Microfluidic protein crystallography
First Claim
1. A method of preparing a plurality of chemical mixtures from a set number of inlets, the method comprising:
- metering a volume of a first chemical from a first inlet to a microfluidic mixing structure;
metering a volume of a second chemical from a second inlet to the microfluidic mixing structure to create a first mixture comprising the first and second chemicals;
flowing the first mixture out of the microfluidic mixing structure;
again metering the volume of the first chemical from the first inlet to the microfluidic mixing structure; and
metering a volume of a third chemical from a third inlet to the microfluidic mixing structure to create a second mixture comprising the first and the third chemicals.
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Abstract
The use of microfluidic structures enables high throughput screening of protein crystallization. In one embodiment, an integrated combinatoric mixing chip allows for precise metering of reagents to rapidly create a large number of potential crystallization conditions, with possible crystal formations observed on chip. In an alternative embodiment, the microfluidic structures may be utilized to explore phase space conditions of a particular protein crystallizing agent combination, thereby identifying promising conditions and allowing for subsequent focused attempts to obtain crystal growth.
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Citations
51 Claims
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1. A method of preparing a plurality of chemical mixtures from a set number of inlets, the method comprising:
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metering a volume of a first chemical from a first inlet to a microfluidic mixing structure;
metering a volume of a second chemical from a second inlet to the microfluidic mixing structure to create a first mixture comprising the first and second chemicals;
flowing the first mixture out of the microfluidic mixing structure;
again metering the volume of the first chemical from the first inlet to the microfluidic mixing structure; and
metering a volume of a third chemical from a third inlet to the microfluidic mixing structure to create a second mixture comprising the first and the third chemicals. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. The method of 1 in which the volume of each of the first and second mixtures comprises about 20 nL or less.
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12. The method of 1 further comprising repeatedly preparing additional mixtures in the mixing structure to systematically characterize a chemical or biological response as a function of a composition of the mixture.
- 13. The method of 12 wherein macromolecule solubility is characterized.
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19. The method of 15 in which characterization of macromolecule solubility and the batch screening experiments are performed on a single microfluidic device.
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20. The method of 15 in which characterization of macromolecule solubility and batch screening experiments are performed on different microfluidic devices.
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21. An apparatus comprising:
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a microfluidic flow channel network formed in a first elastomer layer, the microfluidic flow channel network comprising a first set of inlet branches in fluid communication with a junction and with a reagent source, a second set of inlet branches in fluid communication with the junction and with a buffer source, and a mixing structure in fluid communication with the junction and with an outlet;
a first control channel network formed in a second elastomer layer adjacent to the first elastomer layer, the first control channel network adjacent to the first inlet branch set to define a first multiplexer structure configured to flow a select reagent into the junction; and
a second control channel network formed in the second elastomer layer, the second control channel network adjacent to the second inlet branch set to define a second multiplexer structure configured to flow a select buffer into the junction. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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35. A method of identifying conditions conducive to crystallization comprising:
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preparing a first solution including a solvent and a sample at a first concentration;
mixing with the first solution, a second solution including a crystallizing agent;
noting a first condition at which solid phase first appears in the mixture;
adding additional solvent to the mixture;
noting a second condition at which solid phase disappears in the mixture; and
identifying a hysteresis between the first and second conditions. - View Dependent Claims (36, 37, 38, 39, 40)
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41. A method of identifying conditions conducive to crystallization comprising:
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detecting scattering of light as a crystallizing agent is mixed with a solution containing a sample;
correlating the detected scattered light with a known range of a second virial coefficient characteristic of a protein crystal-containing solution. - View Dependent Claims (42, 43)
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44. A method of controlling flow through a microfluidic device comprising:
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disposing a fluid in a flow channel;
applying a pressure to a control channel adjacent to and separated from the flow channel to cause an intervening elastomer membrane to deflect into the flow channel; and
maintaining a baseline pressure of the fluid in the flow channel at greater than 5 psig while relaxing the pressure applied to the control channel to bias the elastomer membrane out of the flow channel. - View Dependent Claims (45, 46, 47)
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48. A method of identifying conditions conducive to solubilization of a membrane protein, the method comprising:
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introducing a solid membrane protein into a microfluidic closed circuit mixing structure;
introducing an amphiphilic moiety into the microfluidic closed circuit mixing structure;
exposing a mixture of the membrane protein and the amphiphilic moiety to a detectable ligand configured to bind only with a form of the membrane protein as folded in a membrane; and
detecting the ligand to identify the folded form of the membrane protein in solution. - View Dependent Claims (49, 50, 51)
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Specification