Assay apparatus and methods

• US 10,384,207 B2
• Filed: 07/19/2016
• Issued: 08/20/2019
• Est. Priority Date: 07/21/2015
• Status: Active Grant

• Current Assignee: Neuro Probe Incorporated
• Original Assignee: Neuro Probe Incorporated
• Inventors: Goodwin, Jr., Richard H.
• Primary Examiner(s): Dines, Karla A
• Application Number:
  US15/213,632
• Publication Number:
  US 20170021350A1
• Time in Patent Office: 1,127 Days
• Field of Search: 506 10
• CPC Class Codes: B01L 2200/0694 Creating chemical gradients in a fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/161 Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0472 Diffusion
• CPC Class Codes: B01L 3/502723 characterised by venting arrangements
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Abstract

Apparatus and methods for determining whether a test compound induces cell activity, changes cell activity, prevents cell activity, or inhibits cell activity. An embodiment comprises placing a test compound solution in contact with a cell suspension media containing cells, diffusing the test compound solution into the cell suspension from one or more sides, and detecting activity in the cells with respect to their distance from the side from which the test compound is diffusing. Embodiments may provide an apparatus that allows a side source, a point source, or both, from which a test compound solution diffuses into a cell suspension media and contacts cells. Detecting cell activity may involve detecting activity in a first cell group proximate to the side from which the test compound is diffusing, and detecting activity in a second cell group farther than the first cell group from the side from which the test compound is diffusing.

Claims

1. An apparatus for exposing cells to a test compound solution to determine whether the test compound solution induces progressive cell activity response in the cells, the apparatus comprising:

a substrate having an inner surface and an outer surface opposite to the inner surface;

a plate disposed over the substrate such that a cavity is defined between the plate and the substrate,

wherein the plate has a first surface and a second surface opposite to the first surface, the second surface facing the inner surface of the substrate,

wherein the plate defines a through-well extending from the first surface of the plate to the second surface of the plate,

wherein the through-well exposes the inner surface of the substrate, and

wherein the through-well is in fluid communication with the cavity; and

a hydrophobic barrier on the substrate and/or the plate,

wherein the hydrophobic barrier defines a test site enclosing the through-well when the apparatus is viewed from a plan view perpendicular to a plane defined by the plate,

wherein the hydrophobic barrier allows passage of gas outside of the test site while limiting passage of liquid outside of the test site,

wherein the substrate, the plate, and the hydrophobic barrier are configured to contain the cells within the test site,

wherein the through-well is disposed at a first side portion of the test site when viewed from the plan view, the test site having a second side portion opposite to the first side portion,

wherein, when viewed from the plan view, the test site has a length from the first side portion to the second side portion, and the length is greater than a width of the test site,

wherein the plate and the substrate define a gap between the substrate and an edge of the through-well at the second surface of the plate, on a side of the through-well facing the second side portion of the test site, and

wherein the gap extends substantially across the width of the test site.

2. The apparatus of claim 1, wherein the hydrophobic barrier comprises a groove in the second surface of the plate and/or the inner surface of the substrate.

3. The apparatus of claim 1, wherein the hydrophobic barrier comprises a chemical hydrophobic barrier applied to the inner surface of the substrate and/or the second surface of the plate.

4. The apparatus of claim 1, wherein the gap extends across at least ⅗

of the width of the test site.

5. The apparatus of claim 1, wherein the test site is oval-shaped.

6. The apparatus of claim 1, wherein the through-well is circular when viewed from the plan view.

7. The apparatus of claim 1, wherein the plate defines a second through-well extending from the first surface of the plate to the second surface of the plate,

wherein the second through-well is disposed at the second side portion of the test site,

wherein the second through-well exposes the inner surface of the substrate,

wherein the second through-well is in fluid communication with the cavity,

wherein the plate and the substrate define a second gap between the substrate and an edge of the second through-well at the second surface of the plate, on a side of the second through-well facing the first side portion of the test site, and

wherein the second gap extends substantially across the width of the test site.

8. The apparatus of claim 7, wherein the plate defines a through-hole extending from the first surface of the plate to the second surface of the plate,

wherein the through-hole is disposed at a middle portion of the test site between the first side portion and the second side portion of the test site, and

wherein at the second surface of the plate, the through-hole is substantially smaller in width than the through-well at the first side portion of the test site and the second through-well at the second side portion of the test site.

9. The apparatus of claim 8, wherein the through-hole comprises a tapered through-hole having a wider dimension at the first surface of the plate and a smaller dimension at the second surface of the plate.

10. The apparatus of claim 1, wherein between the gap and the second side portion of the test site, the bottom surface of the plate has a recessed portion bounding the cavity.

11. The apparatus of claim 1, wherein the substrate is transparent and has a thickness from the inner surface to the outer surface within a range of about 0.1 mm to 2.0 mm.

12. The apparatus of claim 11, wherein the substrate comprises sapphire.

13. The apparatus of claim 11, wherein the substrate comprises glass.

14. The apparatus of claim 1, further comprising a fastener holding the substrate and the plate together in contact.

15. A method for determining whether a test compound solution induces progressive cell activity response, the method comprising:

depositing a media containing cells into a cavity defined between a plate and a substrate,

wherein the substrate has an inner surface and an outer surface opposite to the inner surface,

wherein the plate has a first surface and a second surface opposite to the first surface, the second surface facing the inner surface of the substrate,

wherein the plate defines a through-well extending from the first surface of the plate to the second surface of the plate,

wherein the through-well exposes the inner surface of the substrate,

wherein the through-well is in fluid communication with the cavity,

wherein a hydrophobic barrier on the substrate and/or the plate defines a test site enclosing the through-well when the substrate and the plate are viewed from a plan view perpendicular to a plane defined by the plate,

wherein, when viewed from the plan view, the test site is elongated, having a length greater than a width,

wherein the hydrophobic barrier allows passage of gas outside of the test site while limiting passage of liquid outside of the test site,

wherein the substrate, the plate, and the hydrophobic barrier are configured to contain the cells within the test site,

wherein the through-well is disposed at a first side portion of the test site when viewed from the plan view, the test site having a second side portion opposite to the first side portion,

wherein the plate and the substrate define a gap between the substrate and an edge of the through-well at the second surface of the plate, on a side of the through-well facing the second side portion of the test site, and

wherein the gap extends substantially across the width of the test site;

allowing the media containing cells to wick into the cavity out to the hydrophobic barrier and form a meniscus at the gap;

placing the test compound solution in the through-well and in contact with the media containing cells at the meniscus at the gap;

diffusing into the media containing cells the test compound solution in a substantially linear front originating from the gap, spanning the width of the test site, and moving across the length of the test site from the first side portion of the test site to the second side portion of the test site,

wherein a first cell of the media is closer to the gap than a second cell of the media;

allowing the test compound solution to diffuse into the media for a predetermined duration such that the first cell is exposed to the test compound solution longer than the second cell is exposed to the test compound solution;

after the predetermined duration, detecting a degree of activity of the first cell and a degree of activity of the second cell; and

comparing the degree of activity of the first cell to the degree of activity of the second cell to determine the presence or absence of a progressive cell activity response by the cells of the media to determine whether the test compound solution induces progressive cell activity response.

16. The method of claim 15, further comprising depositing a gel in the cavity.

17. The method of claim 15, wherein the media containing cells comprises a gel.

18. The method of claim 15, wherein the length of the test site is at least two times greater than the width of the test site, to provide the substantially linear front.

19. The method of claim 15, wherein depositing the media containing cells into the cavity defined between the plate and the substrate comprises depositing the media containing cells in the through-well.

20. The method of claim 15, wherein the plate defines a second through-well in fluid communication with the cavity, and wherein depositing the media containing cells into the cavity comprises depositing the media containing cells in the second through-well.

21. The method of claim 15, wherein the hydrophobic barrier comprises a groove in the second surface of the plate and/or the inner surface of the substrate.