Electrochemical determination of uric acid in urine and serum with uricase/carbon nanotube /carboxymethylcellulose electrode.

The detection of uric acid in blood and urine is clinically important in terms of suitable diagnosis and self-healthcare. An amperometric thin film biosensor composed of carbon nanotube and uricase enzyme is presented. The CNT is successfully dispersed in aqueous solution with carboxymethylcellulose surfactant. This enables thin film formation by a simple drop-casting layer-by-layer process. The uricase/carboxymethylcellulose dispersed carbon nanotube/gold thin film biosensor shows the best sensing performance compared to that with sodium cholate surfactant in terms of higher current and lower detection potential. The presented procedure shows good performance with neither electron transfer mediator nor complicated process. Cyclic voltammetry exhibited a sensitivity of 233 µA mM-1 cm-2 at +0.35 V, a linear range of 0.02-2.7 mM, and a detection limit of 2.8 µM. We quantify and graph uric acid data in actual physiological samples (serum and urine) for the first time and detection values showed good agreement with those obtained by a conventional analytical method (enzymatic colorimetry kit).

Comparison of Direct and Mediated Electron Transfer in Electrodes with Novel Fungal Flavin Adenine Dinucleotide Glucose Dehydrogenase.

Direct and mediated electron transfer (DET and MET) in enzyme electrodes with a novel flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from fungi are compared for the first time. DET is achieved by placing a single-walled carbon nanotube (CNT) between GDH and a flat gold electrode where the CNT is close to FAD within the distance for DET. MET is induced by using a free electron transfer mediator, potassium hexacyanoferrate, and shuttles electrons from FAD to the gold electrode. Cyclic voltammetry shows that the onset potential for glucose response current in DET is smaller than in MET, and that the distinct redox current peak pairs in MET are observed whereas no peaks are found in DET. The chronoamperometry with respect to a glucose biosensor shows that (i) the response in DET is more rapid than in MET; (ii) the current at more than +0.45V in DET is larger than the current at the current-peak potential in MET; (iii) a DET electrode covers the glucose concentration range for clinical requirements and is not susceptible to interfering agents at +0.45 V; and (iv) a DET electrode with the novel fungal FAD-GDH does not affect sensing accuracy in the presence of up to 5 mM xylose, while it often shows a similar response level to glucose with other conventionally used fungus-derived FAD-GDHs. It is concluded that our DET system overcomes the disadvantage of MET.

Chemistry of bisulfite genomic sequencing; advances and issues.

Methylation at position 5 of cytosine in DNA plays a major role in epigenetic gene control. The methylation analysis can be performed by bisulfite genomic sequencing. Conventional procedures in this analysis include a treatment of single stranded DNA with 3-5 M sodium bisulfite at pH 5 and at 50-55 degrees for 4-20 hr. This will convert cytosine into uracil, while 5-methylcytosine resists this deamination. Amplification by PCR of the bisulfite-treated DNA followed by sequencing reveals the positions of 5-methylcytosine in the gene. We reported recently that the whole procedure can be speeded up by use of a highly concentrated bisulfite solution, 10 M ammonium bisulfite. We also reported that urea, which has been often added to the reaction mixture with the purpose of facilitating the reaction, may not work as anticipated. This time, we would like to address the need for further investigating the chemistry of the bisulfite modification of DNA. Particularly important is to study side reactions that may occur due to the exhaustive bisulfite treatment required for achieving complete deamination of all the cytosine residues in a given sample of DNA.

Does urea promote the bisulfite-mediated deamination of cytosine in DNA? Investigation aiming at speeding-up the procedure for DNA methylation analysis.

Methylation of cytosine in DNA at position 5 plays important roles in gene functions. Changes in the methylation status are linked to cancer. These studies have been developed on the basis of determining 5-methylcytosine residues [mC] in DNA. This analytical procedure uses the principle that bisulfite deaminates cytosine [C] but it deaminates mC only very slowly. Thus, 'bisulfite genomic sequencing' involves treatment of a given DNA sample with bisulfite followed by PCR amplification and sequencing, through which C residues in the original DNA are found as T and mC as C. In this procedure, a treatment with 3-5 M sodium bisulfite for 12-16 hr at 55 degrees C has been conventionally used. Recently, we were able to improve the efficiency of this procedure by introducing a highly concentrated (10 M) bisulfite solution. Aiming at further improvement of the procedure, we have now explored the effect of adding urea in this bisulfite treatment, as urea was reported to improve the deamination efficiency. Using 7.5 M ammonium bisulfite (pH 5.4) at 70 degrees C with or without the presence of 6 M urea, we performed deamination and sequencing of a DNA sample having known multiple CpG sites with mC. The deaminated DNAs were then subjected to PCR amplification followed by sequencing. In the 15 min-treated sample, the deamination extents were; C 96.5%, mC 1.1% for "bisulfite-only"; and C 90.3%, mC 1.4% for "bisulfite + urea". In the 30 min-treated sample, these values were; C 99.7%, mC 3.6% for "bisulfite only"; and C 99.7%, mC 2.1% for "bisulfite + urea". These results indicate that urea did not enhance the deamination efficiency. In the PCR, we did not observe significant improvements regarding the amounts of DNA necessary to obtain adequate amplification. Urea at 2 M, 4 M, and 8 M, showed no improvements. We conclude that urea gave no significant effect in the bisulfite genomic sequencing of the DNA used.