The ability to pattern and then release biomolecules, cells, tissues, organisms, colonies, embryos, and other biological samples at specific sites on a plate is important for many areas of scientific research and medicine. By placing such samples at known locations, researchers may study many samples under identical and controlled conditions, while monitoring each sample independently and repeatedly over time. Since each sample is in a known location, large numbers of samples may be studied, resulting in statistically significant data sets. Arrays of isolated biological samples can be used to assist in diagnosis by observing the samples under controlled conditions in the presence of unknown agents or pathogens. Furthermore, it has additional advantages if the samples on specific location can be isolated from the array. Release and collection permits the patterned material to be analyzed further as well as by a variety of other techniques.
To accomplish patterning combined with release of biological samples, conventional technologies rely on four major strategies: (1) patterned surface treatments on a flat plate, (2) structured microwells, (3) physical stenciling, (4) a micro-bubble plate, all of which have shortcomings overcome by the embodiments described herein. Only the microbubble plate permits release of the arrayed samples.
(1) Patterned surface treatments on a flat plate create surfaces with specific chemistry at predetermined locations. This is performed by lithographic methods (i.e., UV radiation through a photographic mask), or by rubber stamp approaches, where chemicals are physically transferred to the plate surface by a patterned rubber stamp. The result of surface treatments is that regions are made favorable or unfavorable for sample attachment (e.g., cell growth, protein binding). After incubation with the media, the media and buffer are washed away. These methods are not highly selective—patterning of the media is not particularly good, and each media/plate requires a different surface treatment specific to that media/plate combination. In addition, the samples attached on specific location can not be isolated from the array.
(2) Structured microwells may be patterned into the surface by lithographic etching, lithographic photopolymerization or molding cavities in the surfaces. Such microwells are of limited use because cells will readily grow out of the cavities and media may readily coat all sides of the microwells. For best results, high aspect ratio wells are required (to contain the biological media), increasing manufacturing difficulty and cost. In addition, the samples attached inside a specific microwell can not be isolated from the array.
(3) Physical stenciling techniques use a rigid temporary barrier that is placed over the plate of interest. This barrier is in the form of a stencil or microfluidic device. Following this, media is introduced and allowed to attach to the surface at locations allowed by the stencil. After attachment, the stencil is removed leaving patterned media. This method suffers because it is difficult (and expensive) to produce stencils of high resolution, with large numbers of precision holes. The stencils must remain in intimate contact with the surface during the entire period of incubation (attachment). Generally, stencils leak between openings further reducing pattern resolution. Finally, such stencils are expensive, and a single stencil must be used for the entire incubation period for each surface that is to be patterned. In addition, the samples attached on a specific location cannot be isolated from the array.
(4) A micro-bubble plate is an array of releasable micropallets with the cavities that trap gas, usually in the form of bubbles, when the plate is submerged in liquid. Gas bubbles provide contiguous, impenetrable barriers blocking access of biological sample to the cavities. Biological samples placed on the array are forced to attach only on the tops of the micropallets, i.e., the surfaces wetted by aqueous media. In addition, the samples attached on a specific micropallet can be isolated from the array by a mechanical force generated by a focused laser pulse. However, the micro-bubble plate has several limitations. In order to trap air bubbles, a hydrophobic silane layer is coated on the surface of the plate. The trapped air tends to become unstable in some micropallet geometries, such as in big cavities. The trapped air tends not to be stable on a long term in some wetting solutions, such as in pure water, in solution with low surface tension, and in some protein solutions.
USPTO Backgrounds
USPTO Backgrounds