Device design and function

The device for the production, collection and long-time storage of aqueous mi-crofluidic drops is illustrated in Fig. 4.1. It is equipped with one oil inlet, another oil supply at the bottom that functions either as inlet or as outlet and six outlets (Fig. 4.1). Additionally, there are three aqueous inlets that jointly compose the dispersed phase of the emulsion. [118] Typical flow velocities during the usage of the device are listed in Tab. 4.1. For the three aqueous components, we choose multivalent salt solution on the left side (aq 1) and at the opposite side the pro-tein solution (aq 3) (Fig. 4.1b). In between, there is the inlet that contains only buffer (aq 2). This way, the first contact of multivalent salt and protein solution is retarded (Sec. 5.1). Due to the laminar flow regime, the three solutions flow side-by-side. When the aqueous fluid reaches the cross channel, it is focused by the oil phase and – as a result of the interplay between shear and interfacial forces – drops pinch off (Sec. 2.3.2). [7] A constant flow rate is applied for the protein

procedure u_{top oil} u_{bottom oil} u_{aq 1} u_{aq 2} u_{aq 3}

startup 23 +4 4.7 4.7 4.7

drop collection 23 -2 zig-zag: 0-9.4 9.4-u_{aq 1} 4.7

drop content imaging 0 0 0 0 0

Table 4.1: Flow velocities. Typical average ow velocitiesu in mm s⁻¹ applied during the dierent procedures of device operation. In the zig-zag pattern, u_{aq 1} is increased linearly from 0 to 9.4 mm s⁻¹ within 7.5 s, and subsequently, it is decreased again to 0 in the same time. The pattern is repeated leading to a periodicity of 15 s. Details on the zig-zag ow velocities are further discussed in Sec. 6.1.

Device C: Concentration dependence 4.1

Figure 4.1: Concept of the device C. The aim is to image vimentin protein laments at dierent magnesium concentrations. For this purpose, the device consists of several modules for uid manipulation. In the drop composer (b), three aqueous uids (aq 1, aq 2, aq 3) join up shortly before they are encapsulated into drops by focusing them with a lateral oil ow. [7,118] c) A zig-zag ow prole is applied for aq 1 and aq 2. d) The drops pass a serpentine channel, to ensure fast mixing of the drop content. [27] e) In a drop basin, the drops are densied and excess drops are rejected laterally. The bottom of the basin leads to a `collecting channel'. At the beginning of the experiments, there is an oil inow from this channel into the basin. Consequently, no drops enter. Drop collection is initiated when this oil ow is inverted (Fig. 4.2). Drops are collected for about one minute into a delay line (f), which ensures smooth drop collection. From the delay line, the drops are conducted to the `drop storage' (g). In this storage, there are 500 constrictions in the channel walls. [109]

When the overall ow in the device is stopped, these constrictions immobilize the drops.

The content of the drops is then imaged with uorescence microscopy. (Adapted from reference [36] with permission from AIP Biomicrouidics)

4 MICROFLUIDIC DEVICES FOR PROTEIN STUDIES

containing fluid (aq 3). The flow rates of the other two components follow a pe-riodic zig-zag course (Fig. 4.1c) and their flow rates taken together are constant.

This way monodisperse drops are produced. At the same time, the concentration of the multivalent salt is changed from drop to drop in a defined manner. The drops pass an approximately 3 mm long serpentine channel (Fig. 4.1d). During this passage they undergo strong internal mixing by means of chaotic advection (Sec. 2.3.3). [27]

The drops are produced at a rate of about 400 Hz and mixed directly afterwards.

The microfluidic system has to be considered as an entity of the syringe pump, the tubing and the channels. For the zig-zag flow rate profiles we have to choose a minimum periodicity of about 15 s, so that the microfluidic system has enough time to respond to the set flow rate profiles. In one period of 15 s, 6000 drops are produced. This number is too large to observe the content in all of these drops with the method we use (see below). Therefore, the number of drops is reduced as the drops are conducted into a region with 18 lateral channels and one central channel (Fig. 4.1e). Most of the drops are rejected in this ‘basin’ as they flow into one of the lateral channels, which lead to outlets. The most important channel is the channel at the very bottom of the basin. At the beginning of an experiment, we apply an oil inflow to this channel (Fig. 4.2a). As a result, no drops can

en-a) b) c)

d)

1^st drop

oil inflow oil outflow 40 µm

0 20 40 60 80

Figure 4.2: Drop collection process. The collection process of drops is initiated when the inow into the drop basin from the collecting channel (a) is inverted to an outow (b). After a short delay time the drops begin to enter the collecting channel. This process is recorded in detail with brighteld microscopy. (b and c). The details of the collection process are important for calculating the composition of each collected drop later on. d) For this purpose, the number of collected drops as a function of time is analyzed (Sec. 6.1).

Device C: Concentration dependence 4.1

ter. A drop ‘collection process’ is initiated as the oil inflow is changed to negative values. Then the drops are collected for about one minute (Fig. 4.2b/c) and enter a straight channel, which is used as a delay line (Fig. 4.1f). Its straight channel walls (compared to the drop storage region, see below) allows for a smooth drop collection. The drops enter collection channel at a rate of 5 Hz. Therefore about 1%of the initially produced drops is collected. Since there are at maximum three drops in parallel in the basin, the chronological drop order is preserved to a great extent in the collection process. [34]

The drop collection process is recorded in brightfield microscopy (Fig. 4.2b-d).

This image series is used to determine the exact drop composition for each inves-tigated drop (Sec. 6.1). When enough drops have been collected into the delay line, the oil syringe at the bottom is controlled by hand and the drops are con-ducted into a channel with constrictions at the channel walls (Fig. 4.1g and 4.3) giving the ability to trap 500 drops at maximum (see Sec. 2.3.2). [109]. All tubings are then cut-off, which stops the flow all over the device. The drop content can be imaged for a few hours, due to the water saturation of the PDMS device (Sec.

3.2.2 and 5.2).

With this procedure not all of the positions are occupied by the drops. For the experiments with vimentin we have about half the positions in the drop storage occupied (Fig. 4.3b). When the drops are stored in the constrictions, up to eight drops are imaged in parallel. All drops are recorded, as we take the images con-secutively (Fig. 4.3b). This procedure takes about 1-2 h time when the whole storage region is imaged. Orientation marks next to the channel walls (Fig. 4.1f) have proven necessary during image acquisition and data analysis. [34]