A Personal Glucose Meter for Label-Free and Washing-Free Biomolecular Detection.

We developed a label-free and washing-free method for biomolecular detection using a personal glucose meter (PGM). ATP was selected as a model target, and cascade enzymatic reactions promoted by hexokinase and pyruvate kinase were adopted to link the amount of ATP to glucose that is detectable by a hand-held PGM. In principle, the presence of target ATP enables hexokinase to catalyze the conversion of glucose to glucose 6-phosphate by providing a phosphate group to glucose, and thus the amount of glucose is decreased in proportion to the amount of ATP. In addition, adenosine 5'-diphosphate (ADP), which is generated after hexokinase-catalyzed enzymatic reaction, is recovered to ATP by a pyruvate kinase enzyme. The regenerated ATP is again supplemented to catalyze multiple rounds of cascade enzymatic reactions, leading to signal amplification. As a result, the change of glucose amount that is inversely proportional to ATP amount is simply measured by a hand-held PGM. By employing this strategy, we successfully determined ATP down to 49 nM with high selectivity even in real samples such as tap water, human serum, and bovine urine. Importantly, the developed system does not require expensive modification and washing steps but is conveniently operated with a commercially available PGM, which would pave the way for the development of a simple and cost-effective sensing platform.

Sensitive electrochemical aptasensor by coupling "signal-on'' and "signal-off'' strategies.

A simple electrochemical aptasensor for sensitive and selective determination of adenosine triphosphate (ATP) has been developed on the basis of a new dual-signaling amplification strategy. This aptasensor features both ''signal-on'' and ''signal-off'' elements. The ferrocene (Fc)-labeled aptamer probe (Fc-P) is designed to hybridize with the thiolated methylene blue (MB)-modified DNA probe (MB-P) on gold electrode to form rigid duplex DNA. In the presence of ATP, the interaction between ATP and the aptamer leads to the dissociation of the duplex DNA structure and thereby the release of the Fc-P from the sensing interface. The single-stranded MB-P could thus tend to form a hairpin structure through the hybridization of the complementary sequences at both its ends. Such conformational changes result in the oxidation peak current of Fc decreases and that of MB increases, and the changes of dual signals are linear with the concentration of ATP. When "ΔI = ΔI(MB) + |ΔI(Fc)|" (ΔI(MB) and ΔI(Fc) are the change values of the oxidation peak currents of MB and Fc, respectively.) is used as the response signal for quantitative determination of ATP, the detection limit (1.9 nM) is much lower than that by using either MB-P or Fc-P alone. This new dual-signaling aptasensor is readily regenerated and shows good response toward the target. It will have important applications in the sensitive and selective electrochemical determination of other small molecules and proteins.

Renal bioenergetics during early gram-negative mammalian sepsis and angiotensin II infusion.

PURPOSE: To measure renal adenosine triphosphate (ATP) (bioenergetics) during hypotensive sepsis with or without angiotensin II (Ang II) infusion. METHODS: In anaesthetised sheep implanted with a renal artery flow probe and a magnetic resonance coil around one kidney, we induced hypotensive sepsis with intravenous Escherichia coli injection. We measured mean arterial pressure (MAP), heart rate, renal blood flow RBF and renal ATP levels using magnetic resonance spectroscopy. After 2 h of sepsis, we randomly assigned sheep to receive an infusion of Ang II or vehicle intravenously and studied the effect of treatment on the same variables. RESULTS: After E. coli administration, the experimental animals developed hypotensive sepsis (MAP from 92 ± 9 at baseline to 58 ± 4 mmHg at 4 h). Initially, RBF increased, then, after 4 h, it decreased below control levels (from 175 ± 28 at baseline to 138 ± 27 mL/min). Despite decreased RBF and hypotension, renal ATP was unchanged (total ATP to inorganic phosphate ratio from 0.69 ± 0.02 to 0.70 ± 0.02). Ang II infusion restored MAP but caused significant renal vasoconstriction. However, it induced no changes in renal ATP (total ATP to inorganic phosphate ratio from 0.79 ± 0.03 to 0.80 ± 0.02). CONCLUSIONS: During early hypotensive experimental gram-negative sepsis, there was no evidence of renal bioenergetic failure despite decreased RBF. In this setting, the addition of a powerful renal vasoconstrictor does not lead to deterioration in renal bioenergetics.

Mechanisms by which a phytotherapeutic drug influences bladder activity in rats.

PURPOSE: We investigated the mechanisms by which Eviprostat, a phytotherapeutic drug for benign prostatic hyperplasia, influences bladder activity in rats. MATERIALS AND METHODS: A total of 42 female rats were divided into a control group and an Eviprostat group. Rats in the control group were fed a standard diet, while animals in the Eviprostat group were fed a diet containing 0.1% Eviprostat. After 2 weeks 14 rats (7 rats per group) underwent continuous cystometry with physiological saline or 0.1% acetic acid solution and bladder activity was recorded. Body weight, blood pressure, plasma monoamines and adenosine triphosphate were measured in another 14 rats (7 per group). In the remaining 14 rats (7 per group) 0.1% acetic acid solution was infused into the bladder and urinary adenosine triphosphate was measured before and after stimulation. RESULTS: During cystometry with acetic acid the interval between bladder contractions was shorter and maximum bladder contraction pressure was higher in the control group compared with results obtained using physiological saline but such differences were not seen in the Eviprostat group. Plasma adrenaline and noradrenaline were lower in the Eviprostat group than the control group but no difference in blood pressure was observed. Urinary adenosine triphosphate was higher in the 2 groups than before stimulation but the increase was smaller in the Eviprostat group than in the control group. CONCLUSIONS: These results suggest that Eviprostat acts to maintain low catecholamine and also inhibit pathological bladder activity by decreasing adenosine triphosphate release from the bladder.