Patent Application
20200360922
The present disclosure relates generally to innovations in microfabrication, microbiology, analytical chemistry. More specifically, the present disclosure relates to systems methods for high throughput screening and identification of biological entities on microfabricated devices.
Identification and screening microorganisms in microbiomes has been gaining more attention and interest in the chemical, pharmaceutical, agricultural, and other industries as people realize the importance of microbiomes in our health, food production and environment. Useful information can be derived from such efforts, for example, for disease diagnosis and discovery of particular species of microorganisms having certain property of interest or can produce substances of interest.
Recently, various technology platforms utilizing plates or panels containing high density of wells for performing microorganism cultivation and screening have been developed. Due to the extremely small sizes of the wells, and small volume of material involved in each individual well, it is challenging to assay individual wells in-situ. Furthermore, to perform many assays, the contents of the wells need to be accessed and gathered, which disrupts the biological process ongoing in the wells. This often hinders, or even makes it impossible for the well contents to be used for other assays. Although prior to the assay, a replicate of the well contents can be made and transferred to another plate, such a step may require the use of sophisticated equipment and/or techniques, and could introduce errors.
In one aspect, a method of screening for at least one biological entity of interest in a sample using a microfabricated device is provided. The microfabricated device has a top surface defining an array of microwells. The method comprises: loading, into at least one microwell of the array of microwells, at least one cell from the sample and a nutrient; applying a gas permeable membrane to the microfabricated device to retain the at least one cell in the at least one microwell; incubating the microfabricated device at predetermined conditions for a duration of time to grow a plurality of cells from the at least one cell in the at least one microwell; detecting, by mass spectrometry, a gaseous substance in a sampling region exterior and corresponding to the at least one microwell; and determining a presence or absence of at least one biological entity of interest in the at least one microwell based on detection or non-detection of the gaseous substance.
In some embodiments, the detection by mass spectrometry comprises irradiating an area with the sampling region to thereby generate an ionized species from the gaseous substance, if any; and transporting an aliquot of air in the sampling region to a mass analyzer for detection of the existence of any ionized species, to thereby determine the presence of the gaseous substance in the sampling region.
In some embodiments, the at least one microwell includes a plurality of microwells, and detecting the gaseous substance comprises detecting the gaseous substance over an area atop the plurality of microwells.
In some embodiments, the method further comprises correlating the pattern of the detected gaseous substance with the locations of the microwells on the microfabricated chip to thereby determine one or more microwells that have produced the gaseous substance.
The at least one biological entity of interest can comprise a eukaryotic cell, a bacterial cell, etc.
If a biological entity of interest is determined to be present in the at least one microwell, the method can further comprise transferring at least one or some of the plurality of cells from the microwell to a target location.
In some embodiments, the at least one microwell includes a plurality of microwells, and loading the at least one cell comprises loading into each of the plurality of microwells no more than one cell.
In some embodiments, each microwell of the array of microwells has a diameter of about 25 μm to about 500 μm. In some embodiments, the surface density of the array of microwells is at least 750 microwells per cm2. In some embodiments, the distance between two neighboring microwells in the array of the microwells of the microfabricated device is less than 500 μm.
In another aspect, a method of screening for at least one biological entity of interest in a sample using a microfabricated device having a top surface defining an array of microwells is provided. The method comprises: loading, into each of a plurality of microwells of the array of microwells, at least one cell from the sample and a nutrient; applying a gas permeable membrane to the microfabricated device to retain the at least one cell loaded in each of the plurality of microwells; incubating the microfabricated device at predetermined conditions for a duration of time to grow a plurality of cells from the at least one cell in each of the plurality of microwells; detecting, by mass spectrometry, a gaseous substance in a sampling region exterior and corresponding to each of the plurality of microwells; and determining a presence or absence of at least one biological entity of interest in each of the plurality of microwells based on detection or non-detection of the gaseous substance. In some embodiments, the microfabricated device is mounted on a stage movable in a plane parallel to a major surface of the microfabricated device, and the detection comprises: positioning an irradiating source to create a focal spot in a first sampling region corresponding to a first microwell to thereby generate an ionized species from the gaseous substance, if any, in the first sampling region; transporting an aliquot of air in the first sampling region to a mass analyzer for detection; laterally moving the stage such that the focal spot of the irradiating source falls in a second sampling region corresponding to a second microwell to thereby generate an ionized species from the gaseous substance, if any, in the second sampling region; and transporting an aliquot of air in the second sampling region to a mass analyzer for detection.
The present disclosure relates generally to systems and methods for isolation, culturing, sampling, and/or screening of biological entities. A microfabricated device (or a “chip”) is used for receiving a sample comprising at least one biological entity (e.g., at least one cell). The term “biological entity” may include, but is not limited to, an organism, a cell, a cell component, a cell product, and a virus, and the term “species” may be used to describe a unit of classification, including, but not limited to, an operational taxonomic unit (OTU), a genotype, a phylotype, a phenotype, an ecotype, a history, a behavior or interaction, a product, a variant, and an evolutionarily significant unit.
As used herein, a microfabricated device or chip may define a high density array of microwells (or experimental units). For example, a microfabricated chip comprising a “high density” of microwells may include about 150 microwells per cm2 to about 160,000 microwells or more per cm2 (for example, at least 150 microwells per cm2, at least 250 microwells per cm2, at least 400 microwells per cm2, at least 500 microwells per cm2, at least 750 microwells per cm2, at least 1,000 microwells per cm2, at least 2,500 microwells per cm2, at least 5,000 microwells per cm2, at least 7,500 microwells per cm2, at least 10,000 microwells per cm2, at least 50,000 microwells per cm2, at least 100,000 microwells per cm2, or at least 160,000 microwells per cm2). A substrate of a microfabricated chip may include about or more than 10,000,000 microwells or locations. For example, an array of microwells may include at least 96 locations, at least 1,000 locations, at least 5,000 locations, at least 10,000 locations, at least 50,000 locations, at least 100,000 locations, at least 500,000 locations, at least 1,000,000 locations, at least 5,000,000 locations, or at least 10,000,000 locations. The arrays of microwells may form grid patterns, and be grouped into separate areas or sections. The dimensions of a microwell may range from nanoscopic (e.g., a diameter from about 1 to about 100 nanometers) to microscopic. For example, each microwell may have a diameter of about 1 μm to about 800 μm, a diameter of about 25 μm to about 500 μm, or a diameter of about 30 μm to about 100 μm. A microwell may have a diameter of about or less than 1 μm, about or less than 5μm, about or less than 10 μm, about or less than 25 μm, about or less than 50 μm, about or less than 100 μm, about or less than 200 μm, about or less than 300 μm, about or less than 400 μm, about or less than 500 μm, about or less than 600 μm, about or less than 700 μm, or about or less than 800 μm. In exemplary embodiments, the diameter of the microwells can be about 100 μm or smaller, or 50 μm or smaller. A microwell may have a depth of about 25 μm to about 100 μm, e.g., about 1 μm, about 5 μm, about 10 μm, about 25 μm, about 50 μm, about 100 μm. It can also have greater depth, e.g., about 200 μm, about 300 μm, about 400 μm, about 500 μm. The microfabricated chip can have two major surfaces: a top surface and a bottom surface, where the microwells have openings at the top surface. Each microwell of the microwells may have an opening or cross section having any shape, e.g., round, hexagonal, square, or other shapes. Each microwell may include sidewalls. For microwells that are not round in their openings or cross sections, the diameter of the microwells described herein refer to the effective diameter of a circular shape having an equivalent area. For example, for a square shaped microwell having side lengths of 10×10 microns, a circle having an equivalent area (100 square microns) has a diameter of 11.3 microns. Each microwell may include a sidewall or sidewalls. The sidewalls may have a cross-sectional profile that is straight, oblique, and/or curved. Each microwell includes a bottom which can be flat, round, or of other shapes. The microfabricated chip (with the microwells thereon) may be manufactured from a polymer, e.g., a cyclic olefin polymer, via precision injection molding or some other process such as embossing. The chip may have a substantially planar major surface.
The high density microwells on the microfabricated chip can be used to conduct various experiments, such as growth or cultivation or screening of various species of bacteria and other microorganisms (or microbes) such as aerobic, anaerobic, and/or facultative aerobic microorganisms. The microwells may be used to conduct experiments with eukaryotic cells such as mammalian cells. Also, the microwells can be used to conduct various genomic or proteomic experiments, and may contain cell products or components, or other biological substances or entities, such as a cell surface (e.g., a cell membrane or wall), a metabolite, a vitamin, a hormone, a neurotransmitter, an antibody, an amino acid, an enzyme, a protein, a saccharide, ATP, a lipid, a nucleoside, a nucleotide, a nucleic acid (e.g., DNA or RNA), etc.
A cell may be Archaea, Bacteria, or Eukaryota (e.g., fungi). For example, a cell may be a microorganism, such as an aerobic, anaerobic, or facultative aerobic microorganisms. A virus may be a bacteriophage. Other cell components/products may include, but are not limited to, proteins, amino acids, enzymes, saccharides, adenosine triphosphate (ATP), lipids, nucleic acids (e.g., DNA and RNA), nucleosides, nucleotides, cell membranes/walls, flagella, fimbriae, organelles, metabolites, vitamins, hormones, neurotransmitters, and antibodies.
A nutrient may be defined (e.g., a chemically defined or synthetic medium) or undefined (e.g., a basal or complex medium). A nutrient may include or be a component of a laboratory-formulated and/or a commercially manufactured medium (e.g., a mix of two or more chemicals). A nutrient may include or be a component of a liquid nutrient medium (i.e., a nutrient broth), such as a marine broth, a lysogeny broth (e.g., Luria broth), etc. A nutrient may include or be a component of a liquid medium mixed with agar to form a solid medium and/or a commercially available manufactured agar plate, such as blood agar.
A nutrient may include or be a component of selective media. For example, selective media may be used for the growth of only certain biological entities or only biological entities with certain properties (e.g., antibiotic resistance or synthesis of a certain metabolite). A nutrient may include or be a component of differential media to distinguish one type of biological entity from another type of biological entity or other types of biological entities by using biochemical characteristics in the presence of specific indicator (e.g., neutral red, phenol red, eosin y, or methylene blue).
A nutrient may include or be a component of an extract of or media derived from a natural environment. For example, a nutrient may be derived from an environment natural to a particular type of biological entity, a different environment, or a plurality of environments. The environment may include, but is not limited to, one or more of a biological tissue (e.g., connective, muscle, nervous, epithelial, plant epidermis, vascular, ground, etc.), a biological fluid or other biological product (e.g., amniotic fluid, bile, blood, cerebrospinal fluid, cerumen, exudate, fecal matter, gastric fluid, interstitial fluid, intracellular fluid, lymphatic fluid, milk, mucus, rumen content, saliva, sebum, semen, sweat, urine, vaginal secretion, vomit, etc.), a microbial suspension, air (including, e.g., different gas contents), supercritical carbon dioxide, soil (including, e.g., minerals, organic matter, gases, liquids, organisms, etc.), sediment (e.g., agricultural, marine, etc.), living organic matter (e.g., plants, insects, other small organisms and microorganisms), dead organic matter, forage (e.g., grasses, legumes, silage, crop residue, etc.), a mineral, oil or oil products (e.g., animal, vegetable, petrochemical), water (e.g., naturally-sourced freshwater, drinking water, seawater, etc.), and/or sewage (e.g., sanitary, commercial, industrial, and/or agricultural wastewater and surface runoff).
After a sample is loaded on a microfabricated device, a membrane may be applied to at least a portion of a microfabricated device.
A membrane may cover at least a portion of a microfabricated device including one or more experimental units, wells, or microwells. For example, after a sample is loaded on a microfabricated device, at least one membrane may be applied to at least one microwell of a high density array of microwells. A plurality of membranes may be applied to a plurality of portions of a microfabricated device. For example, separate membranes may be applied to separate subsections of a high density array of microwells.
A membrane may be connected, attached, partially attached, affixed, sealed, and/or partially sealed to a microfabricated device to retain at least one biological entity in the at least one microwell of the high density array of microwells. For example, a membrane may be reversibly affixed to a microfabricated device using lamination. A membrane may be punctured, peeled back, detached, partially detached, removed, and/or partially removed to access at least one biological entity in the at least one microwell of the high density array of microwells.
A membrane may be impermeable, semi-permeable, selectively permeable, differentially permeable, and/or partially permeable to allow diffusion of at least one nutrient into the at least one microwell of a high density array of microwells. For example, a membrane may include a natural material and/or a synthetic material. A membrane may include a hydrogel layer and/or filter paper. In some embodiments, a membrane is selected with a pore size small enough to retain at least some or all of the cells in a microwell. For mammalian cells, the pore size may be a few microns and still retain the cells. However, in some embodiments, the pore size may be less than or equal to about 0.2 μm, such as 0.1 μm. An impermeable membrane has a pore size approaching zero. It is understood that the membrane may have a complex structure that may or may not have defined pore sizes
In one aspect of the present invention, there is provided a nondestructive, spatially-sensitive assay of a biological entity of interest of the samples loaded in the microfabricated chip based on the detection of a gaseous substance (or substances) produced in individual wells by mass spectrometry. The assay can be performed in a highly parallelized manner, enabling fast screening of very large numbers of microbes (e.g. microbiomes) in a single experiment. Compared with alternative methods for assaying contents of microwells, which may involve adding reagents into the well contents, and therefore cause disruption of the normal cell growth/proliferation or even destroy the cells in the microwells (a lot of reagents are harmful or toxic to the cells), the method of the present disclosure separates the site of detection (in the cover film atop of the microwell) from the well contents, therefore does not interfere with cell growth or proliferation in the microwells. This allows the well contents to be available for further assays or tests.
In some embodiments, a method of screening for at least one biological entity of interest in a sample using a microfabricated device is provided. At least one cell from the sample and a nutrient are loaded into at least one microwell of the array of microwells. A gas permeable membrane is applied to the microfabricated device to retain the at least one cell in the at least one microwell. The microfabricated device is incubated at predetermined conditions for a duration of time to grow a plurality of cells from the at least one cell in the at least one microwell. Mass spectrometry is used to detect gas in a sampling region for the at least one microwell. This MS detection can be performed during the incubation or after incubation, and can be done at a single time point, a multiple time points over a time course, or continuously over a period of time. A presence or absence of at least one biological entity of interest, e.g., a eukaryotic cell or a bacterial cell, in the at least one microwell is then determined based on the detection.
The microwell array on the microfabricated chip of the present disclosure is suited for imaging by mass spectrometry. The microwells can be constructed with desired pattern with well-defined parameters (e.g., overall layout, dimension of individual wells, inter-well spacing, etc.) which provide a clear way to relate chemical signals back to the microwell(s) of origin and the location of the microwells. In some embodiments, the microfabricated chip can be made with similar dimensions as a microscope slide, and can be used by standard equipment associated with mass spectrometry without modification. Individual microwells can be resolved by commercial instruments, which routinely achieve a spot size of 10 microns or smaller.
While H2S is used as an example, it is understood that other gas such as oxygen, carbon monoxide, nitric oxide, and ammonia, may be produced in the metabolic activities during the lifecycles of cells (growth, division, multiplication, death), or the reactions or interactions of cells with the micro-environment they are in, e.g., the nutrient loaded in the microwells. For example, sulfate reducing bacteria (SRB) such as Desulfovibrio can react to sulfate contained in a nutrient, producing H2S as a product which can permeate through the membrane and be detected by mass spectrometry.
It is understood that different gases would have different molecular sizes and affinity to different membrane materials, and their transport rates or behavior through the membrane may also depend on other microstructural parameters of the membrane (which in turn may depend on how the membrane is manufactured, e.g., the cast and drying process, how the pores are created inside the membrane, etc.). The overall gas permeability of the membrane with respect to any particular gas also depends on the thickness of the membrane. The particular membrane (e.g., with specific permeability, thickness and other parameters) selected for any assay can be determined based on the identity and amount of the gas compounds expected to be produced by the microwell contents. In some examples, the gas permeable membrane can be polyurethane sealing membrane with FDA-approved acrylic adhesive commonly used to seal multi-well plates.
The gas permeable membrane forms a tight seal over the microwells. In typical cases, the thickness of the membrane material (e.g., a few microns) will make gas migration from one microwell to another by diffusion through the membrane negligible. In some embodiments, the gas permeable membrane can have a microstructure that enables an anisotropic diffusion of the gaseous substance. The microstructure of the membrane can be such that the gaseous substances can diffuse more rapidly along the thickness direction of the membrane (in other words, perpendicular to the top surface 501 of the microfabricated chip 500) than along its transverse direction (or the direction parallel to the top surface 501). As an example, the gas permeable membrane can have a plurality through channels that are substantially normal to the major surface of membrane. In such a structure, gas diffusion will be predominantly along the direction normal to the membrane surface.
In the example shown in
The focal spot of the laser or the ion beam or e-beam can be in the sampling region as illustrated in
More complex laser-based methods can be used. For example, muti-photon ionization, particularly with crossed beams, can be used to restrict the probe volume (down to a spot size of a few cubic microns). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) or matrix free laser desorption mass spectrometry (LDS) can also be used to generate images of the array of microwells (based on the detected gas in the detection area outside the corresponding microwells) in one or more mass-to-charge (m/z) values, providing the capability of mapping specific gas molecules emerged from the microwells to two-dimensional coordinates of these microwells. For example, the matrix in the MALDI MS can be a thin coating applied on the membrane while keeping the gas permeability of the membrane. Alternatively, the laser can be tuned to be in resonance with the membrane material so that the additional matrix coating is not needed. In such instances, the sampling region can be on the outer surface of the membrane (part of the membrane, not off the membrane). Other ionization methods such as plasma or electron guns have even more spatial resolution, where the ionizing beams can be directed down to tens of nanometers.
As shown in
Based on gas detected (or non-detection) by the mass spectrometer and the location of the detection relative to the microfabricated chip, the microwell or microwells producing the gas can be determined, and the presence or absence of a biological entity of interest in such microwell or microwells can be determined. If a biological entity of interest is determined to be present in such microwell(s), at least parts of the contents of such microwell(s) (including some of the plurality of cells) can be further sampled or transferred to a target location for further growth, cultivation, and/or analysis.
The microfabricated chip can be loaded with a sample containing a plurality of species, strains or genera of microorganisms or eukaryotic cells and the cells can be loaded such that each of the plurality of microwells can contain, on average, one cell, two cells, three cells, four cells, five cells, six cells, seven cells, eight cells, and so on. This can be accomplished using a cell sorter or other available techniques. In some embodiments, the cells can be loaded such only a small subset of the plurality of microwells contain cells to make it unlikely any microwell would contain more than one cell. This is to ensure that if a diverse sample is used, e.g., a sample containing a plurality of microbial cells of different species or genera, each microwell will most likely only contain a single species of cells after incubation.
While various embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
This application claims priority to U.S. Provisional Application No. 62/849,103 filed May 16, 2019, the disclosure of which is incorporated by reference herein by its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62849103 | May 2019 | US |
