**3. Electrical and electrochemical methods**

Detection and analysis of droplets using electrical or electrochemical transduction mechanisms have the potential to reach speeds difficult to achieve by conventional optical means. Furthermore, implementing miniaturized transducers that can be applied in low-cost portable devices such as the lab-on-a-chip approach can be more easily implemented by getting rid of the optical elements. The nature of the detection is label-free, as compared to certain optical techniques like fluorescence, which also suffers from photobleaching. Performing multiple parallel measurements by using various sensors in a single chip is also enabled more easily. **Figure 5** shows examples on their positioning, either as sensors at the bottom of the channel or parallel plates to surround the droplets, or some examples of the types of transducers that can be used, from simple planar or parallel electrodes to field-effect transistors (FETs), resistors, etc.

As shown by early demonstrations, access to basic information such as presence, size and ionic concentration [34] is possible through the direct contact of conductive droplets with coplanar electrodes at the bottom surface of the microfluidic channel, providing a signal in opposition to the insulating nature of the carrier phase, either air

#### **Figure 5.**

*Electrical transducers in droplet microfluidics: (a) examples of their integration in the bottom of the channel or as parallel plates, including narrowing the channel for increased sensor coverage, and (b) examples of transducer types.*

[35] or oil [36]. Size and velocity can be deduced measuring the time taken to travel between or through electrodes and the dimensions and distance of these electrodes. Differences in thermal conductivity between the two phases can also provide the information at the cost of a more difficult fabrication approach requiring resistive serpentine resistors [34]. Although some of such early works could implement the droplet sensing systems in microdevices where complex biological experiments were carried out (*e.g.* yeast cell electroporation [36] or DNA amplification through PCR [37]), the analysis of the biological and biochemical events occurring inside the droplets was still done via traditional microscopy or cameras. The sensors could for example be used to indicate the location of the droplets, and an automated position control generated pneumatic pulses to prevent the escape of the droplets from the reaction area. Such work reported that biological material can be lost through such direct contact with the sensor area, which could cause cross-contamination as well as decrease in the yield of the reactions.

The passing of droplets nearby electrical sensors resembles to some extent the impedance flow cytometry, where cells change the resistive and capacitive components of the signal [38]. Inspired by such technology, variations in the different components of the impedance can be used as source of information for droplet analysis. Another early demonstration [39] reported a capacitive detection system that could analyze droplet composition depending on its dielectric constant, in addition to droplet size and speed. Here, the detection system was coupled to a feedback loop for droplet sorting up to 10 kHz. This detection rate was not limited by the measurement speed, but by the upper pressure limit to fabricate the droplets without leading to leakage at the inlets ports. The capacitance of parallel electrodes on the side walls of microfluidic channels changed with the passing of the droplets due to the difference in the dielectric constant between droplets and carrier liquid. Such difference is a requirement for this type of sensing, which is the case for droplet microfluidic applications consisting of water-in-oil emulsions. In order to fabricate sidewall electrodes, a multistage photolithographic process was necessary where one of the steps involved filling photoresist cavities with a PDMS gel containing silver micro/nanoparticles to form the electrodes. The final microfluidic chip was integrated in a custom made circuit board containing an ac waveform generator, an L-C resonance circuit, an amplifier, an ac-dc converter and a comparator to generate the feedback control for the droplet sorting. When droplets have a correctly developed symmetric geometry, the sizing and speed calculation is straightforward with one simple pair of electrodes by evaluating the time taken to reach a plateau in the signal and the duration of the plateau. Since the difference between long and short droplets is mostly the signal amplitude, the size and speed can also be measured by evaluating the time taken to pass from one pair of electrodes to the next pair. Similar results can be obtained if a single electrode pair with fork shape is used (**Figure 6a.i**). Here, the plateau shows a central peak for large droplets and a dip for the small ones, helping to determine size and velocity. Since the principle of the technique is the difference in dielectric constant between the carrier fluid and the droplets, the chemical composition will also lead to signal variations, as shown by the authors using water and ethylene glycol droplets as proof-of-principle (**Figure 6a.ii**). In their device, the authors used a first pair of electrodes to determine velocity, size and content, while further pairs with different polarization (determined by the output signal of the first pair) attracted or repelled droplets to the different branches in the microfluidic channel layout. The required minimum effective electric field was given by the hydrodynamic flow resistance, while for too high electric fields the water droplets were stretched into satellite droplets.

*Real-Time Tracking of Individual Droplets in Multiphase Microfluidics DOI: http://dx.doi.org/10.5772/intechopen.106796*

#### **Figure 6.**

*Application examples of electrical droplet sensing: (a) type of information to target: i. size and velocity, ii. Composition of the liquid forming the droplet, iii. Presence of entities in the droplet (cells, particles, etc.). (b) Type of content to measure depending on transduction mechanism: i. redox reactions with electrochemical sensors, ii. pH or ions with FETs, iii. Cells with impedance sensors. (Adapted with permission from ref. Niu et al. [39]; Kemna et al. [40]; Schutt et al. [41]; Han et al. [42] and Spencer et al. [43].)*

Although parallel electrodes are expected to have better sensitivity, capacitive sensors based on coplanar electrodes can perform the task as seen before. Their fabrication is simpler, faster, and they are already commercially available. A previously reported work demonstrated the implementation of commercial interdigitated electrodes with an analytical model to consider the effect of the passivation layer that prevents the crosscontamination issue through the direct contact between droplet and electrodes [44].

By a more complex analysis of the signal which includes not only capacitance, but the whole impedance signal including the resistive component as well, deeper insights on the droplet interior can be obtained beyond size, velocity and composition of the liquid forming the droplet. If the electrical detection of droplets functions similarly to impedance flow cytometry for cell detection and analysis, the presence of cells in droplets should also be feasible (**Figure 6a.iii** and **b.iii**). This application was demonstrated for the first time using planar electrode pairs functioning at 100 kHz for high speed detection. The authors used the technique to discriminate droplets containing viable mouse myeloma cells from non-viable ones. The 100% yield of viable cell-containing droplet generation is not possible if a fast detection technique is not used to make the sorting possible. They showed that empty droplets with low conductive medium could not be distinguished from those containing non-viable cells, due to the resistive effect of their membrane. On the contrary, droplets containing viable cells resulted in lower impedance signals due to the presence of the conductive cytoplasm. As aforementioned for the capacitance sensors, when a droplet appears the capacitance values change. In addition, the resistance is also different for the oil and buffer. Additional equivalent circuit elements appear with the presence of cells, which include the capacitance of the membrane and resistance of the cell interior. Under the megahertz frequencies, the membrane of the cells usually imposes a barrier to the current flow and the change in the impedance signal will be dependent on cell size [45]. However, if a low conductivity buffer is used, the current can flow through the cell at lower frequencies [46].

Considering that at low frequencies the double layer capacitance dominate, the measurements should then be made at intermediate frequencies where the resistance due to liquid composition (including cytoplasm) play a role. The authors could demonstrate as well that the electric field did not negatively affect cell viability.

Certain biological and chemical processes (*e.g.* enzymatic reactions) present rapid kinetics in the milliseconds range and with especial prominence in their very first moments. To achieve maximum detail in the kinetics, fast detection techniques are a must here. In this context, chronoamperometry can be a faster alternative compared to impedance, requiring as well a simpler operation and readout setup [47]. The signal in amperometric sensors is generated by the exchange of electrons between the electrode and the analyte or the receptor at the electrode surface interacting with the analyte. The analyte is involved in a redox reaction, changing its oxidation state and producing a measurable electron flux, proportional to the amount of species transformed [48]. The targeted type of samples is therefore limited to electroactive species. An additional limitation comes from the contact time between electrodes and droplets. When this time is too short, the faradaic current originated from the redox reaction is convoluted with a large capacitive current [49]. To ensure enough contact time, large droplets or fluid plugs can be generated (therefore decreasing the throughput), or the microchannel section can be narrowed down at the area where the electrodes are located, stretching the droplets and improving as well the intradroplet mass transfer characteristics [50]. Another solution was found by enhancing the wettability of the detection area to convert the droplet flow into a vertical laminar flow, where the oil phase flows on top of a continuous aqueous stream only during the measurement [51]. The chronoamperometric technique has been used for example for the measurement of the Michaelis–Menten kinetics of the hydrogen peroxide decomposition by the enzyme catalase [42]. Here, the route of the droplets was controlled by pneumatic valves, from short to long, allowing to measure at different time intervals of the reaction. The H2O2 was oxidized at the surface of platinum electrodes polarized at 600 mV, decomposing into protons, electrons and oxygen. The diffusion was not a limiting factor, as observed by an independence of the current peak from the flow rate. The current amplitude depended from the concentration of the analyte. A time resolution of 0.05 s was achieved, consuming less than 50 μL (**Figure 6b.i**). The amperometry technique combined with microfluidics allows to get deeper insights into enzyme analysis such as inhibition assays, which would otherwise require time consuming serial dilution experiments involving 1000-fold larger reagent volume. The microfluidic channel can be modified to perform additional tasks such as the generation of concentration gradients in order to analyze the dose–response assay with enzymes and inhibitors at different concentrations, as shown with acetylcholinesterase and various drugs (pesticides and therapeutic drugs for Alzheimer's disease) [52]. Here, the measurement principle consisted on the oxidation of thiocholine as product of the enzymatic activity. The authors suggested that the presence of a surfactant as droplet stabilizer can be an important factor to consider. Surfactants can cause protein adsorption at the interface between oil and droplet, decreasing the efficiency of the reactions. The droplets can remain stable for several minutes, which can be enough for enzymatic activity assays, but the presence of a surfactant is necessary in longer incubation periods, needing investigations in order to choose the most appropriate one. The authors could determine the half maximal inhibitory concentration (IC50) in the μM concentration range during a final assay time of only 6 minutes. When nanomaterials are part of the electrode, the signal has been observed to enhance at least an order of magnitude [53, 54].

#### *Real-Time Tracking of Individual Droplets in Multiphase Microfluidics DOI: http://dx.doi.org/10.5772/intechopen.106796*

Potentiometry can also be considered as a fast technique offering time resolutions comparable to those obtained by amperometry. Certain works report the use of lightaddressable potentiometric sensors, where a light source excites and generates carriers on a semiconductor device, producing a potential that will be modified by the presence of charged species at the surface [55, 56]. However, in this section we will focus on the optics-less systems which get rid of the optical elements for an easier miniaturization. Potentiometric sensors that have been used for droplet detection can be mainly categorized into the next two types: ion-selective electrodes and ion-sensitive field-effect transistors (FETs). The fabrication process and the setup used in the first type can be as simple as for the amperometric sensors, requiring just a simple set of electrodes with the necessary surface modification to make them selective towards the ion of interest [57]. The measurements here are done at zero current condition (open circuit), by comparing the potential of the working electrode with a reference electrode and quantifying its changes with the presence of the target ions. A reported example made use of platinum electrodes modified with Mg2+ ionophores to study RNA–Mg2+ binding kinetics by measuring the concentration of magnesium ions [58]. A similar set of pneumatic valves comparable to those mentioned in the amperometric approach was used to study the reaction at different time points, with a time resolution in the milliseconds range and utilizing less than 20 μL for a single experiment.

The second potentiometric sensor type (FETs) consists of semiconductor channels whose switching voltage depends on the content of the surrounding ionic species, providing a highly sensitive way to measure surface potential changes [59]. Analysis of droplets with FETs (**Figure 6b.ii**) was proposed for the first time in 2016 using silicon nanowires as semiconductor channel [41]. Silicon nanowires are excellent candidates for sensing in microfluidics, with ultrasensitivity and CMOS compatibility. First, the authors probed the content of all droplets up to 10 Hz resolving pH and ionic strength values through measured variations of the current through the nanowires. They observed that droplets required a minimum length equal to the linear dimensions of the sensor. The gate potential was influenced by the oil–water interface using short droplets, which could be useful to detect interfacial charges. As proof-of-concept of a biological assay, the activity of the glucose oxidase enzyme was monitored by measuring the produced acidification. The enzyme activity was monitored in parallel with an integrated optical setup with fiber optics and a portable spectrophotometer, providing such dual detection for the first time. For this, the enzyme reaction was coupled to a second one consisting of horseradish peroxidase which reduced the produced hydrogen peroxide while oxidizing the liquid colorimetric substrate 3,3′, 5,5′-tetramethylbenzidine. In a following work, the same group demonstrated that the pH change is not a requirement for the monitoring of enzymatic reactions in droplets [60]. Here, they encapsulated β-galactosidase, whose activity could be monitored due to the different ionic content after the reaction. Some authors observed that extended exposure of basic pH could degrade the sensitivity of droplet sensing with FETs due to degradation of the gate oxide [61], which would require further investigation of more suitable materials.
