Patent Application
20110071051
The present invention is directed to the field of microfluidic devices for the separation and detection of analytes, such as proteins, metabolites, glycoproteins and/or peptides.
Antibody arrays represent one of the high-throughput techniques that are able to detect multiple proteins and antigens simultaneously. These arrays can be used for example for the measurement of changes in expression of disease-related proteins or posttranslational modifications. This allows for diagnosis, prognosis, measurements of drug response, characterization of signaling pathways, and testing for modifications associated with disease development and progression.
Many different antibody array technologies have been developed, each with particular advantages, disadvantages, and optimal applications. The methods have been demonstrated on various sample types, such as serum, plasma, and other bodily fluids; cell culture supernatants; tissue culture lysates; and resected tumor specimens.
However, the use of antibody microarrays is still challenging due to several limitations:
Manual pre-fractionation of a sample (for example according to size, charge, isoelectric point, polarity, . . . ) is a means to reduce the sample complexity and by that decrease the problem of specificity and cross-reactivity. However, this is very labour intensive, requires manual handling steps between fractionation and detection and is therefore prone to errors.
US 2006/0292649 A1 proposes a biochip wherein one or more analytes, such as proteins of a biological sample, are resolved by isoelectric focusing in a capillary. According to US 2006/0292649 A1 the resolved analytes are immobilized in the capillary by photomimmobilization and detection agents, such as antibodies, are flowed through the capillary which bind to or interact with the analytes, forming antibody-protein complexes. Subsequently, a chemiluminescent substrate is flowed through the capillary and detected with a photon detector.
US 2006/0292558 A1 describes a biochip wherein one or more analytes are resolved by isoelectric focusing in a capillary. Afterwards a serum from a human or non-human subject under analysis is flowed through the capillary and antibodies specific to the immobilized analytes bind to the analytes. Subsequently, a secondary antibody including a detectable marker is introduced, and binds to the immobilized antibody-analyte complexes. By means of the detectable markers, the locations of the antibody-analyte complexes are detected.
However, the biochips disclosed in US 2006/0292649 A1 and US 2006/0292558 A1 require a relatively big analyte volume. As a result, antibodies with a high affinity or a high amount of antibodies with a low affinity must be used, which is in both cases expensive. Furthermore, the antibodies will encounter many different analytes (basically each different antibody species will come in contact with all analyte proteins present in the sample) and therefore cross-reactivity cannot be excluded. Moreover, the photo-immobilization of the analytes to the capillary might disturb analyte/antibody binding, for example if the not well defined immobilization site is identical to the antibody binding site, and therefore decrease sensitivity and accuracy.
The object of the present invention is to overcome the mentioned above problems, to increase the analyte concentration, the accuracy and reproducibility and to provide a fast, improved and automatable solution.
The present invention relates to a biochip for fractionating and detecting analytes, such as proteins, protein-complexes, metabolites, glycoproteins, peptides, DNA, RNA, lipids, fatty acids, carbohydrates and/or other ampholytes, comprising
whereas the isoelectric focusing channel is connectable to the buffer reservoir by opening the first flow barrier and to the detection chamber by opening the second flow barrier, whereas the first and the second flow barrier are arranged on opposite sides of the isoelectric focusing channel.
The first and the second flow barrier have for example the advantage that the sample does not interact with a capture probe in a detection chamber during isoelectric focusing. This is in particular advantageous, if the biochip comprises several detection units for detecting post-translational protein modifications having different isoelectric points, since the post-translational protein modifications can first be separated by isoelectric focusing and then for example be detected by binding to same antibodies located in different detection chambers.
Within the scope of the present invention, the term “pH gradient between a first pH value (pH1) and a second pH value (pH2)” can not only mean that the pH value continuously, for example linearly or exponentially, increases (or decreases) from a first pH value (pH1) to a second pH value (pH2), but also that the pH value incrementally, for example stepwise or stairwise, increases (or decreases) from a first pH value (pH1) to a second pH value (pH2).
For example pH gradient between a first pH value (pH1) and a second pH value (pH2) according to the invention may be realized by at least two, in particular several, gels (gel pads) of which each gel has a particular pH value, wherein the gels are aligned with respect to each other to that effect the pH value increases (or decreases) from gel to gel in the alignment.
According to the invention, the gels can thereby adjacently be aligned. However, within the scope of the present invention it is also understood as pH gradient, if between two gels, in particular between all gels, a fluid, for example water or a buffer, is positioned. Insofar the pH value of the gels increases (or decreases) from gel to gel in the alignment, such an arrangement of, in particular alternating, gels and fluids is also understood as pH gradient within the scope of the present invention, if a liquid has a pH value, which is not between the pH values of the neighboring gels.
The term “micro fluidic” denotes within the context of the present invention that the means characterized by this adjective has a volume of the order of micro liters, for example of ≧0.01 μl to ≦50 μl, in particular of ≧0.1 μl to ≦10 μl.
For example, the sample channel and/or the buffer reservoir/s and/or the detection chamber/s and/or the detection probe reservoir/s (explained below) can have a volume of about ≧0.1 μl to about ≦50 μl, in particular of about ≧1 μl to about ≦10 μl, and/or a width of about ≧0.2 mm to about ≦5 mm, in particular of about ≧0.5 mm to about ≦1.5 mm, and/or a height of about ≧1 μm to about ≦500 μm, in particular of about ≧10 μm to about ≦200 μm, and/or a length of about ≧1 mm to about ≦100 mm, for example of about ≧1 mm to about ≦50 mm, in particular of about ≧5 mm to about ≦20 mm. The isoelectric focusing channel can for example have a volume of about ≧0.1 μl to about ≦50 μl, in particular of about ≧1 μl to about ≦10 μl, and/or a width of about ≧0.2 mm to about ≦5 mm, in particular of about ≧0.5 mm to about ≦1.5 mm, and/or a height of about ≧1 μm to about ≦500 μm, in particular of about ≧10 μm to about ≦200 μm, and/or a length of about ≧1 mm to about ≦100 mm, for example of about ≧1 mm to about ≦50 mm, in particular of about ≧5 mm to about ≦20 mm. The anode and cathode can for example comprise, in particular consist of, platinum, gold, copper, aluminum or doped silicon, preferably coated with a platinum layer.
A biochip according to the invention is capable to fractionate a sample via isoelectric focusing (IEF) in several pH ranges and to detect the fractioned analytes in a second step in the detection unit via immunoassay/microarray techniques, for example by binding to (labeled) antibodies.
The pre-fractionation of the sample via isoelectric focusing has the advantage that the amount of contaminants is decreased as contaminants having a different pI range than the analytes of interest are separated, the analytes of interest are up-concentrated and the reaction volume of the binding reaction is decreased. Due to the increased analyte concentration, the lowered contaminant amount and the lowered reaction volume antibodies with lower affinity can be used. Simultaneously, specificity and sensitivity of the detection step is increased. Thus, crossreactivity can be circumvented or tremendously reduced. Furthermore, the biochip according to the invention is automatable and can be used for rapid digital diagnostic testing (RDT). All needed functions are therefore advantageously performed on one chip without manual handling steps, whereby accuracy and reproducibility is advantageously increased. Moreover, a biochip according to the invention advantageously enables the realization of portable biochemical systems for point-of-care testing. The biochip according to the invention therefore provides a portable, automatable, fast and improved assay without manual handling steps and with low time consumption for the operator.
Within the scope of the present invention, the first and the second flow barrier are each in particular arranged on one side of the isoelectric focusing channel that is parallel to the pH gradient, whereas the first and the second flow barrier are positioned opposite to each other.
According to the invention, the pH gradient may be a mobilized or immobilized pH gradient.
Within the scope of one embodiment of the present invention, the isoelectric focusing channel comprises, in particular is filled with, a fluid having a pH gradient between a first pH value (pH1) and a second pH value (pH2) generated by ampholytes. Alternatively, the isoelectric focusing channel comprises, in particular is filled with, a gel(strip) having a pH gradient between a first pH value (pH1) and a second pH value (pH2) generated by ampholytes.
Within the scope of another embodiment of the present invention, the isoelectric focusing channel comprises, in particular is filled with, a gel having a pH gradient between a first pH value (pH1) and a second pH value (pH2), whereas the pH gradient is generated by polymerizing at least two, in particular adjacent, formulations based on (meth)acrylate(s), in particular acrylate(s), such as acrylamide, N,N′-methylenebisacrylamide, hydroxyethylacrylate, polyethyleneglycolacrylate, diethyleenglycol diacrylate and/or triethyleneglycol diacrylate, methacrylates, such as hydroxyethylmethacrylate, polyethyleneglycolmethacrylate, diethyleenglycol dimethacrylate and/or triethyleneglycol dimethacrylate, thiolene(s) and/or epoxides, having one or more pH-buffering subunits. For example, the pH gradient is generated by polymerizing at least two, in particular adjacent, formulations comprising at least
acrylamide monomers of the formula (I):
N,N′-methylenebisacrylamide monomers of the formula (II):
and
monomers, in particular acrylamide monomers, having one or more pH-buffering subunits (immobiline monomers), whereas the formulations comprise different pH-buffering monomers resulting in a different pH value.
For example, the gel may be generated by polymerizing at least three or at least four, in particular a plurality of, adjacent formulations, whereas the pH value increases or decreases from the first to the last formulation. This can be obtained by using a so-called gradient mixer, into which two formulations with different pH values are inserted and mixed in a certain ratio and subsequently injected into the isoelectric focusing area of the biochip. When during filling of the isoelectric focusing area the ratio of the formulations is continuously changed the pH will vary in the filling direction, the pH being closest to the pH of formulation 1 in the beginning and closest to the pH of formulation 2 at the end of the channel. Then the liquid is polymerized to form the pH-gradient gel.
The gel formulations are generally made by mixing ≧0% by weight to ≦20% by weight, in particular ≧2% by weight to ≦10% by weight, of monomers in deionized water. The ratio acrylamide to bisacrylamide is for example in a range of ≧20:1 to ≦100:1, for example about 40:1. To obtain a good buffering capacity at pH of the used immobiline monomers, the concentration of the immobiline monomers can for example be in a range of ≧1 mM to ≦50 mM, for example about 25 mM.
In the biochip, the pH value of the gel or the fluid preferably increases from the area of the anode to the area of the cathode. In particular, the pH gradient of the gel or the fluid is a positive pH gradient and/or increases from the area of the anode to the area of the cathode.
Within the scope of a preferred embodiment of the present invention, the isoelectric focusing channel is provided with an anode and a cathode inlet. By this means, an electric contact of the electrodes and the fluid or gel in the isoelectric focusing channel can simply be achieved by introducing the anode and cathode, respectively into the inlets. Furthermore, the user can advantageously inject ampholytes of his preference through the anode and cathode inlet and thereby generate a custom made pH gradient.
Within the scope of another preferred embodiment of the present invention, the isoelectric focusing channel has no rectangular shape. For example, the width of the isoelectric focusing channel may vary along the pH gradient. In particular, the width of the isoelectric focusing channel may vary along the pH gradient and symmetrically to the axis of the pH gradient or rather the longitudinal axis of the isoelectric focusing channel or rather the axis of the electric flux lines of the anode-cathode pair. Preferably, the isoelectric focusing channel has a greater width at pH ranges where a high amount of analytes is concentrated by isoelectric focusing or rather is expected after isoelectric focusing and has a smaller width at pH ranges where a low amount of analytes is concentrated by isoelectric focusing or rather is expected after isoelectric focusing.
Tuning the geometry of the isoelectric focusing channel by this way advantageously improves the pre-fractionation efficiency and allows easy transfer into a detection chamber arranged at such a position (see
Within the scope of the present invention, the buffer reservoir preferably comprises at least one buffer. For applying a pressure to the buffer, the buffer reservoir is for example connected to a pressure means.
By such an arrangement, it is possible to transfer the analyte from the isoelectric focusing channel into the detection chamber by applying a pressure to the buffer in the buffer reservoir, opening the flow barriers and flushing the buffer through the isoelectric focusing channel into the detection chamber.
The detection chamber preferably comprises at least one capture probe. According to the invention, a capture probe is capable to interact with the analyte, for example via antibody-antigen, protein-protein, protein-metabolite, DNA-sense/antisense, RNA-DNA, RNA-RNA or receptor-ligand interaction. A capture probe may be a capture antibody, a capture antigen, a capture protein, a capture metabolite, an oligo-DNA, an oligo-RNA or another molecule having a high affinity to an analyte, for example a single chain variable fragments (scFv), biotin or avidin. The capture probe may also be a suitable/matching topography/molecular imprint technology as known in the art.
Preferably, the capture probe is immobilized to the biochip, in particular to the wall/s, for example the side, bottom and/or top wall/s, of the detection chamber. For example, the capture probe is covalently attached to biochip in a modular way by simple chemistry (for example click chemistry). This advantageously allows to use the biochip according to the invention in a flexible way for many purposes. Alternatively, the capture probe is adsorbed/physisorbed to surface, or sterically and/or kinetically and/or magnetically trapped, or embedded in gel/gel-matrix.
Within the scope of a further preferred embodiment of the present invention, the detection chamber or at least one, in particular each, detection chamber of a set of multiple detection chambers comprises at least two or at least three, for example at least four or at least five or at least six, in particular a plurality of, different capture probes. In a preferred embodiment the capture probes are arranged separated form each other, in particular in different/individual spots, for example like an array/microarray.
Advantageously, in this way several analytes, for example proteins, characterized by an identical pI value can be assayed, in particular distinguished and detected, simultaneously in a single run.
Within the scope of the present invention, the detection of analytes can be achieved by sandwich assay/s as well as competitive assay/s. The detection is thereby preferably carried out optically, for example by using fluorescence, surface plasmon resonance or evanescent field detection.
A detection probe, preferably a labeled detection probe, for example a labeled detection antibody, can be applied to the analytes by several ways:
In one embodiment of the present invention, the buffer reservoir comprises at least one detection probe. Thereby the detection probe can bind directly to the analyte, for example the protein, after isoelectric focusing and after opening the first valves but before reaching the capture probe in the detection chamber.
In another embodiment, the detection chamber comprises at least one detection probe.
In yet another embodiment of the present invention, the detection unit further comprises an, in particular microfluidic, detection probe reservoir, whereas the detection probe reservoir is connected or connectable by opening a third flow barrier to the detection chamber. Thereby, the detection probe reservoir preferably comprises at least one detection probe.
Within the scope of these three mentioned above embodiments, the sample channel and/or the buffer reservoir and/or the detection chamber and/or the detection probe reservoir can be provided with an inlet and/or a further flow barrier, for example a septum, through which the detection probe can be inserted manually or automatically, to allow the detection of user defined analytes and/or the removal of fractionated analytes.
These means have the advantage that the addition the detection probe may be achieved by forming a covalent bond (for example by simple click-chemistry, cross-linking, NHS-chemistry or surface grafting), an adsorption, or a DNA/RNA-oligo-interactions, allowing a versatile use of the biochip according to the invention.
Preferably, the buffer reservoir and the detection chamber or the buffer reservoir and the detection probe reservoir are provided with inlets and/or outlets and/or flow barriers. By this means the capture probe area can be washed, for example after applying the detection probe, by flow of a washing buffer from the buffer reservoir through the detection chamber. Within the scope of a preferred embodiment of the present invention, the detection chamber and/or the detection probe reservoir is connected, for example via an outlet, and/or connectable, for example by opening a flow barrier, to a waste chamber, in particular for collecting the washing buffer after washing.
Within the scope of a further preferred embodiment of the present invention, the biochip according to the invention comprises at least two or at least three, for example at least four or at least five or at least six, in particular a plurality of, detection units positioned, in particular well separated from each other, at different pH ranges of the pH gradient of the isoelectric focusing channel. For example, each detection unit is positioned at a different pH range having a width in a range of a hundreds pH value to 2 pH units, preferably of 5 hundreds pH value to 1 pH unit, most preferably of a tenth pH unit to 0.5 pH units. Moreover, according to the invention a detection unit is for example spaced from the neighboring detection unit by a hundreds pH value to 4 pH units, preferably by 5 hundreds pH value to 2 pH units, most preferably by a tenth pH unit to 1 pH unit. However, within the scope of the present invention, it is possible that several detection units are positioned within a range of 1 pH unit.
By other words, each of the detection units is characterized by a pre-defined, in particular narrow, pI range. For example, the pI range at of one detection unit has a width of about a tenth pI value.
By this means only proteins in a very narrow pI range can enter a dedicated detection chamber. This has the advantage that many analytes characterized by several pI values can be assayed, in particular distinguished and detected, simultaneously in a single run.
A post-translational modification can change the isoelectric point of an analyte, in particular a protein or protein-complex. Therefore, the different species of modified and unmodified analyte would be focused at a different pI during isoelectric focusing. By isoelectric focusing the different species can therefore be separated and detected in different detection units of the biochip according to the invention, for example by the same or a different antibodies.
For example, Mitogen-activated protein kinase 1 (also known as ERK2) can by be separated and detected by a biochip according to the invention, is. Mitogen-activated protein kinase 1 is a serine/threonine kinase that phosphorylates MAP2 and myelin basic protein. Mitogen-activated protein kinase 1 and is a member of the Mitogen-activated protein (MAP) kinase family. Mitogen-activated protein kinases, also known as extracellular signal-regulated kinases (ERKs), act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. Mitogen-activated protein kinase 1 in particular is an important proximal component of the Mitogen-activated protein kinase pathway involved in transmitting the signals from growth factors, neurotransmitters and hormones at the cell surface to the transcriptional events in the nucleus. The activation of Mitogen-activated protein kinase 1 requires its phosphorylation by upstream kinases. Thereby, Mitogen-activated protein kinase 1 is activated by mitogen-activated protein kinase kinase 2 (also known as MAP2K2 or MEK2), which phosphorylates neighboring threonine 183 and tyrosine 185 residues, whereas the structures of inactive, unphosphorylated and active, phosphorylated structure was published 1997 by Canagarajah et al. With standard antibody based immuno-detection techniques these two species can not be distinguished and quantified in a simple manner. Therefore disease related correlations can not easily be detected by known devices and methods. But, as the unphosphorylated form has a pI value of 6.523 and the phosphorylated form has a lower pI of 6.373, it is advantageously possible to separate the two forms by a biochip according to the invention and to detect the separated forms in different detection chambers by the same antibody.
Furthermore, the biochip according to the present invention advantageously allows the comparison of the intensities of the signals of several/different analytes, such as proteins, or rather the detection of patterns of presence or absence, respectively, of several/different analytes in the same sample. Moreover, ratios between different analytes, such as proteins, in particular post-translational modified and unmodified proteins, can be assayed. This enables a quantitative and/or semi-quantitative analysis of the ratio of modified and unmodified analyte. Advantageously, the biochip according to the invention can thereby be used for detecting, in particular “fingerprinting”, certain diseases.
However, within the scope of the present invention, the capture probes and/or the corresponding detection probes of the different detection units can at least partially differ to each other.
According to a preferred embodiment the biochip comprises a set of capture and detection probes, in particular a set of capture and detection antibodies, for example an antibody array, distinguishing between different post-translational modifications, for example phosphorylation and ubiquitination, of an analyte, in particular a protein. This advantageously also allows a (semi)-quantitative determination of the ratio of modified and unmodified analyte.
According to another preferred embodiment the biochip comprises a set of capture and detection probes, in particular a set of capture and detection antibodies, for example an antibody array, specific for several proteins and/or enzymes belonging to a certain signaling pathway, for example a pathway up or down regulated in certain diseases.
Generally, all known flow barriers for microfluidic channels, for example microvalves, can be used for a biochip according to the invention.
Within the scope of another embodiment of the present invention, at least one flow barrier is a hydrophobic stop barrier. In particular, the first, the second and/or the third flow barrier of a detection unit is a hydrophobic stop barrier.
A hydrophobic stop barrier can be achieved by coating at least one area inside a capillary, such as the sample channel or a detection chamber or a buffer reservoir or a detection probe reservoir, with a water repellant agent, such as 1H,1H,2H,2H-perfluoroalkyltrihalogenosilanes, for example 1H,1H,2H,2H-perfluorohexyltrichlorosilane, 1H,1H,2H,2H-perfluorooctyltrichlorosilane, 1H,1H,2H,2H-perfluorodecyltrichlorosilane and/or 1H,1H,2H,2H-perfluorododecyltrichlorosilane, in particular 1H,1H,2H,2H-perfluorodecyltrichlorosilane, and/or 1H,1H,2H,2H-perfluoroalkyltrialkoxysilanes, for example 1H,1H,2H,2H-perfluorohexyltrimethoxysilane, 1H,1H,2H,2H-perfluorooctyltrimethoxysilane, 1H,1H,2H,2H-perfluorodecyltrimethoxysilane, 1H,1H,2H,2H-perfluorododecyltrimethoxysilane, 1H,1H,2H,2H-perfluorohexyltriethoxysilane, 1H,1H,2H,2H-perfluorooctyltriethoxysilane, 1H,1H,2H,2H-perfluorodecyltriethoxysilane and/or 1H,1H,2H,2H-perfluorododecyltriethoxysilane, in particular 1H,1H,2H,2H-perfluorodecyltrimethoxy-silane and/or 1H,1H,2H,2H-perfluorodecyltriethoxysilane, and/or Teflon (poly-perfluoroethylene) based compounds, for example Teflon AF1600, and/or a compound of the formula (III):
Such a coating ensures that a liquid, for example the sample, a fraction of the sample or a buffer or a liquid comprising a detection probe or a gel formulation, is stopped at the position of the coating (see
Within the scope of a specially preferred embodiment of the present invention, the biochip comprises a first and a second substrate, whereas the first substrate is slidably abutting the second substrate, whereas the channel/s, reservoir/s, chamber/s and flow barriers of the biochip are realized, in particular at least partially, by recesses in the abutting faces of the first and a second substrate, whereas the flow barriers are openable and closable by shifting one of the substrates with respect to the other from a first to a second position. For example, the isoelectric focusing channel and the flow barriers are realized by in alternation overlapping recesses in the abutting faces of the first and a second substrate.
According to the present invention, the pH gradient of the isoelectric focusing channel can thereby not only realized by filling the isoelectric focusing channel formed by the in alternation overlapping recesses in the abutting faces of the first an the second substrate with a liquid or gel having a continuous pH gradient between a first pH value (pH1) and a second pH value (pH2), but also by filling at least two recesses of the in alternation overlapping recesses in the abutting faces of the first and a second substrate with gels of different pH values, whereas the pH values of the gels increase (or decrease) from gel to gel. Thereby, the other recesses can be filled with a liquid, such as water or a buffer, which for example may have a pH value which is not between the pH values of the neighboring gels.
Within the scope of one embodiment of the present invention, only the recesses in the second substrate forming the isoelectric focusing channel by alternately overlapping with recesses in the first substrate or only the recesses in the first substrate forming the isoelectric focusing channel by alternately overlapping with recesses in the second substrate, in particular only the recesses in the second substrate, are filled with gels of particular pH values, whereas the pH value increases (or decreases) from gel to gel, whereas the recesses in the other substrate, in particular the first substrate, are filled with a liquid, such as water or a buffer.
According to the present invention, preferably both the recesses in the first substrate and the recesses in second substrate are spaced to recesses in the same substrate.
The inlet/s is/are for example realized by an inlet hole or holes in the first and/or the second substrate merging into a recess in the substrate.
For example, the flow barrier, the isoelectric focusing channel, the buffer reservoirs, the detection chambers of the biochip are realized by that,
whereas the recesses are shaped and arranged to that effect that
For example, the first substrate comprises at least two or at least three, for example at least four or least five or at least six, in particular a plurality of, recess pairs and the second substrate comprises at least two or at least three, for example at least four or at least five or at least six, in particular a plurality of, recess triplets.
By shifting the first and the second substrate from the first to the second position, the first and the second recess of a recess pair serve as the first and the second flow barrier of the detection unit.
The first outer recess and the second outer recess of a recess triplet thereby serves as buffer chamber and detection chamber, respectively.
In one embodiment of the present invention, at least two recesses selected from the group of first recesses of recess pairs and middle recesses of recess triplets are filled with gels of different pH values, whereas the pH values of the gels increase (or decrease) from gel to gel.
In a preferred embodiment of the present invention, all first recesses of recess pairs are filled with gels of different pH values, whereas the pH values of the gels increase (or decrease) from gel (or recess) to gel (or recess), whereas all middle recesses of recess triplets are filled with a liquid, such as water or a buffer; or all middle recesses of recess triplets are filled with gels of different pH values, whereas the pH values of the gels increase (or decrease) from gel (or recess) to gel (or recess), whereas all first recesses of recess pairs are filled with a liquid, such as water or a buffer.
For additionally realizing the detection probe reservoir/s and third flow barrier/s
whereas are shaped and arranged to that effect that
For example, the first substrate comprises at least two or at least three, for example at least four or at least five or at least six, in particular a plurality of, recess triplets and the second substrate comprises at least two or at least three, for example at least four or at least five or at least six, in particular a plurality of, recess quartets.
By shifting the first and the second substrate from the first to the second position, the middle, the first outer, and the second outer recess of a recess triplet serve as the first, second and third flow barrier of the detection unit.
The first outer, the second middle and the second outer recess of a recess quartet thereby serves as buffer chamber, detection chamber and detection probe reservoir, respectively.
In one embodiment of the present invention, at least two recesses selected from the group of first outer recesses of recess triplets and first middle recesses of recess quartets are filled with gels of different pH values, whereas the pH values of the gels increase (or decrease) from gel to gel.
In a preferred embodiment of the present invention, all first outer recesses of recess triplets are filled with gels of different pH values, whereas the pH values of the gels increase (or decrease) from gel (or recess) to gel (or recess), whereas all first middle recesses of recess quartets are filled with a liquid, such as water or a buffer; or all first middle recesses of recess quartets are filled with gels of different pH values, whereas the pH values of the gels increase (or decrease) from gel (or recess) to gel (or recess), whereas all first outer recesses of recess triplets are filled with a liquid, such as water or a buffer.
In these embodiments of the present invention, the connection between the microfluidic sample channel and the isoelectric focusing channel may be realized by an inlet recess in the second substrate overlapping in the first position with a first recess of a recess pair, or a first middle recess of a recess triplet.
For providing the isoelectric focusing channel with two inlets, for example for injecting gel formulations and/or ampholytes for generating and/or adjusting the pH gradient, the first and the second substrate may each comprise at least one inlet recess and/or inlet hole, whereas the inlet recesses and/or inlet holes are shaped and arranged to that effect that
Preferably, the inlet recesses of the first and the second substrate do not overlap in the second position a recess of a recess pair, triplet or quartet.
Another subject of the present invention is the use of a biochip according to the invention in
Additional details, features, characteristics and advantages of the object of the invention are disclosed in the subclaims, the figures and the following description of the respective figures and examples, which—in an exemplary fashion—show several preferred embodiments of a chip according to the invention.
a shows a schematic top view of a biochip according to a first embodiment of the present invention having one detection unit.
b to 1d show enlarged schematic top views of the biochip shown in
a shows a schematic top view of a biochip according to a third embodiment of the present invention having an adapted isoelectric focusing channel.
b shows a schematic top view of a biochip according to another form of the third embodiment of the present invention having an adapted isoelectric focusing channel.
a shows a schematic top view of the first position a biochip according to a forth embodiment of the present invention comprising a first and a second substrate.
b shows a schematic cross sectional view of the first position of the biochip shown in
c shows a schematic top view of the second position of the biochip shown in
d shows a schematic cross sectional view of the second position of the biochip shown in
a shows a schematic top view of the first position a biochip according to a fifth embodiment of the present invention comprising a first and a second substrate.
b shows a schematic top view of the second position a biochip shown in
a to 6c show schematic cross-sectional views of a hydrophobic stop barrier according to the present invention.
a shows a schematic top view of a biochip according to a first embodiment of the present invention comprising an isoelectric focusing channel 1 having a pH gradient between a first pH value (pH1) and a second pH value (pH2) and a microfluidic sample channel 2. In the embodiment shown in
However, within the scope of the present invention, it is also possible that the sample channel 2 is designed connectable to the isoelectric focusing channel 1 (not illustrated in
a shows that according to the present invention, the sample channel 2 is preferably connected (or connectable) to the center part of the isoelectric focusing channel 1.
Furthermore
a shows that the biochip according to the present invention comprises an anode-cathode pair 12, 13. To enable isoelectric focusing of analytes 14 in the isoelectric focusing channel 1, the isoelectric focusing channel 1 is at least partially arranged between the anode 12 and the cathode 13 of the anode-cathode pair 12, 13.
The isoelectric focusing channel 1 is preferably filled with a gel such that a pH gradient is formed in which the isoelectric focusing of the analytes 14 can take place. The pH gradient is for example built up between different pH values in the area of the anode 12 and the area of the cathode 13. Preferably, the pH gradient of the gel is positive and/or increases from the area of the anode 12 to the area of the cathode 13.
To achieve an electric contact of the anode and cathode, respectively with the fluid in the isoelectric focusing channel 1 and to inject gel formulations and/or ampholytes for generating and/or adjusting the pH gradient, the isoelectric focusing channel 1 is preferably provided with a not illustrated anode and a cathode inlet.
Upon applying an electric field between the anode 12 and the cathode 13, the analytes 14 in the sample will at least partially move to a place where their isoelectric point (pI) equals the pH value of the gradient in the isoelectric focusing channel 1. There, the net charge, and therefore the net force on the analyte 14, is zero and all analytes with that respective pI will be concentrated.
For detecting analytes 14 concentrated via isoelectric focusing, the biochip according to the invention comprises at least one detection unit 3.
The buffer reservoir 4 preferably comprises at least one buffer. After the focusing step flow barriers 5, 6 are opened to allow buffer in the reservoir 4 to transport the analyte 14 to the detection chamber 7. The detection chamber 7 comprises preferably at least one capture probe 10, which binds to the analyte 14.
In the embodiment shown in
The enlarged schematic top views in
Advantageously, all required sample handling steps are therefore integrated into a single biochip according to the invention.
Moreover
a and 3b show schematic top views of a biochip according to two forms of a third embodiment of the present invention having an adapted isoelectric focusing channel. As shown in
a and 4b show a schematic top view or rather a schematic cross sectional view of a biochip according to a forth embodiment of the present invention comprising a first (upper) 20 and a second (lower) 22 abutting flat substrate. Thereby the two substrates 20, 22 have a shape which allows to shift the abutting sides of the substrates with respect to each other. In particular,
Said substrates 20, 22 comprise a plurality of recesses indicated as 25a, 25b, 25c, 25d, 25e, 26a, 26b, 26c, 26d, 26e in the first substrate 20 and indicated as 21a, 21b, 21c, 21d, 21e, 24a, 24b, 24c, 24d, 24e, 27a, 27b, 27c, 27d, 27e in the second substrate 22, respectively, realizing the flow barriers 25a, 25b, 25c, 25d, 25e, 26a, 26b, 26c, 26d, 26e, the isoelectric focusing channel 21a, 21b, 21c, 21d, 21e, the buffer reservoirs 24a, 24b, 24c, 24d, 24e, the detection chambers 27a, 27b, 27c, 27d, 27e of the biochip.
a shows that the first substrate 20 in particular comprises five recess pairs 25a, 26a, . . . , 25e, 26e having a first 25a, 25b, 25c, 25d, 25e and a second 26a, 26b, 26c, 26d, 26e recess, whereas the second substrate 22 comprises five recess triplets 23a, 23b, 23c, 23d, 23e having a middle recess 21a, 21b, 21c, 21d, 21e, a first outer recess 24a, 24b, 24c, 24d, 24e and a second outer recess 27a, 27b, 27c, 27d, 27e.
Moreover,
The second position of the first 20 and the second 22 substrate is illustrated in
The first outer recess 24a, 24b, 24c, 24d, 24e and the second outer recess 27a, 27b, 27c, 27d, 27e of a recess triplet 23a, 23b, 23c, 23d, 23e thereby serves as buffer chamber and detection chamber, respectively.
Furthermore,
Moreover, in the embodiment shown in
a and 4d show that in the first position, the first axis through the first recesses 25a, 25b, 25c, 25d, 25e of the recess pairs 25a, 26a, . . . , 25e, 26e is arranged parallel above or under the forth axis through the middle recesses 21a, 21b, 21c, 21d, 21e of the recess triplets 23a, 23b, 23c, 23d, 23e.
a and 5b show a schematic top view of the first and second position a biochip according to a fifth embodiment of the present invention. This embodiment of a biochip according to the present invention comprises additionally to the embodiment illustrated by
The recesses are thereby shaped and arranged to that effect that in a first position, the first outer recess 25a of a recess triplet 25a, 26a, 29a overlaps with the first middle recess 21a of one recess quartet 23a or the first outer recess 25b, 25c, 25d, 25e of a recess triplet 25b, 26b, 29b . . . , 25e, 26e, 29e overlaps with the first middle recesses 21a, 21b, 21c, 21d, 21e of two neighboring recess quartets 23a, 23b, 23c, 23d, 23e, forming the isoelectric focusing channel 1.
b shows that in the second position, the first outer recess 25a, 25b, 25c, 25d, 25e of a recess triplet 25a, 26a, 29a . . . , 25e, 26e, 29e overlaps with the first outer recess 24a, 24b, 24c, 24d, 24e and the first middle recess 21a, 21b, 21c, 21d, 21e of a recess quartet 23a, 23b, 23c, 23d, 23e, whereas the middle recess 26a, 26b, 26c, 26d, 26e of the recess triplet 25a, 26a, 29a . . . , 25e, 26e, 29e overlaps with the first middle recess 21a, 21b, 21c, 21d, 21e and the second middle recess 27a, 27b, 27c, 27d, 27e of the recess quartet 23a, 23b, 23c, 23d, 23e, whereas the second outer recess 29a, 29b, 29c, 29d, 29e of the recess triplet 25a, 26a, 29a . . . , 25e, 26e, 29e overlaps with the second middle recess 27a, 27b, 27c, 27d, 27e and the second outer recess 28a, 28b, 28c, 28d, 28e of the recess quartet 23a, 23b, 23c, 23d, 23e, forming five chambers.
The first outer 24a, 24b, 24c, 24d, 24e, the second middle 27a, 27b, 27c, 27d, 27e and the second outer 28a, 28b, 28c, 28d, 28e recess of a recess quartet 23a, 23b, 23c, 23d, 23e thereby serves as buffer chamber, detection chamber and detection probe reservoir, respectively.
Similar to embodiment illustrated in
Thereby, the first middle recesses 21a, 21b, 21c, 21d, 21e, the first outer recesses 24a, 24b, 24c, 24d, 24e, the second middle recesses 27a, 27b, 27c, 27d, 27e and the second outer recess 29a, 29b, 29c, 29d, 29e of several recess quartets 23a, 23b, 23c, 23d, 23e are arranged along a forth, a fifth axis, a sixth or an eighth axis, respectively, whereas the forth, the fifth, the sixth and the eighth axis are parallel to each other and form the same angle, in particular a rectangular angle, with the third axes.
In the first position, the first axis is arranged parallel above or under the forth axis and in the second position, the axis through the recesses of a recess triplet 25a, 26a, 29a . . . , 25e, 26e, 29e is parallel above or under the third axis. The switch between the first and the second position is thereby also achieved by displacing the first substrate 20 with respect to the second substrates 22 for a certain distance, for example corresponding to the width of the first recess in the direction the first axis, along the first or rather forth axis and for another certain, for example corresponding to half the width of the first recess in the direction of the third axis, along the third axis.
a to 6c show schematic cross-sectional views of a hydrophobic stop barrier 5 according to the present invention. As illustrated by
| Number | Date | Country | Kind |
|---|---|---|---|
| 08104112.1 | May 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB09/52089 | 5/19/2009 | WO | 00 | 11/17/2010 |
