Effect of surfactant type and redox polymer type on single-walled carbon nanotube modified electrodes.

Electrodes modified with single-walled carbon nanotubes (SWNTs) offer a number of attractive properties for developing novel electrochemical sensors. A common method to immobilize SWNTs onto the electrode surface is by placing a droplet of a SWNT suspension onto the electrode surface and allowing the solvent to evaporate. In order to maximize the properties of individual SWNTs, surfactants are normally present in these suspensions to provide stable and homogeneous SWNT dispersions. In this study we investigated the effect of different surfactants on the electrochemical and enzymatic performance of SWNT modified glassy carbon electrodes (GCEs). Amperometic biosensors for glucose were fabricated by a two-step procedure. In the first step, SWNT films were deposited onto GCEs by solution casting suspensions of SWNTs in water, Triton X-100, Tween 20, sodium cholate or sodium dodecylbenzenesulfonate (NaDDBS). In the second step, hydrogels containing a redox polymer and the enzyme, glucose oxidase (GOX), were deposited and cross-linked onto the SWNT-modified GCE. Three different redox polymers were tested: 3-ferrocenylpropyl-modified LPEI, (Fc-C3-LPEI), 6-ferrocenylhexyl-modified LPEI, (Fc-C6-LPEI), and poly[(vinylpyridine)Os(bipyridyl)2Cl](2+/3+)(PVP-Os). Biosensors constructed with SWNT films from suspensions of Triton X-100 or Tween 20 generally produced the highest electrochemical and enzymatic responses, with Triton X-100 films producing current densities of ~1.7-2.1 mA/cm(2) for the three different redox polymers. In contrast, biosensors constructed with SWNT films from sodium cholate suspensions resulted in significant decreases in the electrochemical and enzymatic response and in some cases showed no enzymatic activity. The results with SWNT films from NaDDBS suspensions were dependent upon the specific redox polymer used, but in general gave reduced enzymatic responses (~0.05-0.4 mA/cm(2)). These results demonstrate the importance of surfactant type in fabricating SWNT-modified electrode films.

Incorporation of single-walled carbon nanotubes into ferrocene-modified linear polyethylenimine redox polymer films.

In this study, we describe the effects of incorporating single-walled carbon nanotubes (SWNTs) into redox polymer-enzyme hydrogels. The hydrogels were constructed by combining the enzyme glucose oxidase with a redox polymer (Fc-C(6)-LPEI) in which ferrocene was attached to linear poly(ethylenimine) by a six-carbon spacer. Incorporation of SWNTs into these films changed their morphology and resulted in a significant increase in the enzymatic response at saturating glucose concentrations (3 mA/cm(2)) as compared to films without SWNTs (0.6 mA/cm(2)). Likewise, the sensitivity at 5 mM glucose was significantly increased in the presence of SWNTs (74 µA/cm(2)·mM) as compared to control films (26 µA/cm(2)·mM). We demonstrate that the increase in the electrochemical and enzymatic response of these films depends on the amount of SWNTs incorporated and the method of SWNT incorporation. Furthermore, we report that the presence of SWNTs in thick films allows for more of the ferrocene redox centers to become accessible. The high current densities of the hydrogels should allow for the construction of miniature biosensors and enzymatic biofuel cells.

High-throughput DNA sequencing on a capillary array electrophoresis system.

A capillary array electrophoresis apparatus capable of running and analyzing 48 DNA sequencing samples simultaneously has been constructed. The instrument uses a replaceable sieving buffer and incorporates a convenient method for introducing the buffer into the capillaries. Data from laser-induced fluorescence are collected as four separate images, one for each optical channel. The integrated data analysis software employs an open architecture that allows use of any DNA base-calling algorithm. DNA sequencing runs are completed in approx. 1 hr (approximately 500 bases), and instrument turnaround time between runs is less than 15 min. Overall, the instrument throughput is on the order of 720 templates/day, or 360,000 bases/day.

Increase in asthma and a high prevalence of bronchitis: results from a population study among adults in urban and rural Vietnam.

BACKGROUND: While a large amount of data about the epidemiology of asthma, COPD, chronic bronchitis and respiratory symptoms are available from developed countries, the information about these diseases in developing countries in south-east Asia are scarce. AIM: Assess the prevalence of respiratory diseases and symptoms and their relation with demographic data including smoking habits among adults in rural and urban Vietnam. METHODS: A random sample of subjects aged 21-70 years were invited; 3008 subjects living in an inner city area of Hanoi and 4000 in a rural area of Bavi in northern Vietnam. An internationally used questionnaire was delivered by field workers to the study subjects. The questionnaire was completed by the subjects, or when necessary, by the field workers after reading the questions for the study participants. RESULTS: The response rate was 92% in Bavi and 70% in Hanoi. Of men in Bavi 67.8% (Hanoi 49.7%; p < 0.001) were smokers, while of women 4.2% were smokers in Hanoi (Bavi 1.2%; p < 0.001). The prevalence of ever asthma was in Hanoi 5.6% (Bavi 3.9%; p = 0.003) with no major gender difference. The most common symptom was longstanding cough (Hanoi 18.1%, Bavi 12.0%; p < 0.001) followed by sputum production, while the prevalence of symptoms common in asthma was considerably lower. Although the large difference in smoking habits, respiratory symptoms tended to be only slightly more common in men than women. Family history of asthma and chronic bronchitis, respectively, were strongly associated with both diseases. CONCLUSIONS: The prevalence of asthma in adults may have increased in both urban and rural Vietnam, as the few previous estimates have found 2% of adults having asthma. Half of men in Hanoi and two-thirds in Bavi were smokers versus a few percent of women in both areas. Bronchitic symptoms were common in both men and women.

High-sensitivity amperometric biosensors based on ferrocene-modified linear poly(ethylenimine).

Amperometric biosensors for glucose and hydrogen peroxide have been built by immobilizing glucose oxidase (GOX) and horseradish peroxidase (HRP) in cross-linked films of ferrocene-modified linear poly(ethylenimine). At pH 7, the glucose sensors generated limiting catalytic current densities of 1.2 mA/cm2. These current densities are approximately 4 times higher than those with other ferrocene-based redox polymers and are comparable to the highest reported values for osmium-based redox polymers with GOX. Because of the high sensitivity of these films (73 nA/cm2.microM), glucose concentrations in the micromolar range could be detected. Similarly, sensors were constructed with HRP-generated current densities of 0.9 mA/cm2 under saturation conditions and sensitivities of 500 nA/cm2.microM. The results show that the ability of Fc-LPEI to effectively communicate with a variety of enzymes has potential applications in measuring low substrate concentrations in implantable biosensors and producing high current outputs in enzymatic biofuel cells.

Automated carboxy-terminal sequence analysis of polypeptides containing C-terminal proline.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on automation of the thiocyanate chemistry using diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J. M., Nikfarjam, F., Shenoy, N. S., and Shively, J. E. (1992) Protein Sci. 1, 1622-1633). A major limitation of this approach was the inability to derivatize C-terminal proline. We now describe chemistry based on the DPP-ITC/pyridine reaction which is capable of derivatizing C-terminal proline to a thiohydantoin. The reaction of DPP-ITC/pyridine with C-terminal proline rapidly forms an acyl isothiocyanate which is capable of forming a quaternary amine containing thiohydantoin. Unlike formation of peptidylthiohydantoins with the other 19 commonly occurring amino acids in which cyclization to a thiohydantoin is concomitant with loss of a proton from the amide nitrogen, proline has no amide proton and as a result the newly formed proline thiohydantoin contains an unprotonated ring nitrogen. This cyclic structure if left unprotonated will regenerate C-terminal proline during the cleavage reaction. However, if protonated by the addition of acid, the proline thiohydantoin ring is stabilized and can be readily hydrolyzed to proline thiohydantoin and a shortened peptide by the addition of water vapor or alternatively by sodium or potassium trimethylsilanolate, the reagent normally used for the cleavage reaction. By introducing vaporphase trifluoroacetic acid (TFA) for the protonation reaction and water vapor for the hydrolysis reaction we have been able to automate the chemistry required for derivatization of C-terminal proline.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.

Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.

Short tandem repeat typing by capillary array electrophoresis: comparison of sizing accuracy and precision using different buffer systems.

Polymorphic microsatellite markers are widely used in gene discovery and mapping, human identification, agricultural genetics, and diagnosis of triplet-repeat expansion disorders. Reliable genotyping of these markers requires polymerase chain reaction (PCR) amplification and very-high-resolution electrophoresis. Capillary array electrophoresis offers extremely fast, high-resolution separation of DNA and more automated sample processing because labor-intensive slab-gel pouring and sample loading are eliminated. We report a simple, reliable procedure for preparing PCR samples for electrokinetic injection into capillaries using a 96-well tray and float dialysis. We developed an improved sizing standard for genotyping and used it to evaluate systematically the sizing accuracy and precision of low-viscosity, replaceable matrix formulations. Our study sizing over 28,000 alleles yielded an average precision of +/- 0.12 bp for fragments up to 350 bp. Low-viscosity formulations permit low-pressure matrix injection (40 psi) and a turnaround time of 70 min for 48-96 samples.