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 treatment with bioactive chemical substances.

In recent years, many analytical platforms have been developed that facilitate the analysis of single living cells or the chemical composition of the cell content. While fluorescent activated cell sorting (FACS) is the gold standard for very high-throughput analysis of single living cells, the method cannot be employed for the quantification of intracellular or secreted compounds. The emergence of microfluidic platforms has promised novel analytical strategies for positioning, treatment and observation of single cells. A milestone in microfluidics was reached with the integration of flexible PDMS valves realized by Quake and coworkers^14,15. These valves are useful since they can isolate regions on chip, e.g. separate two cultures ¹⁶. Furthermore, they are especially applicable for single cell analysis and therefore help to reduce analyte dilution problems. The power of this approach for single-cell analysis has been recently demonstrated by Hansen and coworkers, who analyzed the gene expression from hundreds of single cells in parallel¹⁷.

When targeting proteins and metabolites, the analysis is very difficult due to the lack of suitable amplification methods, the large number of different compounds present, and their variations in chemical nature. Furthermore, most intracellular biomolecules are expected to be present in low copy numbers in the order of a few ten thousands¹⁸, hence the analytical method used must have a high sensitivity. More powerful assays such as immunoassays and enzyme-linked immunoassays (ELISA) are difficult to integrate into microfluidic devices since they require several washing and incubation steps as well as surface immobilization.

Due to these challenges, it is not surprising that only a few examples have been reported where proteins or metabolites were quantified on the single-cell level. For example, studies on the secretion of fluorescent compounds have been reported^19,20. Recently, the implementation with ELISA was presented for the analysis of secreted (nonfluorescent) proteins from a cell culture (THP-1 cells)²¹ and single (immune) cells¹⁰. Targeting intracellular proteins, Shi et al. developed a microfluidic device that facilitated the identification of intracellular proteins for the analysis of signaling pathways in tumor cells by means of an immunoassay¹¹. However, only relative amounts of proteins were determined and no enzymatic amplification was used to increase the signal for low abundance proteins.

Recently, we were able to combine a single-cell trapping microdevice with fluorescence assays⁸ and immunoassays²² (Figure 1). Cells are passively trapped in microsized hurdle structures, which allow supply and (rapid) exchange of medium and other chemical agents without any movement of the cells. A ring-shaped valve around each trap enables isolation of the cell in a very small volume (“the microchamber”). This valve is actuated immediately after introducing a cell-lysing (hypoosmolar) buffer, hence preventing intracellular molecules or secreted molecules to diffuse away. Most importantly, due to the small size of the volume (625 pl) large dilution of the molecules is avoided. Furthermore, since lysis and analysis are performed in at the same position in the chip, there is no loss of analytes due to transportation. The chip design described here comprises 8 alternating rows of either 7 or 8 microchambers, totaling 60 microchambers. The chambers are actuated in rows, so that cross-contamination along a line is precluded.

The platform can be used in combination with fluorescence assays as well as immunological assays (Figure 1d). For the latter, we established protocols for immobilization of the antibodies, which are compatible with the chip production and assembly process. Hence the platform opens the way for sensitive, reliable and quantifiable assays at the single cell level. Up to now, we have used the device for the analysis of intracellular and secreted enzymes (relative quantification by enzymatic assays), intracellular cofactors, proteins and small molecules (absolute quantification by endpoint assays or ELISA). In the following, we describe the process of chip fabrication by means of multilayer soft lithography and the protocols for patterning of the antibodies by means of microcontact printing and surface chemistry. Additionally, some examples of chip use and operations are given.

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 resist over the whole wafer).
   3. Spin wafer at 1,750 rpm for 30 sec (defines the height of the resist to 20 µm).
2. Remove resist from the edge of the wafer with acetone (use a swab) as this will otherwise stick to the hotplate in the next step. Transfer the wafer to a hotplate at 95 °C and heat for 4 min. After the softbake, expose the resist through a photomask taped to sodalime glass in a mask aligner with UV light (150 mJ/cm², measured at 365 nm).
3. After the exposure, heat the wafer again at 95 °C for 5 min on a hotplate. Cool down the wafer to RT and then immerse it into a bath of SU-8 developer for 4 min. Shake gently to remove unexposed SU-8. Wash the wafer with clean-room grade isopropanol and blow dry with nitrogen. Check under a microscope if the development was successful (especially the cell traps). If the cell traps are completely developed, the reflection of the silicon wafer should be visible. If necessary, develop the wafer again for a few minutes.
4. Bake the wafer in an oven for 2 hr at 180 °C to remove all residual solvent. Check the height of the channels with a step profiler. If the height differs from the desired height, repeat this protocol starting at step 1.1 with a new wafer and change the spin-speed (step 1.1.3). Finalize the fabrication of the master mold by silanizing the wafer with 1H,1H,2H,2H-Perfluorodecyl-dimethylchloro-silane in a desiccator overnight.

2. Master Fabrication for Microcontact Printing

1. In order to prepare master molds for microcontact printing, dehydrate a silicon wafer for 10 min at 180 °C. Spin-coat 1 ml HDMS onto this wafer at 7,500 rpm for 30 sec (the resist adheres better to a silanized surface). Spin-coat 2 ml of AZ1518 positive resist with this protocol:
   1. Static dispense of the resist on to the wafer.
   2. Spin wafer for 5 sec at 500 rpm.
   3. Spin wafer for 60 sec at 4000 rpm.
2. After a 50 sec softbake at 100 °C on a hotplate, expose the resist to UV-light (21 mJ/cm² at 365 nm) through a photomask and on a mask aligner. Develop the resist in a bath of AZ 726 for 75 sec. Rinse the developed wafer with DI-water and blow dry with nitrogen. Remove residual solvent by heating the wafer at 115 °C for 50 sec. Silanize the wafer under vacuum overnight.

3. Fabrication of the Microfluidic Chip

A two-layer device design is used for these experiments. The two layers are prepared separately and are bonded together, before the chip is finally bonded onto a glass slide (Figure 3b). This step describes the production of the PDMS layers and the PDMS stamp for microcontact printing.

1. Prepare a 10:1 mixture of PDMS and curing agent. Prepare approximately 80 g PDMS in total (using our designs, this will result in 10 chips). Mix both parts vigorously and degas the PDMS until the mixture is bubble free (~30 min).
2. Place the wafer with the control layer inside a Petri dish and tape it onto the bottom. Next, pour  ~50 g of PDMS on top and put the wafer for at least 2 hr in an oven at 80 °C to assure complete curing of the PDMS. Repeat the same procedure for the microcontact printing wafer, for this ~20 g PDMS is needed.
3. Spin-coat PDMS on the wafer with the fluidic layer at a rotational speed of 2,000 rpm to form an approximately 40 µm high PDMS layer. Cure the PDMS in an oven for at least 1 hr at 80 °C.
4. When both parts are cured, remove the control layer from the wafer and cut into pieces with a razor blade. Punch pressure connection holes by using a 1 mm biopsy puncher. For storage, it is advisable to put a piece of tape over the channels.
5. To bond the two layers, take the spin-coated wafer and the top part and place them in a plasma cleaner²³. After plasma treatment, quickly align both parts under a microscope with large working distance. Add some spare PDMS around the placed top parts. This step is not mandatory, however it facilitates the removal of the PDMS from the wafer. Place the wafer in an oven at 80 °C for at least 1 hr.
6. Use a scalpel to carefully remove the PDMS from the wafer and to cut the microchips. Punch access holes for the fluidic connections with a 1.5 mm biopsy puncher.
7. The PDMS chip is now finished and can be stored for months. Optional: If the chip is used with a reservoir, cut the upper part of a 200 µl pipette tip and use some spare, semi-cured PDMS to glue it on top. Put the chip in the oven again at 80 °C for at least 1 hr.

4. Bonding to Glass Slide

To use the device for direct enzymatic assays, the only step required after bonding is the blocking of surfaces. For this protocol, proceed to A. However, to use the microfluidic chip for immunoassays or ELISAs, please refer to protocol B to restrict the binding sites only to the glass slide (i.e. for TIRF microscopy or SPR). If this restriction is not important, refer to A, but use biotinylated conjugates in step 4.3 and continue with step 4.8 in protocol B to create a fully functional surface.

Prior to performing protocol A and B: It is advisable to filter all of the used protein solutions prior to introducing them into the chip. Debris and protein aggregates may otherwise block cell traps, thereby reducing the number of chambers that can be used for analysis. For this purpose, use a nonprotein adsorbing filter unit to filter all solutions before introducing them into the channels.

Protocol A (enzymatic assays)

1. To finalize the microfluidic device, bond the PDMS parts onto a glass slide. To do this, first clean a glass slide with soap, distilled water and ethanol. Dry the slide using a nitrogen stream. Clean the PDMS part with scotch tape.
2. Put the PDMS part and the cleaned glass slide into the plasma cleaner for 45 sec at 18 W. To assure a tight bonding, put the chip on a hot plate (100 °C) for 5 min.
3. After bonding, fill the chip with fluids. An easy way to achieve this is to use centrifugal force. Cut the lower part of 200 µl pipette tip. For the fluidic channels, fill them with the blocking solution (bovine serum albumin (4% w/v) or poly-L-lysine) grafted polyethylene glycol (0.05% w/v) and place the tips into the inlets. Fill the control layer inlets with either water or with an isosmotic solution, e.g. PBS. Place the chip into the centrifuge (800 x g) for 5 min. The channels should be now filled with fluid completely. If not, repeat this step.
4. Incubate the blocking solution for at least 30 min at room temperature. Wash the solution out of the fluid layer with PBS at a flow rate of 10 µl/min using a syringe pump. The device is now ready for cell experiments.

Protocol B (immunoassays)

For a complete overview of the surface modification from step 4.7 onwards, please refer to Table 1.

5. Incubate PDMS stamps for microcontact printing with a bBSA/BSA solution (e.g. 1-100). After 30 min, clean the stamps thoroughly with distilled water and dry the stamp under a stream of nitrogen. Quickly place the stamps on a cleaned glass slide (Figure 4).
6. Expose the glass slide with the stamp together with the top part of the microfluidic chip to oxygen plasma for 45 sec at maximum power (18 W) in a plasma cleaner. After plasma treatment, remove the stamp and align the printed surface underneath the microchambers of the device. Place the chip for 30 min on a hot plate at 50 °C.
7. To block the remaining surface, introduce sterile-filtered BSA solution (4% (w/v) in PBS) into the chip with a centrifuge (see step 4.2). Incubate for 1 hr and wash the solution out of the fluid layer with PBS (see step 4.3). The chip can then be used directly or stored for up to 2 weeks at 4 °C in a humid box.
8. For a fully functional binding surface, introduce avidin (0.025% (w/v) in PBS) into the reservoir. Use a flow rate of 5 µl/min for 30 min to withdraw the avidin solution through the fluid channels. Afterwards, flush PBS for 10 min. Rinse the reservoir thoroughly.
9. Afterwards, add Protein G (0.0025% (w/v) in PBS) and flush for another 30 min. Wash with PBS for another 10 min afterwards. Next, add the antibody of interest (0.0001% (w/v) in PBS) for 10 min. After 10 min, stop the flow for 15 min to allow binding. Next, wash with PBS for 10 min.

5. Cell Experiments

The protocol is written in general terms because of the variety of possible assays. As an example the reagents needed for the G6PDH toxicity assay are given. The initial cell trapping efficiency of the device is around 2.5%, i.e. 5 out of 200 cells will be trapped. The final occupancy of the cell traps strongly depends on the used cell line, the protocol to suspend the cell line and the time the cells are flushed through the channel. For cells naturally growing in suspension (such as U937), single cells can be found in about 75% of the chambers after a few minutes. The other chambers are either not filled, or occupied by two or more cells. In general, the single-cell occupancy is smaller for adherent cell lines. When using the rather mild suspending protocol presented here, the percentage decreases to about 30% (HEK, MLT cells). The single-cell occupancy can be improved by trypsin treatment of the cells, but this may also alter the results of the experiments.

Depending on the target molecule, adsorption or absorption to PDMS can influence the results. Adsorption can be reduced by blocking the surfaces with BSA or PLL-g-PEG. Absorption is unlikely for most hydrophilic biomolecules, but it should be checked for (small) lipophilic cell components.

1. Prepare the cells according to the specific protocol (e.g. drug addition). For suspension cells, add cells directly to the chip. For adhesive cell lines, use enzyme-free dissociation buffer to produce a cell suspension. Filter the cells before introducing them to the chip to reduce the amount of cell clusters.
2. Load the cells onto the chip using a syringe pump and forward flow, rather than to withdraw the cell suspension. Use a flow rate of 5 µl/min for suspension cell lines and higher flow rates of 10-20 µl/min for adhesive cells since to reduce nonspecific attachment of the cells onto the channel walls.
3. When the traps are filled, close the chambers. If many cells adhere nonspecifically to the surface, try to wash them away with cell dissociation buffer at a flow rate of 10-30 µl/min.

Protocol A (enzymatic assays)

4. Withdraw lysis buffer through the chip. For this example, this buffer is a hypoosmolar buffer with added Tween 20 (10 mM Tris-HCl, 10 mM KCl, 1.5 mM MgCl₂, 1% (v/v) Tween 20). For lysis, quickly open and close chambers (i.e. 500 msec for flow rates of 10-50 µl/min ^7,21). For the G6PDH assay, add 2 mM glucose-6-phosphate, 0.5 mM NADP, 0.5 U/ml diaphorase and 0.3 mM resazurin to the lysis buffer. Start monitoring the kinetic reaction immediately after lysis.

Note: Choose any buffer which is suitable for the assay, however bare in mind that strong detergents (like Triton, SDS) lyse cells immediately and will lead to a loss of analyte during chamber opening.

Protocol B (immunoassays)

For a short description of the ELISA cell experiments (flow rates, chamber status, etc.), please refer to Table 1.

5. For ELISA, add the required detection reagent (e.g. secondary antibody, labeled antigen) directly to the lysis buffer. The herein added Tween 20 reduces the nonspecific adsorption. Lyse the cells according to step 5.4. Incubate the mixture for 10-15 minutes to allow binding (incubation time depending on the used antigen and antibody).
6. Antibody enzyme conjugates that adsorbed nonspecifically to the channel walls can lead to a conversion of the detection reagent already inside the reservoir and channels. To reduce issues from this adsorption, flush a permanent inhibitor of the used detection enzyme through the chip during the incubation time (chambers closed, before adding the detection reagent). For HRP, use 20 mM HCl in water to permanently inactivate the nonspecifically adsorbed enzymes.
7. After incubation time, introduce the detection reagent (e.g. Amplex Red and hydrogen peroxide for HRP) to the chip. Open the chambers shortly again to wash the nonbound secondary antibody away and to add the detection reagent. Start monitoring the kinetic reaction immediately.
8. Calibration: Immobilize the antibody against the target of interest. Close chambers. Prepare different concentrations of analyte in buffer or in cell lysate off-chip. Apply a concentration to the chip and quickly exchange the volume inside the chambers (500 msec opening time). This opening time is sufficiently short to ensure that no additional accumulation occurs from analyte flushing, and only a specific number of molecules from the volume of the microchamber are detected. From the known concentration of the antigen inside the solution and the volume of the chamber (625 pl), calculate the amount inside the chamber. Afterwards, continue with step 5.5 and introduce the detection moiety.

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.