WO2010023589A1

WO2010023589A1 - Method for preparing a ph gradient in an isoelectric focusing biochip

Info

Publication number: WO2010023589A1
Application number: PCT/IB2009/053596
Authority: WO
Inventors: Roel Penterman; Christopher J. Backhouse
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2008-08-28
Filing date: 2009-08-14
Publication date: 2010-03-04

Abstract

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.

Description

METHOD FOR PREPARING A PH GRADIENT IN AN ISOELECTRIC FOCUSING BIOCHIP

FIELD OF THE INVENTION

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). In iso-electric focusing (IEF) 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.

BACKGROUND OF THE INVENTION

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.

SUMMARY OF THE INVENTION

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. When two adjacent regions are filled with the gel mixtures and they merge prior to the polymerization, diffusion of the different gel monomers results in a pH-gradient in the region around the interface. Within the scope of the present invention, the term "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).

For example 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.

The term "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 μl to < 50 μl, in particular of > 0.1 μl to < 10 μl. The term "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. For example, 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

In one embodiment of the inventive method 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.

Within the scope of another embodiment of the present invention, 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, N₅N'- 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. For example, the pH gradient is generated by polymerizing at least two formulations comprising at least acrylamide monomers of the formula (I):

N,N'-methylenebisacrylamide monomers of the formula (II):

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.

In another preferred embodiment of the present inventive method the pH- buffering monomers are immobiline monomers. Particularly preferred examples of such 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:

For example, 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.

In other words, 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. 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. 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. Preferably, 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. In this case, 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.

In a preferred embodiment of the present inventive method 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.

Like that, 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.

In another preferred embodiment of the present invention a hydrophobic stop can be provided at one of the top or bottom substrate.

The term "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.

Like that, the position of the interface as well as its shape can be controlled. For example, the first liquid pH buffered monomeric gel can be filled until the hydrophobic stop is reached. Subsequently, 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. Preferably, in order to facilitate the merging of the two liquid fronts, at least one of the liquids may be pushed in under pressure. Alternatively, 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. In another alternative, electrostatic attraction may be achieved between the two gels by applying a voltage over the channel.

According to another preferred embodiment of the present invention a hydrophobic stop can be provided between the top and bottom substrate in a diagonal orientation regarding the horizontal axis of the microfluidic channel. By that, the interface area of the two adjacent gels is increased and the diffusion distances perpendicular to the horizontal axis of the gel strip is much smaller than the length of the formed gradient region. As a result, the diffusion time t can be reduced as well as a much extended pH gradient region can be achieved.

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.

To ensure that the pH gradient gels in the biochip prepared according to the inventive method stay hydrated during storage, 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,

- a 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,

- a biosensor, in particular microfluidic biosensor, used for molecular diagnostics, - a high throughput screening chip for chemistry, pharmaceuticals or molecular biology,

- a protein diagnostic biochip for cardiology, infectious diseases, new born screening, oncology, food, environment and/or metabolomics, and/or - a biochip for the detection and quantitation of proteins with posttranslational modifications and/or ratios between modified and unmodified species of the same protein.

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

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.

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.

DETAILED DESCRIPTION OF EMBODIMENTS

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 μl. 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. Like in figure 1, 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. In this embodiment 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. Like that, 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. To control the position of the interface as well as its shape 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. Subsequently, 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. To facilitate the liquid fronts merge, at least one of the liquids may be pushed in under a pressure. Alternatively, the capillary force in the stop region can be increased by temporarily reducing the spacing between the two substrates by pressing the top substrate. Yet another alternative is using electrostatic attraction by applying a high voltage over the channel 2. 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.

10 min. UV-ozone UVP- 100

^■ Methacryloxypropyltrimethoxysilane (Fluka) deposition in a dessicator (lmBar) for 1 hour

^■ Masking: scotch tape. ■ 15 min. UV-ozone UVP-100

^■ Perfluorodecyltrichlorosilane (ABCR) deposition in dessicator (lmBar) for 1 hour)

^■ Removal of scotch tape

^■ Applying 200 μm spacers (Scotch tape) on bottom substrate ■ Aligning of top and bottom substrate under a microscopy (patterns on both substrates were visible).

^■ PAGE Hydrogel mix: 7 % acrylamide/bis-acrylamide (ratio 37.5:1), dissolved in de-ionized water with 1 wt% photoinitiator Irgacure 2959. Moreover, to the pH 4.6 gel mixture 25mM Immobiline pH 4.6 (Fluka) was added and to the pH 8.5 gel mixture 25mM Immobiline pH

8.5 (Fluka) was added.

^■ Filling of channel by using Eppendorf 1 - 10 μl syringe, .

^■ UV-exposure (Philips PLlO, 3 mWcm^"2) in N2-chamber for 20 minutes. Iso-electric focusing of "IEF standard protein mix" (BioRad).

^■ Anode buffer: 7mM phosphoric acid

^■ Cathode buffer: 2OmM arginine + 2OmM lysine ^■ Voltage: max 50V

^■ Current: max 20 μA

^■ Time: approx. 1 hour

This resulted in multiple visible bands in the gradient region that can be ascribed to Myoglobin, Human hemoglobin A and Human hemoglobin C. Moreover, at the anode side Phycocyanin (pH 4.5) was observed and close to the cathode Cytochrome C (pH 9.5) was found.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

Claims

CLAIMS:

1. 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.

2. A method according to claim 1 characterized in that the diffusion time t is chosen between 1 minute and 2 hours and in particular between 10 minutes and 1 hour.

3. A method according to claim 1 or 2 characterized in that the gel is forming physical and or chemical bonds with the top and/or bottom substrates of the IEF biochip defining the microfluidic channel.

4. A method according to claim 1 characterized in that 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) are chosen from the group comprising acrylamide, N₅N'- methylenebisacrylamide, hydroxyethylacrylate, polyethyleneglycolacrylate, diethyleenglycol diacrylate and/or triethyleneglycol diacrylate, and the methacrylates are 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 after a diffusion time t.

5. A method according to claim 4 characterized in that the pH gradient is generated by polymerizing at least two formulations comprising at least acrylamide monomers of the formula (I):

o AA ,

N,N'-methylenebisacrylamide monomers of the formula (II):

H H

bA ^υ , and monomers having one or more pH-buffering subunits, whereas the formulations comprise different pH-buffering monomers resulting in a different pH value.

6. A method according to claim 5 characterized in that the pH-buffering monomers are immobiline monomers.

7. A method according to claim 3 characterized in that the top and/or bottom substrate of the isoelectric focusing biochip are patterned with hydrophilic/hydrophobic patterning technique involving silane compounds.

8. A method according to claim 7 characterized in that a hydrophobic stop is provided at one of the top or bottom substrate.

9. A method according to claim 1 characterized in that at least one of the liquids is pushed into the channel under pressure.

10. A method according to claim 1 characterized in that the space between the top and bottom substrate which define the microfluidic channel is temporarily reduced.

11. A method according to claim 1 characterized in that a voltage is applied over the channel.

12. A method according to claim 8 characterized in that a hydrophobic stop is provided between the top and bottom substrate in a diagonal orientation regarding the horizontal axis of the micro fluidic channel.

13. A isoelectric focusing biochip comprising at least one microfluidic channel at least partly filled with a polymer pH gradient gel which can be prepared by filling at least two adjacent regions of the mirco fluidic 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.

14. A biochip according to claim 13 characterized in that the biochip is sealed with a removable seal for storage.

15. Use of an isoelectric focusing biochip prepared according to any of the claims 1 to 12 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, - a 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, - a biosensor, in particular microfluidic biosensor, used for molecular diagnostics, a high throughput screening chip for chemistry, pharmaceuticals or molecular biology, a protein diagnostic biochip for cardiology, infectious diseases, new born screening, oncology, food, environment and/or metabolomics, and/or a biochip for the detection and quantitation of proteins with posttranslational modifications and/or ratios between modified and unmodified species of the same protein.