Patent Application
20130295683
The present invention is related to devices, methods, and structural solutions that are used to hold a liquid sample for optical examination and analysis. Especially the present invention is related to the use of non-specific interacting surfaces for allowing the sample to take part in reactions, indications of which may be measured optically.
Optical analysis of liquid samples frequently involves placing small amounts of the sample in spots or wells on a carrier, where it comes in touch with so-called interacting surfaces. Luminophores that are present within the sample and/or within the spots or wells, or are otherwise introduced into the process, become bound in different ways depending on the interactions between the sample and the interacting surfaces. The subsequent optical measurement involves selectively subjecting the spots or wells to optical excitation signals, and measuring the luminescent response. Identifying and quantifying the constituents of a sample, and/or identifying a sample as being equal to a previously measured reference sample, is possible by comparing the observed luminescent response to a library of previously measured responses.
Prior art publications that describe devices and structural solutions for holding a sample in the way considered above are for example the patent applications number PCT/FI2010/050354 and EP10185964.3, which at the time of writing this description are not available to the public.
Setting up the interacting surfaces by coating or otherwise chemically treating specific locations (like spots or wells) in the carrier has some inherent disadvantages. For example, in order to make the liquid sample flow smoothly and easily over the carrier surface, or in some cases through channels defined by the carrier, the carrier material (or at least a surface thereof) should be highly hydrophilic. This, however, may be contradictory to requiring that the coatings used to implement the interacting surfaces should stick and remain tightly in place in the appropriate spots or wells. Additionally it may prove difficult to offer sufficiently large interacting surfaces within the spots or wells to properly detect some relatively weak interactions. Yet another disadvantage is the relatively large number of distinct spots or wells on a single carrier (or the need for multiple carriers) that are required for implementing a versatile analysis program that could be optimized for various specialized needs.
An objective of the present invention is to present a device for holding a sample for optical fingerprinting examination and analysis that would allow easily controlling the flow of a liquid sample. Another objective of the present invention is to present such a device that would allow versatile optimization of the measurement configuration for various specialized needs. Yet another objective of the present invention is to present such a device that would allow offering a relatively large interacting surface for samples despite the relatively compact size of the device itself. Yet another objective of the present invention is to present a device that allows the sample to undergo changes within the device itself.
Yet another objective of the present invention is to provide a method for utilizing a device of the above-described kind in an effective and advantageous manner.
The objectives of the present invention are achieved by using a distribution of non-specific interacting surfaces extending across a carrier, and by ensuring that said carrier allows optical fingerprinting analysis of interactions between the sample and the non-specific interacting surfaces at multiple locations. Either particles or an essentially continuous membrane can be used to define said non-specific interacting surfaces.
A device according to the present invention is directed at holding a sample for optical fingerprinting analysis, the device comprising a carrier, a distribution of non-specific interacting surfaces extending across said carrier, at least one fluidic channel configured to allow a fluid sample to flow through at least a part of said carrier to get in touch with one or more of said non-specific interacting surfaces, and one or more optical windows adjacent to said non-specific interacting surfaces for enabling optical analysis of results of said sample getting in touch with said non-specific interacting surfaces at multiple locations of the carrier, where the device with said fluidic channel and distribution of said interacting surfaces is configured to introduce said fluid sample with at least one interacting surface in a sequential manner so that at least one first portion of the interacting surface is configured to interact with said sample before at least one other portion of the same interacting surface, and/or at least one first interacting surface is configured to interact with said sample before at least one other interacting surface.
In some aspects, the device comprises a membrane that extends across or constitutes said carrier, and one or more surfaces of said membrane offer said non-specific interacting surfaces to said sample.
In some aspects, the device comprises a plurality of particles immobilized within said carrier, and surfaces of said particles offer said non-specific interacting surfaces to said sample.
In some aspects, a structural material of the carrier has the form of an essentially planar layer, said non-specific interacting surfaces have an essentially two-dimensional distribution within a plane defined by said structural material, the device comprises non-specific interacting surfaces of several different types, and non-specific interacting surfaces of same type form continuous areas that extend across a region of said structural material.
In some aspects, a layer of the device that is transparent to at least one wavelength of optical radiation and parallel to said essentially planar layer defines said one or more optical windows.
In some aspects, the carrier defines a compartment, and said non-specific interacting surfaces are enclosed within said compartment.
In some aspects, the device comprises particles of several different types, wherein the type of particle is defined by the type of non-specific interacting surfaces it offers to a sample, the carrier defines a single compartment, and particles of different types are confined together in said single compartment.
In some aspects, particles of different types are separated from each other within said single compartment by further immobilizing particles of particular type within an immobilizer, such as hydrogel.
In some aspects, the device comprises particles of several different types, wherein the type of particle is defined by the type of non-specific interacting surfaces it offers to a sample, the carrier defines a number of separate compartments, and particles of a particular type are confined in a compartment separate from particles of other types.
In some aspects, the carrier comprises a combination of compartment cells attached together through mechanical attachment means, and the mechanical attachment means define parts of the fluidic channel for allowing a fluid sample to flow from one compartment cell to another.
In some aspects, a compartment of said carrier is free of non-specific interacting surfaces for allowing an optical measurement of a fluid sample alone.
A method according to the present invention is directed at performing optical fingerprinting analysis of a fluid sample, the comprising making the sample flow over or through a distribution of non-specific interacting surfaces that extend across a carrier, so that the sample gets in touch with one or more of said non-specific interacting surfaces in a sequential manner so that, introducing the sample with at least one first portion of the interacting surface to interact with said sample before at least one other portion of the same interacting surface, and/or introducing the sample with at least one first interacting surface to interact with said sample before at least one other interacting surface; directing an excitation signal to multiple locations of said carrier where the sample has got in touch with one or more of said non-specific interacting surfaces; collecting and detecting luminescent responses from locations of said carrier subjected to said excitation signal; and producing a combined set of measurement data indicative of said luminescent responses to characterise said sample.
In some aspects, the step of directing optical excitation radiation to multiple locations of said carrier comprises selectively irradiating a number of locations with optical excitation radiation one location at a time.
In some aspects, the step of producing a combined set of measurement data comprises deriving one or more statistical descriptors that characterise a plurality of individual measurement results of luminescent response, and outputting said one or more statistical descriptors as said combined set of measurement data.
In some aspects, the step of directing optical excitation radiation to multiple locations of said carrier comprises irradiating a number of locations simultaneously with selected irradiation intensities.
In some aspects, the present invention is directed at a use of the device for holding a sample for optical fingerprinting analysis.
Using particles to define the interacting surfaces, instead of just chemically treated surfaces of spots or wells, brings literally another dimension into consideration. Particles are three-dimensional by definition, which means that an ensemble of particles in a liquid sample can easily offer a much larger interacting surface per unit volume than any two-dimensional spot or bottom of well. Particles of various kinds are readily available in a variety of sizes, and it is relatively easy to measure and apply a very accurately known amount of particles, which enables tailoring the interacting surfaces and the whole carrier to various sizes, shapes, and needs. Different kinds of particles can be mixed together in accurately controllable proportions, which further facilitates easy variation and tailoring.
Immobilizing particles within a carrier is in many cases easier than trying to establish similar interacting properties directly on surfaces of the carrier. As an example, carriers may be chemically bonded on a surface offered by a structural material of the carrier, or immobilized within a matrix defined by a structural material. The last-mentioned covers e.g. cases where some structural material of the carrier is porous, fibrous or otherwise such that it inherently defines pores or cavities within which the particles can be captured. Yet another approach is to immobilize a plurality of particles in a compartment defined by the carrier, either simply by confining the free particles as such within the compartment or by further immobilizing particles within a hydrogel or similar agent that will be confined in the compartment. In general the term to immobilize means in this description that the particles are kept in place within the carrier, so that no significant portion of the particles is allowed to move along with the sample flow over distances that would be significant compared to the overall dimensions of the carrier.
An essentially continuous membrane offers many advantages similar to those related to particles. It extends the choice of physical locations where interactions can be observed and measured, to cover even the whole carrier. Different kinds of interactions may occur at different locations of the membrane, because the interaction properties of the membrane may be deliberately made different in its various parts and/or because the sample that has already reacted with the first parts of the membrane has a different constitution than what it had when it first entered the carrier. A membrane that extends across a carrier can offer bigger interacting surfaces, i.e. better chances to react, than prior art solutions that were concentrated in spots or wells. Further advantages are described in more detail later in this text.
Using the carrier additionally to define a channel that allows a fluid sample to flow through at least a part of the carrier involves further advantages. This is an easy and well-functioning way of making the sample get in touch with desired ones of the non-specific interacting surfaces, and even in a desired order or cascade, if that is required. The sample may flow through the channel either by itself (e.g. due to capillary forces or natural gravitation) or in an assisted manner (e.g. due to pumping, suction, or centrifuging). If the channel passes through regions of immobilized particles or makes the sample otherwise come into contact with one or more interacting surfaces, the sample that propagates further therefrom is not necessarily any more identical to the sample that entered the carrier, because it may have reacted or otherwise transformed while interacting with the surfaces. Chained regions of non-specific interacting surfaces may thus be used to set up a kind of chromatographic fingerprinting analysis.
Contrary to e.g. known HPLC (high-performance liquid chromatography, or high-pressure liquid chromatography) setups, a carrier according to the present invention should comprise one or more optical windows adjacent to the non-specific interacting surfaces, for enabling optical fingerprinting analysis of the results of the sample getting in touch with the surfaces. This is associated with the fact that the present invention allows interactions to be observed and measured within the carrier and even at multiple locations therein, but also with the fact that the present invention encompasses also embodiments where the sample introduced to the device remains there and does not necessarily flow further, which it does in HPLC where the sample flows always all the way through and out of the column that includes the stationary phase. According to the present invention the carrier should allow optical measurements (excitation with an excitation signal which is typically optical, and the observation of luminescent radiation) at various locations within the carrier, where interactions occur, and/or in some cases even at locations where no interactions occur. This opens the way to the so-called fingerprinting of samples, where a sample is characterized through the luminescent response that can be observed by irradiating various locations of one or more sample-laden carriers according to a known irradiation pattern.
The exemplary embodiments of the present invention presented in this patent application are not to be interpreted to pose limitations to the applicability of the appended claims. The verb “to comprise” is used in this patent application 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.
The novel features which are considered as characteristic of the invention are set forth in particular in the appended claims. The present invention itself, however, both as to its construction and its method of operation, together with additional objects and advantages thereof, will be best understood from the following description of specific embodiments when read in connection with the accompanying drawings, in which:
The term fingerprinting and its derivatives refer to the handling of a set of measured signals as an entity that represents the distinctive, identifying characteristics of the sample. The handling of the measurement signals may involve e.g. weighting, combining, and other kinds of processing of individual outputs of a measurement apparatus. Fingerprinting of non-specific signals refers to the recording of distinctive and identifying characteristics that do not necessarily have a direct physico-chemical explanation but the explanation follows from comparisons to a library of other fingerprints. A fingerprint may consist of one or several distinctive and identifying values.
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. 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 terms non-specific interacting surfaces or non-specific binding refer 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 Preferably the non-specific binders or binding surfaces of the present invention are not specific in any context. In comparison, a specific binder will typically bind to an individual entity; for example an antibody binding to an individual ligand or a single epitope on a protein. Such binders do not bind to chemically and conformationally non-identical molecules with an affinity compariable to their binding to their specific ligand.
Thus a non-specific interacting surfaces have low selectivity and will exhibit the capability to bind multiple entities, typically greater than 10, where as a specific binder has high selectivity for a single ligand and typically not more that 3. Further a non-specific binder along with low selectivity will typically have a binding affinity of less that 107 M−1, where as a specific binder typically along with high selectivity has an affinity of greater than 107 M−1.
Particles can be placed at arbitrary locations of the carrier, and different types of particles can be placed differently. The result is a distribution of non-specific interacting surfaces extending across the carrier. As an important difference to prior art structures that were based on spots or wells, the distribution of non-specific interacting surfaces can be made essentially continuous across the carrier, or at least across relatively large portions of the carrier. By controlling the way in which different kinds of particles are placed, it is possible to create any kinds of transitions between regions, including both sharp jumps from one particle type to another and gradient-type transitions where the predominant particle type changes smoothly from region to region. It is also possible to use intermediate material between regions of particles, so that the transition involves the end of one region, then no particles at all, and only thereafter a new region. The intermediate material may have an interacting function, or it may be just inert material in e.g. porous or fibrous form so that it allows the liquid sample to pass smoothly. One possible interacting function that an intermediate material may have is filtering. Also a region of the carrier where interacting surfaces do occur may have additional functions: for example, a region (typically at that side of the carrier where the incoming sample is introduced) may have a filtering function to remove unwanted matter such as cells or solid particles from the sample.
What the particles actually are may or may not be particularly important to the present invention. On the allegedly continuous scale from individual atoms and molecules through macromolecules, clusters, colloids, particles, and solids, particles of in the traditional sense of the word lie between macroscopic sizes and a diameter of approximately 50 nanometres. At diameters smaller than that the individual grains of matter become small enough to be called nanoparticles, and their interactions with other substances and with environmental conditions become predominantly nanotechnical. As such, the present invention allows the use of nanoparticles, and they should for this purpose be included under the general interpretation of the word particle. A practical upper limit at the larger end of the size scale may come from the fact that the larger the particle, the less advantage can be gained from the available interacting surface per unit volume.
A very large variety of particles can come into question. Examples of particle types indude but are limited to metal (such as gold) particles, glass spheres, polymer particles, silica particles, liposomes, and living organisms like bacteria or even fruit flies. Gel particles made of a cross-linked polysaccharide gel have been successfully tested, as well as carboxy microparticles. At the time of writing this description, an extensive selection of particles are commercially available from companies like Seradyn, Inc., Particle Technology Division, Indianapolis, USA.
One device for holding a sample may comprise particles of only one kind, or particles of different kinds. In many cases it is advantageous to have within the device particles that offer different kinds of interacting surfaces to the sample, so that the sample reacts in different ways at different parts of the carrier. Some typical reactions between the sample, the interacting surfaces, and the possible additional luminophore labels involved include, but are not limited to:
direct binding: one of sample molecules and luminophore labels bind to the interacting surface much better than the other, resulting in either a luminophore-rich region or a region where essentially no luminophores appear,
indirect binding: luminophore labels do not bind to the interacting surface, but sample molecules bind to both the interacting surface and the luminophore labels, resulting in a relatively high concentration of luminophores,
selective binding: of two or more different kinds of luminophore labels, only one or only some bind to the interacting surface, either directly or indirectly,
inhibition: sample molecules keep the luminophore labels from binding to the interacting surface,
dissolving: sample dissolves a labeled substance from the interacting surface.
In the context of the present invention the term luminophore refers to an atom or atomic grouping in a chemical compound that manifests luminescence. The term luminopore label refers to the chemical compound that comprises the luminophore. The luminophore label may consist any luminophore with any Stokes shift. In a preferred embodiment the luminophore consist of lanthanide complex and/or a Stokes shift of more than 50 nm or more preferably more than 100 nm or even more preferably more than 150 nm or most preferably more than 200 nm.
In preferred embodiment the luminophore is in solution and is solution-based. The luminophore which is detected may not be immobilized on a surface. The luminophore is introduced to the reaction separately or together with other reactants or it is dried on the reaction chamber. Essential is that the luminophore essentially is soluble and interacts in the solution phase with the reactants such as sample and surfaces. In one embodiment of the present invention, one or multiple luminophores is applied. Preferably four luminophores or more preferably three luminophores or even more preferably two luminophores or most preferably one luminophore is applied in the method of invention.
According to one embodiment of the present invention, the luminescene reading is performed with a single measurement. In an alternative embodiment, the reading is performed using two or more separate measurements and the ratio of the measurements are used to create the fingerprint. The optical analysis is according to this embodiment a ratiometric measurement.
In a preferred embodiment of the present invention, the sample flows through a carrier. The flow facilitates the introduction of the sample through the carrier efficiently. The flowthrough is coupled to the detection where two or more detection windows are applied. In one embodiment, the flow is stopped for the detection.
The arrow 103 in
According to an embodiment of the present invention, the device comprises one or more optical windows adjacent to the particles for enabling optical analysis of results the sample getting in touch with the particles. Here we may draw a difference between optical windows for entering the radiation stimuli and optical windows for collecting the luminescent response. As a limiting case at least one of these may be the open surface of the device. For example in the schematic illustration of
Typically it is more advantageous to have the carrier covered with a layer that e.g. keeps volatile components of the sample from evaporating in uncontrolled fashion. A layer of the carrier that is transparent to at least one wavelength of optical radiation is a practical part to be used to define said one or more optical windows. The optical windows for entering the radiation stimuli may comprise regions of the covering layer that are essentially transparent to electromagnetic radiation at the wavelength used for excitation.
In the schematic illustration of
According to one embodiment, the particles are transparent to electromagnetic radiation to facilitate the penetration of the electromagnetic radiation to the particles and from the particles. In another embodiment, semi or non-transparent particles are applied.
In one embodiment particles or porous materials are used according to the present invention. In another embodiment, a mixture of different surface is utilized. Particles and/or porous and planar surfaces can be mixed to versatile the fingerprinting.
The porous or fibrous structural material is in the embodiment of
The intensity of electromagnetic radiation that is available for optical excitation in a reader apparatus is typically far greater than the intensity of luminescent response that will be obtained. Therefore with a carrier that has a structure according to
In
An alternative measurement geometry might include excitation means and light collection optics on the same side of the carrier, in which case no transparency of the carrier would be required. This applies to all embodiments of the invention where the carrier has a generally planar form. An alternative to having multiple excitation means would be the use of very strictly located excitation means, e.g. only a single LED, together with a controllable x-y table that was configured to move the device so that each time the appropriate location of the device was subjected to excitation and detection of luminescent responses.
In the embodiment of
Although all regions are here shown with different hatching, the device may have also multiple regions with the same type of non-specific interacting surfaces. Additionally it is not necessary to have only one type of non-specific interacting surfaces within a region, but within at least some of the regions there may be a mixture of two or more different types of surfaces.
In the exemplary embodiment of
The embodiment of
Concerning all embodiments of the present invention discussed so far, it should be noted that the device may have optical windows also at locations where no non-specific interacting surfaces are located, for the purpose of making optical measurements of the sample as it is at the respective location. According to an embodiment of the present invention there are two such windows: a “preliminary” optical window may be located before any non-specific interacting surfaces in the sample flow direction, to enable making optical measurements of the sample before it has undergone any interactions. An “end result” optical window may be located after all non-specific interacting surfaces in the sample flow direction, to enable making optical measurements of the sample after it has undergone all possible interactions that it can within the device. An optical window after all interactions have taken place enables also measurements known from prior art, e.g. from liquid chromatography, to be combined with fingerprinting analysis according to the invention.
The fluid sample should be let to flow into and through at least part of the carrier. To that purpose the carrier has a tubular form that defines a sample flow direction, which is essentially parallel with a longitudinal axis 1003 of said tubular form. The longitudinal axis is horizontal in
The tubular form offers a variety of possibilities to implement one or more optical windows adjacent to said particles for enabling optical analysis of results of said sample getting in touch with said particles at multiple locations of the carrier. A very straightforward alternative is to make the whole carrier of a material that is transparent to electromagnetic radiation at wavelengths of both excitation radiation and luminescent responses. Such a solution would give maximal freedom in selecting the physical configuration of radiating means and detector(s) in the reader apparatus.
Details of the form shown in
It should be noted that the present invention does not actually require separating different types of particles sharply, or even separating them at all. An example of other than sharp separation would be diffuse separation, in which particles of different types would be allowed to slightly mix with each other at and close to the plane separating them.
Using a compartment of the carrier to immobilize particles underlines the appropriate interpretation of the term to immobilize in the framework of the present invention. For example in the embodiment of
A carrier of the generally tubular kind makes it possible to have a larger cross-section of the fluid channel than a device of the generally planar kind. One reason is because in the latter the fluid channel is close to two-dimensional, while in a tubular device it is truly three-dimensional. Even if the carrier is filled with particles, if the particles are large enough, a relatively large amount of free space will be left around them. A larger cross-section of the fluid channel enables a significant flow of the fluid sample all the way through the device, which in turn makes it possible to couple a number of devices together to form chains and/or networks and to still get a good flow of the fluid sample through each device.
The “snap-together” type carrier of
In the snap-together approach of
Step 2003 involves directing optical excitation radiation to multiple locations of the carrier where the sample has got in touch with at least some of the non-specific interacting surfaces. Previously in the description it has been already pointed out that excitation radiation may be deliberately directed also to locations where it is known that no interactions occur, for example to a compartment where there are no particles; to a portion of a planar carrier where there is no membrane, no coating, or no particles; or to an otherwise provided part of the carrier where the sample is found in essentially “pure” form. Embodiments of the invention may differ from each other with respect to whether the excitation is performed one location at a time or at multiple locations simultaneously, which will be described in more detail later.
Concerning the relation of steps 2002 and 2003 it should be noted that the present invention encompasses embodiments where significant portions of the sample are still there when the measurement begins, but also embodiments where the device has been essentially drained and in some cases even carefully dried so that the measurement takes place in a dry state. Also some embodiments of the present invention may involve repeated rounds through step 2002, either with the same sample or with another sample that should be analysed in combination of it. There may also be rinsing and washing steps, if required.
Step 2004 involves collecting and detecting luminescent responses from locations of the carrier which were subjected to the optical excitation radiation. Time-gated detection is advantageously used, so that the actual detection of luminescent responses is only started after the excitation means have been shut off. One reason for time-gating is that otherwise some of the excitation radiation might enter the detector and become misinterpreted as luminescent emission radiation. It is also possible that some short-lived autofluorescence in the sample (or some other involved material, like the structural materials of the carrier) takes place during and immediately after the excitation radiation pulse, which should not be confused with the desired emission radiation from the luminophore label(s). Autofluorescence decays typically much faster than the emission radiation from luminophore labels, so it can be avoided by time-gated detection. Yet another possible reason to use time-gated detection is the possibility of measuring the emission radiation during two or more different time intervals of a single emission period, which gives information about the decaying rate and lifetime of the emission radiation.
Additionally the Rayleigh and Raman scattering of the excitation radiation must be taken into account. Raman scattering involves a Stokes shift due to the inelastic scattering mechanism, and is thus susceptible of introducing interference to such fluorometric measurements where the detector is active simultaneously with the introduction of optical excitation. It is believed that Raman scattering will set an absolute limit of about 10−10 M to the sensitivity of simultaneous fluorometric measurements unless sophisticated techniques from e.g. single molecule detection are used.
Step 2005 involves producing a combined set of measurement data that is indicative of the luminescent responses. The aim is to use the combined set of measurement data to characterise the sample. In many cases the measurement is made for sample identification, i.e. to identify, which one of a set of previously known samples did this particular sample come from. A slightly different possibility is the comparison of a sample to one or more known samples, so that differences to the known samples are looked for through the fingerprinting. The combined set of measurement data should be descriptive enough so that for each time a particular sample (for example: a particular brand of a drinkable liquid) comes to the measurement, the same or very similar combined set of measurement data is obtained.
On the left side in
Concerning the last-mentioned aim,
We may recall that using either particles or a membrane to set up a distribution of non-specific interacting surfaces brings about a particular “mobility” with respect to where within the carrier the interesting interactions will occur: not only in some predetermined spots or wells, but somewhere at an arbitrary location within said distribution. For example, it may occur that neither of two pure interacting surfaces produces quite satisfactory results for measurements, but at a border zone that exhibits a smooth transition from one particle type to another, or one region of a membrane to another, a very significant interaction takes place.
If there are sufficiently many separately controllable sources of excitation radiation in the reader apparatus, the analysis may involve a phase where a device holding a sample is selectively irradiated at a large number of different locations in turn, and the respective luminescent responses are detected and measured. The combined response that describes, what kind of luminescence was obtained as a result to each different excitation stimulus, constitutes the so-called fingerprint of the sample. From a statistical analysis of responses to a number of excitation rounds with individual excitation sources it may be possible to derive one or more statistical descriptors that characterise a plurality of individual measurement results of luminescent response. If nothing else than individual excitation is aimed at also in the future, these statistical descriptors can be output as the combined set of measurement data.
However, the statistical descriptors are also a way to constitute subgroups of excitation sources. The definition of each subgroup would precise, which excitation sources should be switched on and at which intensity. The combination of luminescent responses to excitation with well-defined subgroups of excitation sources may prove to be quite as accurate in identifying samples as the fingerprints that list responses to excitations with individual excitation sources. Performing the excitation by subgroups, rather than by one excitation source at a time, may significantly speed up analyses, because fewer excitation rounds would be needed. If the irradiation intensities have been selected to reflect the relative significance of luminescent responses obtained from corresponding locations of a measured reference sample, a list of luminescent response measurement results from the irradiating of a number of locations simultaneously can be directly output as the combined measurement data that characterises the sample.
In some cases the fingerprint of a sample may be a combination of measurement data from analysing two or more devices, where the same sample has been subjected to the same or different kinds of interactions. Using two identical devices for parallelly analysing the same sample adds redundancy and decreases the effect of statistical fluctuation of interactions on the analysis. Using two devices with different kinds of interactions, for example with the same interacting surfaces but in a different order, gives more dimensions and versatility to the analysis. Yet another possibility for “combinatorial fingerprinting” is to make one measurement with a portion of the sample, then let the sample age in documented conditions for a given time, and to make another measurement (with a similar or different selection of interacting surfaces) thereafter.
Variations to the exemplary embodiments of the present invention are possible without parting from the scope of protection defined by the appended claims. As an example, most of the examples above have illustrated a multitude of optical windows, each optical window corresponding with a location of the device where results of the sample getting in touch with the non-specific interacting surfaces should be analyzed optically. As an alternative it is possible to have fewer optical windows, or e.g. only one optical window which is large enough to optically analyze all locations of interest within the device.
A further class of variations to the above-described embodiments involves using something else than electromagnetic radiation in the optical range as an excitation signal. An optical response such as luminescence can be invoked for example by using a localized magnetic field as an excitation signal. Paramagnetic particles can be used on the carrier, so that their properties perturb the magnetic field. Also excitation signals in electric form can be used, for example by subjecting the sample-laden carrier to an electric field, connecting it to an electric voltage, or inducing electric currents within the carrier, particles, or membrane. Silicon-based particles may be doped to have P- or N-type conductivity like in electronic semiconductor components.
2 mL of 10% solids of carboxylated microparticles (3.1 μm in diameter, Seradyn, Indianapolis, Ind.) were incubated with 5 mL of 2% solutions of epomin 1,800 (Nippon Shokubai, Osaka, Japan) or Triton X-100 (Sigma-Aldrich, St. Louis, Mo.) at room temperature for 2 hours. The particle suspensions were transferred to 1.5 mL centrifugal tubes and centrifuged using 5,000 rpm for 1 min. The solution was removed and 1 mL of MilliQ water was added, mixed and centrifuged. The protocol was repeated four times. In addition, Sephadex-25 particles from NAP-5 columns (GE Healthcare, Waukesha, WI) and silica particles (6.84 μm in diameter, Bangs Laboratories, Fishers, Ind.) were applied to the microtiter plate assays. These particles were washed 3 times with MilliQ water and no coating was applied.
Amberlite MB-1 (MB-1, Sigma-Aldrich), AG-501 silica (AG, Bio-Rad, Hercules, Calif.) and concanavalin coated Sepharose 4B (Con, GE Healthcare).
10 μL of 10 μM BSA solution in MilliQ water and 1% solutions of epomin 1,800 (Nippon Shobukai), polyacetic acid, polyallyl amine hydrochloride, d-sucrose and Triton X-100 (Sigma-Aldrich) were applied as a lane (approximately 5×20 mm) on a nitrocellulose membrane (HiFlow Plus, Millipore, Billerica, Mass.) having a dimension of 90×20 mm. The 20-mm dimension of the lane was equal to the width of the membrane. The solutions were dried and used immediately to the membrane assays.
One milliliter plastic syringes (Plastipak, Becton Dickinson, Franklin Lakes, N.J.) were used as columns. First a stopper was inserted to the column to hold particles inside or to separate particles from one particle lane to another. Columns having stoppers at both ends were prepared (column without intermediate stoppers). Columns having stoppers at both ends and in between each particle lane were also prepared (column with intermediate stoppers). Black towel from Luhta (Aalto, 100% cotton, Lahti, Finland) was chosen as a stopper material due to a low time-resolved fluorescence background signal. Each particle lane in a column was approximately 10 mm in length.
Single particle cartridges were prepared using 1000 μL clear graduated pipette tips (VWR, Finland). Black towel was used to hold particles in each tip.
Water samples: deionized MilliQ water was from MilliQ (Academic, Millipore), Evian (Natural mineral water, Evian , France) and tap water (Turku, Finland)
Tea samples: Twinings Earl Grey (Twinings, Valora Trade Finland, Helsinki) and Lipton Earl Grey (Unilever, Finland) teas (1.9 g) were added to boiling water and incubated without heating for 5 min. The samples were decanted twice and the supernatant was diluted to MilliQ water for analysis.
Twinings is a registered trade mark of R. Twining and Company Ltd., London, United Kingdom. Lipton is a registered trade mark of Unilever N.V., Rotterdam, the Netherlands. Evian is a registered trade mark of Société anonyme des eaux minérales d'Evian, Evian, France.
First, second, third, fourth, and fifth interacting surfaces were created using non-coated (1), Triton X-100 coated (2) and epomin 1,800 coated (3) carboxy, Sephadex (4) and silica (5) microparticles (5 μL of 5% solids). These were incubated with MilliQ water, Evian bottled water and Turku tap water samples in a total volume of 365 μL containing 300 μL of water sample, 30 μL of 12 nM Eu chelate ((2,2′,2″,2″′-[(4′-phenyl-2,2′,6′2″-terpyridine-6,6″-diyl)bis(methylenenitrilo)]tetrakis (acetic acid))) and 30 μL of 5 mM phosphate buffer pH 3. The suspension was incubated for 5 min, transferred to a clear maxisorp NUNC microtiter plate (Roskilde, Denmark) and measured for long-lived luminescence signal at 614 nm using a Victor2 time-resolved fluorescence plate reader. The excitation wavelength was 340 nm, the delay time of the measurement was 400 μs and the integration time was 400 μs.
First, second, third, fourth, and fifth interacting surfaces were created using non-coated (1), Triton X-100 coated (2) and epomin 1,800 coated (3) carboxy, Sephadex (4) and silica (5) microparticles (5 μL of 5% solids). These were incubated with Twinings Earl Grey tea and Lipton Earl Grey tea samples in a total volume of 345 μL containing 300 μL of sample (1:10 dilution to MilliQ water), 10 μL of 11 nM Eu chelate ((2,2′,2″,2″′-[(4′-phenyl-2,2′,6′2″-terpyridine-6,6″-diyl)bis(methylenenitrilo)]tetrakis (acetic acid))) and 30 μL of 5 mM phosphate buffer pH 3. The suspension was incubated for 5 min, transferred to a clear maxisorp NUNC microtiter plate and measured for long-lived luminescence signal at 614 nm using a Victor2 time-resolved fluorescence plate reader. The excitation wavelength was 340 nm, the delay time of the measurement was 400 μs and the integration time was 400 μs.
Samples of 1500 μL containing 150 μL of Twinings Earl Grey tea and Lipton Earl Grey tea and 0.5 μL of 1 mM Eu chelate ((2,2′,2″,2″′-[(4′-phenyl-2,2′,6′2″-terpyridine-6,6″-diyl)bis(methylenenitrilo)]tetrakis (acetic acid))) in MilliQ water was injected to the column in two 750 μL volumes using a Biohit pipette (Helsinki, Finland). Approximately 1400 μL of the solution was injected to the particles and the injection was stopped before all solution reached the particles (approximately 100 μL of the solution remained in the column not reaching the particles). Columns contained particles with interacting surfaces in the following order, MB-1 (1), AG (2) and Con (3). Each particle lane was measured for long-lived luminescence signal at 614 nm using a Victor2 time-resolved fluorescence plate reader. The excitation wavelength was 340 nm, the delay time of the measurement was 400 μs and the integration time was 400 μs. Graph 2501 in
Samples of 1500 μL were prepared containing 150 μL of MilliQ water, Twinings Earl Grey tea and Lipton Earl Grey tea, each with 0.5 μL of 1 mM Eu chelate ((2,2′,2″,2″′-[(4′-phenyl-2,2′,6′2″-terpyridine-6,6″-diyl)bis(methylenenitrilo)]tetrakis (acetic acid))) in MilliQ water. The were injected to the column in two 750 μL volumes using a Biohit pipette. Approximately 1400 μL of the solution was injected to the particles and the injection was stopped before all solution reached the particles (approximately 100 μL of the solution remained in the column not reaching the particles). Columns contained, in the following order, MB-1 (2), AG (3) and Con (4). Each particle lane and separately the sample solutions before (1) and after (5) the column on a NUNC (Roskilde, Denmark) microtiter well were measured for long-lived luminescence signal at 614 nm using a Victor2 time-resolved fluorescence plate reader. The excitation wavelength was 340 nm, the delay time of the measurement was 400 μs and the integration time was 400 μs.
Samples of 250 μL were prepared containing 25 μL of MilliQ water, Twinings Earl Grey tea and Lipton Earl Grey tea, each with 0.5 μL of 0.1 mM Eu chelate ((2,2′,2″,2″′-[(4′-phenyl-2,2′,6′2″ -terpyridine-6,6″-diyl)bis(methylenenitrilo)]tetrakis (acetic acid))) in MilliQ water. These were applied through the particle columns using a Biohit pipette. The columns contained a mixture of MB-1, AG and Con particles, and the respective photon counts are illustrated corresponding to the number 2 on the horizontal axis in
Samples of 500 μL were prepared containing 50 μL of MilliQ water, Twinings Earl Grey tea and Lipton Earl Grey tea, each with 0.5 μL of 0.1 mM Eu chelate ((2,2′,2″,2″′-[(4′-phenyl-2,2′,6′2″-terpyridine-6,6″-diyl)bis(methylenenitrilo)]tetrakis (acetic acid))) in MilliQ water. These were injected through each cartridge using a Biohit pipette in the following order, MB-1 (2), AG (3) and Con (4). Each particle lane and separately the sample solutions before (1) and after (2) the column on a NUNC microtiter well were measured for long-lived luminescence signal at 614 nm using a Victor2 time-resolved fluorescence plate reader. The excitation wave-length was 340 nm, the delay time of the measurement was 400 μs and the integration time was 400 μs. The photon counts are shown in
The sample solutions faced the coating lanes of the membranes in the following order:
1. membrane: nitrocellulose (1), BSA (2), epomin 1,800 (3), polyacetic acid (4), polyallyl amine hydrochloride (5), d-sucrose (6) and Triton X-100 (7). The photon counts are illustrated in
2. membrane: nitrocellulose (1) BSA (2), polyallyl amine hydrochloride (3), d-sucrose (4), Triton X-100 (5) and epomin 1,800 (6). The photon counts are illustrated in
The membrane was placed to a 50 mL tube (Falcon, BD, two oak park, Bedford Mass.) containing 4000 μL of sample solution including 200 μL of MilliQ water, Twinings Earl Grey tea, or Lipton Earl Grey tea, each with 0.5 μL of 0.1 mM Eu chelate ((2,2′,2″,2′″-[(4′-phenyl-2,2′,6′2″-terpyridine-6,6″-diyl)bis(methylenenitrilo)]tetrakis (acetic acid))) in MilliQ water. In
| Number | Date | Country | Kind |
|---|---|---|---|
| 10190221.1 | Nov 2010 | EP | regional |
This application is a National Phase entry of PCT Application No. PCT/FI2011/050980, filed Nov. 7, 2011, which claims priority from EP Application No. 10190221.1, filed Nov. 5, 2010, the disclosures of which are hereby incorporated by referenced herein in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FI2011/050980 | 11/7/2011 | WO | 00 | 7/17/2013 |
