Another major domain of microdroplet detection techniques is electrical detection amongst which impedimetric (Figure 5
a) and electrochemical (Figure 5
b) detection are the most widely used methods. Electrical detection of droplets can be both contact and non-contact based. Analytical parameters such as limit of detection, sensitivity and dynamic range are primarily affected by signal-to-noise ratio and sampling rate, which depend on the detection instrument. Hence, it is difficult to give overarching values for the performance of electrical droplet sensing studies. Each work should be evaluated individually taking into account the specific type of electrical detection method used, the type of instrument and the choice of electrodes.
5.1. Impedimetric Sensing
Chen and Troian et al. performed one of the most fundamental studies for capacitive detection of microdroplets using a digital droplet system that uses thermocapillary droplet actuation [66
]. They used coplanar electrodes for impedimetric sensing (Figure 5
a) that are originally used as resistive heaters to generate the thermocapillary movement of the droplets for electrical sensing. They provided the analytical model for the semicircular electric field generated by coplanar thin film electrodes and verified it both numerically and experimentally. They also showed droplet sensing results for symmetric two-prong electrodes and interdigitated electrodes. They demonstrated preliminary results of sensing of droplet position, volume, and content setting the groundwork for the upcoming studies.
In order to increase the sensitivity, Srivastava and Burns used conductivity sensing with electrodes that are in contact with the solution [67
]. In addition to detecting the presence of the electrodes, they placed the electrodes in the most common configuration which is fabricated underneath the microchannel and placed perpendicularly to the channel. They showed a second configuration where the two electrodes were placed parallel to the channel to detect the volume of the droplet by measuring the conductivity change between the two electrodes that are placed at the two sides of the microchannel.
Building on Chen’s work, Elbuken and Ren et al. developed a high-sensitivity droplet capacitive system using off-the-shelf electronic components significantly reducing the cost and entry barrier for the implementation of capacitive detection [68
]. They showed that droplet size and speed can be detected by using an electronic circuit that costs less than $
20. Additionally, the sensing unit was very small and can be applied to portable applications. Isgor and Elbuken et al. showed that using the same electrical circuitry droplet, content can also be measured [69
]. The change in the dielectric content of the droplet, due to the mixing of ethanol and water at varying ratios, can be detected when a nanometer-thick SiO2
layer is used as a passivation layer between the droplets and the planar electrodes.
Kemna and van den Berg et al. reported the impedimetric detection of viable single cells inside segmented droplets at moderate throughputs (112 Hz) [70
]. They have modeled the system using lumped electric circuit models with an analogy to electrical circuits. The optimal frequency and electrical properties of materials can be determined by such a model. They showed that viable mouse myeloma cells present in water droplets yield a detectable signal using a high-end impedance spectrometer.
Simon and Lee et al. showed an interesting application of impedimetric detection by detecting polymerized DNA chains inside segmented droplets [71
]. Using a microfluidic system and a detection circuitry similar to the previous study, they combined on-chip thermocycling to demonstrate the impedance signal difference between droplets with amplified and non-amplified DNA content. Although, the measurement is susceptive to slight changes in droplet size, once perfected it can be used for digital droplet PCR to alleviate the need for the fluorescent tagging [72
Marcali and Elbuken showed impedimetric detection of hemagglutination inside segmented microdroplets [73
]. As a model assay, they used blood typing to form agglutination positive and negative droplets using a microfluidic chip as schematically shown in Figure 6
a. Using narrow electrodes placed perpendicular to the microchannel, they created a minimized detection zone to obtain a steady-state reading from droplets. Using the impedance data, they were able to differentiate between droplets of different content as well as droplets with agglutinated red blood cells.
Impedimetric droplet sensing was implemented on other droplet fluidic platforms, as well. Digital droplet systems already integrate coplanar electrodes with fluidics; hence, such an implementation is relatively easy for these systems due to minimal hardware changes required. Sadeghi and van Dam et al. modified a digital droplet system by simply adding a resistor to the AC actuation circuitry [74
]. They showed that they can detect droplet volume, droplet type (DMSO or water) and droplet content (concentration of potassium fluoride) without sacrificing the droplet actuation and droplet handing performance of the digital droplet platform. Shih and Wheeler et al. showed the very first implementation of detection of mammalian cells inside droplets on a digital droplet system [75
]. Instead of turning all actuation cells into sensors, they placed dedicated detection electrodes in between the actuation electrodes. Also, in order to obtain a statistically significant data to distinguish droplets with varying cell concentrations, they used a buffer exchange method. Just before the impedimetric analysis, they changed the cell buffer solution inside the droplet with low conductivity solution that was replaced by the original solution after the measurement.
Ernst et al. showed an integrated capacitive droplet measurement system for a dispensing droplet platform [76
]. The sensor was very well integrated into the print head. Sensing of droplet volume was achieved by 3D electrodes that are placed through a custom designed printed circuit board (PCB). The electronics are also placed on the same PCB minimizing the signal loss due to electrical contacts. The fabrication of the sensor was achieved with a little tweak to the standard PCB fabrication process. The droplets were dispensed through a via which is used for inter-layer connections in PCB designs. After PCB fabrication, the via is sliced into two, forming symmetrical semi-cylindrical electrodes creating an axially symmetric 3D electric field. The droplets were dispensed through the center of the via and real-time droplet measurement was achieved. The system was not tested for any analyte sensing. It was mostly designed for droplet size control for bioprinting applications, but conceptually it can be applied to detect the content of droplets as well.
Impedance detection of droplets was also implemented on stationary droplets on a dispensed droplet detection system. Ebrahimi and Alam et al. showed a system which is composed of an array of nanostructured hydrophobically coated electrodes [22
]. The sample was dispensed on the electrodes and real-time impedance measurement was obtained from stationary droplets. A very nice feature of this work was that they utilized the concept of evaporation induced concentration increase. Evaporation of droplets causes a decrease in droplet volume that in turn increases the analyte concentration beating the diffusion transport limit. The evaporation time was around 20 min when 3 µL droplets were used; 850 bp synthetic DNA fragments were used for the experiments. Using a lumped circuit model, they modeled the electrical circuit interpretation of DNA concentration increase during the experiment. They showed that using evaporation induced sample enrichment, an order of magnitude improvement can be obtained in the limit of detection. This enrichment concept has been applied to many other systems and is proven to be a very effective method when the experiment time and instability of volume are not concerns for the assay. The enrichment technique is especially well suited for biological applications that require cell culturing which inherently takes time so that the waiting time for droplet evaporation is not a concern. Such an exemplary application was shown by Ebrahimi and Alam in 2016 [26
]. They showed that using the droplet impedance monitoring system, the osmotic response of bacterial cells to varying medium conditions can be measured as an alternative method to determine cell viability. The osmoregularity response of bacteria (E. coli
, S. epidermidis
, and S. typhimurium
) were generated by evaporation of the droplet while monitoring the impedance change and deriving the electrical properties of the solution and cells. For dead cells, the cell membrane was compromised and solutes were free to transport across the membrane whereas in viable cultures active osmotic response of cells modulated the solution conductivity which was continuously monitored with the integrated electrodes.
5.2. Electrochemical Sensing
Electrical sensing of droplets was also shown using electrochemical sensing (Figure 5
b). Electrochemical sensing is closely related to impedimetric change and can be confused with impedimetric studies. In this article, we discuss electrochemical sensing articles that utilize electroactive species [77
]. There are three main types of electrochemical detection: amperometric, potentiometric and conductometric. As the name implies, the electrical property that is monitored is different for these techniques.
An early work on electrochemical sensing of microdroplets was carried out by Cai and Cooper et al. [78
]. They showed a microchamber with two integrated electrodes (counter and reference electrodes were combined) for amperometric analysis of lactate. The sample solution was pipetted into the chamber that is covered by oil to prevent evaporation. Measurements were performed with droplets containing lactate, either spiked or generated as a metabolite by individual heart cells inside droplets. The study demonstrated that lactate can be detected at very low volumes by miniaturizing the electrodes and the working sample.
Label-free electrochemical detection of droplets was mostly used for segmented droplet systems. A major challenge in such systems is to have a nice contact between the liquid sample, i.e., the droplet, and the measurement electrodes. The early examples of such systems used electrodes that are pierced into the microchannel that probe the droplet that is aligned with the electrodes. Han and Zheng et al. [79
] and Gu and Ding et al. [80
] used such an approach to integrate amperometric sensing with PDMS microfluidic segmented droplet devices using platinum and gold electrodes, respectively. Han et al. used guidance microchannels to facilitate the insertion of the electrodes into the microchannel.
Sassa and Suzuki et al. showed a segmented flow system for coulometric detection inside droplets by squeezing the droplets into the sensing region for better contact [81
]. Liquid droplets of discrete volumes were formed using syringe pumps and an auxiliary side channel inside microchannels. The droplets were transferred to the sensing region that has a shallower channel section to allow for better positioning of the sample over the electrochemical sensing region. Different types of sensor geometries were fabricated and coulombic detection of hydrogen peroxide was shown for analysis of oxidase substrates. The working electrodes designed as an array of thin electrodes gave the best results in terms of sensitivity.
Lin and Chen et al. used a very novel approach to get around the contact problem [82
]. They used standard glass/PDMS devices and a T-junction geometry to form segmented droplets. In the sensing section, they applied selective surface coatings so that the two-phase flow is converted into a laminar flow where the aqueous phase gets into contact with the bottom surface of the chip with the oil phase contacting the upper channel. The electrodes were placed underneath the channel and made a strong contact with the droplets. As an example of enzymatic kinetic study, they studied oxidation of glucose inside droplets by hydrogen peroxide (H2
) measurement. By measuring the resultant H2
concentration for six different input glucose concentration levels, they were able to obtain the Lineweaver–Burke curve in 20 min.
Itoh and Suzuki et al. showed a very interesting application of amperometric detection using segmented flow droplets on a portable microfluidic device [83
]. They have implemented a system for measurement of ATP concentration in fish (jack mackerel) extracts for quantification of freshness. They used a glass/PDMS device and formed aqueous droplets separated by air. By avoiding the use of an oil-based continuous solution, they avoided the contact problem between the sample and the electrodes. However, this sacrificed the control of sample integrity which heavily depends on the hydrophobicity of the channels. The droplets containing the sample and the substrate solutions were formed separately and merged before the detection region using the commonly used enlarging channel geometry. Detection was achieved using a three-electrode sensor with the enzymes (glycerol kinase and glycerol-3-phosphate oxidase) immobilized on the sensor itself which contains a Nafion membrane for additional selectivity. The system measures the amount of H2
concentration formed at the end of a two-step enzymatic reaction. The turn-around time of the system (from obtaining the sample to result) is around 30 min. Their results show that fish start losing ATP and hence, freshness, significantly after 5 h of death even if they are refrigerated.
The researchers have extended their study by further developing this system by measuring the K-value which is a metric used for quantitative assessment of ATP breakdown to uric acid through the concentration of byproducts of a series reactions [84
]. They used two sensing sites to measure multiple reaction byproducts (Figure 6
b). The freshness of jack mackerel, yellowtail, and sea bream was measured by comparing the results with HPLC measurements. Although it depends on the type of the fish, 5 h after death can be generalized as a threshold value for fresh fish.
Rattanarat and Chailapakul et al. have demonstrated that sensitivity of label-free droplet electrochemical sensing can be improved by modification of electrodes [85
]. They used graphene/polyaniline nanocomposite to coat screen printed carbon paste electrodes to determine the amount of residual 4-aminophenol (4-AP) in commercial paracetamol formulations. The measurement potential voltage was optimized for chronoamperometric measurement. The system gave a very linear response to 4-AP concentration and was able to provide the required limit of detection determined by universal guidelines.