Karan V. I. S. Kaler

Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR) is facilitated by leveraging droplet microfluidic (DMF) system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points). This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs). The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind) panels of extracted patient samples with acceptably high PCR efficiency (>94%). The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.

Droplet microfluidics (DMF) is a fluidic handling technology that enables precision control over dispensing and subsequent manipulation of droplets in the volume range of microliters to picoliters, on a micro-fabricated device. There are several different droplet actuation methods, all of which can generate external stimuli, to either actively or passively control the shape and positioning of fluidic droplets over patterned substrates. In this review article, we focus on the operation and utility of electro-actuation-based DMF devices, which utilize one or more micro-/nano-patterned substrates to facilitate electric field-based handling of chemical and/or biological samples. The underlying theory of DMF actuations, device fabrication methods and integration of optical and opto-electronic detectors is discussed in this review. Example applications of such electro-actuation-based DMF devices have also been included, illustrating the various actuation methods and their utility in conducting chip-based laboratory and clinical diagnostic assays.

Miniaturized bio-diagnostic devices have the potential to allow for rapid pathogen screening in clinical patient samples, as a low cost and portable alternative to conventional bench-top equipment. Miniaturization of key bio-diagnostic techniques, such as: nucleic acid detection and quantification, polymerase chain reaction (PCR), DNA fingerprinting, enzyme linked immunosorbent assay (ELISA), results in substantial reduction of reaction volumes (expensive samples/reagents) and shorter reaction times. Droplet microfluidics (DMF) is one of several miniaturized bio-sample handling techniques available for manipulating clinical samples and reagents in microliter (10⁻⁶ L) to picoliter (10⁻¹² L) volume regime. Electro-actuation of sample and reagent in the form of droplets in the aforementioned volume regime, using dielectrophoresis (DEP) and/or Electrowetting (EW) are achieved by means of patterned, insulated metal electrodes on one or more substrates. In this work, we have utilized electro-actuation based DMF technology, integrated with suitably tailored resistive micro-heaters and temperature sensors, to achieve chip based real-time, quantitative PCR (qRT-PCR). This qRT-PCR micro-device was utilized to detect and quantify the presence of influenza A and C virus nucleic acids, using in-vitro synthesized viral RNA segments. The experimental analysis of the DMF micro-device confirms its capabilities in qRT-PCR based detection and quantification of pathogen samples, with accuracy levels comparable to established commercial bench-top equipment (PCR efficiency ∼95%). The limit of detection (LOD) of the chip based qRT-PCR technique was estimated to be ∼5 copies of template RNA per PCR reaction.

Droplet based microfluidic systems, in recent years, have demonstrated numerous advantages and exciting potential for Lab-On-a Chip applications. In order to fully realize the potential benefits of this technology, one requires precision dispensing and manipulation of droplets of known volume and sample concentration, in a rapid and controlled manner. In this article, we demonstrate the rapid and controlled microactuation of aqueous samples and subsequent dispensing of variable volume droplets in nanolitre to picolitre regime by using a coplanar tapered electrode structure that leverages the phenomena of liquid dielectrophoresis (L-DEP). The transient behavior of the tapered liquid jet departs significantly from that of a uniform liquid jet case and is not adequately explained in terms of a simplified lumped capacitance model as in the case of the uniform jet, during the L-DEP actuation. A more generalized numerical model is developed for the tapered actuation scheme to account for the experimental observations. We furthermore demonstrate that the density of the dispensed droplets can be proactively controlled by the judicious placement of electrode bumps and pinches in the electrode structure thus overcoming the limitations imposed by Rayleigh's instability criterion. The proposed droplet dispensing schemes are superior to existing L-DEP based dispensing schemes which are restricted in size and spacing of the dispensed droplets by Rayleigh's instability criteria and furthermore mostly restricted to equi-volume droplets.

Liquid dielectrophoresis (L-DEP), when deployed at microscopic scales on top of hydrophobic surfaces, offers novel ways of rapid and automated manipulation of very small amounts of polar aqueous samples for microfluidic applications and development of laboratory-on-a-chip devices. In this article we highlight some of the more recent developments and applications of L-DEP in handling and processing of various types of aqueous samples and reagents of biological relevance including emulsions using such microchip based surface microfluidic (SMF) devices. We highlighted the utility of these devices for on-chip bioassays including nucleic acid analysis. Furthermore, the parallel sample processing capabilities of these SMF devices together with suitable on- or off-chip detection capabilities suggest numerous applications and utility in conducting automated multiplexed assays, a capability much sought after in the high throughput diagnostic and screening assays.

Liquid Dielectrophoresis (L-DEP) has been successfully leveraged at microscopic scales and shown to provide a controllable means of on-chip precision dispensing and manipulation of sub-nanoliter single emulsion droplets. In this paper, we report on the dynamics of a DEP actuated emulsion jet prior to break-up and compare its characteristic behavior based on the lumped parameter model of Jones et al. (R. Ahmed and T. B. Jones, J. Micromech. Microeng., 2007, 17, 1052). Furthermore, features and aspects of these emulsion jets, their break-up and formation of sub-nanoliter emulsion droplets is studied in further detail. Applications of the proposed scheme in dispensing encapsulated sub-nanoliter droplets is envisioned in various fields including microTAS, on-chip handling and storage of cells and other biological samples for longer duration in controlled environments as well as solving the more general encapsulation issues in surface microfluidic devices. Scalability of the proposed scheme is shown by producing controlled sample–oil single emulsion droplets (aqueous samples in oil) in the range of 50–400 picoliters.