Rohit Mishra

CD-Based Microfluidics for Primary Care in Extreme Point-of-Care Settings

We review the utility of centrifugal microfluidic technologies applied to point-of-care diagnosis in extremely under-resourced environments. The various challenges faced in these settings are showcased, using areas in India and Africa as examples. Measures for the ability of integrated devices to effectively address point-of-care challenges are highlighted, and centrifugal, often termed CD-based microfluidic technologies, technologies are presented as a promising platform to address these challenges. We describe the advantages of centrifugal liquid handling, as well as the ability of a standard CD player to perform a number of common laboratory tests, fulfilling the role of an integrated lab-on-a-CD. Innovative centrifugal approaches for point-of-care in extremely resource-poor settings are highlighted, including sensing and detection strategies, smart power sources and biomimetic inspiration for environmental control. The evolution of centrifugal microfluidics, along with examples of commercial and advanced prototype centrifugal microfluidic systems, is presented, illustrating the success of deployment at the point-of-care. A close fit of emerging centrifugal systems to address a critical panel of tests for under-resourced clinic settings, formulated by medical experts, is demonstrated. This emphasizes the potential of centrifugal microfluidic technologies to be applied effectively to extremely challenging point-of-care scenarios and in playing a role in improving primary care in resource-limited settings across the developing world.

Lipophilic-membrane based routing for centrifugal automation of heterogeneous immunoassays

We demonstrate centrifugal [liquid handling] automation of an Enzyme-Linked Immuno-Sorbent Assay (ELISA) for the detection of anti-p53 antibodies in whole blood. On this “Lab-on-a-Disc” (LoaD) platform, all unit operations were implemented by event-triggered rotational flow control. In order to avoid interference during absorbance measurement from the solid phase in this heterogeneous assay format, it is pivotal that the intermediate reaction product is eventually forwarded from the incubation chamber to a distinct optical measurement chamber. To this end we have devised routing of flows by lipophilic film valves (LFVs) which remain intact in aqueous and selectively dissolve when exposed to an ancillary, oleophilic solvent.

Wirelessly powered and remotely controlled valve-array for highly multiplexed analytical assay automation on a centrifugal microfluidic platform

In this paper we present a wirelessly powered array of 128 centrifugo-pneumatic valves that can be thermally actuated on demand during spinning. The valves can either be triggered by a predefined protocol, wireless signal transmission via Bluetooth, or in response to a sensor monitoring a parameter like the temperature, or homogeneity of the dispersion. Upon activation of a resistive heater, a low-melting membrane (Parafilm™) is removed to vent an entrapped gas pocket, thus letting the incoming liquid wet an intermediate dissolvable film and thereby open the valve. The proposed system allows up to 12 heaters to be activated in parallel, with a response time below 3 s, potentially resulting in 128 actuated valves in under 30 s. We demonstrate, with three examples of common and standard procedures, how the proposed technology could become a powerful tool for implementing diagnostic assays on Lab-on-a-Disc. First, we implement wireless actuation of 64 valves during rotation in a freely programmable sequence, or upon user input in real time. Then, we show a closed-loop centrifugal flow control sequence for which the state of mixing of reagents, evaluated from stroboscopically recorded images, triggers the opening of the valves. In our last experiment, valving and closed-loop control are used to facilitate centrifugal processing of whole blood.

Automated assembly of microfluidic "lab-on-a-disc"

Point-of-care (POC) testing attracts more and more attention in the medical health sector because of their specific property to perform the diagnostic close to the patient. The fast diagnosis right at the hospital or the doctor’s office improves the medical reaction time and the chances for a successful healing process. One of this POC test systems is a “Lab-on-a-Disc” (LoaD) which looks like a compact disc crisscrossed with microfluidic tubes and cavities. The fluid to be analysed is placed in the LoaD and an external device then rotates the LoaD. The cavities inside the LoaD and the centrifugal force ensure a clearly defined sequence of the analysis. Furthermore, we aim for an inexpensive manufacture of the medical product without neglecting its quality and functionality. Therefore, the Fraunhofer IPT works on an assembly cell to implement dissoluble films concisely into the disc. This dissoluble film demonstrates its successful usage as a gate for the fluid, which opens after a predefined moment in the cycle. Furthermore, we investigate to integrate a laser welding process into our gantry system and demonstrate its efficiency with the welding of polymer discs. This procedure is clinically safe because no further laser absorption material is needed in the sealing process, which might pollute the LoaD. Moreover, this process allows the alignment of several discs before the welding and therefore leads to precisely manufactured LoaDs in large quantities. All these methods together enable a fast, costefficient and reliable mass production to bring POC testing among the people.

Label-free, spatially multiplexed SPR detection of immunoassays on a highly integrated centrifugal Lab-on-a-Disc platform

As a direct, label-free method, Surface Plasmon Resonance (SPR) detection significantly reduces the needs for liquid handling and reagent storage compared to common enzyme-linked immunosorbent assays (ELISAs), thus enabling comprehensive multiplexing of bioassays on microfluidic sample-to-answer systems. This paper describes a highly integrated centrifugal Lab-on-a-Disc (LoaD) platform for automating the full process chain extending between plasma extraction and subsequent aliquoting to five parallelized reaction channels for quantitative SPR detection by an inexpensive smartphone camera. The entire, multi-step / multi-reagent operation completes within less than 1 h. While the emphasis of this work is on the fluidic automation and parallelization by previously introduced, very robust event-triggered valving and buoyancy-driven centripetal pumping schemes, we successfully implement an immunoglobulin G (IgG) assay; by specific functionalization of the detection surfaces, the same disc layout can readily be customised for immunoassays panels from whole blood.

Nucleic acid purification on a Lab-on-a-Disc with time-controlled incubation

Here we present integrated Lab-on-a-Disc (LoaD) cartridges which are applied to the purification of nucleic acid using the silica bead based method. We utilize a novel combination of ‘event-triggered’ dissolvable film (DF) valving [1] and a centrifugo-pneumatic siphon valve (CPSV) [2] to permit timing of sample incubations, washes and DNA elution. We present two systems; one in with a conventional ‘wash through’ elution which achieves a purification yield of 32.3% ± 5.4% (n = 4). The second system, which uses a CPSV to enable extended wash and elution steps, provides an increased efficiency of 58.4 ± 7.5% (n = 3).