A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

Summary

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays. 

Abstract

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

Introduction

Many studies in the past have revealed cell-to-cell differences within a large cell population^1-3, in particular signaling processes⁴, or the amounts of intracellular biomolecules such as proteins^5,6, metabolites, and cofactors^7,8. These heterogeneities are considered to be fundamentally important for cell adaptation and evolution⁹, but also play a key role in the emergence and treatment of diseases such as cancer^10-13. Therefore, studies on the single-cell level are of high interest in biological and pharmacological research, particularly if these studies reveal the different cell responses after ....

Protocol

1. SU-8 Master Fabrication

Prepare both master molds for the channels (fluidic and control, for schematics and dimensions see Figure 2) with the following protocol but with different mask patterns. The process is shown in Figure 3a.

1. Start by heating a 4 inch silicon wafer for 10 min at 180 °C. Load the dehydrated wafer on a spin-coater and use the following protocol for spin-coating SU-8 2015:
   1. Spin wafer at 100 rpm for 20 sec (open lid of spin-coater, dispense SU-8 during this step, close lid after dispensing).
   2. Spin wafer at 500 rpm for 10 sec (this will spread the resis....

Results

Our platform is able to analyze a variety of intracellular as well as secreted molecules present in or produced by single cells. Here, we would like to present different example studies to underline the variety of possible assays. We will give an example for a secreted enzyme (Figure 5a) as well as an intracellular enzyme (Figure 5b) and protein (Figures 5c and d). For more examples, such as cofactors or small molecules, please refer to Eyer et al.

Discussion

Microfluidics technology has opened new and fascinating possibilities for single-cell analysis. In particular, the possibility to trap and immobilize cells individually by microfluidic tools has allowed systematic short and long-term studies on single-cell properties and response. Additionally, encapsulation of cells in high frequency microdroplets, generated on a microchip, has enabled single cell secretion studies, which cannot be performed with conventional cytometry devices. The microdroplet approach, however, has so.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS:
Name of the Reagent	Company	Catalogue Number	Comments (optional)
SU-8 2015	MicroChem Corp. (Netwon, MA)	n.a.
1H,1H,2H,2H-Perfluorodecyl-dimethylchloro-silane	ABCR (Karlsruhe, Germany)	AB103608
4-Methylumbelliferyl β-D-N,N′-diacetylchitobioside hydrate	Sigma Aldrich	M9763
Acetone	Merck VWR (Darmstadt, Germany)	100014
Avidin	AppliChem (Axon Lab AG)	A-2568
AZ 1518	AZ Electronic Materials (Wiesbaden, Germany)	n.a.
AZ 726 developer	AZ Electronic Materials (Wiesbaden, Germany)	n.a.
Bovine serum albumin	Sigma Aldrich	A-4503
Bovine serum albumin, biotin	Sigma Aldrich	A-8549
Cell dissociation buffer	Invitrogen	13151-014
Hexamethyldisilazane (HDMS)	Sigma Aldrich	40215
Hydrochloric acid	Fluka	84422
Isopropanol	Merck VWR (Darmstadt, Germany)	109634
Magnesium chloride hexahydrate	Fluka	63068
MR developer 600	Microresist technology GmbH (Berlin, Germany)	n.a.
PBS	Invitrogen	10010-031
PLL-g-PEG grafted	SuSoS, (Dübendorf, Switzerland)	n.a.
PLL-g-PEG grafted biotin	SuSoS, (Dübendorf, Switzerland)	n.a.
Potassium chloride	Fluka	60132
Protein G, biotin	Sigma Fine Chemicals	41624
Silicon wafer	Si-Mat (Kaufering, Germany)	n.a.
Sylgard 184 Silicone Elastomer Kit (PDMS)	Dow Corning	39100000
Tris(hydroxymethyl)-aminomethan	Biorad	1610716
Tween 20	Biorad	1706531
EQUIPMENT:
Material Name	Company	Catalogue Number	Comments (optional)
0.22 µm PES syringe filter	TRP	99722
1/1.5 mm biopsy puncher	Miltex, York PA	33-31AA/33-31A
Cell Trics filter 20 µm	Partec	04-004-2325
Centrifuge Sigma 3-18K	Kuehner	n.a.
Hotplate HP 160 III BM	Sawatec, Sax, Switzerland	n.a.
MA-6 mask aligner	Karl Suess	n.a.
Multizoom AZ100M microscope	Nikon Corporation	n.a.
Photomask	Microlitho, Essex, U.K.	n.a
Plasma Cleaner PDC-32G	Harrick	n.a.
Spin coater Modell WS-400 BZ-6NPP/LITE	Laurell	n.a.
Spin Modules SM 180 BM	Sawatec, Sax, Switzerland	n.a.
Step profiler Dektak XT Advanced	Bruker	n.a.
Syringe pump neMESYS	Cetoni	n.a.