GB2452101A - A microfluidic device for thermal shift assays - Google Patents
A microfluidic device for thermal shift assays Download PDFInfo
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- GB2452101A GB2452101A GB0724206A GB0724206A GB2452101A GB 2452101 A GB2452101 A GB 2452101A GB 0724206 A GB0724206 A GB 0724206A GB 0724206 A GB0724206 A GB 0724206A GB 2452101 A GB2452101 A GB 2452101A
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- 239000007788 liquid Substances 0.000 claims abstract description 27
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- 238000001212 derivatisation Methods 0.000 description 1
- SYELZBGXAIXKHU-UHFFFAOYSA-N dodecyldimethylamine N-oxide Chemical compound CCCCCCCCCCCC[N+](C)(C)[O-] SYELZBGXAIXKHU-UHFFFAOYSA-N 0.000 description 1
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- 229920002313 fluoropolymer Polymers 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
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- SBWGZAXBCCNRTM-CTHBEMJXSA-N n-methyl-n-[(2s,3r,4r,5r)-2,3,4,5,6-pentahydroxyhexyl]octanamide Chemical compound CCCCCCCC(=O)N(C)C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO SBWGZAXBCCNRTM-CTHBEMJXSA-N 0.000 description 1
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- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- 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
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
- B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
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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
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
- B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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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
- B01L2200/06—Fluid handling related problems
- B01L2200/0642—Filling fluids into wells by specific techniques
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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
- B01L2300/0627—Sensor or part of a sensor is integrated
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- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
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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
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- B01L2300/16—Surface properties and coatings
- B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
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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
- B01L2300/16—Surface properties and coatings
- B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
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- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dispersion Chemistry (AREA)
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Abstract
A microfluidic device for carrying out thermal assays comprises a conduit 3 where the temperature varies along the length of the conduit. Droplets 5 including the molecules to be tested, e.g. macromolecules, are dispensed into an immiscible carrier liquid in the conduit 3. By moving the carrier liquid these droplets 5 can be moved along the conduit 3, exposing the droplets and the macromolecules to changing temperatures. If the properties of the droplets are monitored, thermal assays can be performed. Frequently the molecules to be tested will be biological macromolecules, thus using static light scattering, fluorescent dyes etc., or simply by viewing and analyzing images, the temperature at which a macromolecule denatures can be determined. This allows assays known as "thermal shift assays" (TSA) to be performed. The information obtained from TSAs can be used for binding studies, searching for ligands, stability studies, etc.
Description
New microfluidic device for thermal assays
TECHNICAL FIELD
This invention relates to microfluidic systems for performing thermal assays. Assays that can be performed include thermal shift assays (TSA, or differential scanning fluorimetry, DSF) where a parameter corresponding to the temperature at which a macromolecule denatures is measured.
BACKGROUND OF THE INVENTION
Microfluidic systems have the advantages of using very small volumes, consuming small amounts of samples and reagents, and compact size.
Important information about the structure, behavior and stability of biological macromolecules can be determined by the quantification of certain temperature dependent transitions of the macromolecule. One such transition is the denaturation of a biological molecule such as a protein. A simple model of a protein considers it being in one of two configurations, folded and denatured. The equilibrium state of the protein is dependent on the physical and chemical environment of protein. Various chemicals can trigger a denaturation of a protein when present in high concentrations -examples include urea and guanidinium chloride. Temperature is also an important determinant of the protein equilibrium state and for most proteins when the temperature increases above a certain level, Tm, the equilibrium state shifts from being mostly folded to being mostly denatured. This transition temperature can be affected by chemical agents.
Denaturants such as urea will tend to lower the Tm whereas ligands that bind strongly to the protein will tend to increase the Tm. Most ligands that bind tightly increase the Tm, while some decrease Tm, and a few tightly-binding ligands have no effect on Tm.
The first objective of the invention is to provide a device for determining temperature profiles for different properties of various sample mixtures, which device is has a low sample consumption and a high throughput.
A second objective of the invention is to provide simple methods for operating the device.
These objectives have been achieved by the invention as it is defined in the claims. And as it will be explained below, the invention and embodiments of the invention exhibit further beneficial properties
compared with prior art devices and methods.
BRIEF SUMMARY OF THE INVENTION
One aspect of the invention is a microfluidic device that contains a conduit through which liquid can flow. The liquid can be either uniform or multiphasic. An example of a multiphasic liquid is aqueous droplets that are carried by an immiscible carrier fluid such as mineral oil. The liquid may contain a biological macromolecule and/or other indicator molecules, denaturants and ligands. Biological macromolecules include various proteins, DNA, RNA. carbohydrates and lipids. This liquid containing indicator molecules is referred to as the target molecule mixture.
The device can be designed such that different regions are maintained at different temperatures resulting in different regions of the conduit to be kept at different temperatures. Thus, by flowing the target molecule mixture through the conduit its temperature will be changed to the corresponding temperatures of the device. As the temperature of the target molecule mixture is changed. properties of the target molecule mixture can be quantified resulting in temperature profiles for the corresponding target molecule mixture. Examples of properties to be quantified include: fluorescence, absorbance, refractive index, birefringence, static light scattering, dynamic light scattering, viscosity and density. Any sort of measurement of a property of the target molecule mixture performed on the device is referred to as a temperature profile. This term is used for a single measurement or a senes of measurements of a property of the target molecule mixture at one or more temperatures.
One embodiment of the device may have a means for maintaining the temperature of the device in any chosen arbitrary pattern, enabling complete control over the temperature at different regions of the conduit, such that the target molecule mixture flowing through the conduit will be subjected to predetermined temperature patterns.
An alternative embodiment of the device may have a means for maintaining the temperature essentially constant throughout the device for a given period of time, such that the target molecule mixture in the conduit will be maintained at the same temperature.
Another embodiment of the device may have a means for maintaining the temperature of two or more regions at different levels allowing for a temperature gradient to form across the device. The target molecule mixture flowing through the conduit will experience a temperature gradient as it flows through the conduit of the device.
Another embodiment of the device may have a means for formulating the target molecule mixture that flows through the device. By metering and mixing various solutions in the device, different target molecule mixtures may be formulated in the device and subjected to a particular temperature patterns as they flow through the conduit enabling the determination of temperature profiles for the target molecule mixtures. Furthermore, the device may have a mechanism for adding solutions to the device from an external container, such as a microwell plate or a cartridge.
The devices described above may have a mechanism for the determination of the following and other properties of the target molecule mixture: fluorescence, absorbance, refractive index, birefringence, static light scattering, dynamic light scattering, viscosity and density at one or multiple points throughout the conduits of the device.
The invention also relates to methods of using the device for determining temperature profiles using a device as described above.
A method of the invention comprises: 1) Creating a particular temperature pattern in the device 2) Flowing that target molecule mixture through the conduit of the device. The target molecule mixture may be formulated on the device.
3) Quantifying along the conduit one or more of the following properties of the target molecule mixture: fluorescence, absorbance, refractive index, birefringence, static light scattering, dynamic light scattering, viscosity and density resulting in temperature profiles for the target molecule mixture.
A second method of the invention comprises; I) Determining multiple temperature profiles for different target molecule mixtures that differ in their composition in various ways such as the presence of particular ligands.
2) Comparing these temperature profiles to each other to identify the effect of the various components of the target molecule mixture of the temperature profile.
An important application of the invention is the determination of the temperature at which a protein or macromolecule denatures in the presence of small molecules or fragments. This denaturation can be detected by fluorescent dyes, static or dynamic light scattering, or by detecting precipitation in images.
BRIEF DESCRIPTION OF THE DRAWINGS 2.
Figure Ia shows a schematic view of a simple embodiment of the invention that is capable of determining thermal profiles of target molecule mixtures in droplets (5) that are contained and moved by an immiscible carrier liquid (3).
Figure lb is labeled with one example of temperatures that could be used.
Figure Ic shows a non multiphasic target molecule mixture flowing through the device.
Figure 2 shows an apparatus that is similar to the apparatus of figure 1 with two innovations: the apparatus possesses a side-arm (21), and the conduit has a serpentine form which allows a compact system to have a relatively long conduit.
Figure 3 shows an apparatus that is similar to that of figure 2, but which has several side-arms (21).
Figure 4 shows that the apparatus of any previous figure can be placed within one or more blocks (41, 42).
Figure 5 shows that the conduits of block 41 of figure 4 can be similar to the side arms of the appaiatus of figure 3.
DETAILED DESCRIPTION OF THE INVENTION
The invention will now be described in detail, with reference to the accompanying drawings.
In the accompanying drawings, like features are denoted by like numerals.
Figure Ia shows a schematic view of a simple embodiment of the invention that is capable of temperature profiles with target molecule mixtures in droplets (5) that are contained and moved by an immiscible carrier liquid (3).
The apparatus disclosed in figure 1 a comprises a conduit, having a proximal and a distal end, with an inlet or outlet (I) at the proximal end, and an inlet or outlet (2) at the distal end. The temperature may vary along the length of the conduit. The temperature near one end may be low, while the temperature near the opposite end may be high. A temperature gradient may be established between these two extremes.
The conduit contains a liquid (3) that is immiscible with the target molecule mixture to be tested. This immiscible liquid will be referred to hereinafter as the "canier liquid". As an example, if the target molecule mixtures are aqueous, the carrier liquid may be a paraffin oil, a liquid silicone or a liquid fluorocarbon. Moreover, for aqueous target molecule mixtures, the walls of the conduit (4) can advantageously be constructed from or coated with a hydrophobic material as described below. Another approach is to use surface-acting solutes such as detergents, which can prevent the droplets from adhering to the conduit walls and other interfaces.
Aqueous target molecule mixtures are introduced into the conduit via the inlet (1) in the form of droplets (5). By moving the carrier liquid (3) the droplets (5) can be moved along the conduit. By moving the droplets from the colder end of the conduit to the hotter end, the temperature in the droplets can be increased. By quantifying a particular property of the target molecule mixture at one or more positions along the conduit temperature profiles for the target molecule mixture can be determined. For example, protein denaturation can be detected by using fluorescent dyes or by observing precipitation etc. Precipitation can be detected by monitoring light scattering.
Advantageously, droplets can be individually dispensed and the ingredients in each droplet controlled independently of all other droplets. The ingredients can be controlled by dispensing precise volumes into droplets. The droplets in figure Ia are clustered in two groups of 4 and one of 3. This has no particular significance other than to emphasize that droplets can be precisely controlled in terms of both composition and position.
Figure lb is labeled with examples of high and low temperatures that could be used. Purely as an example, the proximal end of the conduit in figure I is labeled 20°C while the distal end of the conduit in figure 1 is labeled 70°C. A temperature gradient may be established along the conduit between these two extremes.
Figure Ic shows that it is not essential for a target molecule mixture to be a multiphasic liquid. The target molecule mixture (6) can instead be introduced into the conduit in the absence of any immiscible carrier.
The target molecule mixture can be moved from colder to hotter areas, or from hotter to colder areas, by causing it to flow along the conduit, and thermal transitions can be monitored.
Figure 2 shows an apparatus that is similar to the apparatus of figure 1 with two innovations: the apparatus possesses a side-arm (21), and the conduit has a serpentine form which allows a compact system to have a relatively long conduit. The side-arm (21) can be filled with a target molecule mixture, which caii be moved volumetrically out of the side-arm and into the conduit. Then the carrier liquid can be moved, forming a droplet. Alternatively, droplets can be brought in through the inlet, and extra ingredients can be added to droplets from the side-arm (21). For example, each droplet introduced through the inlet can contain a different component of the target molecule mixture, and protein, buffers and fluorescent dyes or other components of the target molecule mixture can he added from the side-arm.
In this example the effect of different components in the target molecule mixture on the temperature profile of the target molecule mixture can be measured. The temperature of the two ends of the device may be kept at 20°C and 70°C, respectively. The target mixture may be multiphasic as shown.
Figure 3 shows an apparatus that has several side-arms (21). Liquids from these side-arms can be mixed together to investigate the effect on the temperature profile of the target molecule mixture of varying the composition of the target molecule mixture. In the area where the side-anns join the conduit, the conduit can advantageously be tapered as shown (31). This increases the accuracy of dispensing and also increases the range of volumes that can be dispensed. The target mixture may be multiphasic as shown.
Using the apparatus of figure 3, complicated mixtures can be dispensed. For example, experiments using the well-known Central Composite and Box-Behnken designs can be carried out.
Figure 3 shows solution from the fourth side-arm being added to a droplet (5). This droplet might already contain solution from any of channels I to 3. (Channels are here numbered from left to right).
Figure 4 shows that the apparatus of any previous figure can be placed within one or more blocks (41, 42). In figure 4, the metering part (conduits not shown) of the apparatus is placed on a block (41) that is separate from the block where the temperature profiles are determined (42). The two blocks can be joined by a length of tubing (43) or another fluidic connection. The blocks can advantageously be constructed by clamping or joining sheets or plates.
Figure 5 shows that the metering part of the apparatus of figure 4 can be similar to the metering part of the apparatus of figure 3 with multiple side arms leading into the main conduit. However, many other configurations and methods can be used for metering instead.
Any of the apparatuses in figures 1 to 5 can be used in several different modes. For example, by moving the droplets slowly so that they can reach thermal equilibrium, and by knowing or estimating the temperature at every point along the conduit the temperature profile of the target molecule mixture can be determined. Also, the temperature at which a thermal transition in the target molecule mixture takes place can be measured or estimated. U.
Alternatively, the temperature of the various parts of the microfluidic device can be kept constant but otherwise disregarded. Instead, the physical position along the conduit at which measurements of particular properties of the target molecule mixture takes place can be recorded. These measurements may also result in a temperature profile of the target molecule mixture -however, the absolute temperature may not be known. If the assay is repeated with known variations of the target molecule mixture, different temperature profiles can be determined. By accurately recording this positional information, the relative effect of these variations can be quantified with no exact knowledge of the temperatures at which the measurements are performed. With this approach, the temperature at which a thermal transition tales place is not known, hut the position at which the transition takes place is known and can be compared to the position for other target molecule mixtures.
An important application of the invention is the determination of the temperature at which a protein or other macromolecule denatures in the presence of one or more small molecules or molecular fragments.
Useful methods of detecting denaturation include the use of an environmentally-sensitive fluorescent dye, and static or dynamic light scattering. For example, these methods can detect the presence of protein precipitation. Another useful method is to identify precipitation directly in images. This approach comprises recording images of samples, and examining or analyzing these images to identify samples with precipitation. Precipitation often appears as dark grains, particles or clouds against a lighter background. It can also appear as light grains, particles or clouds against a darker background. These variations in hue, saturation or luminescence can be can be identified automatically by image recognition algorithms and computer programs.
The walls of the microfluidic device can be constructed from hydrophobic materials including fluoropolymers such as PTFE, PES, PAl, PPS, PEEK, PFA, FEP, PCTFE, ETFE. ECTFE, or from polycarbonate, polypropylene, COC, polystyrene, nylon, acetal, or a silicone such as PDMS.
Alternatively, the walls can advantageously be made from a hydrophilic material such as glass or metal and coated with a hydrophobic material, for example by silanization or by derivatization with fluorine or other atoms to produce a hydrophobic surface. Advantageously, at least part of the microfluidic device can be made from a transparent material such as FEP, COC, polystyrene or glass.
Detergents can advantageously be added to the droplets or to the carrier liquid to reduce the tendency for droplets to stick to the walls of the microfluidic device or for droplets to coalesce with other droplets.
Examples of useful surfactants are l-s-Heptyl-13-D-thioglucoside, l-s-Nonyl-B-D-thiOglucoside, 1-s-Octyl-B-D-thioglucoside, ABIL EM9O, ABIL WEO9, Anapoe� 20, Anapoe� 35, Anapoe� 58, Anapoe� 80, Anapoe� CIOE6, Anapoe� C1OE9, Anapoe� C12E1O, Anapoe� C13E8, Anapoe� X-1 14, Anapoe� X-305, Anapoe� X-405, BAM, C12E8, C12E9, C8E5, CHAPS, CHAPSO, C-HEGA-l0, C-HEGA-il, C-HEGA-9, CTAB, Cymal�-l. Cymal�-2, CYMAL�-3, Cymal�-4, CYMAL�-5, CYMAL�-6, CYPFOS-3, DDAO, DDMAB, Deoxy BigChap, FOS-Choline�-10, FOS-Choline�-12, FOS-Choline�-8, FOS-Choline�-9, HECAMEG. HEGA-10. HEGA-8, HEGA-9, Heptyl-13-D- thioglucoside, IPTG, LDAO, MEGA-8, MEGA-9, n-Decanoylsucrose, n-Decyl-13-D-maltosjde, n-Decyl- B-D-thiomaltoside, n-Dodecyl-N,N-dimethylglycine. n-Dodecyl-13-D-rnaltoside, n-Dodecyl-B-D-maltotrioside, n-Hexadecyl-B-D-maltoside, n-Hexyl-B-D-glucoside, n-Nonyl-13-D-maltoside, n-Nonyl-13- D-maltoside, n-Octanoylsucrose, n-Octyl-13-D-glucoside. n-Octyl-l3-D-thiomaltoside. Nonyl-B-D-glucoside, n-Tetradecyl-B-D-maltoside. n-Tridecyl-B-D-maltoside, n-Undecyl-B-D-maltoside, Pluronic� F-68, SPAN 80, Sucrose monolaurate, Thesit�, TRITON� X-100, Tween 80, Zonyl FSN, ZWITTERGENT� 3-10, ZWITTERGENT� 3-12, Zwittergent� 3-14, Zwittergent�-3-08. In addition, useful detergents can be made by substituting hydrogen atoms with fluorine atoms or vice versa in the detergents listed above. Also, adding polymers such as polyethyleneglycol or polydimethylsiloxane can lead to useful properties of the detergents.
Claims (44)
- What we claim is: I. A device containing one or more conduits passing through one or more regions that can be maintained at particular temperatures such that the contents of the conduits can be kept at an arbitrary temperature at any given time and place.
- 2. A device as described in claim I in which the temperature of the conduit(s) is kept at discrete temperatures such that different sections of the device are kept at specific different temperatures.
- 3. A device as described in claim 1 in which different parts of the conduit(s) can be set to any arbitrary temperatures.
- 4. A device as described in claim I in which the temperature of the conduit(s) is continuously varying with length along the conduit due to a temperature gradient or gradients formed between two or more regions maintained at specific different temperatures.
- 5. A method for using a device as described in any of the claims above in which a liquid is flowed through the conduit(s) such that the liquid will be kept at essentially the same temperature as the conduit(s).
- 6. A method for using a device as described in any of the claims above in which the liquid is a target molecule mixture that changes its properties as the temperature changes.
- 7. A method for using a device as described in any of the claims above in which a carrier liquid containing droplets of a target molecule mixture that changes its properties as the temperature changes is flowed through the conduit(s) such that the droplets will be kept at essentially the same temperature as the conduit(s).
- 8. A device as described in any of the claims above which contains a mechanism for formulating the target molecule mixture by metering and mixing multiple solutions in the device.
- 9. A device as described in any of the claims above which contains a mechanism for formulating the target molecule mixture into droplets by metering and mixing multiple solutions in the device.
- 10. A device as described in any of the claims above which contains a mechanism for introducing various solutions from an external container.
- 11. A device as described in any of the claims above which contains a mechanism for introducing various liquids from a microwell plate.
- 12. A device as described in any of the claims above which contains a mechanism for introducing various liquids from a pretilled cartridge.
- 13. A method for using a device as described in any of the claims above in which the concentration of at least one of the components of the target molecule mixture is varied using the mechanism for formulating the target molecule mixture in the device.
- 14. A device as described in any of the claims above in which properties of the target molecule mixture can be measured along the conduit(s) as the target molecule mixture is flowed through the conduit(s).
- 15. A device as described in any of the claims above in which the fluorescence of the target molecule mixture can be measured along the conduit(s) as the target molecule mixture is flowed through the conduit(s).
- 16. A device as described in any of the claims above in which the absorbance of the target molecule mixture can be measured along the conduit(s) as the target molecule mixture is flowed through the conduit(s).
- 17. A device as described in any of the claims above in which the refractive index of the target molecule mixture can be measured along the conduit(s) as the target molecule mixture is flowed through the conduit(s).
- 18. A device as described in any of the claims above in which the birefringence of the target molecule mixture can be measured along the conduit(s) as the target molecule mixture is flowed through the conduit(s).
- 19. A device as described in any of the claims above in which the static light scattering of the target molecule mixture can be measured along the conduit(s) as the target molecule mixture is flowed through the conduit(s).
- 20. A device as described in any of the claims above in which the dynamic light scattering of the target molecule mixture can be measured along the conduit(s) as the target molecule mixture is flowed through the conduit(s).
- 21. A device as described in any of the claims above in which the viscosity of the target molecule mixture can be measured along the conduit(s) as the target molecule mixture is flowed through the conduit(s).
- 22. A device as described in any of the claims above in which the density of the target molecule mixture can be measured along the conduit(s) as the target molecule mixture is flowed through the conduit(s).
- 23. A method for using a device as described in any of the claims above in which the target mixture contains a biological molecule.
- 24. A method for using a device as described in any of the claims above in which the target mixture contains a protein.
- 25. A method for using a device as described in any the claims above in which the target mixture contains a nucleic acid.
- 26. A method for using a device as described in any of the claims above in which the target mixture contains a polymer.
- 27. A method for using a device as described in any of the claims above in which the target mixture contains a molecule that that changes the optical characteristics in response to a change in a property of the target molecule.
- 28. A method for using a device as described in any of the claims above in which the target mixture contains a molecule that changes the fluorescence of the target molecule mixture in response to a change in a property of the target molecule.
- 29. A method for using a device as described in any of the claims above in which the target mixture contains a molecule that that changes the absorbance of the target molecule mixture in response to a change in a property of the target molecule.
- 30. A method for using a device as described in any of the claims above in which the target mixture contains a molecule that that changes the refractive index of the target molecule mixture in response to a change in a property of the target molecule.
- 31. A method for using a device as described in any of the claims above in which the target mixture contains a molecule that that changes the birefringence of the target molecule mixture in response to a change in a property of the target molecule.
- 32. A method for using a device as described in any of the claims above in which the target mixture contains a molecule that that changes the physical characteristics in response to a change in a property of the target molecule.
- 33. A method for using a device as described in any of the claims above in which the target mixture contains a molecule that that changes the viscosity characteristics in response to a change in a property of the target molecule.
- 34. A method for using a device as described in any of the claims above in which the target mixture contains a molecule that that changes the density characteristics in response to a change in a property of the target molecule.
- 35. A method for using a device as described in any of the claims above in which a change in one of the measured properties of the target molecule mixture is correlated to the temperature of the target molecule mixture at the time of the measurement generating temperature profiles.
- 36. A method for using a device as described in any of the claims above in which a change in one of the measured properties of the target molecule mixture is correlated to the folding state of the target such that the folding of the target molecule can be characterized at different point along the conduit(s) leading to a thermal denaturation profile of the target molecule.
- 37. A method for using a device as described in any of the claims above in which the concentration of at least one of the components of the target molecule mixture is varied using the mechanism for formulating the target molecule mixture in the device and where the change in the properties of the target molecule mixture is measured along the conduit(s).
- 38. A method for using a device as described in any of the claims above in which the effect of varying the concentration of a particular component of the target molecule mixture on the thermal denaturation profile is quantified.
- 39. A method for using a device as described in any of the claims above in which one or more of different compounds are added to the target molecule mixture using the mechanism for adding solutions from an external container and their effect on the properties of the target molecule mixture is measured.
- 40. A method for using a device as described in any of the claims above in which one or more of different compounds are added to the target molecule mixture using the mechanism for adding solutions from an external container and their effect on the thermal denaturation profile of the target molecule is quantified.
- 41. A method of quantifying the thermal stability of at least one macromolecule in solution using a device as described in any of the claims above where the method comprises the steps of: a. providing one or more solutions of at least one macromolecule b. providing a microfluidic device c. maintaining a cooler region of said microfluidic device at a temperature that is below the temperature where the macromolecules to be tested are expected to denature d. heating a hotter region of said microfluidic device to a temperature that is above the temperature where the macromolecutes to be tested are expected to denature e. allowing a thermal gradient to become established between said cooler and hotter regions f. providing a conduit that passes from said cooler region to said hotter region g. filling said conduit with a liquid that is immiscible with said solution(s) of macromolecules h. dispensing small volumes of said solution(s) of macromolecules into said conduit in the form of at least one droplet in said immiscible liquid i. moving said immiscible liquid in the conduit so that said droplet(s) move from the cooler region to the hotter region while monitoring said droplet(s) in order to detect denaturation j. observing and recording the position or range of positions where denaturation takes place k. repeating steps 9 and 10 with other solutions of macromolecules I. interpreting differences in said observed positions where denaturation takes place
- 42. A method as described in claim 41 where the temperature at at least one position along said conduit is determined prior to making said observations so that the temperature of denaturation can be recorded or estimated.
- 43. A method as described in claim 41 where the temperature in the region where denaturation takes place is not known, but where the position of denaturation is recorded and interpreted.I
- 44. A method as described in any previous claim where the temperature at which a macromolecule denatures is determined by identifying precipitation of that macromolecule in one or more images. (0
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0716382A GB2452057A (en) | 2007-08-23 | 2007-08-23 | A microfluidic device for thermal assays |
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| GB0724206D0 GB0724206D0 (en) | 2008-01-23 |
| GB2452101A true GB2452101A (en) | 2009-02-25 |
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| GB0716382A Withdrawn GB2452057A (en) | 2007-08-23 | 2007-08-23 | A microfluidic device for thermal assays |
| GB0724206A Withdrawn GB2452101A (en) | 2007-08-23 | 2007-12-12 | A microfluidic device for thermal shift assays |
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| Application Number | Title | Priority Date | Filing Date |
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| GB0716382A Withdrawn GB2452057A (en) | 2007-08-23 | 2007-08-23 | A microfluidic device for thermal assays |
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| GB (2) | GB2452057A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CA2767113A1 (en) * | 2010-03-25 | 2011-09-29 | Bio-Rad Laboratories, Inc. | Detection system for droplet-based assays |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002081729A2 (en) * | 2001-04-06 | 2002-10-17 | California Institute Of Technology | Nucleic acid amplification utilizing microfluidic devices |
| US20030203497A1 (en) * | 1996-05-09 | 2003-10-30 | Pantoliano Michael W. | Microplate thermal shift assay apparatus for ligand development and multi-variable protein chemistry optimization |
| EP1574586A2 (en) * | 2004-03-12 | 2005-09-14 | Samsung Electronics Co., Ltd. | Method and apparatus for amplifying nucleic acids |
| WO2007062666A1 (en) * | 2005-12-02 | 2007-06-07 | Technical University Of Denmark | Fluidics device for assay |
-
2007
- 2007-08-23 GB GB0716382A patent/GB2452057A/en not_active Withdrawn
- 2007-12-12 GB GB0724206A patent/GB2452101A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030203497A1 (en) * | 1996-05-09 | 2003-10-30 | Pantoliano Michael W. | Microplate thermal shift assay apparatus for ligand development and multi-variable protein chemistry optimization |
| WO2002081729A2 (en) * | 2001-04-06 | 2002-10-17 | California Institute Of Technology | Nucleic acid amplification utilizing microfluidic devices |
| EP1574586A2 (en) * | 2004-03-12 | 2005-09-14 | Samsung Electronics Co., Ltd. | Method and apparatus for amplifying nucleic acids |
| WO2007062666A1 (en) * | 2005-12-02 | 2007-06-07 | Technical University Of Denmark | Fluidics device for assay |
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| Publication number | Publication date |
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| GB0716382D0 (en) | 2007-10-03 |
| GB0724206D0 (en) | 2008-01-23 |
| GB2452057A (en) | 2009-02-25 |
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