Paper-based device for the colorimetric assay of bilirubin based on in-situ formation of gold nanoparticles.

A paper-based colorimetric assay for the determination of bilirubin has been developed. The method is based on the in-situ reduction of chloroauric acid to form gold nanoparticles. A chromatographic paper was patterned using a wax printer. Chloroauric acid was drop-cast onto the reagent zone. In the presence of bilirubin, gold(III) ions are reduced and form gold nanoparticles. This leads to a color change from yellow to purple. The intensity of the purple color (peak at 530 nm) increases with bilirubin concentration in the 5.0 to 1000 mg L-1 range. The detection limit is 1.0 mg L-1. For the quantification of bilirubin, images were captured using a digital camera, and data were processed with the help of machine learning-based supervised prediction using Random Forest classification. The method was applied to the determination of bilirubin in urine samples. The spiked urine samples exhibit more than 95% recovery. Graphical abstractSchematic representation of the paper-based colorimetric assay for the detection of bilirubin based on the in-situ formation of gold nanoparticles. A color band is generated for visual interpretation and used for the testing of bilirubin in urine.

Regulation of quantal shape by Rab3A: evidence for a fusion pore-dependent mechanism.

The function of Rab3A, a small GTPase located on synaptic vesicles, is not well understood. Studies in the Rab3A(-/-) mouse support a role in activity-dependent plasticity, but have not reported any effects on spontaneously occurring miniature synaptic currents, except that there is a decrease in resting frequency at the neuromuscular junction. Therefore we were surprised to find an increase in the occurrence of mEPCs with abnormally long half-widths at the neuromuscular junctions of Rab3A(-/-) mice. The abnormal miniature endplate currents (mEPCs), which have significantly greater charge than the average mEPCs for the same fibres, could arise from larger vesicles. However, the type of mEPC most increased in Rab3A(-/-) mice has a slow rise, which suggests it is not the result of full collapse fusion. To test if the slow mEPCs increased after loss of Rab3A could be due to malfunctioning fusion pores, we used carbon fibre amperometry to record pre-spike feet, which have been shown to correspond to the initial opening of a narrow fusion pore, in adrenal chromaffin cells of wild-type and Rab3A(-/-) mice. We found that small amplitude pre-spike feet with abnormally long durations were increased in Rab3A(-/-) cells. The correspondence between mEPC and amperometric data supports our interpretation that slow rising, long half-width mEPCs are caused by reduced diameter fusion pores that remain open longer. These data could be explained by a direct action of Rab3A on the fusion pore, or by Rab3A-dependent control of vesicles with unusual fusion pore characteristics.

Enhancement of asynchronous and train-evoked exocytosis in bovine adrenal chromaffin cells infected with a replication deficient adenovirus.

Bovine adrenal chromaffin cells share many characteristics with neurons and are often used as a simple model system to study ion channels and neurotransmitter release. We infected bovine adrenal chromaffin cells with a replication deficient adenovirus that induces expression of the common reporters beta-galactosidase and Green Fluorescent Protein via a bicistronic sequence. In perforated-patch recordings performed 48-h postinfection, peak calcium currents were reduced 32%, primarily due to loss of omega-conotoxin-GVIA-sensitive current. In contrast, sodium currents were increased 17%. Exocytosis, detected as an increase in membrane capacitance immediately after a single step depolarization, was reduced in proportion to the decrease in calcium influx. However, capacitance continued to increase for seconds after the depolarization. The amplitude of this poststimulus drift, or asynchronous exocytosis, was approximately three times that which occurred in a small fraction of control cells. Exocytosis evoked by repetitive stimulation with a train of brief depolarizations was increased 50%. Intracellular calcium levels measured during and after stimulation were lower, not higher, in adenovirus-infected cells. Electroporated cells showed reduced calcium currents but no enhancement of exocytosis. Cells infected with UV-irradiated virus showed reduced calcium currents and enhancement of exocytosis, but the changes were smaller than those caused by intact virus. Our results are consistent with the idea that adenovirus capsid and adenoviral DNA contribute to a Ca2+ influx- and [Ca2+]i-independent enhancement of exocytosis in bovine chromaffin cells.

Rab3A negatively regulates activity-dependent modulation of exocytosis in bovine adrenal chromaffin cells.

Members of the Rab family of monomeric GTPases have been implicated in vesicle trafficking, and Rab3A, located on synaptic vesicles in neurones and secretory vesicles in neuroendocrine cells, is likely to be involved in vesicle fusion leading to neurotransmitter release. A hydrolysis-deficient mutant of Rab3A, Rab3AQ81L, has been shown to potently inhibit hormone release. Here we show that the inhibition of hormone release by Rab3AQ81L is activity-dependent. Bovine adrenal chromaffin cells were induced to express Rab3AQ81L and green fluorescent protein by adenoviral gene transfer of a bicistronic construct. Fluorescent cells were stimulated with single depolarizations and trains of depolarizing pulses in whole cell perforated patch clamp recordings, and exocytosis was detected with cell capacitance measurements and carbon fibre amperometry. When single depolarizations were used to evoke exocytosis, cells expressing Rab3AQ81L showed a 50% reduction in response amplitude. When trains of brief depolarizations (10 or 40 ms) were used to evoke exocytosis, responses rapidly declined to zero in cells expressing Rab3AQ81L. Wild-type Rab3A had effects similar to Rab3AQ81L, causing significant inhibition of exocytosis only during repetitive stimulation. Expression of Rab5A did not alter exocytosis evoked by single depolarizations or repetitive stimulation. Applying a long duration depolarization in the middle of a stimulus train revealed that exocytotic efficacy (capacitance increase per amount of calcium influx) was not decreased in Rab3AQ81L-expressing cells. Instead, the activity-dependent increase in exocytotic efficacy observed in control cells did not occur in Rab3AQ81L-expressing cells. Our results suggest that Rab3A in the GTP bound conformation prevents activity-dependent facilitation.