WO2010023589A1 - Procédé de préparation d’un gradient de ph dans une biopuce à focalisation isoélectrique - Google Patents

Procédé de préparation d’un gradient de ph dans une biopuce à focalisation isoélectrique Download PDF

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Publication number
WO2010023589A1
WO2010023589A1 PCT/IB2009/053596 IB2009053596W WO2010023589A1 WO 2010023589 A1 WO2010023589 A1 WO 2010023589A1 IB 2009053596 W IB2009053596 W IB 2009053596W WO 2010023589 A1 WO2010023589 A1 WO 2010023589A1
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WIPO (PCT)
Prior art keywords
biochip
monomers
gradient
channel
value
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Application number
PCT/IB2009/053596
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English (en)
Inventor
Roel Penterman
Christopher J. Backhouse
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Koninklijke Philips Electronics N.V.
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Publication of WO2010023589A1 publication Critical patent/WO2010023589A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing

Definitions

  • 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.
  • the separation is carried out by so called iso-electric focusing (IEF).
  • IEF iso-electric focusing
  • analytes are separated on the basis of their iso-electric point by applying an electric field over a pH gradient gel which contains the protein sample.
  • a very effective method to establish a pH gradient in a gel is the use of so-called immobiline monomers. These are acrylamide-monomers with buffering capacity that are co -polymerized with the acrylamide gel matrix.
  • Current pH gradient gels are made by using a gradient mixer containing different pH-mixtures. The resulting gel is freeze-dried and subsequently sliced into strips and glued on a plastic backing. These strips are commercially available as IPG-strips, typically 7 to 24 cm long with both narrow (e.g. pH 6-7) and wide pH gradients (e.g. pH 3-12).
  • Iso-electric focusing is a major tool in protein analysis. However, it requires many manual handling steps and therefore reproducibility is an issue. Moreover it is time-consuming, partly because of the long rehydration times of the freeze-dried IPG strips before the actual separation. In order to overcome these issues miniaturized systems are being developed that ultimately would handle all the required steps to speed up the analysis and improve reproducibility. However, currently there is no microfluidic equivalent of the (macro-) IPG-strips reported. IEF is mainly done by using natural methods or by applying ampholites. The object of the present invention is to overcome the above mentioned problems, to miniaturize the IEF system, to speed up the analysis and to improve reproducibility in order to provide a fast, improved and automatable solution for the preparation of a pH gradient gel.
  • the present invention relates to a method for preparing a pH gradient between a first pH value (pHl) and a second pH value (pH2) in an isoelectric focusing biochip comprising the steps of: a) providing an electrophoresis biochip comprising at least one microfluidic channel and at least one cathode-anode pair of electrodes, b) filling at least two adjacent regions of the mircofluidic channel with monomers of a pH buffered gel each having a different pH value (pHl, pH2) to form an interface between the at least two monomeric gels, c) polymerizing the monomeric pH buffered gels after a diffusion time t.
  • pH gradient between a first pH value (pHl) 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 (pHl) 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 (pHl) to a second pH value (pH2).
  • pH gradient between a first pH value (pHl) 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.
  • microfluidic 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 ⁇ l to ⁇ 50 ⁇ l, in particular of > 0.1 ⁇ l to ⁇ 10 ⁇ l.
  • diffusion time t denotes within the context of the present invention the time starting from the first contact of the at least two monomeric pH buffered gels each having a different pH value (pHl, pH2) at the mutual interface up to the initiation of the polymerization of the gels. The diffusion time is varied according to the intended width of the resultant pH gradient region within the microfluidic channel.
  • the diffusion time t can be chosen between a few seconds and up to 24 hours. Preferably, the diffusion time t is chosen between 1 minute and 2 hours and in particular between 10 minutes and 1 hour.
  • the method for preparing a pH gradient in an IEF microfluidic channel of an IEF biochip according to the invention is automatable and the biochip can be readily 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, the method allows rapid handling because no rehydration step is needed compared to the (macro-) IPG strips.
  • the method according to the invention therefore provides an automatable, fast and improved preparation of a pH gradient in a microfluidic IEF channel without manual handling steps or the need of rehydration of the gel and with low time consumption for the operator.
  • the inventive method can therefore not only be advantageously implemented in pre-fractionation of the sample via isoelectric focusing but also in 2D electrophoresis separation where IEF is followed by a PAGE step and as a single test in itself.
  • Pre-fractionation generally 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
  • the gel may be forming physical and/or chemical bonds with the top and/or bottom substrates of the IEF biochip defining the microfluidic channel.
  • the microfluidic channel is filled with monomers of at least two different gels having a first pH value (pHl) and a second pH value (pH2), respectively, whereas the pH gradient is generated by polymerizing at least two formulations based on (meth)acrylate(s), where the acrylate(s) can be chosen from the group comprising acrylamide, N 5 N'- methylenebisacrylamide, hydroxyethylacrylate, polyethyleneglycolacrylate, diethyleenglycol diacrylate and/or triethyleneglycol diacrylate, where the methacrylates can be chosen from the group comprising hydroxyethylmethacrylate, polyethyleneglycolmethacrylate, diethyleenglycol dimethacrylate and/or triethyleneglycol dimethacrylate, thiolene(s) and/or epoxides, having one or more pH-buffering subunits.
  • the pH gradient is generated by polymerizing
  • 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.
  • the pH- buffering monomers are immobiline monomers.
  • immobiline monomers are immobiline A (buffering the gel at ⁇ pH 4.5) of the formula III: and immobiline B (buffering the gel at ⁇ pH 8.5) of the formula IV:
  • the gel may be generated by polymerizing at least two, at least three, or at least four, as well as a plurality of, adjacent formulations after a diffusion time t, whereas the pH value increases or decreases from the first to the last formulation.
  • This can be obtained according to the inventive method by letting the monomer mixtures of the gels prior to polymerization diffuse into each other over a diffusion time t.
  • 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, is made obsolete by the method of the present invention.
  • 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.
  • the concentration of the immobiline monomers can for example be in a range of > 1 mM to ⁇ 50 mM, for example about 25 mM.
  • the filling of the microfluidic channel of the IEF biochip with the at least two pH buffered monomeric gels can be carried out either in a subsequent manner from one end of the microfluidic channel.
  • the filling of at least two adjacent regions of the channel is done by injection at the two ends of the microfluidic channel, one at each side.
  • the channel may contain at least one venting means to prevent the air from being trapped between the liquids in order to conduct away any air trapped. This can be achieved by providing holes preferably in the top substrate of the IEF biochip.
  • the top and/or bottom substrate of the IEF biochip are patterned with hydrophilic/hydrophobic patterning technique involving silane compounds to achieve the desired behaviour of the substrates at the different hydrophobic and/or hydrophilic areas.
  • a "virtual" channel can be created without walls so that the trapped air can escape easily.
  • the hydrophobic areas may be coated with, for example, perfluorodecyltrichlorosilane ("repel silane”) while the hydrophilic areas can be created by coating the substrate at the intended areas with, for example, methacryloxypropyltrimethoxysilane ("bind silane”) which is capable of providing a chemical bond between the substrate and the gel strip.
  • repel silane perfluorodecyltrichlorosilane
  • bind silane methacryloxypropyltrimethoxysilane
  • a hydrophobic stop can be provided at one of the top or bottom substrate.
  • hydrophobic stop denotes in the context of the present invention a hydrophobic area interrupting the preferably hydrophilic surface of the top and/or bottom substrate defining the microfluidic channel. Again, that can be achieved by the patterning technique described above involving a "repel silane” as hydrophobic agent.
  • the position of the interface as well as its shape can be controlled.
  • the first liquid pH buffered monomeric gel can be filled until the hydrophobic stop is reached.
  • the second liquid pH buffered monomeric gel is injected and the two liquids touch at the remaining hydrophilic top or bottom side of the channel.
  • the liquids can reduce their surface energy by filling the remaining gap and the two liquids can merge completely.
  • at least one of the liquids may be pushed in under pressure.
  • the capillary force in the region of the hydrophobic stop can be increased by temporarily reducing the space between the top and bottom substrate which define the microfluidic channel, for example by pressing the top substrate.
  • electrostatic attraction may be achieved between the two gels by applying a voltage over the channel.
  • a hydrophobic stop can be provided between the top and bottom substrate in a diagonal orientation regarding the horizontal axis of the microfluidic channel.
  • the invention is also relates to an isoelectric focusing biochip comprising at least one microfluidic channel at least partly filled with a pH gradient gel which can be prepared by filling at least two adjacent regions of the mircofluidic channel with monomers of a pH buffered gel each having a different pH value (pHl, pH2) to form an interface between the at least two monomeric gels, and polymerizing the monomeric pH buffered gels after a diffusion time t.
  • a pH gradient gel which can be prepared by filling at least two adjacent regions of the mircofluidic channel with monomers of a pH buffered gel each having a different pH value (pHl, pH2) to form an interface between the at least two monomeric gels, and polymerizing the monomeric pH buffered gels after a diffusion time t.
  • the biochip preferably is sealed, in particular with a removable seal, and/or is placed in a sealed box, for example filled with water. This has the advantage that the biochip can be used immediately when needed and no time-consuming rehydration step is required.
  • Another subject of the present invention is the use of an isoelectric focusing biochip prepared by a method according to the invention in - rapid and sensitive detection of proteins, protein-complexes, metabolites, glycoproteins, peptides, DNA, RNA, lipids, fatty acids, carbohydrates and/or other ampholytes in complex biological mixtures, such as blood, saliva, urine,
  • testing chip for example proteins, protein-complexes, metabolites, glycoproteins, peptides, DNA, RNA, lipids, fatty acids, carbohydrates and/or other ampholytes, for example for on-site (point-of-need) testing or for diagnostics in centralized laboratories or in scientific research,
  • biosensor in particular microfluidic biosensor, used for molecular diagnostics, - a high throughput screening chip for chemistry, pharmaceuticals or molecular biology,
  • Fig. 1 shows a schematic top view of a microfluidic channel of a IEF biochip according to a first embodiment of the present invention having two pH buffered gels.
  • Fig. 2 shows a schematic top view of a microfluidic channel of a IEF biochip according to a second embodiment of the present invention having three adjacent pH buffered gels.
  • Fig. 3 a shows a schematic top view of a microfluidic channel of a IEF biochip according to a third embodiment of the present invention having a linear pH gradient in a diffusion region.
  • Fig. 3b shows a schematic top view of a microfluidic channel of a IEF biochip according to another form of the third embodiment of the present invention having three adjacent pH buffered gels after a longer diffusion time t.
  • Fig. 4a shows a schematic top view of a microfluidic channel of a IEF biochip according to a forth embodiment of the present invention comprising a hydrophobic stop.
  • Fig. 4b shows a schematic top view of a microfluidic channel of a IEF biochip according to a forth embodiment of the present invention after removal of the hydrophobic stop shown in Fig. 4a.
  • Fig. 5 a shows a schematic top view of a microfluidic channel of a IEF biochip according to a fifth embodiment of the present invention comprising a hydrophobic stop only at the bottom substrate.
  • Fig. 5b shows a schematic top view of a microfluidic channel of a IEF biochip according to a forth embodiment of the present invention after removal of the hydrophobic stop shown in Fig. 5a.
  • Fig. 6a and 6b show schematic top views of a microfluidic channel of a
  • IEF biochip according to a sixth embodiment of the present invention before and after removal of a diagonal hydrophobic stop.
  • Fig. 1 shows a schematic top view of a microfluidic channel 2 of a IEF biochip according to a first embodiment of the present invention having inserted two pH buffered gels pHl and pH2. Insertion of the pH buffered monomeric gels can be accomplished, for example, by Eppendorf pipettes with a volume of 1 to 10 ⁇ l.
  • the diffusion time t was chosen to be very short, for example in the region of a few seconds, so that a sharp pH gradient in form of a pH step is achieved.
  • the pH gradient is schematically shown in the diagram above the top view and increases stepwise from pHl to pH2. After the diffusion time t the polymerization is initiated, preferably by photo- initiation. Like that, a better control of the diffusion time and thereby of the width of the gradient region is given.
  • Fig. 2 shows a schematic top view of a microfluidic channel 2 of a IEF biochip according to a second embodiment of the present invention having inserted three adjacent pH buffered gels pHl, pH2, and pH3.
  • the diffusion time t was chosen to be very short, for example in the region of a few seconds, so that a stairwise pH gradient is achieved.
  • the pH gradient is schematically shown in the diagram above the top view and increases stepwise from pHl to pH2 and to pH3.
  • Fig. 3a shows a schematic top view of a microfluidic channel 2 of a IEF biochip according to a third embodiment of the present invention having a more linear pH gradient in a diffusion region 3 compared to the gradients in figures 1 and 2.
  • the diffusion time was chosen to be longer than in the first embodiments, for example 20 minutes, and the diagram above the top view shows a more linear increase of the pH from a region starting with pHl to a region of pH2.
  • Fig. 3b shows a schematic top view of a microfluidic channel 2 of a IEF biochip according to another form of the third embodiment of the present invention after a longer diffusion time t when three different monomeric pH buffered gels pHl, pH2, and pH3 were inserted into the channel.
  • the resulting pH gradient as shown in the diagram above the top view is at least in parts linear and the gradient region is wider than in the preceding embodiment involving only two pH buffered gels.
  • Fig. 4a shows a schematic top view of a microfluidic channel 2 of a IEF biochip according to a forth embodiment of the present invention comprising a hydrophobic stop 4.
  • the hydrophobic stop is positioned perpendicular to the horizontal axis of the channel and provides a border between the two different pH buffered monomeric gels pHl and pH2 after filling into the channel.
  • the hydrophobic stop can be created by coating a predetermined stop area with a so called repel silane, e.g. perfluorodecyltrichlorosilane.
  • the hydrophobic stop then can be removed, in particular by photo-activation, or the stop area can be floated, e.g. by applying a pressure.
  • the region and the shape of the interface of the two adjacent pH buffered monomeric gels can be controlled.
  • Fig. 4b also shows a schematic top view of a micro fluidic channel 2 of a
  • IEF biochip according to a forth embodiment of the present invention after removal or floating of the hydrophobic stop 4 shown in Fig. 4a.
  • Fig. 5a shows a schematic top view of a micro fluidic channel 2 of a IEF biochip according to a fifth embodiment of the present invention comprising a hydrophobic stop 4 only at the bottom substrate 5.
  • a small hydrophobic strop 4 is patterned to interrupt the hydrophilic area on one of the substrates 5.
  • the first liquid can be filled until the hydrophobic stop.
  • the second liquid is injected and the two liquids touch at the hydrophilic side of the channel.
  • the liquid can reduce its surface energy by filling the remaining gap and the two liquids are completely merged.
  • at least one of the liquids may be pushed in under a pressure.
  • Fig. 5b shows a schematic top view of a micro fluidic channel 2 of a IEF biochip according to a fifth embodiment of the present invention after removal or floating of the hydrophobic stop 4 shown in Fig. 5a.
  • Fig. 6a and 6b show schematic top views of a micro fluidic channel of a IEF biochip according to a sixth embodiment of the present invention before and after removal of a diagonal hydrophobic stop 4.
  • the diagonal shape of the hydrophobic stop 4 can vary in its angle and is dependent upon the intended width of the gradient region 3. With a diagonal shaped hydrophobic stop 4 the interface region between the two pH buffered gels pHl and pH2 is much extended in view of the first embodiment shown in figure 1. Furthermore, the diffusion time t can be reduced to, for example, 5 minutes and still a wide pH gradient region 3 is achieved.
  • the invention is illustrated by the following non-limiting example. Example
  • This example shows the preparation of a pH gradient in an isoelectric focusing biochip according to invention containing a 2 mm- wide pH gradient from pH 4.6 to 8.5 and an IEF experiment using the biochip.
  • An exemplary procedure was carried out according to the following steps: Flow chart:
  • Substrate cleaning glass substrates were cleaned with soap Extran 02 (Merck), rinsed and blow-dried.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
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Abstract

La présente invention concerne un procédé de préparation d’un gradient de pH entre une première valeur de pH (pH1) et une deuxième valeur de pH (pH2) dans une biopuce à focalisation isoélectrique.
PCT/IB2009/053596 2008-08-28 2009-08-14 Procédé de préparation d’un gradient de ph dans une biopuce à focalisation isoélectrique WO2010023589A1 (fr)

Applications Claiming Priority (2)

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EP08105169 2008-08-28
EP08105169.0 2008-08-28

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001068225A1 (fr) * 2000-03-15 2001-09-20 Proteosys Ag Focalisation isoelectrique pour micropreparation
WO2003008977A2 (fr) * 2001-07-16 2003-01-30 Protein, Forest, Inc. Matrices, reseaux, systemes et procedes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001068225A1 (fr) * 2000-03-15 2001-09-20 Proteosys Ag Focalisation isoelectrique pour micropreparation
WO2003008977A2 (fr) * 2001-07-16 2003-01-30 Protein, Forest, Inc. Matrices, reseaux, systemes et procedes

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