Charles Nwankire

Centrifugal microfluidics for cell analysis.

Over the past two decades, centrifugal microfluidic systems have successfully demonstrated their capability for robust, high-performance liquid handling to enable modular, multi-purpose lab-on-a-chip platforms for a wide range of life-science applications. Beyond the handling of homogeneous liquids, the unique, rotationally controlled centrifugal actuation has proven to be specifically advantageous for performing cell and particle handling and assays. In this review we discuss technologies to implement two important steps for cell handling, namely separation and capturing/counting.

Label-free impedance detection of cancer cells from whole blood on an integrated centrifugal microfluidic platform

An electrochemical Lab-on-a-Disc (eLoaD) platform for the automated quantification of ovarian cancer cells (SKOV3) from whole blood is reported. This centrifugal microfluidic system combines complex sample handling, i.e., blood separation and cancer cell extraction from plasma, with specific capture and sensitive detection using label-free electrochemical impedance. Flow control is facilitated using rotationally actuated valving strategies including siphoning, capillary and centrifugo-pneumatic dissolvable-film (DF) valves. For the detection systems, the thiol-containing amino acid, L-Cysteine, was self-assembled onto smooth gold electrodes and functionalized with anti-EpCAM. By adjusting the concentration of buffer electrolyte, the thickness of the electrical double layer was extended so the interfacial electric field interacts with the bound cells. Significant impedance changes were recorded at 117.2 Hz and 46.5 Hz upon cell capture. Applying AC amplitude of 50 mV at 117.2 Hz and open circuit potential, a minimum of 214 captured cells/mm(2) and 87% capture efficiency could be recorded. The eLoaD platform can perform five different assays in parallel with linear dynamic range between 16,400 and (2.6±0.0003)×10(6) cancer cells/mL of blood, i.e. covering nearly three orders of magnitude. Using the electrode area of 15.3 mm(2) and an SKOV3 cell radius of 5 µm, the lower detection limit is equivalent to a fractional surface coverage of approximately 2%, thus making eLoaD a highly sensitive and efficient prognostic tool that can be developed for clinical settings where ease of handling and minimal sample preparation are paramount.

A portable centrifugal analyser for liver function screening

Mortality rates of up to 50% have been reported after liver failure due to drug-induced hepatotoxicity and certain viral infections (Gao et al., 2008). These adverse conditions frequently affect HIV and tuberculosis patients on regular medication in resource-poor settings. Here, we report full integration of sample preparation with the read-out of a 5-parameter liver assay panel (LAP) on a portable, easy-to-use, fast and cost-efficient centrifugal microfluidic analysis system (CMAS). Our unique, dissolvable-film based centrifugo-pneumatic valving was employed to provide sample-to-answer fashion automation for plasma extraction (from finger-prick of blood), metering and aliquoting into separate reaction chambers for parallelized colorimetric quantification during rotation. The entire LAP completes in less than 20 min while using only a tenth the reagent volumes when compared with standard hospital laboratory tests. Accuracy of in-situ liver function screening was validated by 96 separate tests with an average coefficient of variance (CV) of 7.9% compared to benchtop and hospital lab tests. Unpaired two sample statistical t-tests were used to compare the means of CMAS and benchtop reader, on one hand; and CMAS and hospital tests on the other. The results demonstrate no statistical difference between the respective means with 94% and 92% certainty of equivalence, respectively. The portable platform thus saves significant time, labour and costs compared to established technologies, and therefore complies with typical restrictions on lab infrastructure, maintenance, operator skill and costs prevalent in many field clinics of the developing world. It has been successfully deployed to a centralised lab in Nigeria.

Fluidic Automation of Nitrate and Nitrite Bioassays in Whole Blood by Dissolvable-Film Based Centrifugo-Pneumatic Actuation

This paper demonstrates the full centrifugal microfluidic integration and automation of all liquid handling steps of a 7-step fluorescence-linked immunosorbent assay (FLISA) for quantifying nitrate and nitrite levels in whole blood within about 15 min. The assay protocol encompasses the extraction of metered plasma, the controlled release of sample and reagents (enzymes, co-factors and fluorescent labels), and incubation and detection steps. Flow control is implemented by a rotationally actuated dissolvable film (DF) valving scheme. In the valves, the burst pressure is primarily determined by the radial position, geometry and volume of the valve chamber and its inlet channel and can thus be individually tuned over an extraordinarily wide range of equivalent spin rates between 1,000 RPM and 5,500 RPM. Furthermore, the vapour barrier properties of the DF valves are investigated in this paper in order to further show the potential for commercially relevant on-board storage of liquid reagents during shelf-life of bioanalytical, ready-to-use discs.

Rotationally controlled centrifugo-pneumatic valving utilizing dissolvable films

Here we report on a novel, centrifugo-pneumatic valving scheme based on water-dissolvable films. Compared to previous active valving approaches, this valving technique is merely controlled by introducing aqueous solutions to the substrate and allowing for dissolution; therefore lending itself to simple fabrication in microfluidic devices. Furthermore, the ability to tailor films over a range of dissolution times introduces novel capabilities for valving on the centrifugal platform allowing for greater diffusion barriers to liquids even at high burst frequencies. This report outlines the underlying working principle, assembly, and characterization of the novel valving scheme utilizing the dissolvable films.

SIZE- and deformability-based particle sorting by strategic design of obstacle arrays in continuous centrifugal sedimentation mode

This paper describes a novel, smart-grid technique for isolating, sorting and capturing cancer cells based on size and deformability by lateral displacement. In stopped-flow, centrifugal sedimentation mode, the bioparticles are sizeselectively displaced along the dynamically spaced grid of the microstructures in a deterministic fashion. Following displacement, particles are captured in claw-like structures for onward processing. This displacement method was first demonstrated for sorting seven bead populations with diameters between 5 μm and 30 μm from a mixture. Then HeLa and melanoma cells spiked in buffer and whole blood were isolated and captured. Finally, we succeeded to sort normal and fixed HeLa cells according to their deformability.

Novel Microfluidic Analytical Sensing Platform for the Simultaneous Detection of Three Algal Toxins in Water

Globally, the need for “on-site” algal-toxin monitoring has become increasingly urgent due to the amplified demand for fresh-water and for safe, “toxin-free” shellfish and fish stocks. Herein, we describe the first reported, Lab-On-A-Disc (LOAD) based-platform developed to detect microcystin levels in situ, with initial detectability of saxitoxin and domoic acid also reported. Using recombinant antibody technology, the LOAD platform combines immunofluorescence with centrifugally driven microfluidic liquid handling to achieve a next-generation disposable device capable of multianalyte sampling. A low-complexity “LED-photodiode” based optical sensing system was tailor-made for the platform, which allows the fluorescence signal of the toxin-specific reaction to be quantified. This system can rapidly and accurately detect the presence of microcystin-LR, domoic acid, and saxitoxin in 30 min, with a minimum of less than 5 min end-user interaction for maximum reproducibility. This method provides a robust “point of need” diagnostic alternative to the current laborious and costly methods used for qualitative toxin monitoring.

A centrifugal microfluidic-based approach for multi-toxin detection for real-time marine water-quality monitoring

Globally, fresh and brackish water sources are constantly under treat of exposure to toxins. Two of the most prevalent toxins from fresh and brackish water blooms are the cyclic peptide toxins of the microcystin family, formed from cyanobacterial, and the kainic acid analog neurotoxin known as domoic acid. There is therefore a significant need for constant and cost-effective ‘on-site’ algal-toxin monitoring to respond to constantly increasing demand for safe and ‘toxin-free’ freshwater, shellfish and fish stocks. Herein, we describe a Lab-On-A-Disc (LOAD) platform which was developed to assess microcystin and domoic acid concentration levels in-situ. Using recombinant antibody technology, the LOAD platform combines immunofluorescence with centrifugally driven microfluidic liquid handling to achieve a next-generation disposable device for high throughput sampling.

Cluster sizing of cancer cells by rail-based serial gap filtration in stopped-flow, continuous sedimentation mode

In addition detecting circulating tumour cells (CTCs) in blood, the presence of multi-cellular clusters has recently been identified to carry further information pertaining to patient outcome. We present a label-free method of measuring the range and load of clusters in a blood sample using a size-exclusion rail operated by centrifugal microfluidics. A negative selection strategy first enriches clusters from a whole blood sample; these clusters are then processed along the rail where they resolve to a series of collection bins according to size. Analysis of the occupancy of these bins then provides metrics on the cancer-cell load carried in the blood.