A droplet microfluidic system for sequential generation of lipid bilayers and transmembrane electrical recordings

This paper demonstrates a microfluidic system that automates i) formation of a lipid bilayer at the interface between a pair of nanoliter-sized aqueous droplets in oil, ii) exchange of one droplet of the pair to form a new bilayer, and iii) current measurements on single proteins. A new microfluidic architecture is introduced - a set of traps designed to localize the droplets with respect to each other and with respect to the recording electrodes. The system allows for automated execution of experimental protocols by active control of the flow on chip with the use of simple external valves. Formation of stable artificial lipid bilayers, incorporation of α-hemolysin into the bilayers and electrical measurements of ionic transport through the protein pore are demonstrated.


Geometry of the chip
generation of droplets with protein solution, 4 -inlet for the buffer, 5 -inlet for the protein solution, 6 -channels used as a container for buffer, 7 -channels used as a container for the protein solution, 8, 9, 10, 11, 12, 13, 19 -inlets for oil, 14 -channel which supplies oil for the detachment of droplets, 15 -outlet channels, 16 -channels for the pair of electrodes, 17channels used as a container for the sequence of inhibitors, 18 -inlet for the tubing containing a sequence of droplets with various concentrations of inhibitor.The dimensions of a whole chip are 82 x 53 mm.Aldrich]) containing 1 mg mL -1 of DPhPC (Avanti Polar Lipids) from pressurized reservoirs, through the valves and into the steel capillaries and onto the chip.Setting up the experiment requires a considerable volume of the oil (on the order of few mL).Although the cost of lipids is not high (ca. 1 USD per mL at the concentration that we used), the volume of oil can be minimized to a few hundred microliters.The amount of oil used per exchange of the droplets is very small -below 5 L.
The aqueous samples (protein solution and buffer) were deposited into long storage channels on the chip directly from syringes, which were closed with valves after the deposition of the sample.Once the liquids were deposited onto the chip, the flow of oil pushed the samples into the on-chip microdroplet generators -T-junctions.We controlled the pressure applied to the oil reservoirs using manual pressure regulators (Bosch Rexroth PR1-RGP) and monitored the pressure using digital manometers (AZ 82100, AZ Instruments).A reservoir, which supplied the oil to channel no.14 (see Figure S1), had a separate pressure regulator.We used 10 cmlong HPLC PEEK tubing in order to extend the steel capillary connecting the inlet of this auxiliary channel with the valve.As a result, the resistance of the flow in channel no.14 was much greater than for the other inlets.In this way, the reduced fluctuations of oil facilitated the process of separation of droplets during their automated exchange in screening experiments.
During the experiments of controlled separation of droplets we used a syringe pump (Nemesys, Cetoni) in order to apply a flow of oil at a defined rate through the channel no.14.

Screening experiment
During the screening experiment the routing of droplets on chip was automated.Prior to the testing of various concentrations of γ-cyclodextrin, we introduced a sequence of inhibitors onto the chip and deposited them in a storage channel.A solution of α-hemolysin was introduced onto the chip from a syringe and a droplet of ~300 nL was generated and locked in a trap on an electrode.Then, we performed the screening experiment according to the following protocol: Step 1. Introduction of droplet with inhibitor into the trap: the precise positioning of a droplet containing inhibitor was aided with an edge-detection algorithm.We used a camera operated on-line by an image processing software that controlled the valves depending on the detected image.The edge of the droplet before entering the trap and then inside of the trap was detected.This was correlated with the opening and closing of appropriate valves. 1 Step 2. Formation of bilayer and voltage-clamp measurement (in total 180 s): in this step the protein was incorporated into the bilayer and inhibitor binding measured.
Step 3. Removal of droplet with inhibitor and washing of the electrode: the droplets were separated by the short (1 s) application of the flow of oil through the auxiliary channel no.14 .
Then, a droplet of pure buffer of ~350 nL was generated.The electrode was flushed with the wash droplet, which was not stopped in a trap, however its appearance was detected with edge-detection optical feedback.The script was not continued unless a droplet passed through the trap.
The 3 steps were repeated 12 times, giving 2 full experiments in which 6 concentrations of inhibitor were screened.Next, we introduced a fresh solution of protein.
The voltage of + 50 mV was constantly applied.

Estimation of the Bilayer Size
To estimate the bilayer size, we carried out measurements of capacitive current for pairs of droplets of different volumes (in the range of 250-350 nL).Droplets were generated by aspiring the buffer from a 96-well plate with the aid of a precise pump (Nemesys, Cetoni) and a positioning system (Rotaxys, Cetoni).For each volume we analyzed 5 pairs of droplets.For each pair a triangular potential (10 Hz, 50 mV peak-to-peak) was applied and the resulting square-wave current was recorded at a 1 kHz sampling rate.A model cell with a known capacitance of 100 pF (MCB-1U, Molecular Devices) was used to determine dV/dt (= 1).We collected and analyzed traces of about 60 s.We calculated the average level and standard deviation of the capacitance (table S1).By using the specific capacitance of a DPhPC bilayer formed in pure hexadecane 0.64 µF cm -2 , 2 we estimated the contact area between the droplets.
We distinguish two sources of variation -one refers to time dependent fluctuations in the area of an individual bilayer, caused by flows and convection in both phases.There is also variation between pairs of droplets, which may be the result of different positioning of each pair of droplets in the trap and variation of droplet volume.The relative standard deviation (RSD) for the bilayer area is in the range of ~5-10% in the case of smaller volumes (250 and 275 nL) and less than 5% for larger droplets.Smaller droplets, even if not positioned exactly in space by the confinement provided by the traps, are attracted to each other during the formation of a bilayer.Nonetheless, the position of smaller droplets in the trap is constrained to a lesser extent than for largest drops.The obtained value of K d (61 ± 7 µM) is comparable to the value of 47 ± 9 µM, which was calculated by Mantri et al. 3 for αHL heptamer at -50 mV (buffer conditions 1M KCl, 25mM Tris HCl and 50 mM EDTA, pH 8).In the aforementioned work the protein was added to the ground electrode compartment.In our set-up the protein is contained within compartment with working electrode.Therefore, due to the phenomenon of rectification of current, we compared our value of K d (obtained at + 50 mV) to the one obtained at -50 mV.

Image processing
The analysis of electric current traces was performed in Clampfit 10.3.All illustrations were prepared using Inkscape 0.48 software.

Figure S1 .
Figure S1.Detailed diagram of the layout of the microfluidic chip.The numbers indicate: 1 -

Figure S5 .
Figure S5.Electrophysiological measurements showing the blocking events and the

Figure S7 .
Figure S7.Rapid screening of inhibitors against single α-hemolysin pores.Continuous trace of current shows subsequent incorporation of HL pores in bilayers formed between a droplet

Figure S8 .
Figure S8.Measurements of capacitive current during separation of droplets.The insets show in detail three different stages of separation.Before the application of oil stream the amplitude of current is steady.The flow of oil from the channel no.19 (30 nL s -1 , beginning of operation indicated with a vertical dotted red line) causes the gradual decrease in bilayer area and in capacitive current.Finally, the droplets become completely separated -this moment is indicated with an arrow.

Figure S9 .
Figure S9.The separation of droplets during the voltage-clamp measurement of αHL incorporation.We used a high concentration of protein sample in one of the droplets and observed multiple insertions of pore molecules into the membrane.We applied the flow of oil from the channel no.19 which resulted in complete separation of droplets, up to the moment of de-attachment.In a voltage-clamp measurement, the separation is seen as a sudden drop of current to the base level.

Table S1 .
Results of measurements of capacitance and calculations of sizes of bilayers formed between droplets of various volumes.

Table S2 .
Duration of single-channel recordings in screening experiments.The table collects times (in seconds) between the insertion of the first and the second nanopore.In some cases, single-channel recording was terminated by exchange of the droplet, according to the described protocol."x" indicates experiments in which no channel incoroporated into the bilayer.