Eleven Amino Acids of HLA-DRB1 and Fifteen Amino Acids of HLA-DRB3, 4, and 5 Include Potentially Causal Residues Responsible for the Risk of Childhood Type 1 Diabetes.

Next-generation targeted sequencing of HLA-DRB1 and HLA-DRB3, -DRB4, and -DRB5 (abbreviated as DRB345) provides high resolution of functional variant positions to investigate their associations with type 1 diabetes risk and with autoantibodies against insulin (IAA), GAD65 (GADA), IA-2 (IA-2A), and ZnT8 (ZnT8A). To overcome exceptional DR sequence complexity as a result of high polymorphisms and extended linkage disequilibrium among the DR loci, we applied a novel recursive organizer (ROR) to discover disease-associated amino acid residues. ROR distills disease-associated DR sequences and identifies 11 residues of DRB1, sequences of which retain all significant associations observed by DR genes. Furthermore, all 11 residues locate under/adjoining the peptide-binding groove of DRB1, suggesting a plausible functional mechanism through peptide binding. The 15 residues of DRB345, located respectively in the ß49-55 homodimerization patch and on the face of the molecule shown to interact with and bind to the accessory molecule CD4, retain their significant disease associations. Further ROR analysis of DR associations with autoantibodies finds that DRB1 residues significantly associated with ZnT8A and DRB345 residues with GADA. The strongest association is between four residues (ß14, ß25, ß71, and ß73) and IA-2A, in which the sequence ERKA confers a risk association (odds ratio 2.15, P = 10-18), and another sequence, ERKG, confers a protective association (odds ratio 0.59, P = 10-11), despite a difference of only one amino acid. Because motifs of identified residues capture potentially causal DR associations with type 1 diabetes, this list of residuals is expected to include corresponding causal residues in this study population.

Spectrophotometric determination of aqueous sulfide on a pneumatically enhanced centrifugal microfluidic platform.

A pneumatically enhanced centrifugal microfluidic platform was developed for rapid spectrophotometric determination of aqueous sulfide. This platform performs an automated analysis based on the reaction between hydrogen sulfide and N,N-dimethyl-p-phenylenediamine in the presence of iron(III) chloride to form Methylene Blue. The platform design minimizes the number of integrated valves required, compared to other centrifugal systems, significantly improving the ease of fabrication. The sequential analytical procedure and spectrophotometric analyses were performed directly on-disk, demonstrating significant advantages in portability and cost over conventional analytical methods. This method allows for rapid and precise determination of aqueous sulfide in the concentration range of 0.4-2.0 mg L(-1), which can be extended by a pneumatically induced, on-disk serial dilution to 6.0 mg L(-1). A detection limit of 0.4 mg L(-1) was calculated for this pneumatically enhanced method.

Automated liquid-liquid extraction by pneumatic recirculation on a centrifugal microfluidic platform.

In this technical note, a liquid-liquid extraction technique was performed using pneumatic liquid recirculation on a centrifugal microfluidic device. Non-contact pneumatic pumping enabled a multi-cycle liquid-liquid extraction process using aqueous iodine in a potassium iodide solution and hexadecane while requiring a minimal amount of space on the device. The extraction process was completely automated on the device following sample introduction and required only 50 s for each extraction cycle. The pumping rate achieved during liquid recirculation was 120 ± 10 µL/min. A recycling process such as the one demonstrated would be difficult to implement in a conventional centrifugal microfluidic system.

A valveless pneumatic fluid transfer technique applied to standard additions on a centrifugal microfluidic platform.

This paper demonstrates a valveless pneumatic fluid transfer technique applicable to centrifugal microfluidic platforms. The technique involves using compressed gas to generate a pneumatic force, which works together with the centrifugal force to control and direct fluid flow. Fluid can be pneumatically transferred from chamber to chamber, greatly decreasing the number of conventional valves required in a multistep process. By varying the rotational frequency of the centrifugal microfluidic platform while pneumatic force is applied, sequential fluid transfer steps can be achieved. The effectiveness of this fluid transfer method is demonstrated by performing a standard additions calibration. This technique is shown to be robust, easy to implement, and greatly reduces the design limitations traditionally associated with centrifugal microfluidic platforms.

Pneumatic flow switching on centrifugal microfluidic platforms in motion.

This paper describes a flow switching technique applicable to centrifugal microfluidic platforms, using a regulated stream of compressed gas. This pneumatic flow switching technique allows for flow control at a T-shaped junction between one inlet channel and two outlet channels. This technique provides a noncontact, robust, and efficient method for switching the direction of fluid flow while a disk is rotating at relatively low frequencies. The switching operation can be implemented reproducibly with applied gas flow rates between 17 and 58 L min(-1) and rotational frequencies between 400 rpm (6.6 Hz) and 1200 rpm (20 Hz).

Pneumatically pumping fluids radially inward on centrifugal microfluidic platforms in motion.

This paper describes a pumping technique applicable to centrifugal microfluidic platforms, involving the use of a regulated stream of compressed gas to pump liquid radially inward and toward the center of the platform while spinning. This technique provides a noncontact method for pumping fluids and is highly efficient, requiring only approximately 60 s to reach completion. This pumping operation can be attained with an applied gas flow rate of 58.8 L min(-1), while the platform is rotated at frequencies less than 180 rpm (3.0 Hz).