C-reactive protein and interleukin 6 microfluidic immunoassays with on-chip pre-stored reagents and centrifugo-pneumatic liquid control.

We present a fully automated centrifugal microfluidic method for particle based protein immunoassays. Stick-pack technology is employed for pre-storage and release of liquid reagents. Quantitative layout of centrifugo-pneumatic particle handling, including timed valving, switching and pumping is assisted by network simulations. The automation is exclusively controlled by the spinning frequency and does not require any additional means. New centrifugal microfluidic process chains are developed in order to sequentially supply wash buffer based on frequency dependent stick-pack opening and pneumatic pumping to perform two washing steps from one stored wash buffer; pre-store and re-suspend functionalized microparticles on a disk; and switch between the path of the waste fluid and the path of the substrate reaction product with 100% efficiency. The automated immunoassay concept is composed of on demand ligand binding, two washing steps, the substrate reaction, timed separation of the reaction products, and termination of the substrate reaction. We demonstrated separation of particles from three different liquids with particle loss below 4% and residual liquid remaining within particles below 3%. The automated immunoassay concept was demonstrated by means of detecting C-reactive protein (CRP) in the range of 1-81 ng ml-1 and interleukin 6 (IL-6) in the range of 64-13 500 pg ml-1. The limit of detection and quantification were 1.0 ng ml-1 and 2.1 ng ml-1 for CRP and 64 pg ml-1 and 205 pg ml-1 for IL-6, respectively.

Robust temperature change rate actuated valving and switching for highly integrated centrifugal microfluidics.

We present new unit operations for valving and switching in centrifugal microfluidics that are actuated by a temperature change rate (TCR) and controlled by the rotational frequency. Implementation is realized simply by introducing a comparatively large fluidic resistance to an air vent of a fluidic structure downstream of a siphon channel. During temperature decrease at a given TCR, the air pressure inside the downstream structure decreases and the fluidic resistance of the air vent slows down air pressure compensation allowing a thermally induced underpressure to build up temporarily. Thereby the rate of temperature change determines the time course of the underpressure for a given geometry. The thermally induced underpressure pulls the liquid against a centrifugal counterpressure above a siphon crest, which triggers the valve or switch. The centrifugal counterpressure (adjusted by rotation) serves as an independent control parameter to allow or prevent valving or switching at any TCR. The unit operations are thus compatible with any temperature or centrifugation protocol prior to valving or switching. In contrast to existing methods, this compatibility is achieved at no additional costs: neither additional fabrication steps nor additional disk space or external means are required besides global temperature control, which is needed for the assay. For the layout, an analytical model is provided and verified. The TCR actuated unit operations are demonstrated, first, by a stand-alone switch that routes the liquid to either one of the two collection chambers (n = 6) and, second, by studying the robustness of TCR actuated valving within a microfluidic cartridge for highly integrated nucleic acid testing. Valving could safely be prevented during PCR by compensating the thermally induced underpressure of 3.52 kPa with a centrifugal counterpressure at a rotational frequency of 30 Hz with a minimum safety range to valving of 2.03 kPa. Subsequently, a thermally induced underpressure of 2.55 kPa was utilized for robust siphon valving at 3 Hz with a minimum safety range of 2.32 kPa.

Rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic LabDisk using highly sensitive nested PCR with integrated sample preparation.

Diagnosis of infectious diseases suffers from long turnaround times for gold standard culture-based identification of bacterial pathogens, therefore impeding timely specific antimicrobial treatment based on laboratory evidence. Rapid molecular diagnostics-based technologies enable detection of microorganisms within hours however cumbersome workflows and complex equipment still prevent their widespread use in the routine clinical microbiology setting. We developed a centrifugal-microfluidic "LabDisk" system for rapid and highly-sensitive pathogen detection on a point-of-care analyser. The unit-use LabDisk with pre-stored reagents features fully automated and integrated DNA extraction, consensus multiplex PCR pre-amplification and geometrically-multiplexed species-specific real-time PCR. Processing merely requires loading of the sample and DNA extraction reagents with minimal hands-on time of approximately 5 min. We demonstrate detection of as few as 3 colony-forming-units (cfu) of Staphylococcus warneri, 200 cfu of Streptococcus agalactiae, 5 cfu of Escherichia coli and 2 cfu of Haemophilus influenzae in a 200 µL serum sample. The turnaround time of the complete analysis from "sample-to-result" was 3 h and 45 min. The LabDisk consequently provides an easy-to-use molecular diagnostic platform for rapid and highly-sensitive detection of bacterial pathogens without requiring major hands-on time and complex laboratory instrumentation.

Microfluidic vapor-diffusion barrier for pressure reduction in fully closed PCR modules.

Microfluidic systems for polymerase chain reaction (PCR) should be fully closed to avoid vapor loss and to exclude the risk of contaminating the laboratory environment. In closed systems however, the high temperatures of up to 95 °C associated with PCR cause high overpressures up to 100 kPa, dominated by the increase of vapor partial pressure upon evaporation. Such high overpressures pose challenges to the mechanical stability of microfluidic chips as well as to the liquid handling in integrated sample-to-answer systems. In this work, we drastically reduce the pressure increase in fully closed PCR systems by integrating a microchannel that serves as a vapor-diffusion barrier (VDB), separating the liquid-filled PCR chamber from an auxiliary air chamber. In such configurations, propagation of vapor from the PCR chamber into the auxiliary air chamber and as a consequence the increase of pressure is limited by the slow diffusion process of vapor through the VDB. At temperature increase from 23 °C to 95 °C, we demonstrate the reduction of overpressure from more than 80 kPa without the VDB to only 35 kPa with the VDB. We further demonstrate proper function of VDB and its easy integration with downstream processes for PCR based nucleic acid amplification within centrifugal microfluidics. Without integration of the VDB, malfunction due to pressure-induced delamination of the microfluidic chip occurred.