US20160108455A1 - Systems and conductive structures for determining enzymatic activity and methods of formation - Google Patents
Systems and conductive structures for determining enzymatic activity and methods of formation Download PDFInfo
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- US20160108455A1 US20160108455A1 US14/976,124 US201514976124A US2016108455A1 US 20160108455 A1 US20160108455 A1 US 20160108455A1 US 201514976124 A US201514976124 A US 201514976124A US 2016108455 A1 US2016108455 A1 US 2016108455A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/025—Align devices or objects to ensure defined positions relative to each other
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/12—Specific details about manufacturing devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0609—Holders integrated in container to position an object
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0829—Multi-well plates; Microtitration plates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
Definitions
- thermophilic enzyme characterization in various embodiments, relates generally to the field of thermophilic enzyme characterization. More particularly, this disclosure relates to methods and systems for determining enzymatic activity. The methods and systems may be utilized for enzyme characterization studies applied to enzymatic reactions that involve, among others characteristics, high temperatures, insoluble substrates, and heterogeneous substrates.
- Enzymes are biological molecules that catalyze a chemical reaction of a substrate. To understand the reaction mechanism and kinetics of such enzymatic reactions, it is often desirable to characterize an enzyme by determining its activity. Enzyme characterizations are carried out with assays that quantitatively assess enzyme activity based on, for example, resulting concentrations of one or more products of the enzymatic reaction. Thus, enzyme characterization provides information about the enzyme activity that can be used to predict how the enzyme will behave when reaction mixtures are altered, such as by adjusting the amounts of substrate, enzyme, etc., involved. Characterizing enzyme activity at different temperatures or pH levels provides information about the enzyme activity that can be used to predict how the enzyme will behave when reaction conditions are altered.
- enzyme activity is determined by measuring the concentration of a reaction product of an enzymatic reaction over time for a fixed and constant enzyme concentration.
- concentration of a reaction product of an enzymatic reaction is determined by measuring the concentration of a reaction product of an enzymatic reaction over time for a fixed and constant enzyme concentration.
- the higher a reaction product concentration detected, in a period of time the higher the enzyme activity determined.
- the reaction product concentration levels may be impacted by conditions other than enzyme activity. For example, some enzymatic reactions are carried out at high temperatures (e.g., at temperatures above about 50° C.) that can cause reagents of the reaction to evaporate, skewing concentration measurements.
- reagents evaporate during the reaction, resulting concentrations of reaction products may be determined to be artificially higher than they would otherwise be, indicating a false-high enzymatic activity, or the evaporation may concentrate the enzyme in the reaction, resulting, again, in artificially high measured concentrations of reaction products.
- some substrates are generally insoluble in the reagents, and mass transfer properties, such as the diffusivity of the enzyme into the substrate or the diffusivity of a reaction product out of the substrate, may control the rate of the enzymatic reaction and, thus, the rate of production of the reaction product. Therefore, measured reaction product concentrations may be artificially low based on a low diffusivity value, rather than on the actual rate of the enzymatic reaction.
- not all substrates and enzymes readily intermix with one another. Low intermixing may result in less enzymatic activity and, thus, low reaction product concentrations, even if the reaction rate is actually rapid.
- conventional methods for enzyme characterization may not be well suited to evaluate the enzymatic activity using substrates that are of industrial relevance. Not only may some industrially relevant substrates be generally insoluble, but some may additionally or alternatively be generally heterogeneous such that one small sample of the substrate may vary in composition from another small sample of the same substrate. To try to avoid such heterogeneity impacting the results of enzymatic characterization methods, many conventional methods involve the use of a large amount of the substrate. However, use of a large amount of substrate may require use of a large amount of enzyme in the characterization. Such large-volume methods may not be conducive for characterizing enzymes for which only small amounts are available.
- a method for determining enzymatic activity comprises heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes of the plurality, at least one enzyme is substantially simultaneously added to the closed volumes of the plurality. After adding the at least one enzyme, the plurality of closed volumes are agitated at the predetermined reaction temperature. After the agitation, the activity of the at least one enzyme is determined.
- a method for determining enzymatic activity comprises heating a conductive structure, supporting sealed reaction vessels containing substrate, to a predetermined reaction temperature. At least one enzyme is substantially simultaneously injected into the sealed reaction vessels. The sealed reaction vessels, with the substrate and the at least one enzyme, are agitated in a plane of motion parallel to a length of the sealed reaction vessels. The method also comprises determining activity of the at least one enzyme.
- a system for determining enzymatic activity comprises a conductive structure that defines a plurality of wells protruding into the conductive structure from an upper surface of the conductive structure.
- the plurality of wells is configured to receive a plurality of reaction vessels.
- the conductive structure also defines at least one engagement feature on a sidewall of the conductive structure. The at least one engagement feature is configured to engage a counterpart engagement feature to secure the conductive structure to an agitator.
- FIG. 1 is a top, front, and right side perspective view of a conductive structure of a system for analyzing an enzyme, according to an embodiment of the present disclosure.
- FIG. 2 is a bottom, rear, and left side perspective view of the conductive structure of FIG. 1 .
- FIG. 3 is a top, plan view of the conductive structure of FIGS. 1 and 2 .
- FIG. 4 is a front elevation view of the conductive structure of FIGS. 1 through 3 with a lid on the conductive structure.
- FIG. 4 is also a rear elevation view of the conductive structure of FIGS. 1 through 3 with the lid on the conductive structure.
- FIG. 5 is a right side elevation view of the conductive structure of FIGS. 1 through 4 with the lid on the conductive structure.
- FIG. 5 is also a left side elevation view of the conductive structure of FIGS. 1 through 4 with the lid on the conductive structure.
- FIGS. 6 through 17 are views of various stages of a method for processing an enzyme to be characterized, according to an embodiment of the present disclosure, wherein:
- FIG. 6 is a top and front perspective view of a dry bath incubator into which the conductive structure of FIGS. 1 through 5 is configured to be received;
- FIG. 7 is a top and front perspective view of the dry bath incubator of FIG. 6 having received therein two of the conductive structures of FIGS. 1 through 5 with alignment members received in the conductive structures;
- FIG. 8 is a top and front perspective view of a fluid-containing vial that the conductive structure of FIGS. 1 through 5 is configured to receive;
- FIG. 9 is an exploded view of a conductive structure assembly, including a top and rear perspective view of the conductive structure of FIGS. 1 through 5 with a plurality of the fluid-containing vials of FIG. 8 received in wells of the conductive structure and an alignment member received in the conductive structure, a bottom and rear perspective view of a lid to secure to the conductive structure, and a bottom and rear perspective view of a fastener member to secure the lid to the conductive structure;
- FIG. 10 is a top, rear, and left side perspective view of the conductive structure assembly of FIG. 9 , assembled for heating to a predetermined reaction temperature in the dry bath incubator of FIG. 6 , with the lid secured to the conductive structure via the fastener member and another fastener engaged with the alignment member;
- FIG. 11 is a partial, top, front, and left side perspective view of the conductive structure of FIGS. 1 through 5 in the dry bath incubator of FIG. 6 with a plurality of substrate-containing vials, some of which having lids with septa, and a top and front perspective view of a substrate-containing vial with a septum-containing lid to be received in a well of the conductive structure;
- FIG. 12 is a top, front, and right side view of the two conductive structures of FIG. 7 , in the dry bath incubator of FIG. 6 , the conductive structures having received therein substrate-containing vials with septa-including lids, and with an injector support structure aligned over one of the two conductive structures;
- FIG. 13 is a bottom, rear, and right side view of the injector support structure of FIG. 12 ;
- FIG. 14 is a front, cross-sectional, elevation view, taken along section line 14 - 14 of FIGS. 11 and 12 , with injectors received in conduits of the injector support structure of FIG. 12 , needles of the injectors extending through septa of substrate-containing vials, and the lid of FIG. 9 being utilized to simultaneously depress plungers of the injectors to substantially simultaneously inject at least one enzyme from the injectors into a liquid in the substrate-containing vials;
- FIG. 15 is a top, front, and right side perspective view of a support structure secured to a shaker plate of an orbital shaker;
- FIG. 16 is a front, cross-sectional, elevation view, of the conductive structure of FIGS. 4 and 5 , taken along section line 16 - 16 of FIG. 15 , having an engagement feature engaged with a counterpart engagement feature of the support structure of FIG. 15 ;
- FIG. 17 is a top, front, and left side perspective view of a conductive structure assembly such as that of FIG. 10 , but having received therein substrate-and-enzyme containing vials, secured to the shaker plate of an orbital shaker via the support structure of FIG. 15 and being agitated in a plane of motion that is parallel to a length of the substrate-and-enzyme containing vials while a predetermined reaction temperature is maintained.
- FIGS. 18 through 21 graph enzymatic activity results, for a commercially available enzyme, determined using a conventional test tube assay that does not include agitation and sealed reaction vessels.
- FIGS. 22 through 25 graph enzymatic activity results, for the commercially available enzyme, determined using a method according to the present disclosure that includes agitation and sealed reaction vessels.
- Substrate in closed vessels is brought to a predetermined reaction temperature. Without opening the vessels, at least one enzyme is substantially simultaneously added to each of the closed vessels.
- the closed vessels, with the substrate and added enzyme are then agitated to mix the substrate and the enzyme.
- the closed vessels may be maintained at essentially the predetermined reaction temperature throughout the enzyme addition and the agitation. Therefore, the temperature may be controlled, evaporation of reagents may be prevented, and multiple samples may be simultaneously processed, increasing the throughput.
- the enzymatic reactions which take place in the closed vessels, may be analyzed to accurately determine enzyme activity, even for high-temperature enzymatic reactions, insoluble substrates, substrates and enzymes that do not readily intermix, substrates that are heterogeneous, and low sample sizes of the substrate and enzyme.
- the activity of enzymes for enzymatic reactions that involve insoluble substrates, partially soluble substrates, heterogeneous substrates, small substrate amounts, small enzyme volumes, high-temperatures, or mixing challenges may be determined by a reliable and reproducible method.
- the methods and systems of the present disclosure may also provide a high throughput assay for determining the enzymatic activity.
- substrate means and includes a material to be at least partially consumed in a reaction catalyzed by the at least one enzyme to be characterized.
- the structures described herein may be formed by any suitable technique, the samples may be prepared by any suitable technique, and the enzymatic reactions may be analyzed by any suitable technique, which techniques may be selected by a person having ordinary skill in the art.
- FIGS. 1 through 5 illustrate a conductive structure 100 configured to receive and transfer heat to reaction vessels. Substrate samples and at least one enzyme may be inserted into the reaction vessels, and enzymatic reactions may be carried out before the results of the reaction are analyze to characterize the enzyme.
- the conductive structure 100 may include a number of wells 102 that protrude into the conductive structure 100 from an upper surface 104 of the conductive structure 100 . As illustrated in FIG. 4 , the wells 102 may protrude essentially perpendicularly relative to the upper surface 104 and to a lower surface 106 . The wells 102 may be shaped to snugly receive therein the reaction vessels. Therefore, heat may be conductively transferred between the conductive structure 100 and the reaction vessels.
- the conductive structure 100 may be formed of a conductive material, such as, for example and without limitation, a metal (e.g., aluminum), a metal alloy, or other conductive material.
- the conductive material may be selected to have a high heat conductivity, so that the conductive structure 100 may be quickly heated by a heat source, and a high heat capacity, so that the conductive structure 100 remains heated even if the heat source is temporarily interrupted.
- the conductive structure 100 may be formed from an essentially solid block of the conductive material.
- the wells 102 , and other negative-space features may be machined or otherwise formed into the block by conventional techniques, which are not described in detail herein.
- the conductive structure 100 may be molded to define the wells 102 , and other negative-space features, when the conductive material is first formed into the conductive structure 100 .
- the wells 102 may be arranged in an ordered array, e.g., the column-and-row arrangement illustrated in FIG. 1 , or may be arranged without a particular order.
- each of the wells 102 may have essentially the same dimensions and be evenly spaced.
- the wells 102 may vary in dimension from one to another and not be evenly spaced.
- the dimensions and arrangement of the wells 102 may be tailored to enable heat transfer from the conductive structure 100 with consistent temperatures in the wells 102 regardless of the relative position of each well 102 in the conductive structure 100 .
- the conductive structure 100 may also define therein one or more probe openings.
- a thermometer opening 108 may protrude into the conductive structure 100 from the upper surface 104 .
- the thermometer opening 108 may be configured to receive therein a thermometer during heating and/or reaction stages of methods according to embodiments of the present disclosure.
- the thermometer opening 108 may protrude to, e.g., a depth approximately even with a depth of the wells 102 , as illustrated in FIG. 4 .
- temperatures read from a thermometer received in the thermometer opening 108 may be the same temperatures of materials within the reaction vessels received in the wells 102 .
- the thermometer opening 108 may be filled with a heat transfer liquid (e.g., water or other fluid, e.g., oil) before the thermometer is received.
- a heat transfer liquid e.g., water or other fluid, e.g., oil
- the same heat transfer liquid may also be filled, partially or completely, into the wells 102 before the reaction vessels are received therein to, again, enable efficient heat transfer between the conductive material of the conductive structure 100 and the material of the reaction vessels.
- the conductive structure 100 may also define therein a thermocouple opening 110 configured to receive a thermocouple extending from a heating device.
- the thermocouple opening 110 as illustrated in FIGS. 2 and 4 , may protrude into the conductive structure 100 from the lower surface 106 . With reference to FIG. 4 , the thermocouple opening 110 may align with the thermometer opening 108 , though the two openings may not connect.
- the conductive structure 100 may also define therein one or more openings 112 for receiving an alignment member and/or fastener member.
- the openings 112 may be defined to protrude into the conductive structure 100 from the upper surface 104 .
- the openings 112 may be threaded and therefore configured to receive threaded alignment or threaded fastener members therein.
- the openings 112 may protrude to a depth less than that of the wells 102 .
- the openings 112 may protrude deeper or all the way through the height of the conductive structure 100 .
- the conductive structure 100 may include an engagement feature configured to engage with a counterpart engagement feature of another structure to secure the conductive structure 100 to the other structure.
- the engagement feature may include one or more openings 114 defined in a front surface 116 and a back surface 118 of the conductive structure 100 and extending through a length of the conductive structure 100 . Therefore, counterpart engagement features (e.g., engagement features 1510 ( FIG. 15 )) may be received through the openings 114 and may be utilized to secure the conductive structure 100 to another structure such as, for example, an agitator.
- side surfaces 120 of the conductive structure 100 may be free from openings, as illustrated in FIGS. 1 and 5 .
- the conductive structure 100 of FIGS. 1 through 5 is illustrated to be a six-sided block shape, in other embodiments, the conductive structure 100 may be otherwise shaped but nonetheless configured to receive reaction vessels in wells 102 .
- a method, according to an embodiment of the present disclosure, for characterizing an enzyme may include heating the conductive structure 100 .
- Heat may be provided to the conductive structure 100 from a heat source such as a dry bath incubator 600 , illustrated in FIG. 6 .
- the dry bath incubator 600 may include a cavity 602 into which the conductive structure 100 ( FIGS. 1 through 5 ) is received.
- a thermocouple 604 may protrude from a base of the cavity 602 and may be received in the thermocouple opening 110 ( FIG. 4 ) of the conductive structure 100 ( FIGS. 1 through 5 ) when the conductive structure 100 is in place in the cavity 602 .
- the conductive structures 100 are configured such that more than one conductive structure 100 may be received in the cavity 602 of the dry bath incubator 600 , as illustrated in FIG. 7 .
- the upper surface 104 ( FIG. 1 ) of the conductive structure 100 may be flush with an upper surface of the dry bath incubator 600 .
- FIG. 7 further illustrates alignment members 712 received within one of the openings 112 ( FIG. 4 ) defined in the conductive structures 100 .
- the alignment members 712 may be threaded rods that are screwed into one of the openings 112 of the conductive structure 100 in such embodiments in which the openings 112 are also threaded. Therefore, in some embodiments, the alignment members 712 may be removable from the conductive structures 100 . In other embodiments, the alignment members 712 may be permanently affixed to the conductive structures 100 .
- the conductive structures 100 may be heated, using the dry bath incubator 600 , to, e.g., a predetermined reaction temperature.
- the reaction temperature may be selected based on the enzymatic reaction to be carried out.
- the conductive structures 100 may be heated when empty, i.e., without any material received in the wells 102 .
- the wells 102 may be filled with a heat transfer fluid (e.g., water, oil, or other fluid) prior to or during the heating.
- the heat transfer fluid may also be added to the thermometer opening 108 ( FIG. 4 ) as discussed above.
- the heat transfer fluid may be added to vessels 800 ( FIG.
- the vessels 800 may be essentially the same as those to be used as reaction vessels later in the method.
- a glass vial 802 may be used as the vessel 800
- water 804 may be added to the glass vial 802 and used as the heat transfer fluid.
- the volume of water 804 added may be approximately the same volume as the reagents of the enzymatic reaction to be carried out later in the method. Therefore, during the initial heating of the conductive structures 100 ( FIG. 7 ), the heat to be provided to the later-received reaction vessels will be consistent with the heat provided to the vessels 800 with only the water 804 .
- the vessels 800 may be sealed with a lid 806 that secures to the glass vial 802 .
- a vessel 800 may be loaded into each of the wells 102 ( FIG. 7 ) so that the heat profile across the conductive structure 100 will be even.
- Each well 102 may be configured to snugly receive one of the vessels 800 .
- the walls of the glass vials 802 may be fully received in the wells 102 with essentially only the lids 806 protruding above the upper surface 104 of the conductive structure 100 , as illustrated in FIG. 9 .
- an even heat may be provided from the conductive structure 100 to the glass vials 802 ( FIG. 8 ) and thereafter to the water 804 ( FIG. 8 ) within the glass vials 802 .
- heat transfer fluid e.g., additional water, oil
- additional water, oil may be included in the wells 102 ( FIG. 4 ) before the glass vials 802 are received therein to further ensure conductive heat transfer between the conductive structure 100 and the water 804 within the glass vials 802 .
- a lid 900 may be placed over the top of the conductive structure 100 and over the lids 806 of the vessels 800 ( FIG. 8 ).
- the lid 900 may have sidewalls 902 that extend the height of the lids 806 of the vessels 800 such that a lower edge 904 abuts the upper surface 104 of the conductive structure 100 , as illustrated in FIGS. 4 and 5 .
- An interior surface 906 of the lid 900 may abut the tops of the lids 806 of the vessels 800 ( FIG. 8 ), when the lid 900 is secured to the conductive structure 100 .
- the lid 900 When placed on the conductive structure 100 , the lid 900 may reduce or prevent heat from exiting the conductive structure 100 and the vessels 800 ( FIG. 8 ). Thus, the lid 900 may promote efficient heating of the vessels 800 .
- the lid 900 may be formed of a conductive material (e.g., a metal (e.g., aluminum, tin), a metal alloy).
- the sidewalls 902 of the lid 900 may define a space in which each of the lids 806 of the vessels 800 ( FIG. 8 ) may be received, as illustrated in FIG. 9 .
- the lid 900 may be formed as an essentially solid structure of a conductive material with wells configured to receive, in each, the lid 806 of one of the vessels 800 . Thus, the lids 806 of the vessels 800 may be snugly received within the lid 900 on the conductive structure 100 .
- the lid 900 may define therein openings 912 that may align with the openings 112 defined in the upper surface 104 of the conductive structure 100 . Therefore, the alignment member 712 , received within one of the openings 112 of the conductive structure 100 may align with and extend through one of the openings 912 in the lid 900 .
- a fastener such as a nut 1012 (see FIG. 10 ) may be releaseably engaged with the alignment member 712 to secure the lid 900 to the conductive structure 100 .
- a fastener member 920 may be passed through another of the openings 912 in the lid 900 to engage another of the openings 112 of the conductive structure 100 .
- the fastener member 920 may be a threaded rod that corresponds to threading in the openings 112 .
- the fastener member 920 may be screwed into the opening 112 to secure the lid 900 to the conductive structure 100 .
- a conductive structure assembly 1000 may be secured as the dry bath incubator 600 ( FIG. 6 ) heats the conductive structure 100 .
- the lid 900 may define a thermometer opening 908 passing through the lid 900 and corresponding to the thermometer opening 108 of the conductive structure 100 . Therefore, when the lid 900 is secured to the conductive structure 100 , a thermometer may be inserted into the thermometer openings 108 , 908 to monitor a temperature of the conductive structure 100 .
- FIGS. 9 and 10 illustrate the conductive structure 100 out of the dry bath incubator 600 ( FIG. 6 ) for convenience, it is contemplated that the conductive structure assembly 1000 will be assembled while the conductive structure 100 is received within the cavity 602 ( FIG. 6 ) of the dry bath incubator 600 ( FIG. 6 ) to allow the heating to continue without interruption while the lid 900 is secured to the conductive structure 100 .
- the vessels 800 ( FIG. 8 ) with the heat transfer fluid (e.g., water, oil) may be removed from the conductive structure 100 and quickly replaced with other vessels, such as, as illustrated in FIG. 11 , reaction vessels 1100 or control vessels 1101 .
- the reaction vessels 1100 may include one of the glass vials 802 with a substrate sample 1103 therein and, for example, a buffer fluid 1104 .
- the composition and amount of the substrate sample 1103 and the buffer fluid 1104 may be selected according to the enzymatic reaction to be carried out in the reaction vessel 1100 .
- the substrate samples 1103 may be sealed in the reaction vessels 1100 by lids 1106 having septa 1108 that are penetrable by injection needles without unsealing the contents of the reaction vessels 1100 .
- the control vessels 1101 may each include a control composition against which an enzymatic reaction to be carried out in one of the reaction vessels 1100 is to be compared.
- the control vessels 1101 may be sealed by one of the lids 806 that does not include the septum 1108 .
- each of the wells 102 may receive either one of the reaction vessels 1100 or one of the control vessels 1101 .
- Reaction vessels 1100 may occupy all of the wells 102 of the conductive structure 100 ( FIG. 10 ), control vessels 1101 may occupy all of the wells 120 , or a mixed grouping of the reaction vessels 1100 and the control vessels 1101 may be used to occupy the wells 102 .
- the grouping and disposition of the reaction vessels 1100 and/or control vessels 1101 used may be selected to provide the desired number of reaction results and controls against which to compare the reaction results.
- the same substrate sample 1103 may be included in each reaction vessel 1100 .
- a variety of substrate samples 1103 may be included in the reaction vessels 1100 and enzymatic reactions carried out in each simultaneously.
- the substrate samples 1103 will be selected so that reaction conditions, such as temperature, may be consistent for each of the reactions.
- the vessels 800 ( FIG. 8 ) used during the initial heating of the conductive structure 100 to the predetermined reaction temperature may be quickly replaced with the reaction vessels 1100 and/or the control vessels 1101 while the lid 900 ( FIG. 10 ) is removed from the conductive structure 100 . With minimal time used to switch out the vessels, minimal heat may be lost from the conductive structure 100 . In some embodiments, e.g., such as those embodiments in which the disclosed methods are automated, all of the vessels 800 ( FIG. 8 ) may be simultaneously removed and may be simultaneously replaced with the reaction vessels 1100 and/or the control vessels 1101 , which may minimize the transition time and the heat lost.
- the volume of the water 804 ( FIG. 8 ) in the vessels 800 ( FIG. 8 ), used for the initial heating of the conductive structure 100 to the predetermined reaction temperature and later replaced with the controlled volumes (e.g., the reaction vessels 1100 and/or control vessels 1101 ), will be substantially the same as the volume of the buffer fluid 1104 , and any other solid or liquid material, in the vessels (e.g., the reaction vessels 1100 and/or control vessels 1101 ). Therefore, the heat profile of the vessels 800 ( FIG. 8 ), used for the initial pre-heating, will be substantially similar to the heat profile of the vessels (e.g., the reaction vessels 1100 and/or control vessels 1101 ), prior to enzyme injection. In other embodiments, the volumes may vary.
- the lid 900 may be again secured to the conductive structure 100 ( FIG. 10 ) and the temperature of the conductive structure 100 monitored until the conductive structure assembly 1000 ( FIG. 10 ) returns to the predetermined reaction temperature. Because the conductive structure 100 ( FIG. 10 ) may be pre-heated to the predetermined reaction temperature before the control volumes (e.g., the reaction vessels 1100 and/or the control vessels 1101 ) are received in the wells 102 ( FIG. 1 ) of the conductive structure 100 ( FIG. 10 ), the time needed for the conductive structure 100 to return to the predetermined reaction temperature after the vessels (e.g., the reaction vessels 1100 and/or the control vessels 1101 ) are inserted may be minimal.
- the conductive structure 100 may be first heated to the predetermined reaction temperature with the substrate-free vessels 800 ( FIG. 8 ) before the substrate samples 1103 are added to the system, in other embodiments, the conductive structure 100 may be first heated to the predetermined reaction temperature with the other vessels (e.g., the reaction vessels 1100 and/or the control vessels 1101 ) in the wells 102 ( FIG. 1 ), including the substrate samples 1103 . Whether or not the substrate samples 1103 are included in the initial heating of the conductive structure 100 may depend on whether the material of the substrate samples 1103 will decompose during the heating.
- the lid 900 may again be removed and at least one enzyme substantially simultaneously added to each of the reaction vessels 1100 to begin an enzymatic reaction in each of the reaction vessels 1100 .
- the enzyme may be added into the reaction vessels 1100 through the septa 1108 such that the reaction vessels 1100 remain sealed. Keeping the reaction vessels 1100 sealed may reduce or prevent evaporation of reagents from the closed volumes of the reaction vessels 1100 and, therefore, prevent skewing of resulting concentration measurements.
- an injector support structure 1200 may be utilized to enable simultaneous addition of at least one enzyme to the reaction vessels 1100 ( FIG. 11 ).
- the injector support structure 1200 may include a supportive body 1201 and extensions 1202 protruding from a bottom of the supportive body 1201 .
- the injector support structure 1200 may be made of a solid material that is conductive (e.g., metal, metal alloy) or nonconductive (e.g., wood, plastic).
- the extensions 1202 may protrude a height at least as great as the height of the lids 1106 of the reaction vessels 1100 ( FIG. 11 ) and control vessels 1101 ( FIG. 11 ).
- the extensions 1202 may be spaced from one another by a width that is at least as great as a width of the reaction vessels 1100 and control vessels 1101 in the conductive structure 100 ( FIG. 1 ). Therefore, the injector support structure 1200 may be positioned over the conductive structure 100 ( FIG. 1 ) and the reaction vessels 1100 and control vessels 1101 retained therein, as illustrated in FIG. 12 . In some embodiments, the extensions 1202 are configured to sit on an upper surface of the dry bath incubator 600 , flush with the upper surface 104 of the conductive structure 100 ( FIG. 1 ).
- the injector support structure 1200 may be formed as a unitary body comprising the supportive body 1201 and the extensions 1202 .
- the supportive body 1201 and the extensions 1202 may be separately formed and then assembled together by, for example and without limitations, fasteners (e.g., nails, screws, adhesive).
- the supportive body 1201 may be substantially solid except for a number of conduits 1204 ( FIG. 14 ) extending through a height of the supportive body 1201 .
- Each of the conduits 1204 may extend between an upper opening 1206 and a lower opening 1208 .
- a width of the upper opening 1206 may be greater than a width of the lower opening 1208 .
- the width of the upper opening 1206 may be selected to receive therein a body 1401 of an injector (e.g., a syringe 1400 ), while the width of the lower opening 1208 may be selected to receive therein a needle 1404 extending from the injector (e.g., the syringe 1400 ).
- the width of the conduit 1204 may transition, for example, step-wise from the width of the upper opening 1206 to the width of the lower opening 1208 , such that a ledge 1407 surrounds the lower opening 1208 .
- a lower end 1402 of the body 1401 of the injector e.g., the syringe 1400
- the ledges 1407 of an arrangement of the conduits 1204 may be at equal heights, relative to the upper surface 104 of the conductive structure 100 when the injector support structure 1200 is positioned overhead.
- the number and relative positioning of the conduits 1204 may correspond to the number and relative positioning of the wells 102 ( FIG. 1 ) of the conductive structure 100 .
- the conduits 1204 and the reaction vessels 1100 or control vessels 1101 ( FIG. 11 ) in the wells 102 align.
- the supportive body 1201 of the injector support structure 1200 further defines therein an alignment opening 1212 , as illustrated in FIGS. 13 and 14 .
- the alignment opening 1212 may protrude upward, into the supportive body 1201 and be configured to receive therein an upper portion of the alignment member 712 extending from one of the openings 112 in the conductive structure 100 .
- the injector support structure 1200 may be positioned over the conductive structure 100 such that the alignment member 712 is received in the alignment opening 1212 of the supportive body 1201 .
- an alignment member e.g., the alignment member 712
- the alignment member 712 may be first received in the alignment opening 1212 of the supportive body 1201 and then received within the opening 112 of the conductive structure 100 when the injector support structure 1200 is positioned over the conductive structure 100 .
- the alignment member 712 enable securing of the lid 900 to the conductive structure 100 during heating, but the alignment member 712 may enable alignment of the injector support structure 1200 over the conductive structure 100 during enzyme addition.
- injectors e.g., syringes 1400
- injectors may be positioned in the injector support structure 1200 such that the needles 1404 pass through the septa 1108 in the lids 1106 of the reaction vessels 1100 and down into the buffer fluid 1104 before at least one enzyme is simultaneously added to the reaction vessels 1100 via the injectors (e.g., syringes 1400 ) and into the buffer fluid 1104 .
- Positioning the needles 1404 to a depth internal to the buffer fluid 1104 may inhibit the enzyme or enzymes from denaturing when passing through the narrow needles 1404 .
- a flat surface that simultaneously contacts the tops of plungers 1403 of the injectors may be used to simultaneously depress the plungers 1403 and expel the enzyme into the buffer fluid 1104 .
- an upper surface of the lid 900 may be used, as illustrated in FIG. 14 .
- the enzyme or enzymes may be substantially simultaneously added to the reaction vessels 1100 to initiate the enzymatic reactions therein.
- the injectors may be pre-loaded into the conduits 1204 of the injector support structure 1200 , because the injectors (e.g., the syringes 1400 ) may be positioned simultaneously as a group over the conductive structure 100 , and because the injectors (e.g., the syringes 1400 ) may be simultaneously depressed to inject the contents thereof into the reaction vessels 1100 , the addition of the enzyme or enzymes to the reaction vessels 1100 may be accomplished quickly. Thereafter, e.g., immediately thereafter, the injector support structure 1200 may be removed from over the conductive structure 100 and the lid 900 returned and secured to the conductive structure 100 .
- the predetermined reaction temperature may be substantially maintained before, during, and after the enzyme addition.
- the temperature of the conductive structure 100 may be monitored, during the enzyme addition, via the thermocouple 604 in the thermocouple opening 110 of the conductive structure 100 .
- the closed volume of the reaction vessels 1100 remains sealed even during the enzyme addition. Thus, evaporation of reagents is prevented even during high-temperature processes.
- FIG. 14 illustrates a row of four syringes 1400 received in a row of four conduits 1204 over four reaction vessels 1100 , it is contemplated that even as few as one syringe 1400 may be utilized in the injector support structure 1200 over one reaction vessel 1100 .
- each syringe 1400 utilized in the injector support structure 1200 corresponds and aligns with one of the reaction vessels 1100 in the conductive structure 100 , the number and relative positioning of the syringes 1400 may vary in different rows or columns of the injector support structure 1200 and/or in different runs using the injector support structure 1200 .
- More than one injector support structure 1200 may be simultaneously positioned and utilized in embodiments in which more than one conductive structure 100 is received in the dry bath incubator 600 . Therefore, while FIG. 12 , for example, illustrates one injector support structure 1200 over one of the two conductive structures 100 ( FIG. 7 ) in the dry bath incubator 600 , a second injector support structure 1200 may be positioned, simultaneously or sequentially, over the other of the conductive structures 100 ( FIG. 7 ) and utilized sequentially or simultaneously. Alternatively, one injector support structure 1200 may be used to substantially simultaneously inject enzyme into reaction vessels 1100 of one of the conductive structures 100 ( FIG. 7 ) and then repositioned over the other of the conductive structures 100 ( FIG.
- the injector support structure 1200 can be quickly and easily positioned and repositioned, thus minimizing the time to add the enzyme and minimizing the time with the lid 900 ( FIG. 14 ) off of the conductive structure 100 .
- the lid 900 may be re-secured to the conductive structure 100 , forming the conductive structure assembly 1000 ( FIG. 10 ), which may then be agitated to encourage mixing of the substrate samples 1103 ( FIG. 14 ) and the added enzyme.
- the conductive structure assembly 1000 ( FIG. 10 ) may be moved from the dry bath incubator 600 to an agitator (e.g., an orbital shaker, a reciprocal shaker) that may also be configured to provide heat during the agitation.
- the dry bath incubator 600 and agitator may be integrated such that the conductive structure assembly 1000 may not need to be removed from a heating device (e.g., the dry bath incubator 600 ) to be agitated.
- the agitator may support the dry bath incubator 600 with the conductive structure assembly 1000 such that the conductive structure assembly 1000 may be agitated without removing the conductive structure assembly 1000 from the heating device (e.g., the dry bath incubator 600 ).
- Such agitator may be a movable surface (e.g., a shaker plate) supporting the dry bath incubator 600 , a movable surface of the dry bath incubator 600 itself, or a container containing the dry bath incubator 600 .
- the agitator may be configured to agitate the conductive structure assembly 1000 through a plane that is parallel to a length (e.g., a height) of the vessels (e.g., the reaction vessels 1100 and/or control vessels 1101 ) while the vessels are in the conductive structure assembly 1000 in the dry bath incubator 600 .
- the agitator may move the dry bath incubator 600 and the conductive structure assembly 1000 up and down, either vertically or along an orbit about a horizontal axis.
- the system may be configured to rotate the dry bath incubator 600 , the conductive structure assembly 1000 , and, thus, the vessels (e.g., the reaction vessels 1100 and/or control vessels 1101 ) in the conductive structure assembly 1000 to align the length of the vessels along a substantially horizontal plane before horizontally agitating the conductive structure assembly 1000 , either linearly or along an orbit about a vertical axis.
- the vessels e.g., the reaction vessels 1100 and/or control vessels 1101
- the agitator may include a shaker plate 1500 to which the conductive structure 100 ( FIG. 16 ) may be releaseably connected during agitation.
- a support structure 1502 may be connected (e.g., releaseably connected) to the shaker plate 1500 .
- the support structure 1502 may include a base plate 1504 mountable to the shaker plate 1500 via one or more fasteners 1506 extending through openings 1507 ( FIG. 16 ) in the base plate 1504 and into openings 1508 in the shaker plate 1500 .
- the fasteners 1506 may be configured as screws with threading corresponding to threads in the openings 1508 .
- the shaker plate 1500 may be a component of a conventional and commercially available orbital shaker, such as a New Brunswick Scientific Model Innova 44R, and the openings 1507 in the base plate 1504 may be positioned to correspond to the openings 1508 in the shaker plate 1500 as acquired from its manufacturer.
- the fasteners 1506 utilized may have a greater length, to accommodate a height of the base plate 1504 , than those sold for use with the shaker plate 1500 .
- the fasteners 1506 may be flush with a surface of the base plate 1504 when the base plate 1504 is secured to the shaker plate 1500 , as illustrated in FIG. 15 .
- One or more engagement features 1510 may extend from the base plate 1504 .
- the engagement features 1510 may be releaseably secured to the base plate 1504 .
- the engagement features 1510 may be threaded rods that may be screwed into threaded openings 1512 ( FIG. 16 ) defined in the base plate 1504 .
- the engagement features 1510 may be permanently affixed to the base plate 1504 .
- the engagement features 1510 may be positioned to align with the openings 114 in the conductive structure 100 .
- the engagement feature or features (e.g., the openings 114 ) of the conductive structure 100 may be selectively, slideably engaged (e.g., in the direction of arrows 15 ) with the counterpart engagement features (e.g., the engagement features 1510 ) on the support structure 1502 to mount the conductive structure 100 to the shaker plate 1500 during the agitation.
- threaded caps 1514 may be attached and tightened over the ends of the engagement features 1510 , which may protrude above the conductive structure 100 , to further secure the conductive structure 100 in place.
- the engagement features (e.g., the openings 114 ) of the conductive structure 100 are defined in sidewalls (e.g., the front surface 116 and the back surface 118 ) of the conductive structure 100
- the upper surface 104 ( FIG. 1 ) of the conductive structure 100 is essentially perpendicular to the shaker plate 1500 ( FIG. 15 ).
- the reaction vessels 1100 ( FIG. 11 ) and control vessels 1101 ( FIG. 11 ) within the conductive structure 100 are positioned such that their length is parallel to the shaker plate 1500 ( FIG. 15 ).
- the shaker plate 1500 orbits, e.g., in the direction of arrows 17 (e.g., in the direction of arrows 17 ′, in the direction of arrows 17 ′′, or both alternatingly) in an x-y plane parallel to the surface of the shaker plate 1500 , the conductive structure 100 and the reaction vessels 1100 ( FIG. 11 ) and control vessels 1101 ( FIG. 11 ) are agitated in a plane that is parallel to a length (e.g., a height) of each. Therefore, the substrate samples 1103 ( FIG. 11 ) and the enzyme are mixed along one of the greatest available volume widths to promote better intermixing of the materials than may be achieved if the reaction vessels 1100 ( FIG. 11 ) were agitated parallel to their width.
- Heat may be provided while the conductive structure assembly 1000 is agitated so that the predetermined reaction temperature is maintained.
- the orbital shaker in which the shaker plate 1500 is located, may be pre-heated to the predetermined reaction temperature before or while the conductive structure 100 is initially heated, the reaction vessels 1100 ( FIG. 11 ) are heated, and the enzyme added. Therefore, as soon as the enzyme has been added to the conductive structure 100 and the lid 900 secured, the conductive structure assembly 1000 may be quickly moved to and positioned on the shaker plate 1500 without substantial heat loss during the transition.
- the orbital shaker may be closed around the conductive structure assembly 1000 during the agitation to retain the heat in the system.
- Reaction time following addition of the enzyme, may be monitored and samples taken from the reaction vessels 1100 ( FIG. 11 ) and/or the control vessels 1101 ( FIG. 11 ) at desired times to measure the enzymatic activity.
- the reaction vessels 1100 ( FIG. 11 ) may be transferred to ice or may be injected with a reaction-stopping agent to cease the enzymatic reaction at a desired time, and then contents of the reaction vessels 1100 ( FIG. 11 ) may be analyzed.
- the stop time for one or more reaction vessels 1100 ( FIG. 11 ) of the group of reaction vessels 1100 ( FIG. 11 ) from the conductive structure assembly 1000 may be spaced from the stop time of others so as to analyze an enzymatic reaction at various times using one conductive structure 100 ( FIG. 1 ). Therefore, the same process may be used and the agitation stage carried out for varying times to gather a range of enzyme reaction times.
- the enzymatic reactions may be halted or substantially slowed, at the desired time, by removing the reaction vessels 1100 ( FIG. 11 ) from the heated conductive structure 100 and moving them to ice, with the addition of reagents configured to halt the reaction, or both.
- Techniques for terminating enzymatic reactions are known in the art and so are not described in detail herein.
- the enzymatic activity may be determined according to techniques known in the art, which are also not described in detail herein. By way of non-limiting example, the enzymatic activity may be determined by a reducing sugar assay, high pressure liquid chromatography (HPLC), the Somogyi method, or the DNS method.
- the methods and systems disclosed herein control the temperature of the system with closed volumes for the reaction vessels 1100 ( FIG. 11 ), even during addition of the enzyme, high temperature reactions may be carried out without evaporation and, therefore, without skewed results. Moreover, because the methods and systems disclosed herein provide substantial intermixing of the substrate samples 1103 ( FIG. 11 ) and the enzyme, insoluble substrates and substrates and enzymes that do not readily intermix may be analyzed without skewed results. Furthermore, because multiple small-volume samples may be simultaneously run in the conductive structure 100 , even heterogeneous substrates and substrates or enzymes for which only small amounts are available may be analyzed for enzymatic activity.
- Endo-1,4- ⁇ -XYLANASE M4 (hereinafter “Megazyme M4”) is an enzyme commercially available from Megazyme International Ireland, Ltd., Wicklow, Ireland. The published specific activity for Megazyme M4, in association with a wheat arabinoxylan substrate, which is partially soluble, averages 79.3 U/mg Protein.
- the specific activity was measured at 83.6 ⁇ 6.4 U/mg.
- the specific activity was measured at 93.4 ⁇ 7.6 U/mg.
- the specific activity was measured at 110.2 ⁇ 10.6 U/mg.
- Table I the conventional standard tube assay yielded enzymatic activities that were, on average, about 16.6 U/mg higher (plus or minus between 6.4 U/mg and 10.6 U/mg) than the enzymatic activity reported by the commercial supplier (79.3 U/mg).
- Enzymatic reactions with various volumes of Megazyme M4 and various amounts of wheat arabinoxylan were then carried out according to the sealed vessel with agitation method according to embodiments of the present disclosure, and then activity was determined at various times according to the standard Somogyi characterization methods. All reactions used a substrate solution at a pH of 4.0 and a reaction temperature of 40° C. Results are shown in FIGS. 22 through 25 , which plot amount of reaction product (in ⁇ moles) against reaction stop time (in minutes). With reference to FIG. 22 , using 0.3048 ⁇ g of the enzyme, the specific activity was measured at 87.1 ⁇ 7.4 U/mg. With reference to FIG.
- the specific activity was measured at 92.6 ⁇ 7.2 U/mg.
- the specific activity was measured at 88.9 ⁇ 9.2 U/mg.
- the specific activity was measured at 91.9 ⁇ 8.5 U/mg.
- the method according to the present disclosure yielded enzymatic activities that were, on average, only about 10.8 U/mg higher (plus or minus between 7.2 U/mg and 9.2 U/mg) than the enzymatic activity reported by the commercial supplier (79.3 U/mg), even at lower enzyme amounts than used with the standard tube assay.
- the methods of the present disclosure yielded enzymatic activities that were, on average, closer to the specific activity of the enzyme reported by the commercial supplier than the enzymatic activities determined using a conventional tube assay. Accordingly, the methods of the present disclosure may yield more accurate results, for a partially soluble substrate in very small amounts, than methods that do not use agitation.
- the demonstrated improvement may be achieved with even moderate-temperature enzymatic reactions such as the 40° C. reactions of the examples of FIGS. 22 through 25 . Therefore, though the methods of the present disclosure may be well suited for high-temperature enzymatic reactions, because the methods may avoid skewing due to evaporation, the example discussed here demonstrates that the methods are also effective for moderate-temperature enzymatic reactions.
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Abstract
Methods for determining thermophilic enzymatic activity include heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes, at least one enzyme is added, substantially simultaneously, to the closed volumes. At the predetermined reaction temperature, the closed volumes are agitated and then the activity of the at least one enzyme is determined. The methods are conducive for characterizing enzymes of high-temperature reactions, with insoluble substrates, with substrates and enzymes that do not readily intermix, and with low volumes of substrate and enzyme. Systems for characterizing the enzymes are also disclosed.
Description
- This application is a divisional of U.S. patent application Ser. No. 14/180,161, filed Feb. 13, 2014, pending, the disclosure of which is hereby incorporated in its entirety herein by this reference.
- This invention was made with government support under Contract No. DE-AC07-05-ID14517 awarded by the United States Department of Energy. The government has certain rights in the invention.
- The present disclosure, in various embodiments, relates generally to the field of thermophilic enzyme characterization. More particularly, this disclosure relates to methods and systems for determining enzymatic activity. The methods and systems may be utilized for enzyme characterization studies applied to enzymatic reactions that involve, among others characteristics, high temperatures, insoluble substrates, and heterogeneous substrates.
- Enzymes are biological molecules that catalyze a chemical reaction of a substrate. To understand the reaction mechanism and kinetics of such enzymatic reactions, it is often desirable to characterize an enzyme by determining its activity. Enzyme characterizations are carried out with assays that quantitatively assess enzyme activity based on, for example, resulting concentrations of one or more products of the enzymatic reaction. Thus, enzyme characterization provides information about the enzyme activity that can be used to predict how the enzyme will behave when reaction mixtures are altered, such as by adjusting the amounts of substrate, enzyme, etc., involved. Characterizing enzyme activity at different temperatures or pH levels provides information about the enzyme activity that can be used to predict how the enzyme will behave when reaction conditions are altered.
- Typically, enzyme activity is determined by measuring the concentration of a reaction product of an enzymatic reaction over time for a fixed and constant enzyme concentration. Thus, the higher a reaction product concentration detected, in a period of time, the higher the enzyme activity determined. However, the reaction product concentration levels may be impacted by conditions other than enzyme activity. For example, some enzymatic reactions are carried out at high temperatures (e.g., at temperatures above about 50° C.) that can cause reagents of the reaction to evaporate, skewing concentration measurements. That is, if the reagents evaporate during the reaction, resulting concentrations of reaction products may be determined to be artificially higher than they would otherwise be, indicating a false-high enzymatic activity, or the evaporation may concentrate the enzyme in the reaction, resulting, again, in artificially high measured concentrations of reaction products. As another example, some substrates are generally insoluble in the reagents, and mass transfer properties, such as the diffusivity of the enzyme into the substrate or the diffusivity of a reaction product out of the substrate, may control the rate of the enzymatic reaction and, thus, the rate of production of the reaction product. Therefore, measured reaction product concentrations may be artificially low based on a low diffusivity value, rather than on the actual rate of the enzymatic reaction. As still another example, not all substrates and enzymes readily intermix with one another. Low intermixing may result in less enzymatic activity and, thus, low reaction product concentrations, even if the reaction rate is actually rapid.
- Additionally, conventional methods for enzyme characterization may not be well suited to evaluate the enzymatic activity using substrates that are of industrial relevance. Not only may some industrially relevant substrates be generally insoluble, but some may additionally or alternatively be generally heterogeneous such that one small sample of the substrate may vary in composition from another small sample of the same substrate. To try to avoid such heterogeneity impacting the results of enzymatic characterization methods, many conventional methods involve the use of a large amount of the substrate. However, use of a large amount of substrate may require use of a large amount of enzyme in the characterization. Such large-volume methods may not be conducive for characterizing enzymes for which only small amounts are available.
- Thus, accurately characterizing enzymes for high-temperature (i.e., greater than about 50° C.) reactions, reactions with insoluble substrates, reactions with substrates and enzymes that do not readily intermix, reactions with heterogeneous substrates, or reactions where large quantities of enzymes or substrates are not available often presents challenges.
- A method for determining enzymatic activity, according to an embodiment of the present disclosure, comprises heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes of the plurality, at least one enzyme is substantially simultaneously added to the closed volumes of the plurality. After adding the at least one enzyme, the plurality of closed volumes are agitated at the predetermined reaction temperature. After the agitation, the activity of the at least one enzyme is determined.
- A method for determining enzymatic activity, according to another embodiment of the present disclosure, comprises heating a conductive structure, supporting sealed reaction vessels containing substrate, to a predetermined reaction temperature. At least one enzyme is substantially simultaneously injected into the sealed reaction vessels. The sealed reaction vessels, with the substrate and the at least one enzyme, are agitated in a plane of motion parallel to a length of the sealed reaction vessels. The method also comprises determining activity of the at least one enzyme.
- A system for determining enzymatic activity, according to an embodiment of the present disclosure, comprises a conductive structure that defines a plurality of wells protruding into the conductive structure from an upper surface of the conductive structure. The plurality of wells is configured to receive a plurality of reaction vessels. The conductive structure also defines at least one engagement feature on a sidewall of the conductive structure. The at least one engagement feature is configured to engage a counterpart engagement feature to secure the conductive structure to an agitator.
-
FIG. 1 is a top, front, and right side perspective view of a conductive structure of a system for analyzing an enzyme, according to an embodiment of the present disclosure. -
FIG. 2 is a bottom, rear, and left side perspective view of the conductive structure ofFIG. 1 . -
FIG. 3 is a top, plan view of the conductive structure ofFIGS. 1 and 2 . -
FIG. 4 is a front elevation view of the conductive structure ofFIGS. 1 through 3 with a lid on the conductive structure.FIG. 4 is also a rear elevation view of the conductive structure ofFIGS. 1 through 3 with the lid on the conductive structure. -
FIG. 5 is a right side elevation view of the conductive structure ofFIGS. 1 through 4 with the lid on the conductive structure.FIG. 5 is also a left side elevation view of the conductive structure ofFIGS. 1 through 4 with the lid on the conductive structure. -
FIGS. 6 through 17 are views of various stages of a method for processing an enzyme to be characterized, according to an embodiment of the present disclosure, wherein: -
FIG. 6 is a top and front perspective view of a dry bath incubator into which the conductive structure ofFIGS. 1 through 5 is configured to be received; -
FIG. 7 is a top and front perspective view of the dry bath incubator ofFIG. 6 having received therein two of the conductive structures ofFIGS. 1 through 5 with alignment members received in the conductive structures; -
FIG. 8 is a top and front perspective view of a fluid-containing vial that the conductive structure ofFIGS. 1 through 5 is configured to receive; -
FIG. 9 is an exploded view of a conductive structure assembly, including a top and rear perspective view of the conductive structure ofFIGS. 1 through 5 with a plurality of the fluid-containing vials ofFIG. 8 received in wells of the conductive structure and an alignment member received in the conductive structure, a bottom and rear perspective view of a lid to secure to the conductive structure, and a bottom and rear perspective view of a fastener member to secure the lid to the conductive structure; -
FIG. 10 is a top, rear, and left side perspective view of the conductive structure assembly ofFIG. 9 , assembled for heating to a predetermined reaction temperature in the dry bath incubator ofFIG. 6 , with the lid secured to the conductive structure via the fastener member and another fastener engaged with the alignment member; -
FIG. 11 is a partial, top, front, and left side perspective view of the conductive structure ofFIGS. 1 through 5 in the dry bath incubator ofFIG. 6 with a plurality of substrate-containing vials, some of which having lids with septa, and a top and front perspective view of a substrate-containing vial with a septum-containing lid to be received in a well of the conductive structure; -
FIG. 12 is a top, front, and right side view of the two conductive structures ofFIG. 7 , in the dry bath incubator ofFIG. 6 , the conductive structures having received therein substrate-containing vials with septa-including lids, and with an injector support structure aligned over one of the two conductive structures; -
FIG. 13 is a bottom, rear, and right side view of the injector support structure ofFIG. 12 ; -
FIG. 14 is a front, cross-sectional, elevation view, taken along section line 14-14 ofFIGS. 11 and 12 , with injectors received in conduits of the injector support structure ofFIG. 12 , needles of the injectors extending through septa of substrate-containing vials, and the lid ofFIG. 9 being utilized to simultaneously depress plungers of the injectors to substantially simultaneously inject at least one enzyme from the injectors into a liquid in the substrate-containing vials; -
FIG. 15 is a top, front, and right side perspective view of a support structure secured to a shaker plate of an orbital shaker; -
FIG. 16 is a front, cross-sectional, elevation view, of the conductive structure ofFIGS. 4 and 5 , taken along section line 16-16 ofFIG. 15 , having an engagement feature engaged with a counterpart engagement feature of the support structure ofFIG. 15 ; and -
FIG. 17 is a top, front, and left side perspective view of a conductive structure assembly such as that ofFIG. 10 , but having received therein substrate-and-enzyme containing vials, secured to the shaker plate of an orbital shaker via the support structure ofFIG. 15 and being agitated in a plane of motion that is parallel to a length of the substrate-and-enzyme containing vials while a predetermined reaction temperature is maintained. -
FIGS. 18 through 21 graph enzymatic activity results, for a commercially available enzyme, determined using a conventional test tube assay that does not include agitation and sealed reaction vessels. -
FIGS. 22 through 25 graph enzymatic activity results, for the commercially available enzyme, determined using a method according to the present disclosure that includes agitation and sealed reaction vessels. - Methods and systems for determining activity of an enzyme are disclosed. Substrate in closed vessels is brought to a predetermined reaction temperature. Without opening the vessels, at least one enzyme is substantially simultaneously added to each of the closed vessels. The closed vessels, with the substrate and added enzyme, are then agitated to mix the substrate and the enzyme. The closed vessels may be maintained at essentially the predetermined reaction temperature throughout the enzyme addition and the agitation. Therefore, the temperature may be controlled, evaporation of reagents may be prevented, and multiple samples may be simultaneously processed, increasing the throughput. The enzymatic reactions, which take place in the closed vessels, may be analyzed to accurately determine enzyme activity, even for high-temperature enzymatic reactions, insoluble substrates, substrates and enzymes that do not readily intermix, substrates that are heterogeneous, and low sample sizes of the substrate and enzyme. Thus, the activity of enzymes for enzymatic reactions that involve insoluble substrates, partially soluble substrates, heterogeneous substrates, small substrate amounts, small enzyme volumes, high-temperatures, or mixing challenges may be determined by a reliable and reproducible method. The methods and systems of the present disclosure may also provide a high throughput assay for determining the enzymatic activity.
- The illustrations presented herein are not meant to be actual views of any particular apparatus, system, or method stage, but are merely idealized representations that are employed to describe embodiments of the present invention.
- As used herein, the term “substrate” means and includes a material to be at least partially consumed in a reaction catalyzed by the at least one enzyme to be characterized.
- The following description provides specific details, such as material types and processing conditions in order to provide a thorough description of embodiments of the present disclosure. However, a person of ordinary skill in the art will understand that the embodiments of the present disclosure may be practiced without employing these specific details. The embodiments of the present disclosure may be practiced in conjunction with conventional enzyme characterization methods known in the industry, utilizing the results of the methods of processing the enzymes disclosed herein and/or the systems for processing the enzymes disclosed herein.
- Unless the context indicates otherwise, the structures described herein may be formed by any suitable technique, the samples may be prepared by any suitable technique, and the enzymatic reactions may be analyzed by any suitable technique, which techniques may be selected by a person having ordinary skill in the art.
-
FIGS. 1 through 5 illustrate aconductive structure 100 configured to receive and transfer heat to reaction vessels. Substrate samples and at least one enzyme may be inserted into the reaction vessels, and enzymatic reactions may be carried out before the results of the reaction are analyze to characterize the enzyme. Theconductive structure 100 may include a number ofwells 102 that protrude into theconductive structure 100 from anupper surface 104 of theconductive structure 100. As illustrated inFIG. 4 , thewells 102 may protrude essentially perpendicularly relative to theupper surface 104 and to alower surface 106. Thewells 102 may be shaped to snugly receive therein the reaction vessels. Therefore, heat may be conductively transferred between theconductive structure 100 and the reaction vessels. - The
conductive structure 100 may be formed of a conductive material, such as, for example and without limitation, a metal (e.g., aluminum), a metal alloy, or other conductive material. The conductive material may be selected to have a high heat conductivity, so that theconductive structure 100 may be quickly heated by a heat source, and a high heat capacity, so that theconductive structure 100 remains heated even if the heat source is temporarily interrupted. - The
conductive structure 100 may be formed from an essentially solid block of the conductive material. Thewells 102, and other negative-space features, may be machined or otherwise formed into the block by conventional techniques, which are not described in detail herein. In other embodiments, theconductive structure 100 may be molded to define thewells 102, and other negative-space features, when the conductive material is first formed into theconductive structure 100. - The
wells 102 may be arranged in an ordered array, e.g., the column-and-row arrangement illustrated inFIG. 1 , or may be arranged without a particular order. In some embodiments, each of thewells 102 may have essentially the same dimensions and be evenly spaced. In other embodiments, thewells 102 may vary in dimension from one to another and not be evenly spaced. The dimensions and arrangement of thewells 102 may be tailored to enable heat transfer from theconductive structure 100 with consistent temperatures in thewells 102 regardless of the relative position of each well 102 in theconductive structure 100. - The
conductive structure 100 may also define therein one or more probe openings. For example, athermometer opening 108 may protrude into theconductive structure 100 from theupper surface 104. Thethermometer opening 108 may be configured to receive therein a thermometer during heating and/or reaction stages of methods according to embodiments of the present disclosure. Thethermometer opening 108 may protrude to, e.g., a depth approximately even with a depth of thewells 102, as illustrated inFIG. 4 . Thus, temperatures read from a thermometer received in thethermometer opening 108 may be the same temperatures of materials within the reaction vessels received in thewells 102. To enable efficient heat transfer between walls of thethermometer opening 108 and the thermometer, thethermometer opening 108 may be filled with a heat transfer liquid (e.g., water or other fluid, e.g., oil) before the thermometer is received. The same heat transfer liquid may also be filled, partially or completely, into thewells 102 before the reaction vessels are received therein to, again, enable efficient heat transfer between the conductive material of theconductive structure 100 and the material of the reaction vessels. - In some embodiments, the
conductive structure 100 may also define therein athermocouple opening 110 configured to receive a thermocouple extending from a heating device. Thethermocouple opening 110, as illustrated inFIGS. 2 and 4 , may protrude into theconductive structure 100 from thelower surface 106. With reference toFIG. 4 , thethermocouple opening 110 may align with thethermometer opening 108, though the two openings may not connect. - The
conductive structure 100 may also define therein one ormore openings 112 for receiving an alignment member and/or fastener member. For example, theopenings 112 may be defined to protrude into theconductive structure 100 from theupper surface 104. In some embodiments, theopenings 112 may be threaded and therefore configured to receive threaded alignment or threaded fastener members therein. With reference toFIG. 4 , in some embodiments, theopenings 112 may protrude to a depth less than that of thewells 102. However, in other embodiments, theopenings 112 may protrude deeper or all the way through the height of theconductive structure 100. - In some embodiments, the
conductive structure 100 may include an engagement feature configured to engage with a counterpart engagement feature of another structure to secure theconductive structure 100 to the other structure. For example, the engagement feature may include one ormore openings 114 defined in afront surface 116 and aback surface 118 of theconductive structure 100 and extending through a length of theconductive structure 100. Therefore, counterpart engagement features (e.g., engagement features 1510 (FIG. 15 )) may be received through theopenings 114 and may be utilized to secure theconductive structure 100 to another structure such as, for example, an agitator. - In some embodiments, side surfaces 120 of the
conductive structure 100 may be free from openings, as illustrated inFIGS. 1 and 5 . - Though the
conductive structure 100 ofFIGS. 1 through 5 is illustrated to be a six-sided block shape, in other embodiments, theconductive structure 100 may be otherwise shaped but nonetheless configured to receive reaction vessels inwells 102. - A method, according to an embodiment of the present disclosure, for characterizing an enzyme may include heating the
conductive structure 100. Heat may be provided to theconductive structure 100 from a heat source such as adry bath incubator 600, illustrated inFIG. 6 . Thedry bath incubator 600 may include acavity 602 into which the conductive structure 100 (FIGS. 1 through 5 ) is received. Athermocouple 604 may protrude from a base of thecavity 602 and may be received in the thermocouple opening 110 (FIG. 4 ) of the conductive structure 100 (FIGS. 1 through 5 ) when theconductive structure 100 is in place in thecavity 602. - In some embodiments, the conductive structures 100 (
FIGS. 1 through 5 ) are configured such that more than oneconductive structure 100 may be received in thecavity 602 of thedry bath incubator 600, as illustrated inFIG. 7 . The upper surface 104 (FIG. 1 ) of theconductive structure 100 may be flush with an upper surface of thedry bath incubator 600. -
FIG. 7 further illustratesalignment members 712 received within one of the openings 112 (FIG. 4 ) defined in theconductive structures 100. Thealignment members 712 may be threaded rods that are screwed into one of theopenings 112 of theconductive structure 100 in such embodiments in which theopenings 112 are also threaded. Therefore, in some embodiments, thealignment members 712 may be removable from theconductive structures 100. In other embodiments, thealignment members 712 may be permanently affixed to theconductive structures 100. - In some embodiments, the
conductive structures 100 may be heated, using thedry bath incubator 600, to, e.g., a predetermined reaction temperature. The reaction temperature may be selected based on the enzymatic reaction to be carried out. In some such embodiments, theconductive structures 100 may be heated when empty, i.e., without any material received in thewells 102. In other embodiments, thewells 102 may be filled with a heat transfer fluid (e.g., water, oil, or other fluid) prior to or during the heating. The heat transfer fluid may also be added to the thermometer opening 108 (FIG. 4 ) as discussed above. In one embodiment, according to the present disclosure, the heat transfer fluid may be added to vessels 800 (FIG. 8 ) before or while they are received in thewells 102. The vessels 800 (FIG. 8 ) may be essentially the same as those to be used as reaction vessels later in the method. For example, aglass vial 802 may be used as thevessel 800, andwater 804 may be added to theglass vial 802 and used as the heat transfer fluid. The volume ofwater 804 added may be approximately the same volume as the reagents of the enzymatic reaction to be carried out later in the method. Therefore, during the initial heating of the conductive structures 100 (FIG. 7 ), the heat to be provided to the later-received reaction vessels will be consistent with the heat provided to thevessels 800 with only thewater 804. Thevessels 800 may be sealed with alid 806 that secures to theglass vial 802. - A
vessel 800 may be loaded into each of the wells 102 (FIG. 7 ) so that the heat profile across theconductive structure 100 will be even. Each well 102 may be configured to snugly receive one of thevessels 800. In some embodiments, the walls of theglass vials 802 may be fully received in thewells 102 with essentially only thelids 806 protruding above theupper surface 104 of theconductive structure 100, as illustrated inFIG. 9 . Thus, an even heat may be provided from theconductive structure 100 to the glass vials 802 (FIG. 8 ) and thereafter to the water 804 (FIG. 8 ) within theglass vials 802. In some embodiments, heat transfer fluid (e.g., additional water, oil) may be included in the wells 102 (FIG. 4 ) before theglass vials 802 are received therein to further ensure conductive heat transfer between theconductive structure 100 and thewater 804 within theglass vials 802. - As illustrated in
FIG. 9 , alid 900 may be placed over the top of theconductive structure 100 and over thelids 806 of the vessels 800 (FIG. 8 ). Thelid 900 may have sidewalls 902 that extend the height of thelids 806 of thevessels 800 such that alower edge 904 abuts theupper surface 104 of theconductive structure 100, as illustrated inFIGS. 4 and 5 . Aninterior surface 906 of thelid 900 may abut the tops of thelids 806 of the vessels 800 (FIG. 8 ), when thelid 900 is secured to theconductive structure 100. When placed on theconductive structure 100, thelid 900 may reduce or prevent heat from exiting theconductive structure 100 and the vessels 800 (FIG. 8 ). Thus, thelid 900 may promote efficient heating of thevessels 800. - In some embodiments, the
lid 900 may be formed of a conductive material (e.g., a metal (e.g., aluminum, tin), a metal alloy). Thesidewalls 902 of thelid 900 may define a space in which each of thelids 806 of the vessels 800 (FIG. 8 ) may be received, as illustrated inFIG. 9 . In another embodiment (not shown), thelid 900 may be formed as an essentially solid structure of a conductive material with wells configured to receive, in each, thelid 806 of one of thevessels 800. Thus, thelids 806 of thevessels 800 may be snugly received within thelid 900 on theconductive structure 100. - The
lid 900 may define thereinopenings 912 that may align with theopenings 112 defined in theupper surface 104 of theconductive structure 100. Therefore, thealignment member 712, received within one of theopenings 112 of theconductive structure 100 may align with and extend through one of theopenings 912 in thelid 900. A fastener, such as a nut 1012 (seeFIG. 10 ) may be releaseably engaged with thealignment member 712 to secure thelid 900 to theconductive structure 100. Afastener member 920 may be passed through another of theopenings 912 in thelid 900 to engage another of theopenings 112 of theconductive structure 100. Thefastener member 920 may be a threaded rod that corresponds to threading in theopenings 112. Thefastener member 920 may be screwed into theopening 112 to secure thelid 900 to theconductive structure 100. Thus, as illustrated inFIG. 10 , aconductive structure assembly 1000 may be secured as the dry bath incubator 600 (FIG. 6 ) heats theconductive structure 100. - The
lid 900 may define athermometer opening 908 passing through thelid 900 and corresponding to thethermometer opening 108 of theconductive structure 100. Therefore, when thelid 900 is secured to theconductive structure 100, a thermometer may be inserted into thethermometer openings conductive structure 100. - Though
FIGS. 9 and 10 illustrate theconductive structure 100 out of the dry bath incubator 600 (FIG. 6 ) for convenience, it is contemplated that theconductive structure assembly 1000 will be assembled while theconductive structure 100 is received within the cavity 602 (FIG. 6 ) of the dry bath incubator 600 (FIG. 6 ) to allow the heating to continue without interruption while thelid 900 is secured to theconductive structure 100. - Once the
conductive structure 100 is heated to the predetermined reaction temperature, as indicated by one or both of a thermometer in the thermometer opening 108 (FIG. 1 ) and a thermocouple in the thermocouple opening 110 (FIG. 4 ), the vessels 800 (FIG. 8 ) with the heat transfer fluid (e.g., water, oil) may be removed from theconductive structure 100 and quickly replaced with other vessels, such as, as illustrated inFIG. 11 ,reaction vessels 1100 orcontrol vessels 1101. Each of thereaction vessels 1100 may include one of theglass vials 802 with asubstrate sample 1103 therein and, for example, abuffer fluid 1104. The composition and amount of thesubstrate sample 1103 and thebuffer fluid 1104 may be selected according to the enzymatic reaction to be carried out in thereaction vessel 1100. Thesubstrate samples 1103 may be sealed in thereaction vessels 1100 bylids 1106 havingsepta 1108 that are penetrable by injection needles without unsealing the contents of thereaction vessels 1100. Thecontrol vessels 1101 may each include a control composition against which an enzymatic reaction to be carried out in one of thereaction vessels 1100 is to be compared. For control compositions into which no enzyme is to be added, thecontrol vessels 1101 may be sealed by one of thelids 806 that does not include theseptum 1108. - As illustrated in
FIG. 11 , each of thewells 102 may receive either one of thereaction vessels 1100 or one of thecontrol vessels 1101.Reaction vessels 1100 may occupy all of thewells 102 of the conductive structure 100 (FIG. 10 ),control vessels 1101 may occupy all of thewells 120, or a mixed grouping of thereaction vessels 1100 and thecontrol vessels 1101 may be used to occupy thewells 102. The grouping and disposition of thereaction vessels 1100 and/orcontrol vessels 1101 used may be selected to provide the desired number of reaction results and controls against which to compare the reaction results. - In some embodiments, the
same substrate sample 1103 may be included in eachreaction vessel 1100. In other embodiments, a variety ofsubstrate samples 1103 may be included in thereaction vessels 1100 and enzymatic reactions carried out in each simultaneously. In such embodiments, it is contemplated that thesubstrate samples 1103 will be selected so that reaction conditions, such as temperature, may be consistent for each of the reactions. - The vessels 800 (
FIG. 8 ) used during the initial heating of theconductive structure 100 to the predetermined reaction temperature may be quickly replaced with thereaction vessels 1100 and/or thecontrol vessels 1101 while the lid 900 (FIG. 10 ) is removed from theconductive structure 100. With minimal time used to switch out the vessels, minimal heat may be lost from theconductive structure 100. In some embodiments, e.g., such as those embodiments in which the disclosed methods are automated, all of the vessels 800 (FIG. 8 ) may be simultaneously removed and may be simultaneously replaced with thereaction vessels 1100 and/or thecontrol vessels 1101, which may minimize the transition time and the heat lost. - It is contemplated that the volume of the water 804 (
FIG. 8 ) in the vessels 800 (FIG. 8 ), used for the initial heating of theconductive structure 100 to the predetermined reaction temperature and later replaced with the controlled volumes (e.g., thereaction vessels 1100 and/or control vessels 1101), will be substantially the same as the volume of thebuffer fluid 1104, and any other solid or liquid material, in the vessels (e.g., thereaction vessels 1100 and/or control vessels 1101). Therefore, the heat profile of the vessels 800 (FIG. 8 ), used for the initial pre-heating, will be substantially similar to the heat profile of the vessels (e.g., thereaction vessels 1100 and/or control vessels 1101), prior to enzyme injection. In other embodiments, the volumes may vary. - After replacing the initial vessels 800 (
FIG. 8 ) with the other vessels (e.g., thereaction vessels 1100 and/or the control vessels 1101), the lid 900 (FIG. 10 ) may be again secured to the conductive structure 100 (FIG. 10 ) and the temperature of theconductive structure 100 monitored until the conductive structure assembly 1000 (FIG. 10 ) returns to the predetermined reaction temperature. Because the conductive structure 100 (FIG. 10 ) may be pre-heated to the predetermined reaction temperature before the control volumes (e.g., thereaction vessels 1100 and/or the control vessels 1101) are received in the wells 102 (FIG. 1 ) of the conductive structure 100 (FIG. 10 ), the time needed for theconductive structure 100 to return to the predetermined reaction temperature after the vessels (e.g., thereaction vessels 1100 and/or the control vessels 1101) are inserted may be minimal. - Though in some embodiments, the
conductive structure 100 may be first heated to the predetermined reaction temperature with the substrate-free vessels 800 (FIG. 8 ) before thesubstrate samples 1103 are added to the system, in other embodiments, theconductive structure 100 may be first heated to the predetermined reaction temperature with the other vessels (e.g., thereaction vessels 1100 and/or the control vessels 1101) in the wells 102 (FIG. 1 ), including thesubstrate samples 1103. Whether or not thesubstrate samples 1103 are included in the initial heating of theconductive structure 100 may depend on whether the material of thesubstrate samples 1103 will decompose during the heating. - Once the system with the
substrate samples 1103 is brought to the predetermined reaction temperature, the lid 900 (FIG. 10 ) may again be removed and at least one enzyme substantially simultaneously added to each of thereaction vessels 1100 to begin an enzymatic reaction in each of thereaction vessels 1100. The enzyme may be added into thereaction vessels 1100 through thesepta 1108 such that thereaction vessels 1100 remain sealed. Keeping thereaction vessels 1100 sealed may reduce or prevent evaporation of reagents from the closed volumes of thereaction vessels 1100 and, therefore, prevent skewing of resulting concentration measurements. - With reference to
FIGS. 12 and 13 , aninjector support structure 1200 may be utilized to enable simultaneous addition of at least one enzyme to the reaction vessels 1100 (FIG. 11 ). Theinjector support structure 1200 may include asupportive body 1201 andextensions 1202 protruding from a bottom of thesupportive body 1201. Theinjector support structure 1200 may be made of a solid material that is conductive (e.g., metal, metal alloy) or nonconductive (e.g., wood, plastic). Theextensions 1202 may protrude a height at least as great as the height of thelids 1106 of the reaction vessels 1100 (FIG. 11 ) and control vessels 1101 (FIG. 11 ). Theextensions 1202 may be spaced from one another by a width that is at least as great as a width of thereaction vessels 1100 andcontrol vessels 1101 in the conductive structure 100 (FIG. 1 ). Therefore, theinjector support structure 1200 may be positioned over the conductive structure 100 (FIG. 1 ) and thereaction vessels 1100 andcontrol vessels 1101 retained therein, as illustrated inFIG. 12 . In some embodiments, theextensions 1202 are configured to sit on an upper surface of thedry bath incubator 600, flush with theupper surface 104 of the conductive structure 100 (FIG. 1 ). - The
injector support structure 1200 may be formed as a unitary body comprising thesupportive body 1201 and theextensions 1202. Alternatively, thesupportive body 1201 and theextensions 1202 may be separately formed and then assembled together by, for example and without limitations, fasteners (e.g., nails, screws, adhesive). - With continued reference to
FIGS. 12 and 13 and with reference toFIG. 14 , thesupportive body 1201 may be substantially solid except for a number of conduits 1204 (FIG. 14 ) extending through a height of thesupportive body 1201. Each of theconduits 1204 may extend between anupper opening 1206 and alower opening 1208. A width of theupper opening 1206 may be greater than a width of thelower opening 1208. The width of theupper opening 1206 may be selected to receive therein abody 1401 of an injector (e.g., a syringe 1400), while the width of thelower opening 1208 may be selected to receive therein aneedle 1404 extending from the injector (e.g., the syringe 1400). - The width of the
conduit 1204 may transition, for example, step-wise from the width of theupper opening 1206 to the width of thelower opening 1208, such that aledge 1407 surrounds thelower opening 1208. Alower end 1402 of thebody 1401 of the injector (e.g., the syringe 1400) may rest against theledge 1407 when the injector (e.g., the syringe 1400) is received within theconduit 1204, as illustrated inFIG. 14 . Theledges 1407 of an arrangement of theconduits 1204 may be at equal heights, relative to theupper surface 104 of theconductive structure 100 when theinjector support structure 1200 is positioned overhead. - The number and relative positioning of the
conduits 1204 may correspond to the number and relative positioning of the wells 102 (FIG. 1 ) of theconductive structure 100. Thus, when theinjector support structure 1200 is positioned over theconductive structure 100, theconduits 1204 and thereaction vessels 1100 or control vessels 1101 (FIG. 11 ) in thewells 102 align. In some embodiments, thesupportive body 1201 of theinjector support structure 1200 further defines therein analignment opening 1212, as illustrated inFIGS. 13 and 14 . Thealignment opening 1212 may protrude upward, into thesupportive body 1201 and be configured to receive therein an upper portion of thealignment member 712 extending from one of theopenings 112 in theconductive structure 100. Therefore, to enable appropriate alignment of theconduits 1204 over the wells 102 (FIG. 1 ), theinjector support structure 1200 may be positioned over theconductive structure 100 such that thealignment member 712 is received in thealignment opening 1212 of thesupportive body 1201. In other embodiments, an alignment member (e.g., the alignment member 712) may be first received in thealignment opening 1212 of thesupportive body 1201 and then received within theopening 112 of theconductive structure 100 when theinjector support structure 1200 is positioned over theconductive structure 100. Thus, not only may thealignment member 712 enable securing of thelid 900 to theconductive structure 100 during heating, but thealignment member 712 may enable alignment of theinjector support structure 1200 over theconductive structure 100 during enzyme addition. - As illustrated in
FIG. 14 , injectors (e.g., syringes 1400) may be positioned in theinjector support structure 1200 such that theneedles 1404 pass through thesepta 1108 in thelids 1106 of thereaction vessels 1100 and down into thebuffer fluid 1104 before at least one enzyme is simultaneously added to thereaction vessels 1100 via the injectors (e.g., syringes 1400) and into thebuffer fluid 1104. Positioning theneedles 1404 to a depth internal to thebuffer fluid 1104 may inhibit the enzyme or enzymes from denaturing when passing through the narrow needles 1404. - In some embodiments, a flat surface that simultaneously contacts the tops of
plungers 1403 of the injectors (e.g., the syringes 1400) may be used to simultaneously depress theplungers 1403 and expel the enzyme into thebuffer fluid 1104. For example, an upper surface of thelid 900 may be used, as illustrated inFIG. 14 . Thus, the enzyme or enzymes may be substantially simultaneously added to thereaction vessels 1100 to initiate the enzymatic reactions therein. - Because the injectors (e.g., the syringes 1400) may be pre-loaded into the
conduits 1204 of theinjector support structure 1200, because the injectors (e.g., the syringes 1400) may be positioned simultaneously as a group over theconductive structure 100, and because the injectors (e.g., the syringes 1400) may be simultaneously depressed to inject the contents thereof into thereaction vessels 1100, the addition of the enzyme or enzymes to thereaction vessels 1100 may be accomplished quickly. Thereafter, e.g., immediately thereafter, theinjector support structure 1200 may be removed from over theconductive structure 100 and thelid 900 returned and secured to theconductive structure 100. - During the enzyme addition, heat may be continuously provided to the
conductive structure 100 via thedry bath incubator 600. Therefore, the predetermined reaction temperature may be substantially maintained before, during, and after the enzyme addition. The temperature of theconductive structure 100 may be monitored, during the enzyme addition, via thethermocouple 604 in thethermocouple opening 110 of theconductive structure 100. - Because the enzyme or enzymes are added to the
reaction vessels 1100 through thesepta 1108 in thelids 1106, the closed volume of thereaction vessels 1100 remains sealed even during the enzyme addition. Thus, evaporation of reagents is prevented even during high-temperature processes. - Though
FIG. 14 illustrates a row of four syringes 1400 received in a row of fourconduits 1204 over fourreaction vessels 1100, it is contemplated that even as few as one syringe 1400 may be utilized in theinjector support structure 1200 over onereaction vessel 1100. Provided each syringe 1400 utilized in theinjector support structure 1200 corresponds and aligns with one of thereaction vessels 1100 in theconductive structure 100, the number and relative positioning of the syringes 1400 may vary in different rows or columns of theinjector support structure 1200 and/or in different runs using theinjector support structure 1200. - More than one
injector support structure 1200 may be simultaneously positioned and utilized in embodiments in which more than oneconductive structure 100 is received in thedry bath incubator 600. Therefore, whileFIG. 12 , for example, illustrates oneinjector support structure 1200 over one of the two conductive structures 100 (FIG. 7 ) in thedry bath incubator 600, a secondinjector support structure 1200 may be positioned, simultaneously or sequentially, over the other of the conductive structures 100 (FIG. 7 ) and utilized sequentially or simultaneously. Alternatively, oneinjector support structure 1200 may be used to substantially simultaneously inject enzyme intoreaction vessels 1100 of one of the conductive structures 100 (FIG. 7 ) and then repositioned over the other of the conductive structures 100 (FIG. 7 ) and used to substantially simultaneously inject enzyme into thereaction vessels 1100 of the other conductive structure 100 (FIG. 7 ). Because positioning theinjector support structure 1200 over oneconductive structure 100 may be quickly accomplished by sliding thealignment opening 1212 over thealignment member 712, theinjector support structure 1200 can be quickly and easily positioned and repositioned, thus minimizing the time to add the enzyme and minimizing the time with the lid 900 (FIG. 14 ) off of theconductive structure 100. - After addition of the enzyme to the
reaction vessels 1100, thelid 900 may be re-secured to theconductive structure 100, forming the conductive structure assembly 1000 (FIG. 10 ), which may then be agitated to encourage mixing of the substrate samples 1103 (FIG. 14 ) and the added enzyme. For example, the conductive structure assembly 1000 (FIG. 10 ) may be moved from thedry bath incubator 600 to an agitator (e.g., an orbital shaker, a reciprocal shaker) that may also be configured to provide heat during the agitation. In other embodiments, thedry bath incubator 600 and agitator may be integrated such that theconductive structure assembly 1000 may not need to be removed from a heating device (e.g., the dry bath incubator 600) to be agitated. - In some embodiments, the agitator may support the
dry bath incubator 600 with theconductive structure assembly 1000 such that theconductive structure assembly 1000 may be agitated without removing theconductive structure assembly 1000 from the heating device (e.g., the dry bath incubator 600). Such agitator may be a movable surface (e.g., a shaker plate) supporting thedry bath incubator 600, a movable surface of thedry bath incubator 600 itself, or a container containing thedry bath incubator 600. The agitator may be configured to agitate theconductive structure assembly 1000 through a plane that is parallel to a length (e.g., a height) of the vessels (e.g., thereaction vessels 1100 and/or control vessels 1101) while the vessels are in theconductive structure assembly 1000 in thedry bath incubator 600. For example, the agitator may move thedry bath incubator 600 and theconductive structure assembly 1000 up and down, either vertically or along an orbit about a horizontal axis. - Alternatively, the system may be configured to rotate the
dry bath incubator 600, theconductive structure assembly 1000, and, thus, the vessels (e.g., thereaction vessels 1100 and/or control vessels 1101) in theconductive structure assembly 1000 to align the length of the vessels along a substantially horizontal plane before horizontally agitating theconductive structure assembly 1000, either linearly or along an orbit about a vertical axis. - With reference to
FIG. 15 , in embodiments in which the agitator is separate from thedry bath incubator 600, the agitator may include ashaker plate 1500 to which the conductive structure 100 (FIG. 16 ) may be releaseably connected during agitation. In some embodiments, asupport structure 1502 may be connected (e.g., releaseably connected) to theshaker plate 1500. Thesupport structure 1502 may include abase plate 1504 mountable to theshaker plate 1500 via one ormore fasteners 1506 extending through openings 1507 (FIG. 16 ) in thebase plate 1504 and intoopenings 1508 in theshaker plate 1500. Thefasteners 1506 may be configured as screws with threading corresponding to threads in theopenings 1508. Theshaker plate 1500 may be a component of a conventional and commercially available orbital shaker, such as a New Brunswick Scientific Model Innova 44R, and theopenings 1507 in thebase plate 1504 may be positioned to correspond to theopenings 1508 in theshaker plate 1500 as acquired from its manufacturer. Thefasteners 1506 utilized, however, may have a greater length, to accommodate a height of thebase plate 1504, than those sold for use with theshaker plate 1500. Thefasteners 1506 may be flush with a surface of thebase plate 1504 when thebase plate 1504 is secured to theshaker plate 1500, as illustrated inFIG. 15 . - One or more engagement features 1510 may extend from the
base plate 1504. The engagement features 1510 may be releaseably secured to thebase plate 1504. For example, the engagement features 1510 may be threaded rods that may be screwed into threaded openings 1512 (FIG. 16 ) defined in thebase plate 1504. In other embodiments, the engagement features 1510 may be permanently affixed to thebase plate 1504. - With reference to
FIG. 16 , the engagement features 1510 may be positioned to align with theopenings 114 in theconductive structure 100. Thus, the engagement feature or features (e.g., the openings 114) of theconductive structure 100 may be selectively, slideably engaged (e.g., in the direction of arrows 15) with the counterpart engagement features (e.g., the engagement features 1510) on thesupport structure 1502 to mount theconductive structure 100 to theshaker plate 1500 during the agitation. In some embodiments, threadedcaps 1514 may be attached and tightened over the ends of the engagement features 1510, which may protrude above theconductive structure 100, to further secure theconductive structure 100 in place. - Because the engagement features (e.g., the openings 114) of the
conductive structure 100 are defined in sidewalls (e.g., thefront surface 116 and the back surface 118) of theconductive structure 100, when theconductive structure 100 is positioned on the engagement features 1510 of thesupport structure 1502, the upper surface 104 (FIG. 1 ) of theconductive structure 100 is essentially perpendicular to the shaker plate 1500 (FIG. 15 ). Thus, the reaction vessels 1100 (FIG. 11 ) and control vessels 1101 (FIG. 11 ) within theconductive structure 100 are positioned such that their length is parallel to the shaker plate 1500 (FIG. 15 ). With reference toFIG. 17 , therefore, when theshaker plate 1500 orbits, e.g., in the direction of arrows 17 (e.g., in the direction ofarrows 17′, in the direction ofarrows 17″, or both alternatingly) in an x-y plane parallel to the surface of theshaker plate 1500, theconductive structure 100 and the reaction vessels 1100 (FIG. 11 ) and control vessels 1101 (FIG. 11 ) are agitated in a plane that is parallel to a length (e.g., a height) of each. Therefore, the substrate samples 1103 (FIG. 11 ) and the enzyme are mixed along one of the greatest available volume widths to promote better intermixing of the materials than may be achieved if the reaction vessels 1100 (FIG. 11 ) were agitated parallel to their width. - Heat may be provided while the
conductive structure assembly 1000 is agitated so that the predetermined reaction temperature is maintained. The orbital shaker, in which theshaker plate 1500 is located, may be pre-heated to the predetermined reaction temperature before or while theconductive structure 100 is initially heated, the reaction vessels 1100 (FIG. 11 ) are heated, and the enzyme added. Therefore, as soon as the enzyme has been added to theconductive structure 100 and thelid 900 secured, theconductive structure assembly 1000 may be quickly moved to and positioned on theshaker plate 1500 without substantial heat loss during the transition. The orbital shaker may be closed around theconductive structure assembly 1000 during the agitation to retain the heat in the system. - Reaction time, following addition of the enzyme, may be monitored and samples taken from the reaction vessels 1100 (
FIG. 11 ) and/or the control vessels 1101 (FIG. 11 ) at desired times to measure the enzymatic activity. For example, the reaction vessels 1100 (FIG. 11 ) may be transferred to ice or may be injected with a reaction-stopping agent to cease the enzymatic reaction at a desired time, and then contents of the reaction vessels 1100 (FIG. 11 ) may be analyzed. The stop time for one or more reaction vessels 1100 (FIG. 11 ) of the group of reaction vessels 1100 (FIG. 11 ) from theconductive structure assembly 1000 may be spaced from the stop time of others so as to analyze an enzymatic reaction at various times using one conductive structure 100 (FIG. 1 ). Therefore, the same process may be used and the agitation stage carried out for varying times to gather a range of enzyme reaction times. - The enzymatic reactions may be halted or substantially slowed, at the desired time, by removing the reaction vessels 1100 (
FIG. 11 ) from the heatedconductive structure 100 and moving them to ice, with the addition of reagents configured to halt the reaction, or both. Techniques for terminating enzymatic reactions are known in the art and so are not described in detail herein. The enzymatic activity may be determined according to techniques known in the art, which are also not described in detail herein. By way of non-limiting example, the enzymatic activity may be determined by a reducing sugar assay, high pressure liquid chromatography (HPLC), the Somogyi method, or the DNS method. - Because the methods and systems disclosed herein control the temperature of the system with closed volumes for the reaction vessels 1100 (
FIG. 11 ), even during addition of the enzyme, high temperature reactions may be carried out without evaporation and, therefore, without skewed results. Moreover, because the methods and systems disclosed herein provide substantial intermixing of the substrate samples 1103 (FIG. 11 ) and the enzyme, insoluble substrates and substrates and enzymes that do not readily intermix may be analyzed without skewed results. Furthermore, because multiple small-volume samples may be simultaneously run in theconductive structure 100, even heterogeneous substrates and substrates or enzymes for which only small amounts are available may be analyzed for enzymatic activity. - Endo-1,4-β-XYLANASE M4 (hereinafter “Megazyme M4”) is an enzyme commercially available from Megazyme International Ireland, Ltd., Wicklow, Ireland. The published specific activity for Megazyme M4, in association with a wheat arabinoxylan substrate, which is partially soluble, averages 79.3 U/mg Protein.
- Enzymatic reactions with various volumes of Megazyme M4 and various amounts of wheat arabinoxylan were carried out, according to a conventional test tube assay that does not include agitation or sealed reaction vessels, and then activity determined at various reaction times according to a standard Somogyi characterization method. All reactions used a substrate solution at a pH of 4.0 and a reaction temperature of 40° C. Results are shown in
FIGS. 18 through 21 , which plot amount of reaction product (in μmoles) against reaction stop time (in minutes). With reference toFIG. 18 , using 0.2018 μg of the enzyme, the specific activity was measured at 96.6±7.5 U/mg. With reference toFIG. 19 , using 0.4035 μg of the enzyme, the specific activity was measured at 83.6±6.4 U/mg. With reference toFIG. 20 , also using 0.4035 μg of the enzyme, the specific activity was measured at 93.4±7.6 U/mg. With reference toFIG. 21 , again using 0.2018 μg of the enzyme, the specific activity was measured at 110.2±10.6 U/mg. As summarized in Table I below, the conventional standard tube assay yielded enzymatic activities that were, on average, about 16.6 U/mg higher (plus or minus between 6.4 U/mg and 10.6 U/mg) than the enzymatic activity reported by the commercial supplier (79.3 U/mg). -
TABLE I (Standard Tube Assay) Difference from Amount of Enzyme Determined Activity Published Activity of (μg) (U/mg) 79.3 U/mg 0.2018 96.6 ± 7.5 (FIG. 18) 17.3 ± 7.5 0.2018 110.2 ± 10.6 (FIG. 21) 30.9 ± 10.6 0.4035 83.6 ± 6.4 (FIG. 19) 4.3 ± 6.4 0.4035 93.4 ± 7.6 (FIG. 20) 14.1 ± 7.6 Average 96.0 16.6 - Enzymatic reactions with various volumes of Megazyme M4 and various amounts of wheat arabinoxylan were then carried out according to the sealed vessel with agitation method according to embodiments of the present disclosure, and then activity was determined at various times according to the standard Somogyi characterization methods. All reactions used a substrate solution at a pH of 4.0 and a reaction temperature of 40° C. Results are shown in
FIGS. 22 through 25 , which plot amount of reaction product (in μmoles) against reaction stop time (in minutes). With reference toFIG. 22 , using 0.3048 μg of the enzyme, the specific activity was measured at 87.1±7.4 U/mg. With reference toFIG. 23 , using 0.1506 μg of the enzyme, the specific activity was measured at 92.6±7.2 U/mg. With reference toFIG. 24 , using 0.3050 82 g of the enzyme, the specific activity was measured at 88.9±9.2 U/mg. With reference toFIG. 25 , using 0.1507 μg of the enzyme, the specific activity was measured at 91.9±8.5 U/mg. As summarized in Table II below, the method according to the present disclosure yielded enzymatic activities that were, on average, only about 10.8 U/mg higher (plus or minus between 7.2 U/mg and 9.2 U/mg) than the enzymatic activity reported by the commercial supplier (79.3 U/mg), even at lower enzyme amounts than used with the standard tube assay. -
TABLE II (Sealed Vial with Agitation Assay) Difference from Amount of Enzyme Determined Activity Published Activity of (μg) (U/mg) 79.3 U/mg 0.1506 92.6 ± 7.2 (FIG. 23) 13.3 ± 7.2 0.1507 91.9 ± 8.5 (FIG. 25) 12.6 ± 8.5 0.3048 87.1 ± 7.4 (FIG. 22) 7.8 ± 7.4 0.3050 88.9 ± 9.2 (FIG. 24) 9.6 ± 9.2 Average 90.1 10.8 - Thus, the methods of the present disclosure yielded enzymatic activities that were, on average, closer to the specific activity of the enzyme reported by the commercial supplier than the enzymatic activities determined using a conventional tube assay. Accordingly, the methods of the present disclosure may yield more accurate results, for a partially soluble substrate in very small amounts, than methods that do not use agitation.
- Notably, the demonstrated improvement may be achieved with even moderate-temperature enzymatic reactions such as the 40° C. reactions of the examples of
FIGS. 22 through 25 . Therefore, though the methods of the present disclosure may be well suited for high-temperature enzymatic reactions, because the methods may avoid skewing due to evaporation, the example discussed here demonstrates that the methods are also effective for moderate-temperature enzymatic reactions. - While the disclosed methods and systems are susceptible to various modifications and alternative forms in implementation thereof, specific embodiments have been shown by way of example in the drawings and have been described in detail herein. However, it should be understood that the present disclosure is not intended to be limited to the particular forms disclosed. Rather, the disclosed methods and systems encompass all modifications, combinations, equivalents, variations, and alternatives falling within the scope of the present disclosure as defined by the following appended claims and their legal equivalents.
Claims (20)
1. A system for determining enzymatic activity, the system comprising:
a conductive structure defining:
a plurality of wells protruding into the conductive structure from an upper surface of the conductive structure, the plurality of wells configured to receive a plurality of reaction vessels; and
at least one engagement feature on a sidewall of the conductive structure, the at least one engagement feature configured to engage a counterpart engagement feature to secure the conductive structure to an agitator.
2. The system of claim 1 , further comprising:
an injector support structure comprising conduits configured to receive injectors and to support the injectors over a corresponding number of wells of the plurality of wells defined in the conductive structure, the injector support structure defining:
upper openings having an upper opening width and defined in an upper surface of the injector support structure; and
lower openings having a lower opening width and defined in a lower surface of the injector support structure, the lower opening width being less than the upper opening width,
each of the conduits extending between one of the upper openings and one of the lower openings.
3. The system of claim 2 , wherein the injector support structure comprises a nonconductive material.
4. The system of claim 2 , wherein the injector support structure further comprises ledges, each of the ledges surrounding one of the lower openings.
5. The system of claim 2 , wherein the injector support structure further comprises extensions protruding from a supportive body, the conduits defined in the supportive body of the injector support structure, the extensions spaced from one another by a width of a row of the plurality of wells.
6. The system of claim 1 , further comprising a support structure configured to be secured to the agitator and comprising the counterpart engagement feature.
7. The system of claim 6 , wherein:
the at least one engagement feature on the sidewall of the conductive structure comprises openings extending from a front surface of the conductive structure to a rear surface of the conductive structure; and
the counterpart engagement feature comprises threaded rods at least partially screwed into the support structure.
8. The system of claim 1 , further comprising a lid for the conductive structure, the lid comprising sidewalls extending a height of lids secured to the reaction vessels of the plurality of reaction vessels.
9. The system of claim 1 , further comprising a lid for the conductive structure, the lid defining therein a thermometer opening and at least one other opening.
10. A conductive structure for supporting and heating a plurality of reaction vessels during agitation, the conductive structure comprising a block of a conductive material defining therein a plurality of wells protruding into the block from a first surface of the block and comprising at least one engagement feature protruding into the block from a sidewall surface of the block.
11. The conductive structure of claim 10 , wherein the conductive material comprises aluminum.
12. The conductive structure of claim 10 , further comprising at least one probe opening protruding into the block from at least one surface of the block.
13. The conductive structure of claim 12 , wherein the at least one probe opening comprises a thermometer opening protruding into the block from the first surface of the block.
14. The conductive structure of claim 13 , wherein the thermometer opening protrudes to a depth approximately even with a depth of the wells of the plurality of wells.
15. The conductive structure of claim 12 , wherein the at least one probe opening comprises a thermocouple opening protruding into the block from a second surface of the block, the second surface opposing the first surface.
16. The conductive structure of claim 10 , wherein the at least one engagement feature comprises at least one opening protruding horizontally into the block from the sidewall surface of the block.
17. The conductive structure of claim 10 , wherein each well of the plurality of wells defines a cylindrical and vertical sidewall and a horizontal floor.
18. A method for forming a conductive structure for supporting and heating a plurality of reaction vessels during agitation, the method comprising:
machining into a block of a conductive material from a first surface to define a plura of wells protruding into the block from the first surface of the block; and
machining into the block from a sidewall surface to define at least one engagement feature protruding into the block from the sidewall surface.
19. The method of claim 18 , wherein machining into the block from a sidewall surface to define at least one engagement feature comprises machining through the block from the sidewall surface to an opposing sidewall surface to define the at least one engagement feature extending through the block.
20. The method of claim 18 , further comprising:
machining into the block from the first surface to define a probe opening protruding into the block; and
machining into the block from a second surface, opposing the first surface, to define another probe opening opposing the probe opening.
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US14/976,124 US20160108455A1 (en) | 2014-02-13 | 2015-12-21 | Systems and conductive structures for determining enzymatic activity and methods of formation |
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US14/180,161 US9284596B2 (en) | 2014-02-13 | 2014-02-13 | Methods for determining enzymatic activity comprising heating and agitation of closed volumes |
US14/976,124 US20160108455A1 (en) | 2014-02-13 | 2015-12-21 | Systems and conductive structures for determining enzymatic activity and methods of formation |
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US14/976,124 Abandoned US20160108455A1 (en) | 2014-02-13 | 2015-12-21 | Systems and conductive structures for determining enzymatic activity and methods of formation |
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DE102015121362B4 (en) * | 2015-12-08 | 2018-05-24 | Analytik Jena Ag | Temperature control device with a reaction vessel |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5961925A (en) * | 1997-09-22 | 1999-10-05 | Bristol-Myers Squibb Company | Apparatus for synthesis of multiple organic compounds with pinch valve block |
US6086831A (en) * | 1998-06-10 | 2000-07-11 | Mettler-Toledo Bohdan, Inc. | Modular reaction block assembly with thermoelectric cooling and heating |
US6171555B1 (en) * | 1995-04-17 | 2001-01-09 | Ontogen Corporation | Reaction block docking station |
US20020072112A1 (en) * | 1990-11-29 | 2002-06-13 | John Girdner Atwood | Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control |
US6556940B1 (en) * | 1999-04-08 | 2003-04-29 | Analytik Jena Ag | Rapid heat block thermocycler |
US20050170493A1 (en) * | 2003-11-07 | 2005-08-04 | Tim Patno | Disposable sample processing module for detecting nucleic acids |
US20100081191A1 (en) * | 2008-09-26 | 2010-04-01 | Marlow Industries, Inc. | Anisotropic heat spreader for use with a thermoelectric device |
US20130265845A1 (en) * | 2010-05-03 | 2013-10-10 | Eppendorf Ag | Connection for a Temperature-Controllable Exchangeable Block |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4708886A (en) | 1985-02-27 | 1987-11-24 | Fisher Scientific Company | Analysis system |
US4788150A (en) | 1985-02-27 | 1988-11-29 | Fisher Scientific Company | Liquid handling |
WO2007022026A2 (en) | 2005-08-11 | 2007-02-22 | Biotrove, Inc. | Apparatus for assay, synthesis and storage, and methods of manufacture, use, and manipulation thereof |
FR2957934B1 (en) * | 2010-03-24 | 2014-10-17 | Centre Nat Rech Scient | BILIRUBIN OXIDASE FROM BACILLUS PUMILUS AND ITS APPLICATIONS |
MY166283A (en) | 2010-10-29 | 2018-06-25 | Thermo Fisher Scientific Oy | Automated system for sample preparation and analysis |
WO2013052318A1 (en) | 2011-09-25 | 2013-04-11 | Theranos, Inc. | Systems and methods for multi-analysis |
US9664702B2 (en) | 2011-09-25 | 2017-05-30 | Theranos, Inc. | Fluid handling apparatus and configurations |
-
2014
- 2014-02-13 US US14/180,161 patent/US9284596B2/en not_active Expired - Fee Related
-
2015
- 2015-12-21 US US14/976,124 patent/US20160108455A1/en not_active Abandoned
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020072112A1 (en) * | 1990-11-29 | 2002-06-13 | John Girdner Atwood | Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control |
US6171555B1 (en) * | 1995-04-17 | 2001-01-09 | Ontogen Corporation | Reaction block docking station |
US5961925A (en) * | 1997-09-22 | 1999-10-05 | Bristol-Myers Squibb Company | Apparatus for synthesis of multiple organic compounds with pinch valve block |
US6086831A (en) * | 1998-06-10 | 2000-07-11 | Mettler-Toledo Bohdan, Inc. | Modular reaction block assembly with thermoelectric cooling and heating |
US6556940B1 (en) * | 1999-04-08 | 2003-04-29 | Analytik Jena Ag | Rapid heat block thermocycler |
US20050170493A1 (en) * | 2003-11-07 | 2005-08-04 | Tim Patno | Disposable sample processing module for detecting nucleic acids |
US20100081191A1 (en) * | 2008-09-26 | 2010-04-01 | Marlow Industries, Inc. | Anisotropic heat spreader for use with a thermoelectric device |
US20130265845A1 (en) * | 2010-05-03 | 2013-10-10 | Eppendorf Ag | Connection for a Temperature-Controllable Exchangeable Block |
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US9284596B2 (en) | 2016-03-15 |
US20150225765A1 (en) | 2015-08-13 |
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