TOOLS AND METHODS FOR ISOLATION AND ANALYSIS OF INDIVIDUAL COMPONENTS FROM A BIOLOGICAL SAMPLE

Abstract

The present invention describes a device(s) and assay(s) for the isolation and analysis of individual components from a sample. The invention provides a means of both isolating a multitude of individual components into an organized array and the subsequent analysis of such components by various detection and analysis methodologies. The invention provides a significant advancement in both the number of individual components that can be individually analyzed as well as enabling the quality and number of analytical methodologies that can be applied to them.

Claims

1. An apparatus for processing biological samples, comprising: at least a substrate with a plurality of arrays of vertical fluidic channels that are formed through the substrate, having definable locations on said substrate, and a multitude of controllable dimensions.

2. The apparatus of claim 1 wherein the isolation of the components in the biological sample is achieved via the application of a centrifugal force.

3. The apparatus of claim 1 wherein the isolation of the components in the biological sample is achieved via the application of capillary force.

4. The apparatus of claim 1 wherein the isolation of the components in the biological sample is achieved via the application of gravity.

5. The apparatus of claim 1 wherein the fluidic channels are opposed on one end with an additional substrate, comprising a substrate that has been selected or modified by a chemical entity to enable further analysis of the isolated component(s).

6. The apparatus of claim 5 wherein the modifying entity on the substrate is a sequence of nucleic acids.

7. The apparatus of claim 5 wherein the modifying entity on the substrate may be analyzed optically.

8. The apparatus of claim 1, wherein the wells have a larger diameter on one face of the substrate and a smaller diameter on the opposite face.

9. The apparatus of claim 1, wherein a unique and distinguishable chemical entity is patterned on regions of the apparatus.

10. The apparatus of claim 1, with wells having a multitude of dimensions no larger than 500 microns and no smaller than 100 nm.

11. The apparatus of claim 1, in which the device is built using silicon, fused silica, glass, polycarbonate, acrylic, PDMS, polyethylene, silicon nitride, polyimide, or polystyrine, polyethylene terephthalate, polyetherketone, polyamide, polyoxymethylene, or polysulphone.

12. A method for analyzing cells, the method comprising: a. Placing cells onto a substrate with a plurality of arrays of vertical fluidic channels formed through the substrate b. Translating cells through the vertical fluidic channels, isolating the cell contents

13. The method of claim 12 wherein the translation of the components is achieved via the application of a centrifugal force.

14. The method of claim 12 wherein the translation of the components is achieved via the application of capillary force.

15. The method of claim 12 wherein the translation of the components is achieved via the application of gravity

16. The method of claim 12 wherein the fluidic channels are opposed on one end with an additional substrate, comprising a substrate that has been selected or modified to enable further analysis of the isolated component(s).

17. The method of claim 16 wherein the modifying entity on the substrate is a sequence of nucleic acids.

18. The method of claim 16 wherein the modifying entity on the substrate may be analyzed optically.

19. The method of claim 12, wherein the wells have a larger diameter on one face of the substrate and a smaller diameter on the opposite face.

20. The method of claim 12, wherein a unique and distinguishable chemical entity is patterned on regions of the apparatus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 illustrates the general layout of the multi-dimensional vertical channel array. The high feature density can be determined by the 200 micron scale bar, with the illustrated layout having over 180,000 individual vertical channels.

[0024] FIG. 2 illustrates the substrate and the vertically integrated capture well and channel. A 3D model illustrates the vertical, through substrate nature of the vertical channels.

[0025] FIG. 3 illustrates a side-view of the device, showing the cross-section of the substrate with vertical channels.

[0026] FIG. 4. illustrates a mode of operation in which the sample is applied to the device and placed under centrifugation to populate the individual features with analyte from the sample. Analyte remaining outside of the capture well can be flushed from the device prior to further processing. Additional reagents can be applied to the device and the device can be placed under a gradient, chemical, thermal or photonic (e.g., application of a light source) exposure.

[0027] FIG. 5. illustrates the ability of the device to discriminate sample components based on physical attributes, including, but not limited to, physical size, Youngs modulus, or plasticity, for example.

[0028] FIG. 6. illustrates the ability of the device to separate cargo from an analyte for further downstream processing by application of force, including, but not limited to, centrifugation, wherein the relationship between the analyte and cargo is maintained based on the addressable individuality of the vertical channels.

[0029] FIG. 7. illustrates the ability of the device to process multiple types of cargo within an analyte, wherein the cargo may be separated by use of applied force as illustrated in FIG. 6, or by the application of a gradient that disrupts the analyte causing release of the cargo. Said cargo can then be processed and or analyzed on an assay chip, wherein the assay chip can be either a separate substrate or the same substrate as the device containing the vertical channels.

DETAILED DESCRIPTION OF THE INVENTION

[0030] Note that, the singular forms a, an, and the, as used both within the application and in the appended claims, include plural referents. Thus, unless the context clearly dictates otherwise, reference to a vertical fluidic channel refers to one or more copies of a vertical fluidic channel, and reference to the isolation of cells . . . includes reference to equivalent steps and methods known to those skilled in the art.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned in the present description are referenced for describing and disclosing articles and methodologies that may be used in relation with the described invention.

[0032] Wherein a range of values is given, it is understood that the intervening values between the upper and lower limit of that range, inclusive of any other stated or intervening value in that stated range is encompassed within the invention.

[0033] In the following description, details are provided to enable a more thorough understanding of the invention that are not requisite in their entirety to enable the present invention, as should be evident to one skilled in the art. Also, features and procedures common to those skilled in the art have not been described.

The Invention

[0034] The present invention provides a device and methods for isolation and analysis of individual components from a biological sample. The invention provides isolation of individual components from a sample by isolating them within a vertical fluidic channel. The vertical nature of the channel enables a higher density of channels to be fabricated within a given area. This ability to array the channels on a substrate both enables a high efficiency in isolation of components as well as provides a powerful means by which said components can be analyzed. The arrayed features enable visualization of the isolated components as well as the ability to introduce chemical entities capable of eliciting a detectable signal based upon interaction or interactions with said isolated component. The arrayed, high density vertical fluidic channels also enable the integration of unique chemical entities, capable of generating a detectable signal, that due to the uniqueness of the chemical entity, can be analyzed separate from the device, but traceable back to the individual channel and or isolated component.

Components

[0035] Biological samples contain many types of components that represent various functions. The present invention describes the isolation of individual cells as the preferred embodiment but is not meant to be limiting as those skilled in the art will recognize that the mechanism of isolation is not enabled by the component being a cell, but rather by the physiochemical nature of the component. It will be also evident to those skilled in the art that there exists methods to vary the effective physiochemical nature of an entity that does not permanently alter is biological function. For example, encapsulation of cells within a droplet of uniform size would enable one to capture a larger range of cell sizes and cell types than direct capture. Another example would be the reversible binding of an entity of interest onto a bead such that the entity could be isolated by the physical size of the bead, rather than the entity of interest.

Vertical Fluidic Channel

[0036] A vertical fluidic channel is defined as being formed through the substrate, as opposed to along the substrate, and having a multitude of dimensions along the long axis of the channel and being definable in its location on said substrate. This feature of a multitude of dimension enables greater functionality than that of a filter type device or a device fabricated by random processes. The vertical fluidic channel, by nature of its predeterminable fabrication, can be arrayed in a multitude of configurations in which each channel is physical addressable in a given coordinate system, such as a Cartesian coordinate system defining both the x-axis and y-axis position of said vertical fluidic channels.

Substrate

[0037] A substrate in this context is any material that can be processed to create a vertical fluidic channel through said substrate in a controlled and predetermined manner. The substrate also enable the introduction to the vertical fluidic channel or array of vertical fluidic channels, of the biological sample containing the individual component. The substrate also enable the incorporation of additional substrates containing chemical entities that may be used to elicit a detectable signal from the individual component isolated within the vertical fluidic channel. For example, silicon provides a substrate in which standard microfabrication processes and methods enable one skilled in the art to form vertical channels through the substrate, in predefined positions, with varying dimensions along said channel. For example the Bosch etch process is well known to be capable of forming vias through a silicon substrate, and that by varying said process the physical dimensions of said via can be controlled. By repeating the process with subsequent lithographic patterning steps and from both the top and bottom side of said substrate, a multitude of dimensions, with varying profiles can be achieved.

Capture Surface

[0038] A capture surface is defined as being a substrate to which either the individual component of interest, or a sub-component which comprises a portion of said individual component has, through the method of utilization of the device, has a physical and or chemical interaction with during the course of operation of the device. For example, a microarray comprising an array of unique sequences of nucleic acid, may be incorporated into the device comprising the vertical fluidic channels, such that individual components isolated in said vertical fluidic channels may be processed by the introduction of chemical reagents to release nucleic material of which, in part, the individual component is comprised of and the interaction of said released nucleic acid material to the complimentary nucleic acid material on the capture surface such that a subsequent detectable signal can be generated by the enzymatic extension of the nucleic material that can be analyzed by sequencing technologies, such as next generation sequencing.

Chemical Entity

[0039] A chemical entity herein describes any molecule or molecules that can generate a detectable signal based upon the presence or absence of either said chemical entity or through interaction with an additional chemical entity. For example, a molecule, such as a fluorophore, that in the presence of an interacting chemical entity, changes its ability to fluoresce would constitute a chemical entity capable of generating a detectible signal based upon the presence of said interacting molecule.

REFERENCES

[0040]

TABLE-US-00001 U.S. PATENT DOCUMENTS 1. 5,837,200 November 1998 Diessel et al. 2. US20150051098A1 February 2015 Chen et al. 3. U.S. Pat. No. 6,767,706 B2 July 2004 Quake et al. 4. US 2005/0053952 A1 March 2005 Hong et al. 5. 6,027,873 February 2000 Schellenberger et al. 6. U.S. Pat. No. 8,309,035 B2 November 2012 Chen et al. 7. U.S. Pat. No. 6,338,802B1 October 1998 Bodner et al. 8. 5,506,141 April 1996 Weinreb et al.

OTHER PUBLICATIONS

[0041] 1. Nicola Aceto et al., Circulating Tumor Cell Clusters Are Oligoclonal Precursors of Breast Cancer Metastasis., Cell 158, no. 5 (Aug. 28, 2014): 1110-22, doi:10.1016/j.cell.2014.07.013. [0042] 2. Isaac S Kohane, Ten Things We Have to Do to Achieve Precision Medicine., Science (New York, N.Y.) 349, no. 6243 (Jul. 3, 2015): 37-38, doi:10.1126/science.aab1328. [0043] 3. Evan Z Macosko et al., Highly Parallel Genome-Wide Expression Profiling of Individual Cells Using Nanoliter Droplets., Cell 161, no. 5 (May 21, 2015): 1202-14, doi:10.1016/j.cell.2015.05.002. [0044] 4. Mirjana Rajer and Marko Kmet, Quantitative Analysis of Fine Needle Aspiration Biopsy Samples, Radiology and Oncology 39, no. 4 (2005): 269-72.