the same grape type but from different areas. Similarly wines 3 and 4 were of the same grape type but from different areas. The wines were divided into two sets. A training set containing 4 samples from each wine (16 samples in total). A test set containing 5 samples from each wine (20 samples in total). The training set was used in training the algorithm used in this test. And the test set was used in verifying the function of the array in discriminating the wines from each other. Parallel to this test a non-expert human panel consisting of 10 individuals was given 2 wines randomly from the test set and a training set (1 wine of each type). The humans had to recognize with all of their senses (smell, test, sight) the two wines by comparing them to given wines A, B, C and D.
The array according to the preferred embodiment of this invention consisted of 9 different selected non-specific binding spots and a protocol of the preferred embodiment of this invention was used. The wine samples for training and testing of the array were diluted in water. A single luminophore was used for detection and no extra references or normalizations were made.
The detection matrix (confusion matrix) for the method (left) and human panel (right) show the number of selected wines against the actual wine.
The tested matrix showed good repeatability with up to 10 fold variance of signal between different wines and surfaces. Both the shape of the fingerprint and the absolute measured intensities can be used for identification and quantification purposes. Taken into account the experimental variance (<10% coefficient of variation) and intensity modulation created by different wines (10 fold). With this variance up to 5 different levels of binding for each array spot can be identified reliably. Thus the theoretical differentiation capability of this 9 spot array is 59=1.9 million different samples.
Even thought the samples of different bottled waters and wines have been determined and characterized above, it should be understood that also other types of samples can be determined or characterized, such as certain components can be determined and characterized from blood, saliva or urine samples. In addition it is to be noted that also micro-organisms can be determined and characterized by the methods of the invention. According to an exemplary embodiment the micro-organisms are at first disintegrated so that the inner structures will advantageously be introduced to the interacting surface e.g. via a dilution or liquid medium as described elsewhere in this document. By the method e.g. drug tests can be performed quickly and accurately. The present invention also enables in situ tests.
It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.
In addition is should be noted that even if the luminophore labels and luminescence are described in many examples above, they are only examples and also other type labels can also be used, such as radioactive label, and/or a label changing at least luminance, absorption, emission, reflectivity, scattering, raman scattering, chemiluminescence, radioactive emission, local electromagnetic field and/or polarization of at least one interacting surface of said array when introducing said labelling reactant to interact (in contact) with said interacting surface. Therefore also other type of radiation detected from the interacting surfaces can be detected than only luminescence, such as reflection, absorption, polarization variation, or radioactive emission, for example.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20095501 | May 2009 | FI | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FI2010/050354 | 4/30/2010 | WO | 00 | 11/3/2011 |
