A Method Of Performing An Assay

Abstract

A method of performing an assay for a compound using a device (390) comprising with a plurality of wells (340), said plurality of wells (340) containing cells, and liquid is transferred liquid from the wells (340) to a substrate, which substrate is used to perform the assay.

The method is performed using a device (390) wherein each well (340) comprises a bottom provided with a through-hole (370) extending from the well (340) to the backside (392) of the device (390). Liquid containing the compound is transferred via the through-holes (370) to the substrate.

Preferably a cell is present in a well (340) and compound excreted by the cell in the well (340) is detected.

Claims

1. A method of performing an assay for a compound using a device, wherein the device comprises a frontside with a plurality of wells, and a backside; the plurality of wells of the device contains cells: said method comprising the steps of transferring liquid from the wells to the substrate, and using the substrate to perform the assay; characterized in that each well of the plurality of wells comprises a bottom provided with a through-hole extending from the well to the backside of the device; wherein the method comprises the steps of contacting the backside of the device with the substrate, performing the step of transferring liquid from the wells to the substrate by transferring liquid from the wells via the through-holes to the substrate, and performing the step of performing the assay using the substrate.

2. The method according to claim 1, wherein the compound is excreted by the cells in the device, following which the step of performing the assay using the substrate is performed.

3. The method according to claim 1, wherein at least 10% of the plurality of wells contain a single cell.

4. The method according to claim 1, wherein the through-hole has dimensions that do not allow the cell to pass through the through-hole.

5. The method according to claim 1, wherein the assay is an immunoassay.

6. The method according claim 1, wherein the substrate comprises a micro-porous substrate.

7. The method according to claim 6, wherein the liquid is transferred using a pressure difference with a relatively low pressure at a side of the substrate opposite of the device.

8. The method according to claim 1, wherein the substrate is a polymer.

9. The method according to claim 1, wherein the device comprises a frontside with the plurality of wells and a backside, the backside comprising a plurality of protruding capillaries, the capillaries running transverse to the main plane of the substrate.

10. The method according to claim 1, wherein the backside of the device is a hydrophobic backside.

11. The method according to claim 1, wherein the compound is an antibody.

12. The method according to claim 1, wherein the cell is selected.

13. The method according to claim 12, wherein the selected cell is propagated.

14. The method according to claim 12, wherein the selected cell contains a poly nucleic acid sequence and the method comprises at least one further step chosen from i) multiplication of the poly nucleic acid sequence, ii) determining the nucleotide sequence of the poly nucleic acid sequence, and iii) isolating the poly nucleic acid sequence.

15. The method according to claim 1, wherein after the step of transferring the liquid to the device and before the use of the substrate for the assay the device is separated from the substrate.

16. The method of claim 8, wherein the polymer is chosen from i) polyvinylidene difluoride, and ii) nitrocellulose.

Description

[0059] The invention will now be illustrated with reference to the example section below, and with reference to the drawing wherein

[0060] FIG. 1 shows a photograph of a model experiment for estimating minimal sensitivity achievable by the method according to the invention;

[0061] FIG. 2 shows a photograph of an experiment demonstrating the feasibility of transfer of fluorescently labeled antibody from a microsieve onto a PVDF membrane; and

[0062] FIG. 3A to FIG. 3O schematically shows a method of manufacturing a micro-sieve suitable for use in the present invention both in top view and cross-sectional view.

[0063] Firstly an envisaged method of manufacturing a micro-sieve 390 (FIG. 3O) suitable for use in the present invention will be described, which micro-sieve 390 has capillaries 360 (tubes) protruding from the backside of the device 390.

[0064] Single crystal silicon can used as a main structural material for the membrane.

[0065] Fabrication Steps:

[0066] FIG. 3A. The process starts with a silicon wafer 300. A {100) silicon wafer is preferential because in that case wet anisotropic etching can be used to release the membrane.

[0067] FIG. 3B. Deposition of a silicon nitride layer 310 using Low Pressure Chemical Vapor Deposition (LPCVD).

[0068] FIG. 3C. Patterning of silicon nitride 310 using Reactive Ion Etching (RIE). Here a 33 matrix is schematically shown, but in practice the matrix is much larger for example 100100. The plurality of wells to be formed is not limited to a particular shape, such as rectangular, although a grid-like placement of the wells is preferred.

[0069] FIG. 3D. Local Oxidation of Silicon (LOCOS) to form a temporary silicon oxide layer 319.

[0070] FIG. 3E. Resist patterning, with resist layer 329.

[0071] FIG. 3F. Selective removal of silicon nitride using RIE.

[0072] FIG. 3G. Deep Reactive Etching of Silicon (DRIE) to form small round holes 330.

[0073] FIG. 3H. Resist removal using O.sub.2-ashingand chemical cleaning with 100% HNO.sub.3.

[0074] FIG. 3I. Selective removal of silicon oxide of the temporary layer 319 using wet chemical etching in hydrofluoric acid (HF) or buffered HF.

[0075] FIG. 3J. Deep Reactive Etching of Silicon (DRIE) to form cups 340 (wells 340) and at the same time to further deepen the holes 330.

[0076] FIG. 3K. Removal of the first silicon nitride layer 310 using HF or hot phosphoric acid (H.sub.3PO.sub.4).

[0077] FIG. 3L. Deposition of a first low stress silicon nitride layer 350 (SiRN) and second low stress silicon nitride layer 350 on the backside of the wafer 300 by LPCVD.

[0078] FIG. 3M. Patterning of the second silicon nitride layer 350 on the backside of the wafer 300 using RIE of silicon nitride.

[0079] FIG. 3N. Backside etching of silicon in order to form a silicon membrane and expose closed silicon nitride tubes 360. Note that the bottom of the cups 340 is not exposed in this step. For this step wet anisotropic etching of silicon in TMAH may be used. DRIE process could be used as well, alone or in combination with wet anisotropic etching.

[0080] FIG. 3O. RIE directional etching of silicon nitride from the backside of the wafer 300 in order to create open capillaries 360 (defining through-holes 370). This is a device 390 suitable for use in the method according to the invention, with a frontside 391 and a backside 392. The through-hole 370 of a capillary 360 connects a well 340 with the backside 392.

[0081] FIG. 3P. Backside etching to expose the bottom of the cups 340 in order to make the membrane locally optically transparent, more specifically the (thin) bottoms of the cups 340.

[0082] The invention also relates to a method of manufacturing a device using the method steps described above, with any step involving the removal of resist or a previously formed layer not being limited to the specific chemicals and/or concentrations thereof mentioned.

Example 1

[0083] Spotting experiment for estimating minimal sensitivity (model experiment not according to the invention) PVDF (Polyvinylidene difluoride) membrane (Immun-Blot low fluorescence PVDF membrane, BioRad Laboratories B.V., Veenendaal, The Netherlands) was activated in methanol and MilliQ according to protocol manufacturer.

[0084] The PVDF membrane was placed onto filter paper (Bio-Rad, included with the purchased PVDF) wetted in PBS.

[0085] A rhEpCAM (recombinant human EpCAM protein, ACRO Biosystems, EPM-H5223, Bethesda, Md., USA) dilution series was prepared in PBS buffer:

[0086] A) 100 ng/L

[0087] B) 10 ng/L

[0088] C) 1 ng/L

[0089] D) 100 g/L

[0090] E) 10 g/L

[0091] F) 1 g/L

[0092] G) 0 g/L

[0093] 1 L of each concentration was pipetted manually onto the pretreated PVDF membrane.

[0094] After 10 minutes to allow the droplets to be absorbed, the PVDF membrane with spots A)-G) was incubated with 2 mL blocking buffer (PBS, 1% BSA) for 15 minutes.

[0095] Preparation Anti-Human EpCAM FITC Solution

[0096] 2.32 L of anti-human EpCAM FITC (0.43 mg/mL from AcZon, Bologna, Italy) was pipetted into 2.32 mL PBS buffer.

[0097] The blocking buffer was removed and the PVDF membrane was incubated for 15 minutes in 2 mL anti-human EpCAM FITC solution.

[0098] The antibody solution was removed and the PVDF membrane was washed in washing buffer (PBS) for 25 minutes.

[0099] The PVDF strip was viewed under a fluorescence microscope. The spot diameter was 2 mm and a concentration of 1 ng/l (C) was clearly visible (FIG. 1) while the 100 g/l spot was discernible with the eye.

[0100] The spot diameter in FIG. 1 is 2 mm, i.e. quite large, indicating that the read-out sensitivity for smaller spots with the same fluorescent-protein concentration is below 1 ng. If the diameter of the spots is smaller, then the sensitivity is even well below 1 ng.

Example 2

[0101] Transfer of Fluorescently Labeled Antibody from a Microsieve onto a PVDF Membrane

[0102] Preparation of the Device

[0103] A microsieve was de-aerated in methanol and washed with MilliQ. The microsieve was obtained from VyCap BV (Deventer, The Netherlands) and used without the plastic holder it is sold in. The microsieve has through-holes with a diameter of 5 m.

[0104] PVDF Membrane Preparation

[0105] PVDF (Polyvinylidene difluoride) membrane (0.20 m pore size, Immun-Blot low fluorescence PVDF membrane, BioRad Laboratories B.V., Veenendaal, The Netherlands) was activated in methanol and MilliQ according to protocol manufacturer.

[0106] Transfer of Fluorescently Labelled Antibody to the PVDF Membrane

[0107] The microsieve without holder was placed onto a wet PVDF membrane floating on MilliQ in a Petri dish. (In another experiment, the microsieve was placed onto filter paper (Bio-Rad, included with the purchased PVDF wetted in MilliQ) which also worked).

[0108] 6 L anti-IgG PE (Sigma-Aldrich, P8547, 0.1-0.3 g/L, diluted 1:20, phycoerythrin labeled) was pipetted onto the microsieve.

[0109] The microsieve was removed by lifting it vertically and the PVDF membrane was imaged under a fluorescence microscope. It was found that the solution was successfully passed through the microsieve and tiny fluorescent spots were visible. These spots had a diameter of about 40 m.

Example 3

[0110] Assay of Single Hybridomas Cells Producing an Antibody Against an Antigen on Antigen-Coated PVDF (According to the Invention)

[0111] PVDF Membrane Preparation

[0112] Activation step: Activate the micro-porous PVDF membrane in methanol and MilliQ according to protocol manufacturer. The PVDF membrane with a pore size of 0.45 m was Immun-Blot low fluorescence PVDF membrane (BioRad Laboratories B.V., Veenendaal, The Netherlands).

[0113] Coating step: Apply 500 L 10 ng/L antigen on PVDF and incubate for 15 minutes.

[0114] Blocking step: Incubate PVDF 15 minutes in PBS, 1% BSA.

[0115] Preparation of Device

[0116] The microsieve suitable for use in these experiments can be obtained from VyCap BV (Deventer, The Netherlands) and its plastic holder will be removed for our experiments.

[0117] This microsieve will be de-aerated in methanol, washed with MilliQ in accordance with the protocol of the manufacturer and placed in PBS.

[0118] Cell Loading of the Microsieve

[0119] Hybridoma cells can be cultivated in a suitable culture medium (such as Gibco CD hybridoma, +4 mM L-Glutamine, +1% penicillin/streptomycin). Prior to loading the microsieve with the hybridoma cells, the hybridoma cells will be washed to remove the antibody already present in the culture medium. The hybridoma cell suspension will be centrifuged at 300 rpm for 5 minutes. Excess medium will be removed and the cells will be re-suspended in fresh medium at a concentration of approximately 3000 cells in 50 L medium. The microsieve will be loaded with cells in accordance to the instructions of the manufacturer, with the microsieve being placed on a sponge (Vycap BV). The cell suspension will be pipetted onto the microsieve and, after the cells are sufficiently loaded into the wells, the microsieve may be submerged fully in medium. Excess medium will be removed before further handling of the microsieve.

[0120] Transfer of Supernatant to the PVDF Membrane

[0121] Wet filter paper soaked in PBS will be placed in a petridish.

[0122] The antigen-coated PVDF membrane will be placed on top of said filter paper taking care that no air is trapped between the filter paper and the membrane.

[0123] The microsieve with cells will be placed on the PVDF membrane and allowed to sit overnight in an incubator at 37 C., 100% humidity, 5% CO.sub.2.

[0124] Incubation with Fluorescently Labelled Anti-Antibody

[0125] After the incubation period the PVDF will be removed and rinsed with PBS (25 minutes) and incubated with 10 ng/L anti-IgG-PE for 15 minutes. Anti-IgG-PE (Sigma-Aldrich Corp., St. Louis, Mo., USA) contains phycoerythrin as a fluorescent label.

[0126] The PVDF membrane can be examined under a fluorescence microscope. The differences in fluorescence intensity are proportional to the antibody production levels of individual hybridomas cells.

Example 4

[0127] Assay of Single Hybridomas Cells Producing Antibody Against the Antigen on Unmodified PVDF (According to the Invention)

[0128] This experiment can be performed analogous to Example 3 except for the PVDF preparation which will not be coated with the antigen. After contacting the PVDF membrane with the liquid from the wells, the PVDF membrane is blocked, e.g. using the BSA solution and then incubated with the fluorescently labelled anti-antibody.

[0129] The PVDF membrane can be examined under a fluorescence microscope. Differences in fluorescence intensity are proportional to differences in antibody production levels of individual hybridomas cells.

Example 5

[0130] Stamping on PVDF (According to the Invention)

[0131] Experiment 3 can be continued as follows. After the overnight incubation, the microsieve of Example 3 can be placed for 5 minutes on a freshly prepared PVDF membrane coated with antigen as described in Example 3.

[0132] Visual examination under a fluorescence microscope can be used to reveal that the fluorescent spots are more localised.