Methods and devices for micro-isolation, extraction, and/or analysis of microscale components
Provided herein are devices and methods for the micro-isolation of biological cellular material. A micro-isolation apparatus described can comprise a photomask that protects regions of interest against DNA-destroying illumination. The micro-isolation apparatus can further comprise photosensitive material defining access wells following illumination and subsequent developing of the photosensitive material. The micro-isolation apparatus can further comprise a chambered microfluidic device comprising channels providing access to wells defined in photosensitive material. The micro-isolation apparatus can comprise a chambered microfluidic device without access wells defined in photosensitive material where valves control the flow of gases or liquids through the channels of the microfluidic device. Also included are methods for selectively isolating cellular material using the apparatuses described herein, as are methods for biochemical analysis of individual regions of interest of cellular material using the devices described herein. Further included are methods of making masking arrays useful for the methods described herein.
1. A method to analyze a biological sample, the method comprising
positioning cellular material on top of a first support,
applying a microisolation apparatus comprising a plurality of first access wells on top of the cellular material, said cellular material defining bottoms for the first access wells;
filling the plurality of first access wells of the microisolation apparatus on top of the cellular material with a mixture comprising digestion agents and components necessary for a desired reaction;
allowing the digestion agents to digest the cellular material thereby releasing cellular contents into the mixture in each well of the plurality of first access wells;
inactivating the digestion agents;
fixing the cellular contents in their respective wells;
filling a plurality of second closed bottom access wells of a second support corresponding to the plurality of first access wells with an analytical reaction mixture;
evaporating a fraction of the mixture to uniformly decrease the reaction mixture level across the plurality of second closed bottom access wells leaving a space at the top of the plurality of second closed bottom access wells;
aligning the first support on top of the second support such that the plurality of first access wells face the plurality of second closed bottom access wells,
securing the first and second support to form a secured support; and
inverting the secured support to allow the analytical reaction mixture to flow from the second closed bottom access wells into the first access wells and via gravity contact the cellular contents in order to perform a reaction simultaneously but separately in each well of the secured support.
2. The method of claim 1 , wherein the analytical reaction comprises one or more reagents for PCR, real-time PCR, RT-PCR, flow cytometry, fluorescent labeling, FRET, DNA sequencing, protein-protein interaction assays, immunoassays, protein-nucleic acid assays.
3. The method of claim 1 , wherein signal detection is accomplished by scanning a completed reaction using a fluorescence scanner and/or a fluorescence microscope.
4. The method of claim 1 , wherein the filling the plurality of second closed bottom access wells of the second support corresponding to the plurality of first access wells with the analytical reaction mixture occurs after the digestion agents have digested the cellular material.
5. The method of claim 1 , wherein the filling the plurality of second closed bottom access wells of the second support corresponding to the plurality of first access wells with the analytical reaction mixture occurs before filling the plurality of first access wells with the mixture comprising digestion agents.
6. The method of claim 5 , wherein the analytical reaction mixture is dried.
7. The method of claim 6 , wherein the dried analytical reaction mixture is resuspended by the reaction mixture comprising digestion agents.
8. The method of claim 1 , wherein the reaction and the digestion of cellular materials occur simultaneously but separately.
9. The method of claim 1 , wherein applying a microisolation apparatus comprises:
depositing a negative photosensitive material on the cellular material;
applying a photomask comprising a blocking region onto the negative photosensitive material;
exposing the negative photosensitive material to photons in order to generate a lithographic pattern on the negative photosensitive material;
removing the photomask; and
applying a developer to the negative photosensitive material in order to define the plurality of first access wells.