Flow-enhanced electrochemical immunosensors on centrifugal microfluidic platforms.

We present a novel fully integrated centrifugal microfluidic device with features for target antigen capture from biological samples, via a bead-based enzyme-linked immune-sorbent assay, and flow-enhanced electrochemical detection. The limit of detection (LOD) of our device for the C-reactive protein (CRP) was determined to be 4.9 pg mL(-1), a 17-fold improvement over quantification by optical density. The complete sample-to-answer protocol of our device is fully automated and takes less than 20 min. Overall, the presented microfluidic disc adds to the comparatively small number of fully integrated microfluidic-based platforms that utilize electrochemical detection and exemplifies how electrochemical detection can be enhanced by flow to successfully detect very low levels of biomarkers (e.g. pg mL(-1)).

Serum complement enhances the responses of genotoxin- and oxidative stress-sensitive Escherichia coli bioreporters.

Bacterial bioreporters are limited in their abilities to detect large polar molecules due to their membrane selectivity. In this study, the activity of serum complement was used to bypass this undesired selectivity. Initially, the serum complement activity was assessed using the responses of a bacterial bioreporter harboring a recA::luxCDABE transcriptional fusion when exposed to the chemotherapy drug, mitomycin C (MMC). Using 50 °C-treated serum, the limit of detection for this bacterial sensor was lowered by nearly 450-fold, from 31 µg/L to 0.07 µg/L MMC. Real-time quantitative PCR demonstrated that serum-treated cultures responded more strongly to 100 µg/L MMC, with 3.1-fold higher recA expression levels. Subsequent experiments with other bioreporter strains also found enhanced sensitivities and responses. Finally, combining each of the above findings, tests were performed to demonstrate the potential application of the recA::luxCDABE bioreporter within a lab-on-a-CD platform as a point-of-care diagnostic to measure chemotherapeutic drug concentrations within blood.

Lab-on-DVD: standard DVD drives as a novel laser scanning microscope for image based point of care diagnostics.

We present a novel "Lab-on-DVD" system and demonstrate its capability for rapid and low-cost HIV diagnostics by counting CD4+ cells isolated from whole blood. We show that a commercial DVD drive can, with certain modifications, be turned into an improved DVD-based laser scanning microscope (DVD-LSM). The system consists of a multi-layered disposable polymer disc and a modified commercial DVD reader with rotational control for sample handling, temperature control for optimized bioassay, a photodiode array for detection, and software for signal processing and user interface - all the necessary components required for a truly integrated lab-on-a-chip system, with the capability to deliver high-resolution images down to 1 µm in size. Using discs modified with antibodies, we specifically captured CD4+ cells from whole blood, demonstrating single cell resolution imaging. The novel integrated DVD platform with sub-micron image resolution brings, for the first time, affordable cellular diagnostic testing to the point-of-care and should be readily applicable at resource-limited settings.

Experimental validation of numerical study on thermoelectric-based heating in an integrated centrifugal microfluidic platform for polymerase chain reaction amplification.

A comprehensive study involving numerical analysis and experimental validation of temperature transients within a microchamber was performed for thermocycling operation in an integrated centrifugal microfluidic platform for polymerase chain reaction (PCR) amplification. Controlled heating and cooling of biological samples are essential processes in many sample preparation and detection steps for micro-total analysis systems. Specifically, the PCR process relies on highly controllable and uniform heating of nucleic acid samples for successful and efficient amplification. In these miniaturized systems, the heating process is often performed more rapidly, making the temperature control more difficult, and adding complexity to the integrated hardware system. To gain further insight into the complex temperature profiles within the PCR microchamber, numerical simulations using computational fluid dynamics and computational heat transfer were performed. The designed integrated centrifugal microfluidics platform utilizes thermoelectrics for ice-valving and thermocycling for PCR amplification. Embedded micro-thermocouples were used to record the static and dynamic thermal responses in the experiments. The data collected was subsequently used for computational validation of the numerical predictions for the system response during thermocycling, and these simulations were found to be in agreement with the experimental data to within ∼97%. When thermal contact resistance values were incorporated in the simulations, the numerical predictions were found to be in agreement with the experimental data to within ∼99.9%. This in-depth numerical modeling and experimental validation of a complex single-sided heating platform provide insights into hardware and system design for multi-layered polymer microfluidic systems. In addition, the biological capability along with the practical feasibility of the integrated system is demonstrated by successfully performing PCR amplification of a Group B Streptococcus gene.

Large-volume centrifugal microfluidic device for blood plasma separation.

BACKGROUND: Microfluidic-based systems are ideal for handling small microliter volumes of samples and reagents, but 'real-world' or clinical samples for bioanalysis are often on the milliliter scale. We aimed to develop and validate a large-volume centrifugal or compact disc-based device for blood plasma separation, capable of processing 2 ml undiluted blood samples. RESULTS: This automated blood sample preparation device was shown to yield high purity plasma in less than half the time of commercial plasma preparation tubes, while enabling integration with downstream analysis and detection steps. CONCLUSION: This article draws upon a novel large-volume device to further illustrate the challenges in combining microfluidics structures with large-volume samples and the implications for sample-driven microfluidics systems.

Centrifugal microfluidics for biomedical applications.

The centrifugal microfluidic platform has been a focus of academic and industrial research efforts for almost 40 years. Primarily targeting biomedical applications, a range of assays have been adapted on the system; however, the platform has found limited commercial success as a research or clinical tool. Nonetheless, new developments in centrifugal microfluidic technologies have the potential to establish wide-spread utilization of the platform. This paper presents an in-depth review of the centrifugal microfluidic platform, while highlighting recent progress in the field and outlining the potential for future applications. An overview of centrifugal microfluidic technologies is presented, including descriptions of advantages of the platform as a microfluidic handling system and the principles behind centrifugal fluidic manipulation. The paper also discusses a history of significant centrifugal microfluidic platform developments with an explanation of the evolution of the platform as it pertains to academia and industry. Lastly, we review the few centrifugal microfluidic-based sample-to-answer analysis systems shown to date and examine the challenges to be tackled before the centrifugal platform can be more broadly accepted as a new diagnostic platform. In particular, fully integrated, easy to operate, inexpensive and accurate microfluidic tools in the area of in vitro nucleic acid diagnostics are discussed.

Numerical modeling and experimental validation of uniform microchamber filling in centrifugal microfluidics.

In this paper, a comprehensive approach to numerical and experimental analysis of microchamber filling in centrifugal microfluidics is presented. In the development of micro total analysis systems, it is often necessary to achieve complete, uniform filling of relatively large microchambers, such as those needed for nucleic acid amplification or detection. With centrifugal devices, these large microchambers must often be orientated perpendicularly to the direction of centrifugal force and are usually bounded by materials with varying surface properties. The resulting fluidic flow in such systems can be complex and is not well characterized. To gain further insight into complex fluidic behavior on centrifugal microfluidic platforms, numerical modeling using the Volume of Fluids method is performed to simulate microchamber filling in a centrifugal microfluidic device with integrated sample preparation, amplification, and detection capabilities. Parametric analyses are performed using numerical models to predict microchamber filling behavior for a range of pressure conditions. High-speed flow visualization techniques are used to track the liquid meniscus during filling of the microchambers, and comparison to the numerical predictions for experimental validation is achieved by analyzing the liquid volume fraction as a function of the non-dimensional temporal profile during filling. When channel filling profiles are compared, the numerical model predictions utilizing static conditions are in strong agreement with the experimental data. When dynamic modeling conditions are used, the numerical predictions are extremely accurate as compared to the experimental data.