Liquid chromatography on a centrifugal platform for separation and collection of water-soluble dyes.

A reversed-phase chromatographic process is developed on a centrifugal platform to separate and collect water-soluble dyes from a mixture. A separation column filled with C18-reversed phase silica gel was used to separate the components from a mixture and the eluate was collected by a series of collecting chambers. The purified components can then be identified and extracted from the collecting chambers. The effects of the silica gel's particle size (7-10, 20-45, and 46-63 µm) and the platform's rotational speed (1000, 1500, 2000 RPM) on the separation and collection efficiency were investigated. Experimental results showed that dye separation could be well performed in the column with smaller-sized silica gels (7-10 µm) under a low rotational speed (1000 RPM). However, for the eluate collection, the high eluent flowrate and long processing time resulted in a convective band-broadening problem in the collecting chambers, which affected the recovery ratio of the dyes. Experimental results showed that the convective band broadening effect can be reduced by reducing the flowrate, shortening the collecting time, and switching the eluent to a different composition. The best recovery ratio of the dyes in the current design can be achieved by using the column with a powder size distribution of 46-63 µm and operating at the rotational speed of 1500 RPM. This platform can process a sample volume of 1 µL and the processing time is about 30 min. Since the only instrument used is a motor, the complete chromatographic process, from separation to fraction collection, can be carried out on a centrifugal platform at a low cost.

The centrifugal viscometer.

In this study, a viscometer, which can measure the viscosity of low-volume liquids (25 µl) within 30 s, was developed on a centrifugal platform. The centrifugal viscometer consists of a disk platform and a motor. Under disk rotation, centrifugal, Coriolis, and viscosity-induced drag forces result in deflection of liquid flow. The viscosity of the liquid sample is determined by the deflection angle of the liquid, which can be examined through image analysis or visual inspection. The viscosities of a series of Newtonian model fluids were tested by the centrifugal viscometer and the results showed good agreement with the ones tested by a conventional rotational viscometer. Since the centrifugal viscometer only requires a motor to function, the microfluidic disk can be produced in large quantities at a low cost through injection molding, and the deflection angle can be detected through visual inspection, it provides an inexpensive, easy to operate, and portable approach to measure low-volume liquid viscosity.

Disk-based enzyme-linked immunosorbent assays using the liquid-aliquoting and siphoning-evacuation technique.

A cost-effective way to carry out multiple enzyme-linked immunosorbent assays (ELISAs) on a centrifugal platform using the liquid-aliquoting and siphoning-evacuation (LASE) technique was developed in this paper. Instead of preloading all the reagents in the reservoirs before testing, each reagent was loaded only one time during testing. The reagent was distributed into equal aliquots and delivered into reaction chambers by the aliquoting fluidic function. In addition, a siphoning-evacuation technique was developed to improve the washing efficiency and simplify the assay protocol. Furthermore, the entire assay protocol can be conducted using a two-step spinning protocol, which greatly reduces the cost of the motor control system. With the LASE technique, a low-cost and user-friendly ELISA system can be achieved.

A Point-of-Care Prothrombin Time Test on a Microfluidic Disk Analyzer Using Alternate Spinning.

In this study, we conducted a fully integrated point-of-care prothrombin time test on a microfluidic disk analyzer. The microfluidic functions integrated on the disk were capable of separating whole blood, decanting plasma, and mixing it with reagents in sequence under alternate spinning. The assay protocol was completed by alternate spinning without using microvalves or surface modification. Clinical sample tests on prothrombin time measurement were conducted by both the microfluidic disk analyzer and the reference instrument used in medical centers. The test results showed a good correlation and agreement between the two instruments.

An enzyme-linked immunosorbent assay on a centrifugal platform using magnetic beads.

An automated, disk-based, enzyme-linked immunosorbent assay (ELISA) system is presented in this work. Magnetic beads were used as the antibody carriers to improve the assay sensitivity and shorten the reaction time. The magnetic module integrated on the system is capable of controlling the magnetic beads to either move in the incubation stage or immobilize at a specific location during washing stage. This controlling mechanism utilizes a passive controlling approach so that it can be performed through disk spinning without the need of active control from external devices. The movement of the magnetic beads was investigated and the optimal rotational speed was found to be related to the ratio of the processing time to the cycle time of the magnetic beads. Comparing to ELISA conducted on microtiter plates, similar test results could be achieved by the disk-based ELISA but the entire protocol can be finished automatically within 45 min with much less reagent consumption.

A sample-to-result system for blood coagulation tests on a microfluidic disk analyzer.

In this report, we describe in detail a microfluidic analyzer, which is able to conduct blood coagulation tests using whole blood samples. Sample preparation steps, such as whole blood aliquoting and metering, plasma separation, decanting, and mixing with reagents were performed in sequence through microfluidic functions integrated on a disk. Both prothrombin time (PT) and activated partial thromboplastin time (aPTT) were carried out on the same platform and the test results can be reported in 5 min. Fifty clinical samples were tested for both PT and aPTT utilizing the microfluidic disk analyzer and the instrument used in hospitals. The test results showed good correlation and agreement between the two instruments.

Insight into mechanisms of cellular uptake of lipid nanoparticles and intracellular release of small RNAs.

PURPOSE: Understanding mechanisms of cellular uptake and intracellular release would enable better design of nanocarriers for delivery of nucleic acids such as siRNA and microRNA (miRNA). METHOD: In this study, we investigated cellular pharmacokinetics of siRNA by co-encapsulating fluorescently labeled siRNA and molecular beacon (MB) in four different formulations of cationic lipid nanoparticles (LNPs). A miRNA mimic was also used as a probe for investigating cellular pharmacokinetics, which correlated well with RNAi activities. RESULTS: We tried to find the best LNP formulation based on the combination of DOTMA and DODMA. When the DOTMA/DODMA ratio was at 5/40, the LNP containing a luciferase siRNA produced the highest gene silencing activity. The superior potency of DOTMA/DODMA could be attributed to higher uptake and improved ability to facilitate siRNA release from endosomes subsequent to uptake. CONCLUSIONS: Our findings may provide new insights into RNAi transfection pathways and have implications on cationic LNP design.

A fully integrated prothrombin time test on the microfluidic disk analyzer.

A fully integrated prothrombin time test, which is capable of measuring the time required for blood to coagulate, is presented in this study. The microfluidic functions integrated on the microfluidic disk were able to extract the plasma from whole blood samples and conduct rapid mixing within 1 second. The factors which affected the plasma decanting mechanism were investigated. The complete fluidic design was applied to prothrombin time tests. Ninety-two whole blood clinical samples were tested by the microfluidic disk analyzer and the Sysmex CA1500 coagulation analyzer, which is the instrument used in medical centers. The test results from these two instruments showed good correlation and agreement.

Supernatant decanting on a centrifugal platform.

This study presents a novel approach to decant supernatant on a centrifugal platform. By manipulating the centrifugally induced pressure and the elastic deformation of the plastic lids in the decanting chamber, fixed amounts of the supernatant can be decanted into the detection chamber at lower rotational speeds. The experimental results showed that decanted volume is affected by the volume of deformation and the operating parameters. Factors that influence the decanting ratio are also discussed. This approach has the advantages of simple design and low manufacturing cost; further, it has no need of surface modification. It has been applied to on-disk separation of plasma from whole blood, and the results showed good stability and repeatability.

Pyogenic liver abscess with Prevotella species and Fusobacterium necrophorum as causative pathogens in an immunocompetent patient.

Pyogenic liver abscess of odontogenic origin in an immunocompetent patient is extremely rare. We report an immunocompetent 25-year-old male hepatitis B carrier with severe dental disease that led to the development of liver abscess. A periapical abscess in the upper left molar area was seen on his dental X-ray. Two sets of blood cultures grew Prevotella species, bacteria that are commonly found inside the oral cavity. Bacterial culture of the liver abscess drainage sample grew both Prevotella and Fusobacterium necrophorum. This led to our diagnosis of pyogenic liver abscess of dental origin, since we found no other source of infection in our patient except for his dental disease. After antibiotic therapy with drainage, abdominal sonography showed resolution of the abscess. The diseased teeth were also extracted. During 1 year of follow-up, there was no sign of abscess recurrence. A diagnosis of pyogenic liver abscess necessitates a complete evaluation to rule out possible biliary, colonic or other associated diseases. However, when a liver abscess is thought to be cryptogenic, we also recommend a careful dental examination to help identify the source of infection.