Patent Application
20070284308
A detailed description of various aspects, features and embodiments of the present invention is provided herein with reference to the accompanying drawings, which are briefly described below. The drawings are illustrative and are not necessarily drawn to scale. The drawings illustrate various aspects or features of the present invention and may illustrate one or more embodiment(s) or example(s) of the present invention in whole or in part. A reference numeral, letter, and/or symbol that is used in one drawing to refer to a particular element or feature may be used in another drawing to refer to a like element or feature.
The present invention provides an immobilized-enzyme device, or microreactor, for characterization of a biomolecule or a component thereof, such as a protein and/or a peptide. The invention further provides a method of preparing such a device and a method of using such a device.
In the description of the invention herein, it will be understood that a word appearing in the singular encompasses its plural counterpart, and a word appearing in the plural encompasses its singular counterpart, unless implicitly or explicitly understood or stated otherwise. Further, it will be understood that for any given component described herein, any of the possible candidates or alternatives listed for that component, may generally be used individually or in combination with one another, unless implicitly or explicitly understood or stated otherwise. Additionally, it will be understood that any list of such candidates or alternatives, is merely illustrative, not limiting, unless implicitly or explicitly understood or stated otherwise.
According to one aspect of the invention, an enzyme is immobilized on the surface of a polymerized sol-gel (PSG) material, and preferably, a photopolymerized sol-gel material. Such polymerized sol-gel materials are porous and are amenable to design or modification to achieve desirable porous characteristics. A photopolymerized sol-gel material is preferred, as it can be prepared and modified at low temperature, such that problems associated with higher temperatures, such as the cracking of the material or the thermal degrading, deactivation, and/or destruction of modifiers, are reduced, minimized, or avoided. The microenvironment in the monolith may facilitate or may be at least partially responsible for enhancement of enzymatic activity associated with the immobilized enzyme.
The enzyme may be immobilized relative to the PSG material by way of covalent bonds or linkages formed between the enzyme and the surface of the PSG material. Covalent bonds are used, as non-covalent bonds or linkages, such as those associated with ionic or electrostatic interactions, or with van der Waals forces, are more likely to push the enzyme out somewhat from the PSG material and are much less permanent. Further, covalent chemical bonds are used in preference to physical enzyme-immobilizing or capturing vehicles, such as enzyme-entrapment or encapsulation. (See for example, D. Avnir et al., Enzymes and Other Proteins Entrapped in Sol-Gel Materials, Chem. Mater. 1994 (Invited Review), 6, 1605-1614.) This is because, comparatively, the covalent chemical bonds provide more robust and stable enzyme immobilization. Additionally, it is believed that chemical immobilization of enzymes may reduce or minimize enzyme autolysis, as the enzymes, by virtue of this chemical immobilization, are spatially distant enough to reduce, minimize, or avoid enzyme self-degradation. Thus, as used herein, any reference to linkage, bonding or immobilization of the enzyme relative to the surface of the PSG material, or any associated or corresponding terms, generally refers to covalent linkage, bonding or immobilization, via chemical means. Further, as used herein, a reference to chemical means encompasses photochemical means.
A greater amount of enzyme covalently bound to the surface of the PSG material generally corresponds to a greater amount of substrate, whether protein, peptide, or artificial substrate, that is hydrolyzed. Even so, even a small amount, such as an amount on the order of less than micromolar, such as nanomolar or picomolar, for example, of enzyme bound to the PSG material in this manner, will result in a higher rate of hydrolysis, as compared to the same amount of enzyme in bulk solution.
In one example, oligomers of methacrylate-substituted silicate reagents are formed using sol-gel chemistry. By way of example, such an oligomer may be an oligomer of tetramethylorthosilicate. The oligomers are photopolymerized to provide a PSG material comprising a porous network of both organic and inorganic components. Alternatively, an inorganic PSG material, such as composed only of tetralkoxysilane, for example, may be used, although in such a case, the PSG monolith itself does not play an active role in separation. Such inorganic PSG material would not be used for CEC, for example. The PSG material is then derivatized with a linker, as further described herein, following which an enzyme is immobilized on the PSG material to provide an immobilized-enzyme microreactor. The photopolymerization process determines the dimensions, such as a length, of the immobilized-enzyme microreactor, as further described herein. When the immobilized-enzyme microreactor is short enough, it may not play a significant role in the separation of digestion products. In such a case, separation may be carried out downstream relative to the microreactor, such as via CE or CEC, for example.
According to one aspect of the present invention, an appropriate PSG material is derivatized with a linker that comprises a reactive functional group. The derivatization process generally involves the functionalization of the PSG monolith, via its free silanol groups, with a suitable linker that has a reactive functional group suitable for interaction with a suitable enzyme. An example of a suitable linker is an alkoxysilane, such as a silicate, that comprises an aldehyde or a succinimide group. An enzyme having an active amine functional group is then reacted with the derivatized PSG material. Examples of suitable enzymes having an active amine functional group include trypsin, pepsin, and chymotrypsin. Any unreacted linker is rinsed from the derivatized PSG material using a suitable rinsing agent, such as toluene or another appropriate organic solvent, such as ethanol, for example.
When the linker comprises an aldehyde functional group, an amine of the enzyme and the aldehyde on the PSG surface react at low temperature, such as from about 4° C. to about 23° C., or room temperature (about 20° C. to about 25° C.), to form an imine bond, typically in a period of from about 30 to about 60 minutes. In general, the amine of the enzyme may not be important to the bioactivity of the enzyme. This reaction is associated with Schiff chemistry. When the linker comprises a succinimide functional group, the amine of the enzyme and the succinimide on the PSG surface readily react at this low temperature. By way of example, the reaction may take place in a relatively short period of from about 30 to about 60 minutes at a temperature of from about 4° C. to about 23° C., or room temperature (from about 20° C. to about 25° C.). Use of a linker with a succinimide functional group may be advantageous in that the resulting PSG-bound enzyme is less susceptible to water hydrolysis than is a PSG-bound enzyme resulting from Schiff chemistry. Regardless of the type of derivatization reaction employed, the reaction between an enzyme and the derivatized PSG material results in the binding of the enzyme to the PSG surface, preferably via enzyme-immobilizing covalent bonds.
According to another aspect of the invention, a device comprising an immobilized-enzyme microreactor that is integrated in a separation channel is provided. The separation channel can be a fused-silica capillary column that is useful for CE separations. Merely by way of example, a 75-μm inner diameter×365-μm outer diameter fused-silica capillary from Polymicro Technologies of Phoenix, Ariz. may be used. The microreactor can be of any length, such as from about 1 cm to about 10 cm in length, merely by way of example. Once the microreactor is integrated into the separation channel, a sample containing a peptide substrate or a protein substrate that is suitable for digestion by the immobilized enzyme is introduced into the microreactor. This introduction may be facilitated by the application of any suitable pressure or voltage. Merely by way of example, a suitable pressure may be from about 0.5 psi to about 20 psi, which may depend on the instrumentation employed, and a suitable voltage may be from about 1 kV to about 5 kV.
The digestion reaction can be completed within seconds or minutes, such as from about 5 seconds and about 60 minutes, such as from about 5 seconds to about 15 minutes, depending on the reaction temperature, which may be from about 15° C. to about 40° C., for example. The digestion reaction may take longer, although this is not generally desired, as one of the advantages of the invention is the speed of the digestion reaction. A higher reaction temperature generally corresponds to a faster reaction. Longer reaction times, such as those from about 15 minutes to about 60 minutes or even longer, merely by way of example, may be used where it is desired to preconcentrate the sample.
Once the digestion reaction is completed, a voltage is applied across the capillary column, such that the digestion product(s) and any remaining substrate are separated by capillary electrophoresis. Merely by way of example, a suitable voltage may be from about 1 kV, such as about 5 kV, to about 30 kV. Under the influence of the applied voltage, the substrate and the product from the microreactor move into the open section of the capillary column for separation by electrophoresis. It will be understood that any other suitable means of moving the solution through the column, such as pressure, may be used. Any remaining substrate or product is detected by ultraviolet radiation absorption detection (UV absorption) when it passes through a detection window.
According to yet another aspect of the invention, a device comprising an immobilized-enzyme microreactor in a micropipet tip is provided. This microreactor is prepared by introducing a volume of a sol-gel reaction solution, as further described herein, into a micropipet tip prior to a photopolymerization reaction. The length of the microreactor is controlled by this volume of solution. The solution is then photopolymerized to form a PSG material and an enzyme is bound to a surface of the PSG material using a procedure similar to that described above. It may be desirable or necessary to modify the inner surface of the micropipet tip, such as via oxidation, for example, in advance of photopolymerization to facilitate or to ensure covalent bonding of the monolith to the inner surface of the tip.
In use, a solution comprising a peptide substrate or a protein substrate is provided and allowed to flow through the microreactor. The solution may be an aqueous solution comprising the substrate in a buffer. The substrate may be provided in a nanomolar, or sub-micromolar to micromolar concentration range, the selection of which may depend on the tolerances of the detection instrumentation employed. Merely by way of example, sub-micromolar to micromolar substrate concentrations may be suitable when UV absorption is used. The eluate from the microreactor, which comprises the digestion product(s) and any remaining substrate, is collected, whereupon it is separated and analyzed, typically using capillary electrophoresis. Any remaining substrate or product is detected, such as by UV absorption, when it passes through a detection window.
The present invention provides a method for preparing an immobilized-enzyme microreactor. In this method, a PSG material is prepared from a metalorganic monomer and other agents or reagents, such as an organic monomer, a catalyst, a pore template or porogen, and/or a photoinitiator, as further described herein. Various physical properties of the resulting PSG material, or monolith, such as its porosity, pore size, and/or hydrophobicity, can be fine-tuned by adjusting various reaction parameters, such as a molar ratio of the reagents.
A schematic representation of a synthesis of a PSG material or monolith is shown in
A suitable metalorganic monomer may also comprise a photoactive group or a non-hydrolyzable photoactive group, such as a methacrylate group. An example of such a metalorganic monomer is trimethoxysilypropyl methacrylate, also known as methacryloxypropyltrimethoxysilane, as shown in
A metalorganic monomer, such as a metalorganic monomer that either does or does not comprise a photoactive group, may be combined with an organic monomer having such a photoactive group or a hydrophilic organic monomer having such a photoactive group, such as polyethylene glycol dimethyacrylate. Further, a metalorganic monomer may be combined with a photoinitiator, as shown in
The metalorganic monomer just described, or the combination of the metalorganic monomer and the organic monomer just described, or the combination of the metalorganic monomer and the photoinitiator just described, is combined with an acid catalyst, such as hydrochloric acid, as shown in
A porogen or a combination of porogens may be combined with the metalorganic monomer, any possible organic monomer, and the catalyst during the above-described hydrolysis process, or may be combined with the above-described combination following the hydrolysis process, to partially condense the metalorganic monomer, as shown in
The porogen may comprise a solvent or a combination of solvents, such as toluene or a 1:1 mixture of hexane and toluene, for example, a polymer or a combination of solvents, such as poly(methyl methacrylate) or polystyrene, for example, an inorganic salt, such as sodium chloride, which may be in the form of a powder, or sodium sulfate, for example, or any suitable combination thereof. Other suitable porogens include, but are not limited to, benzene, acetonitrile, isooctane, hexane(s), alcohol(s), tetrahydrofuran, acetone, and any suitable combination of any of the above-mentioned possible porogens.
The porogen provides a molecular template for the formation of pores within the porous matrix of the ultimate monolith. The porosity, pore size, and/or pore shape of the porous matrix of the ultimate monolith can be controlled by the type of porogen used and its concentration or volume in the above-mentioned combination. Merely by way of example, a molar or volume ratio of monomer to porogen may be selected and/or adjusted to form desirable pores in the porous matrix of the ultimate monolith, such that the monolith has desirable physical properties, such as porosity, pore size, and/or pore shape.
After the above-described combination has been partially condensed, it is polymerized to form a porous matrix. Preferably, the combination is photopolymerized, as shown in
PSG monoliths useful in the present invention are generally easy to produce, as demonstrated above, have high porosity, and can be chemically modified. A suitable PSG monolith may have a pore size in the range of 0.5 μm to about 20 μm in diameter, on average, by way of example. Advantageously, the porous characteristics of such PSG monoliths, such as the size, shape, and number of pores in the polymer matrix of the monolith, may be controlled or adjusted by a number of variables, such as the amount of cross-linking monomer employed, the quality of the porogenic solvent employed, and/or the molar ratio of the monomer and the porogenic solvent employed. It is contemplated that different pore size distributions, such as two or more different pore size distributions, may be provided by a PSG monolith. Merely by way of example, there may be relatively large-sized through-pores, such as through-pores on the order of about 0.5 to about a micron up to several microns, such as up to about 20 microns, for example, and relatively small-sized pores, such as pores of less than about a micron, such as about 0.5 microns or less, for example.
According to one example, a metalorganic monomer and a hydrophilic organic monomer, such as polyethylene glycol dimethacrylate, are mixed for about 15 to about 30 minutes with an acid catalyst, such as hydrochloric acid, to promote hydrolysis and condensation of the metalorganic monomer. A photoinitiator, such as Irgacuare 1800, is added to the resulting solution, and the combination is stirred for about 10 to about 30 minutes at room temperature, or about 20° C. to about 25° C. To the resulting combination, an appropriate volume (such as 70% to 90% of the total solution volume) of a porogen template is added. The resulting sol-gel solution is now ready for photopolymerization, such as that described previously.
According to one application, the PSG material or monolith may be prepared in a capillary column 10, as shown in
A sol-gel solution 30, such as that described in the example above, is introduced into a suitable capillary column 10 and the column is sealed at its inlet end 12 and its outlet end 14. Any suitable sealing means or method (not shown) may be used, such as the use of removable seals, merely by way of example. The sol-gel solution 30 is typically introduced via the inlet 12 of the capillary 10 at some distance D away from the inlet, as shown in
The capillary column 10 may have an outside radiation-blocking coating (not shown), such as a polyimide coating. In such a case, a section of the coating is removed to create a radiation-transparent window 18 through which radiation may be passed to photopolymerize the sol-gel solution. Where the coating remains, radiation is blocked and the sol-gel solution does not photopolymerize. Alternatively, the capillary column may have radiation-transparent walls. In such a case, the column may be covered with a radiation-blocking material to block radiation exposure where desired, while leaving a radiation transparent window 18. In either case, the length of the radiation-transparent window 18 of the capillary controls the length L1 of the PSG material in the column. The length L1 may be on the order of from about 1 cm to about 10 cm, merely by way of example, and may be selected as appropriate relative to the dimensions of the column 10 and/or relative to the particular application.
After the sol-gel solution 30 is introduced in the capillary 10 and the radiation transparent window 18 is appropriately provided, the capillary 10 is exposed to suitable radiation for a time sufficient for formation of the monolith. Merely by way of example, the capillary may be exposed to ultraviolet radiation, at a wavelength of about 360 nm to about 365 nm, or about 365 nm, for about 5 minutes, as described above in relation to
According to another application, the PSG material or monolith may be prepared in a micropipet tip 20, as shown in
The filled micropipet tip 20 is then exposed to suitable radiation for a time sufficient for formation of the monolith. Merely by way of example, the micropipet tip 20 may be exposed to UV radiation, at a wavelength of about 360 nm to about 365 nm, or about 365 nm, for about 5 to about 10 minutes, as described above in relation to
According to the present invention, a surface of a PSG material or monolith, such as any of those described above, is derivatized with a linker, or a reagent, comprising a functional group that facilitates the binding of an enzyme to the surface. An example of such a derivatization is shown in
In the derivatization process, a PSG material is rinsed free of water with a rinsing agent, such as an organic solvent, for example. By way of example, suitable solvents include acetonitrile, toluene, and ethanol, the latter being preferred. A solution comprising a derivatizing reagent, such as the above-described silicate reagent, at an appropriate concentration, such as about 30 volume percent relative to the solution, is prepared. This solution is applied to the PSG material and is allowed to flow through the PSG material under conditions sufficient for suitable derivatization, such as at a temperature of about 4° C. to about 23° C., or room temperature (from about 20° C. to about 25° C.), for a period of about 30 to 60 minutes, merely by way of example, as shown in
In the enzyme-bonding process, the PSG material is conditioned with a conditioning agent, such as a buffer solution that will not interfere with the bioactivity of the enzyme. An example of a suitable buffer solution is a phosphate-buffered saline (PBS) solution (about 50 mM, for example), as shown in
A derivatized PSG material 40 may be incorporated into a microreactor device, such as the integrated microreactor-column 10 (
It will be understood that any enzyme suitable for immobilization on a PSG material and for use in a separation process are contemplated herein. As mentioned above, trypsin, pepsin, and chymotrypsin are merely examples. Further examples include, but are not limited to, malate dehydrogenase, citrate lyase, isocitrate dehydrogenase, and lactate dehydrogenase, which can be used in various assays, such as assays of organic acids. By way of example, malate dehydrogenase, citrate lyase, isocitrate dehydrogenase, and lactate dehydrogenase can be used for the enzymatic analysis of oxaloacetate, citrate, D-isocitrate or succinate, and lactate or pyruvate, respectively. It will be appreciated that there are numerous enzymes that could be immobilized relative to a PSG material as contemplated herein, and numerous resulting immobilized-enzyme PSG materials that could be employed in separation processes, such as biomolecule separation processes involving the separation of one or more proteins, oligonucleotides, peptides, steroids, organic acids, or any combination thereof, by any suitable separation means.
Additionally, it will be understood that any means of immobilizing the enzyme relative to the PSG material, and any combination of such means, are contemplated herein. As mentioned above, a flow-through technique, wherein an enzyme solution flows through a PSG material and the immobilization is allowed to proceed at a low temperature for a number of hours, is one example. Merely by way of example, a further example is a pump-through technique, wherein an enzyme solution is pumped through a PSG material and the immobilization is allowed to proceed at a low temperature for a suitable time, such as a number of hours. Yet another example is incubation, such as incubation of a PSG material in an enzyme solution at an appropriate temperature for an appropriate time, such as about 4° C. to about 40° C. for about 30 minutes to about 24 hours, such as about 15 hours to about 24 hours.
As described above, an immobilized-enzyme microreactor may be incorporated into a device, such as a capillary column or a micropipet tip, for example. A microreactor-integrated CE column 50 is schematically shown in whole or in part in
As mentioned in connection with the integrated capillary column of
The sample may be introduced into the column by the application of pressure and may be moved to the microreactor section of the column by voltage, although any suitable introduction or movement process may be used. In one example, a voltage is applied to the column, as represented by a circled positive (+) sign at the column inlet 52 and a circled negative (−) sign at the column outlet 60, merely by way of illustration, to facilitate electroosmotic flow from the inlet to the outlet. As shown in
It is possible to determine the distance that the substrate bands needs to travel relative to an integrated device, such as the column 50, in order to overlap or be in the immobilized-enzyme microreactor section of the device, based on electroosmotic flow, for example. When the substrate band has traveled the required distance or is otherwise in the correct position relative to the microreactor, the enzymatic reaction is allowed to proceed for a certain amount of time, such as from about 5 seconds to about 60 minutes, such as from about 5 seconds to about 15 minutes, merely by way of example. The amount of time may vary according to the reaction conditions, such as temperature.
As a running buffer solution is applied to the column, the substrate and the reaction product(s) move downstream into the separation area 56 and on to the detection area 58, as shown in
Experimental examples pertaining to the present invention are provided below, starting with the following experimental example.
Reagents and Chemicals. All buffers and solutions were prepared using a Milli-Q water purification system from Millipore (Billerica, Mass.) and degassing by sonication for 5 minutes prior to use. Methacryloxypropyltrimethoxy-silane (MPTMS), polyethylene glycol dimethacrylate with an average molecular weight of 330 Daltons (PEG-DM), toluene, Nα-benzoyl-
Trypsin-PSG Microreactor Preparation. A 30-cm-long, fused-silica capillary column was obtained from Polymicro Technologies (Phoenix, Ariz.). The capillary column was 75-μm in inner diameter and 365-μm in outer diameter and was coated with polyimide. At a point at approximately 5 mm from the inlet of the capillary, a 1-cm-long stripe of polyimide was removed from the outside of the capillary column using a razor blade to provide an irradiation window.
The parent PSG monolith was prepared using Solution A and Solution B. The PSG reaction solution was prepared with Solution B. Solution A was made by mixing 400 mL of MPTMS, 175 μL of PEG-DM, and 100 μL 0.12 N HCl at room temperature and in the dark for 30 min. Solution B was made by first dissolving 40 mg of Irgacure 1800 in 320 μL of toluene followed by the addition of 80 μL of Solution A, which was stirred at room temperature and in the dark for 5 minutes. The above-mentioned capillary column with the 1-cm irradiation window near its inlet was flushed with approximately 200 μL of Solution B. The ends of the capillary were sealed with parafilm plugs and the sealed capillary was irradiated in a Spectrolinker UV crosslinker oven (Spectronix Corp.) with predominantly 365 nm bulbs for 5 minutes. The capillary was unsealed, and unreacted starting materials were pressure-rinsed from the capillary columns using a syringe filled with ethanol. Any monolith columns that were not used were rinsed and stored in acetonitrile.
The parent monolith was derivatized with triethoxysilyl aldehyde (BioConext from United Chemical Technologies, Inc.) or with triethoxysilyl butraldehyde (Gelest, Inc.). In this process, the parent PSG monolith was rinsed with toluene to remove as much water in the capillary column as possible. After rinsing with toluene, approximately 100 μL of a 30% by volume solution of the alkoxysilane reagent (referred to as the “linker”) in toluene was allowed to flow through the 30-cm capillary column for 30 to 60 minutes at room temperature. The alkoxy groups of the linker reacted with free silanol groups on the PSG monolith. Any unreacted linker was removed from the capillary column by rinsing the column with toluene, followed by acetonitrile, if the derivatized PSG monolith was not further reacted with an enzyme solution.
An aldehyde-derivatized PSG monolith was prepared for enzyme immobilization by first flushing the column with 10 mM phosphate-buffered saline (PBS). A freshly prepared solution of trypsin (10 mg/mL) in PBS was then allowed to flow through the capillary column. Approximately 100 μL of the trypsin solution flowed through the capillary. The ends of the capillary were then capped and the enzyme-filled capillary was allowed to sit overnight in a refrigerator at 4° C. overnight (about 17 hours) after which the capillary was flushed with PBS and either stored at 4° C. or loaded into a Beckman P/ACE column cartridge for immediate use.
Multiple immobilized-enzyme, microreactor capillary columns were prepared as described above. When any such capillary column was not put to use, it was stored at 4° C.
The capillary column was checked to see that no unbound trypsin was left in the column. In this determination, the enzymatic activity of the rinse liquid, or the PBS that ran through the column, was checked for the presence of trypsin using BAEE. BAEE was allowed to react with any trypsin that might be left in the rinse liquid. If trypsin was present in the rinse liquid, it reacted with BAEE to produce the enzymatic product, BA, which was then detected in an analysis of the BAEE-rinse-liquid solution by capillary electrophoresis. If BA was no longer detected by CE, it was determined that all of the unbound trypsin was removed from the capillary column. This determination was performed to ensure that any enzymatic reaction occurring in the column involved immobilized or covalently bound trypsin, rather than free trypsin still present in the column.
Trypsin-PSG Device Operation. A trypsin-PSG capillary was installed in a Beckman P/ACE 2000 capillary cartridge (Beckman Coulter, Inc., of Fullerton, Calif.), whereupon about 4.4 cm of the original 30-cm long column was cut off. The total length of the resulting capillary column was about 25.6 cm, with the length from the detection window to the outlet being about 6.7 cm. The 1-cm enzyme microreactor region was approximately 5 mm from the capillary inlet. Prior to operation, the trypsin-PSG capillary column was conditioned by rinsing it with a separation buffer using a 0.5-mL disposable syringe in a manual vise. The separation buffer was 50 mM TRIS (pH 7.4). Electrokinetic conditioning of the column followed using a Beckman P/ACE 2000 CE instrument (Beckman Coulter, Inc., of Fullerton, Calif.) at 5 kV for 30 min.
Multiple trypsin-PSG capillaries were prepared as described above, although the lengths of the capillaries varied. CE or CEC separation conditions varied depending on the length of the microreactor. Typically, an electrokinetic injection of about 5 kV or less was used to put the sample on the column. Separation voltages of from about 8 kV to about 10 kV were typical. Substrate and product(s) were detected at 214 nm at run temperatures of 20° C.
BAEE at a concentration of 2.5 mM was used as an artificial substrate to demonstrate the activity of the microreactor, as described below. Also, bradykinin at a concentration of 1 mg/mL in water was used to demonstrate the effectiveness of the microreactor in the digestion of a peptide, as further described below.
Determination of bound trypsin. A trypsin-PSG monolith, as described above, may be exposed to a rinse liquid of 100 mM NaOH to degrade the PSG monolith and thus remove it from the column. In such a process, the trypsin is generally not degraded, although its three-dimensional structure may no longer be intact. The amount of trypsin that was once bound to the PSG surface may then be determined using a Bradford Assay, which utilizes Coumassie Blue dye. This determination is not affected by whether the three-dimensional structure of the trypsin is intact or not. The resulting trypsin-dye complex may be detected and quantitatively measured by absorption spectroscopy. Such determinations may be used to determine the amount of an enzyme, such as trypsin, that is immobilized or covalently bound to the surface of an immobilized-enzyme PSG monolith.
Results. A column incorporating a 1-cm immobilized-trypsin PSG, such as that described above in relation to
It was further determined that there was an approximately linear relationship (y=0.0004x+0.0003; R2=0.9903) between the amount of time the BAEE spent on the column and the amount of product formed, as reflected by absorption at 214 nm. That is, at least the initial rate of the reaction up to about 5 or so minutes is approximately linear.
The activity of trypsin was also demonstrated by cleaving the peptide bradykinin. The bradykinin (1 mg/mL) was introduced into a column incorporating a 1-cm immobilized-trypsin PSG, in the manner described above. The bradykinin was digested, resulting in the digestion reaction product, arginine. The bradykinin and the arginine were separated, as demonstrated in the graphical representation shown in
As shown in
The experimental example just described is notable for demonstrating that the enzyme, trypsin, is able to cleave protein fragments, and is able to do so quite efficiently. The example is more particularly notable for demonstrating that such an enzyme, as used in connection with a PSG monolith according to the present invention, effectively cleaves protein fragments. In assessing the results, it should be noted that all of the digestion reactions just described were performed at 20° C., which is far from the optimal temperature of 37° C. for activity of the trypsin enzyme. It is contemplated that running the digestion reactions at a higher temperature, such as a temperature approaching or reaching 37° C., would further demonstrate that an enzyme-PSG monolith microreactor is very effective for the digestion of biological samples and their components.
In another experimental example, a first test was performed in which BAEE was enzymatically digested at about 20° C. using trypsin in bulk solution in a CE column. Any remaining BAEE and any product(s) of the digestion reaction were detected via UV absorption. Absorption data was collected over time (ABS at 214 nm versus Retention Time in minutes), and the peak height for BA was used to determine the amount of BA digestion product produced. In a separate test, BAEE was enzymatically digested at about 20° C. using an immobilized-trypsin microreactor that was incorporated into a CE column, according to the present invention. As in the first run, absorption data was collected over time and the height of the absorption peak associated with the BA was used to determine the amount of BA produced. When the results of the two tests were compared, it was determined that there was about a 200-fold increase in the peak height associated with the BA product when the trypsin-immobilized microreactor was employed. This result demonstrates that the bioactivity of the enzyme, trypsin, may be enhanced up to 200-fold by being immobilized onto a PSG material according to the present invention. It is contemplated that running the digestion reactions at a higher temperature, such as a temperature approaching or reaching 37° C., or the optimum temperature for the activity of the trypsin enzyme, would further demonstrate that an enzyme-PSG monolith microreactor is very effective for the digestion of biological samples and their components. Further, it is contemplated that even greater enhancements are possible when using this and other enzymes according to the present invention.
In yet another experimental example, an immobilized-trypsin microreactor was prepared in a micropipet tip, more or less as described herein in relation to
In yet another experimental example, an immobilized-trypsin microreactor was prepared in a capillary column, as described in Dulay et al., Enhanced Proteolytic Activity of Covalently Bound Enzymes in Photopolymerized Sol Gel, Anal. Chem. 2005, 77, 4604-4610, which is incorporated herein in its entirety by this reference. In this example, trypsin was covalently linked to a photopolymerized sol-gel monolith that was modified by incorporating poly(ethylene glycol) (PSG-PEG) for on-column digestion of BAEE and two peptides, neurotensin and insulin chain B. The coupling of the enzyme to the monolith was via room-temperature Schiff chemistry in which an alkoxysilane reagent (linker) with an aldehyde functional group was linked to an inactive amine on trypsin to form an imine bond. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of BA, the digestion product of BAEE. The BA was separated from BAEE by capillary electrophoresis and detected downstream (18.5 cm from the microreactor) by absorption (254 nm). Using the Bradford assay, it was determined that 97 nanograms of trypsin were bound to the 1-cm microreactor located at the entrance of the capillary column. The bioactivity of the trypsin-PSG-PEG microreactor at 20° C. for the digestion of BAEE was found to be 2270 units/mg of immobilized trypsin. The bioactivity of trypsin bound to the capillary wall in the open segment upstream from the monolith was 332 units/mg of immobilized trypsin under the same conditions. In contrast, the activity of free trypsin could not be observed for the digestion of BAEE at 20° C. after 16 hours of incubation time.
The reactive hydrophilic macroporous PSG-PEG monolith was prepared in situ in a fused capillary by using a combination of sol-gel chemistry and photopolymerization, followed by functionalization of the monolith surface with an alkoxysilane reagent comprising an aldehyde group. The activated monolith was transformed into an enzyme microreactor by covalent attachment with the enzyme trypsin. Trypsin was chosen because it is a highly selective protease, although any other suitable enzyme or enzymes could have been employed. The resulting trypsin-PSG-PEG microreactor had long-term stability and high proteolytic activity for the on-column digestion of large peptides. The proteolytic activity of the bound trypsin was enhanced more than 2000 times relative to that of free trypsin in solution. The short digestion times at low temperature associated with bound trypsin digests contrast favorably with the longer reaction times at higher temperatures associated with free trypsin digests using free trypsin in solution. The covalently bound enzyme allowed for easy intervacing downstream to CE. As such, it is contemplated that covalently bound enzyme may facilitate applications using larger biomolecules, such as proteins, for example.
The results of the experimental examples, as well as other results described herein, demonstrate that when a microreactor is used in connection with a micropipet tip, or with a column, according to the present invention, the immobilized enzyme, trypsin, is able to cleave protein fragments. These are promising results, in view of the fact that, as demonstrated above, the bioactivity of the enzyme, trypsin, may be enhanced up to 200-fold to more than 2000-fold, such as up to about 2000-fold, for example, by being immobilized onto a PSG material according to the present invention, even when employed at a temperature that is less than optimum for the activity of the enzyme. It is contemplated that enhancements beyond any associated with the experimental examples provided herein may be achieved according to the present invention. Further, it is believed that results, such as those associated with these experimental examples, would flow from the use of any suitable enzymes, such as any described or contemplated herein, and/or in connection with a microreactor and devices incorporating same, such as any described or contemplated herein, according to the present invention.
Accordingly, the present invention encompasses an immobilized-enzyme microreactor and any such microreactor that may be incorporated into any number of appropriate devices, such as a capillary column or a micropipet tip, as described above, in addition to other devices. Merely by way of example, an integrated microreactor device 80 that comprises a microreactor 90 incorporated into a well-plate device 84 is contemplated herein, as partially shown in
In relation to the above-mentioned integrated microreactor device 80, a microreactor 90 according to the present invention is introduced or inserted in one or more well(s) 82 of a well plate 84, as partially shown in
In relation to the above-mentioned integrated microreactor device 100, a microreactor 110 according to the present invention is prepared, introduced or inserted in one or more channel(s) 102 of a chip device or an integrated circuit device 104, as shown in
The microreactor 110 comprises a polymerized sol-gel material, the surface of which is linked to an enzyme, as previously described. The microreactor 110 may be prepared in any suitable manner, such as any previously described. Merely by way of example, a reaction solution may be introduced into a channel or microchannel 102 by pressure, or in any other suitable manner, and prepared as previously described. This reaction solution may be so introduced via an injection port 114, for example. When the monolith is prepared via irradiation, such as via photopolymerizing irradiation, the chip 104 or a channel 102 thereof may be masked in suitable areas with a suitable radiation-masking or radiation-opaque material that prevents the radiation employed (such as UV or visible radiation, for example) from entering the masked areas of the channel 102, such that no monolith is formed in these masked areas. The unmasked areas of the chip 104 or channel 102 thereof may be irradiated, as previously described, to form a monolith in those unmasked areas, or the polymerized sol-gel (PSG) section 118 shown in
The immobilized enzyme of the microreactor 110 may be any suitable enzyme, such as any of those previously described herein, for example. For example, an enzyme, such as trypsin, may be covalently bound to the polymerized sol-gel of the microreactor 110, and may be used to digest an appropriate substrate that flows (via pressure, electrokinetic flow, electroosmotic flow, and/or the like, for example) into the channel 102 of the chip 104, in a manner such as that previously described in relation to other devices. Merely by way of example, a sample may enter the chip 104 via a sample inlet 106. The sample may be allowed to flow into the channel 102 in any appropriate manner, such as via pressure or voltage, and allowed to react with the immobilized enzyme of the microreactor 110 in any appropriate manner, such as via passage of an appropriate amount of time. Products of the reaction, such as digestion products or fragments, may be allowed to flow through and away from the microreactor 110 in any appropriate manner, such as via pressure or voltage causing flow in a downstream direction, for example, toward one or more detection points 108, which may be compatible with or adapted for use in connection with one or more detection devices (not shown). An appropriate buffer solution may be applied to the column, as previously described, via an inlet 112, and may be allowed to exit the column, via an outlet 114. The outlet 114 may also serve as an injection port for the reaction solution used in the preparation of the polymerized sol-gel, as previously described. A sample outlet 116 is also provided, as shown. Applications of pressure to induce flow, as described above, may be facilitated by use of pressure pumps (not shown) or other appropriate devices, and applications of voltage to induce flow, as described above, may be facilitated by any appropriate means or devices, such as any previously described, and such as any described in Morishima et al.
It will be understood that a suitable chip device that comprises a microreactor, as described herein, may be prepared in any suitable manner, via lithography, masking, etching, material layering, via or channel formation, or otherwise, and may take a variety of forms. For example, such a chip device may be made of any suitable material or materials, and may be of any suitable geometry or configuration, such as any suitable for an application. An example of a suitable chip device is a silicon-on-insulator wafer that comprises a device layer comprising at least one reaction chamber and a lid, such as a glass lid, that is capable of withstanding high pressures (i.e., pressures associated with the suitable or effective movement of fluid through the device). The chip device may be miniaturized, and thus, be referred to as a microchip device, or a microfluidic device, or the like. Relative to larger devices, these smaller devices may be advantageous in terms of improved performance, improved consumption (i.e., use of less sample and/or other fluids and chemicals), lower cost, higher throughput, and/or the like.
In terms of size, a channel of a chip device may be of any suitable size for the application, such as on the order of a capillary column described herein, such as on the order of about 1 centimeter in length and about 75 microns in inside diameter, for example, or smaller, such as on the order of about 1 millimeter in length and about 75 microns in inside diameter, for example. The amount of polymerized sol-gel employed generally depends on the volume of the channel. The modification of the polymerized sol-gel with a linker and further with an enzyme in the channel of a chip device generally follows the procedure described herein in connection with the modification of the polymerized sol-gel with a linker and further with an enzyme in the column of a capillary device. As the channel of a capillary device having a length of about 1 centimeter and an inside diameter of about 75 microns may be used to immobilize about 97 nanograms of enzyme, it is contemplated that a channel of a chip device having similar dimensions may also be suitable to immobilize about 97 nanograms of enzyme, although any suitable amount of enzyme may be immobilized.
The pattern of the chip device may be quite simple, such as that shown in
Merely by way of example, it is contemplated that a circuit pattern may be configured for an application comprising derivatizing digestion products or fragments from the enzymatic digestion using a fluorescent reagent suitable for fluorescence detection, such as laser-induced fluorescence detection, for example. In such a case, the circuit pattern may comprise a section for carrying out this derivatization reaction upstream of or before reaching areas designated for detection. An example of such a patterned chip device 200 is shown in
The invention encompasses a great variety of variations and modifications. For example, the device of the invention, or an integrated device of the invention, may contain one or more enzyme(s) immobilized on one or more PSG material(s), and/or multiple enzymes co-immobilized on one or more PSG material(s), such as in series, for example, for the digestion of a solution containing more than one peptide and/or protein substrate. Immobilization of an enzyme according to the present invention reduces or substantially prevents self-destruction of the enzyme, such that the enzyme is effective in the microreactor device.
Further by way of example, the surface area and pore volumes of the PSG material can be modified to accommodate proteins that are large in size, such as proteins having molecular weights of about 200 Daltons or more, such as 200 kDaltons or more, and/or being on the order of more than nanometer size. The PSG material can also be used in connection with a substrate that is much smaller in molecular weight and/or size. As yet further examples, the immobilized-enzyme microreactor and its preparation can be adapted to devices that comprise well-plates, circuit patterns, and/or that have shapes other than the shape of a capillary or a micropipet tip.
The present invention provides an enzyme that is immobilized by covalent bonding to the surface of a porous PSG material that is versatile in application. For example, the immobilized-enzyme, porous PSG material, or monolith, can be used in a variety of formats, such as a microreactor, a capillary, a micropipette, or a chip or circuit format, as well as other formats. The immobilized-enzyme, porous PSG material provides a suitable, if not ideal, interface for downstream analysis applications and instrumentation, such as a variety of separation applications and tools including HPLC, CEC, and CE, for example. In another example, the immobilized-enzyme, porous PSG material provides an interface for separation by mass spectrometry, wherein the reaction products of the enzymatic digestion process are fed into a mass spectrometer using an electrospray ionization interface, for example.
The immobilized-enzyme, porous PSG material is particularly useful in the separation of proteinaceous and/or peptidic analytes from sample, and may be designed to accommodate such analytes that are relatively large. In a particular application, the bioactivity of the enzyme, trypsin, was enhanced up to 200-fold to more than 2000-fold by being immobilized onto a PSG material, even when the temperature employed was less than the optimal temperature of the activity of that enzyme. Such an immobilized-enzyme, PSG material is thus particularly useful in biomolecule separations, such as separations of one or more proteins, oligonucleotides, peptides, steroids, organic acids, or any combination thereof.
Immobilization of an enzyme with respect to a PSG material, as described herein, can result in an increase in the activity of the enzyme by several or many orders of magnitude. It is believed, without being so bound, that in the immobilization process, the enzyme becomes covalently bound to the surfaces in the pores of the PSG material. When the analyte, or substrate, enters a pore, its concentration in the pore relative to its concentration in the bulk of the material, is greatly increased. As the activity of the enzyme depends on the substrate concentration, typically in direct proportion, it too is greatly increased. As such, the immobilized-enzyme, PSG material of the present invention provides a highly active and effective tool for the breakdown, separation, and analysis of biomolecules and their components.
Various references, publications, provisional and/or non-provisional United States patent applications, and/or United States patents, have been identified herein, each of which is incorporated herein in its entirety by this reference. Various aspects and features of the present invention have been explained or described in relation to understandings, beliefs, theories, underlying assumptions, and/or working or prophetic examples, although it will be understood that the invention is not bound to any particular understanding, belief, theory, underlying assumption, and/or working or prophetic example. Various modifications, processes, as well as numerous structures to which the present invention may be applicable will be readily apparent to those of skill in the art to which the present invention is directed, upon review of the specification. Although the various aspects and features of the present invention have been described with respect to various embodiments and specific examples herein, it will be understood that the invention is entitled to protection within the full scope of the appended claims.
This application is a continuation-in-part of U.S. Application No. ______, which entered the U.S. national phase from International Application No. PCT/US2004/041640, filed on Dec. 13, 2004, which claims priority to U.S. application Ser. No. 10/734,998, filed on Dec. 12, 2003. This application is also related to U.S. application Ser. No. 10/124,654, filed on Apr. 16, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/929,275, filed on Aug. 13, 2001, and of U.S. application Ser. No. 10/008,482, filed on Nov. 13, 2001, the latter of which is a continuation of U.S. application Ser. No. 09/507,707, filed on Feb. 18, 2000. This application is also related to U.S. application Ser. No. 10/674,652, filed on Sep. 29, 2003, which is a continuation of the aforementioned U.S. application Ser. No. 10/008,482, which is a continuation of aforementioned U.S. application Ser. No. 09/507,707. This application is further related to U.S. application Ser. No. 09/978,515, filed on Oct. 15, 2001, which is a continuation-in-part of the aforementioned U.S. application Ser. No. 09/929,275. Each of the foregoing applications is incorporated herein in its entirety by this reference.
