Damien King

Optical detection strategies for centrifugal microfluidic platforms

Centrifugal microfluidic systems have become one of the principal platforms for implementing bioanalytical assays, most notably for biomedical point-of-care diagnostics. These so-called ‘lab-on-a-disc’ systems primarily utilise the rotationally controlled centrifugal field in combination with capillary forces to automate a range of laboratory unit operations (LUOs) for sample preparation, such as metering, aliquoting, mixing and extraction for biofluids as well as sorting, isolation and counting of bioparticles. These centrifugal microfluidic LUOs have been regularly surveyed in the literature. However, even though absolutely essential to provide true sample-to-answer functionality of lab-on-a-disc platforms, systematic examination of associated, often optical, read-out technologies has been so far neglected. This review focusses on the history and state-of-the-art of optical read-out strategies for centrifugal microfluidic platforms, arising (commercial) application potential and future opportunities.

Living photonics: monitoring light propagation through cells (LiPhos)

The LiPhos project (EU FP7 Grant Agreement No.: 317916, www.liphos.eu) aims to develop three different biophotonic diagnosis tools (BDTs), based on living photonics, namely: single layer living photonics (SLLP), single cell analysis (SCA); and multi-layer living photonics (MLLP). By Measuring of what we term the Photonic Fingerprint (or PIN), of the cells in such BDTs, should make it is possible to differentiate between healthy and non-healthy cell or tissue states. Moreover, the effect of specific drugs and pro-inflammatory agents could be assessed. This concept is currently being applied to the diagnosis of cardiovascular diseases (CVD).

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.

Leaky Expression of the TET-On System Hinders Control of Endogenous miRNA Abundance

With the ability to affect multiple genes and fundamental pathways simultaneously, miRNA engineering of Chinese Hamster Ovary (CHO) cells has significant advantages over single gene expression or repression. Tight control of these molecular triggers is desirable as it could in theory allow on/off or even tunable regulation of desirable cellular phenotypes. The present study investigated the potential of employing a tetracycline inducible (TET-On) system for conditional knockdown of specific miRNAs but encountered several challenges. The authors show a significant reduction in cell proliferation and culture viability when maintained in media supplemented with the TET-On induction agent Doxycycline at concentrations commonly reported. Calculation of a mature miRNA and miRNA sponge mRNA copy number demonstrates that leaky basal transgene expression in the un-induced state, is sufficient for significant miRNA knockdown. This work highlights challenges of the TET-On inducible expression system for controlled manipulation of endogenous miRNAs with two examples; miR-378 and miR-455. The authors suggest a solution involving isolation of highly inducible clones and use a single cell analysis platform to demonstrate the heterogeneity of basal expression and inducibility. Finally, the authors describe numerous strategies to minimize leaky transgene expression and alterations to current miRNA sponge design.

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).

Systems Biology in Single Cells

From the beginning of the twenty-first century, there has been a shift towards studying biological processes using a holistic rather than a reductionist scientific paradigm thus establishing the approach now named “systems biology” or “systomics”. This method of biological investigation represents a synergy where life sciences, systems engineering, and information technology examine the interactions between biological pathways, rather than solely focusing on individual pathways in an isolated manner. To date, systems biology has often studied population averages rather than individual characteristics of cells which might display a significant spread. However, as a single cell is the smallest operational biological unit that encompasses all metabolites necessary for maintaining a viable living entity, the application of systems biology approaches to the study of distinct cells is fast becoming a goal of many research groups. In this chapter we will describe some of the technologies that enable the isolation of individual cells in a form that accommodates systomics studies, the biological methods that are then deployed on such isolated cells to generate system-level information, and finally describe some of the bioinformatics that is specifically directed towards single-cell studies.