一种用于CYP3A5基因分型的电化学传感器阵列设计

目的：设计一种用于检测CYP3A5基因分型的电化学传感器阵列及其不同基因型的判别方法。方法：设计的电化学基体由印刷电路板（PCB）组成,该电路板包含一组金电极。每个金电极表面修饰有包含单链捕获探针的自组装单分子膜。设计中使用二茂铁做为电活性指示剂,基因分型检测是通过两种不同电势的二茂铁衍生物分别标记等位基因特异性信号探针来实现。结果：该设计能构建一种快速准确、操作简便的DNA电化学传感器阵列检测系统。结论：本文设计为使用电化学方法检测基因分型提供了一种新方法和新技术。     

一种用于CYP3A5基因分型的电化学传感器阵列设计

目的:设计一种用于检测CYP3A5基因分型的电化学传感器阵列及其不同基因型的判别方法。方法:设计的电化学基体由印刷电路板(PCB)组成,该电路板包含一组金电极。每个金电极表面修饰有包含单链捕获探针的自组装单分子膜。设计中使用二茂铁做为电活性指示剂,基因分型检测是通过两种不同电势的二茂铁衍生物分别标记等位基因特异性信号探针来实现。结果:该设计能构建一种快速准确、操作简便的DNA电化学传感器阵列检测系统。结论:本文设计为使用电化学方法检测基因分型提供了一种新方法和新技术。     

On-line measurement of gas production rates

Gas evolution rates represent an important variable to track in biological and certain electrochemical processes. Accurate gas flow rate sensors exist for gas streams possessing a pressure head, such as when pressurized air or oxygen is delivered to a fermentation process. However, these devices impose pressure heads that can inhibit gas production and, therefore, yield false measurements. Examples of effected processes would include electrochemical production of a gas at the electrode (e.g., electrolysis) or anaerobic fermentation (e.g., anaerobic production of methane). In this work, we present an on-line gas measurement technique that measures on-line gas production from an anaerobic microbial process that is continuously fed simulated food waste over a 6-month period. Commentary is given on the sensor's accuracy and ease of use within the context of long-term operation, ability to measure both low and high gas production rates, as well as its potential for process control and system-health monitoring.     

Electrochemical treatment of graphite to enhance electron transfer from bacteria to electrodes

In this paper, graphite felts were continuously electrochemically oxidized to increase the current generation in microbial fuel cells (MFCs). The treated and untreated graphite felts were utilized as anodes in MFCs and current production was compared. The current production on electrochemically treated graphite felt anodes was about 1.13 mA, 39.5% higher compared with that of MFCs containing untreated anodes. The results demonstrated that the electronic coupling between graphite felt electrodes and electrogenic bacteria could be enhanced by electrochemical oxidization of the electrodes. Further study showed that the newly generated carboxyl containing functional groups from electrochemical oxidization were responsible for the enhanced electron transfer, due to their strong hydrogen bonding with peptide bonds in bacterial cytochromes.     

Microfabricated Microbial Fuel Cell Arrays Reveal Electrochemically Active Microbes

Microbial fuel cells (MFCs) are remarkable “green energy” devices that exploit microbes to generate electricity from organic compounds. MFC devices currently being used and studied do not generate sufficient power to support widespread and cost-effective applications. Hence, research has focused on strategies to enhance the power output of the MFC devices, including exploring more electrochemically active microbes to expand the few already known electricigen families. However, most of the MFC devices are not compatible with high throughput screening for finding microbes with higher electricity generation capabilities. Here, we describe the development of a microfabricated MFC array, a compact and user-friendly platform for the identification and characterization of electrochemically active microbes. The MFC array consists of 24 integrated anode and cathode chambers, which function as 24 independent miniature MFCs and support direct and parallel comparisons of microbial electrochemical activities. The electricity generation profiles of spatially distinct MFC chambers on the array loaded with Shewanella oneidensis MR-1 differed by less than 8%. A screen of environmental microbes using the array identified an isolate that was related to Shewanella putrefaciens IR-1 and Shewanella sp. MR-7, and displayed 2.3-fold higher power output than the S. oneidensis MR-1 reference strain. Therefore, the utility of the MFC array was demonstrated.     

Development of an automated flow-injection system for the determination of trace level ammonia

An in-housed designed computerised flow injection system for low level ammonia analysis is examined. The system features an on-line microdistillation preconcentration unit, which was used as an on-line sample pretreatment step in an ammonia gas-sensing probe flow injection system. A simple, low cost computerised control and data acquisition system was designed using a commercial pH meter with RS-232 interface and in-house designed control system. The system offered a practical and effective means of extending the detection limit of commercial available ammonia gas sensing probes to 5 μg/1 NH₃N.     

Are Seafood PCB Data Sufficient to Assess Health Risk for High Seafood Consumption Groups?

Some populations in Washington's Puget Sound area consume much more seafood than the general population. Mean consumption rates are 61?g/person/day for the Tulalip and Squaxin Island Tribes and 117.2?g/person/day for Asian and Pacific Islanders (API). There is concern about possible health risks from seafood PCB exposure for these groups, but exposure evaluation is difficult due to inadequacies of environmental data. Available seafood PCB data were matched to results from recent seafood consumption surveys. To rate quality of matches and identify data gaps, a ranking system based on species specificity, data quality, and location compatibility was developed. Sensitivity of total PCB and congenerspecific PCB testing (for use in TCDD toxic equivalency approaches) necessary for cancer risk assessment was explored and included in the ranking scheme. For the Squaxin Tribe, appropriate total PCB data for risk assessment were available for 58% of seafood consumed, which is dominated by local salmon. For API, appropriate total PCB data were identified for only 4% of seafood consumed, which is dominated by commercial shellfish. Insufficient sensitivity of commercial seafood PCB analysis and overall lack of sufficiently sensitive PCB congener analysis are major gaps in ability to characterize PCB exposure and risk for these groups.     

High-Throughput RAD-SNP Genotyping for Characterization of Sugar Beet Genotypes

High-throughput single-nucleotide polymorphism (SNP) genotyping provides a rapid way of developing resourceful sets of markers for delineating genetic structure and for understanding the basis of the taxonomic discrimination. In this paper, we present a panel of 192 SNPs for effective genotyping in sugar beet using a high-throughput marker array technology, QuantStudio 12K Flex system, coupled with Taqman OpenArray technology. The selected SNPs were evaluated for genetic diversity among a set of 150 individuals representing 15 genotypes (10 individuals each) from five cytoplasmic male steriles (CMSs), five pollinators, and five commercial varieties. We demonstrated that the proposed panel of 192 SNPs effectively differentiated the studied genotypes. A higher degree of polymorphism was observed among the CMSs as compared to pollinators and commercial varieties. PCoA and STRUCTURE analysis revealed that CMSs, pollinators, and varieties clustered into three distinct subpopulations. Our results demonstrate the utility of the identified panel of 192 SNPs coupled with TaqMan OpenArray technology as a wide set of markers for high-throughput SNP genotyping in sugar beet.     

A Novel Rabbit Immunospot Array Assay on a Chip Allows for the Rapid Generation of Rabbit Monoclonal Antibodies with High Affinity

Antigen-specific rabbit monoclonal antibodies (RaMoAbs) are useful due to their high specificity and high affinity, and the establishment of a comprehensive and rapid RaMoAb generation system has been highly anticipated. Here, we present a novel system using immunospot array assay on a chip (ISAAC) technology in which we detect and retrieve antigen-specific antibody-secreting cells from the peripheral blood lymphocytes of antigen-immunized rabbits and produce antigen-specific RaMoAbs with 10^–12 M affinity within a time period of only 7 days. We have used this system to efficiently generate RaMoAbs that are specific to a phosphorylated signal-transducing molecule. Our system provides a new method for the comprehensive and rapid production of RaMoAbs, which may contribute to laboratory research and clinical applications.     

Acoustic microfluidic chip technology to facilitate automation of phage display selection

Modern tools in proteomics require access to large arrays of specific binders for use in multiplex array formats, such as microarrays, to decipher complex biological processes. Combinatorial protein libraries offer a solution to the generation of collections of specific binders, but unit operations in the process to isolate binders from such libraries must be automatable to ensure an efficient procedure. In the present study, we show how a microfluidic concept that utilizes particle separation in an acoustic force field can be used to efficiently separate antigen-bound from unbound members of such libraries in a continuous flow format. Such a technology has the hallmarks for incorporation in a fully automated selection system for the isolation of specific binders.     

Overcoming challenges in WAVE Bioreactors without feedback controls for pH and dissolved oxygen

The biopharmaceutical industry is increasing its use of the WAVE Bioreactor for culturing cells. Although this disposable bioreactor can be equipped to provide real-time pH and dissolved oxygen (DO) monitoring and control, our goal was to develop a process for culturing CHO cells in this system without relying on pH and DO feedback controls. After identifying challenges in culturing cells without controlling for pH and DO in the WAVE Bioreactor, we characterized O(2) and CO(2) transfer in the system. From these cell-free studies, we identified rock rate and rock angle as key parameters affecting O(2) transfer. We also identified the concentration of CO(2) in the incoming gas and the rate of gas flow into the headspace as key parameters affecting CO(2) transfer--and therefore pH--in the disposable culture chamber. Using a full-factorial design to evaluate the rock rate, rock angle, and gas flow rate defined for this WAVE Bioreactor process, we found comparable cell growth and pH profiles in the ranges tested for these three parameters in two CHO cell lines. This process supported cell growth, and maintained pH and DO within our desired range--pH 6.8-7.2 and DO exceeding 20% of air saturation--for six CHO cell lines, and it also demonstrated comparable cell growth and viability with the stirred-tank bioreactor process with online pH and DO control. By eliminating the use of pH and DO probes, this process provides a simple and more cost-effective method for culturing cells in the WAVE Bioreactor.     

Characterization and electrocatalytic properties of Prussian blue electrochemically deposited on nano-Au/PAMAM dendrimer-modified gold electrode

Gold electrode was modified with 3-mercaptopropionic acid (MPA) and further reacted with poly(amidoamine) (PAMAM) dendrimer (generation 4.0) then attached the nano-Au to obtain films on which Prussian blue (PB) was electrochemically deposited to afford much wider pH adaptive range, much better electrochemical stability and excellent electrochemical response. The microstructure and electrochemical behavior of Au/MPA/PAMAM/nano-Au/PB electrode were investigated by scanning electron microscopy (SEM) and cyclic voltammetry. The electrochemical response of the Au/MPA/PAMAM/nano-Au/PB-modified electrode for the electrocatalytic reduction of hydrogen peroxide was investigated, and it was found that the sensitivity as well as the corresponding detection limits were improved as compared to the voltammetric response of a Au/PB-modified electrode and Au/MPA/PAMAM/PB electrode. Based on this, a new electrochemical sensor for determination of hydrogen peroxide has been developed.     

Next-generation teaching: a template for bringing genomic and bioinformatic tools into the classroom

The recent increase in accessibility and scale of genetic data available through next-generation sequencing (NGS) technology has transformed biological inquiry. As a direct result, the application and analysis of NGS data has quickly become an important skill for future scientists. However, the steep learning curve for applying NGS technology to biological questions, including the complexity of sample preparation for sequencing and the analysis of large data sets, are deterrents to the integration of NGS into undergraduate education. Here, we present a course-based undergraduate research experience (CURE) designed to aid in overcoming these limitations through NGS investigations of prokaryotic diversity. Specifically, we use 16S rRNA sequencing to explore patterns of diversity stemming from student-directed hypothesis development. This CURE addresses three learning objectives: (1) it provides a forum for experimental design hypothesis generation, (2) it introduces modern genomic tools through a hands-on experience generating an NGS data-set, and (3) it provides students with an introductory experience in bioinformatics.     

Microbial catalysis in bioelectrochemical technologies: status quo, challenges and perspectives

Over the past decade, microbial electrochemical technologies, originally developed from an interesting physiological phenomenon, have evolved from a rush of initiatives for sustainable bioelectricity generation to a multitude of specialized applications in very different areas. Genetic engineering of microbial biocatalysts for target bioelectrochemical applications like biosensing or bioremediation, as well as the discovery of entirely new bioelectrochemical processes such as microbial electrosynthesis of commodity chemicals, open up completely new possibilities. Where stands this technology today? And what are the general and specific challenges it faces not only scientifically but also for transition into commercial applications? This review intends to summarize the recent advances and provides a perspective on future developments.     

Electrochemical detection for high-performance liquid chromatography of ketoconazole in plasma and saliva

Ketoconazole, cis-1-acetyl-4-[4[[2-(2,4-dichlorophenyl)-2-(1H-imidazol- 1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine, a clinically used antifungal agent, is also an inhibitor of steroid hormone biosynthesis. A high-performance liquid chromatographic method is described which resolves ketoconazole with selectivity and high sensitivity provided by the use of electrochemical detection. Ketoconazole can be detected in high-performance liquid chromatography by electrochemical oxidation at a glassy carbon electrode at a potential of +1.0 V. Electrochemical detection offers improved sensitivity and selectivity over ultraviolet absorbance or fluorescence detection after derivatization. The method utilizes a volatile buffer system compatible with postcolumn analyses and an internal standard which is electrochemically active. This technique provides a simple method to assay ketoconazole. Ketoconazole can be detected in human plasma and saliva after a single oral therapeutic dose.     

Online electrochemical monitoring of nitric oxide during photodynamic therapy

Photodynamic therapy (PDT), as a novel treatment modality, is based on the use of a photosensitizing agent with an excitation light source for the treatment of various malignancies. Its effect is mediated through reactive oxygen species and nitric oxide (NO), which are shown to be present in apoptosis. Individual differences among patients and even in different areas of the same tumor in one patient may cause a major problem with PDT: dose calculation during application of the light. An electrochemical sensor is proposed for online monitoring of NO generation as a solution of this problem. 5-Aminolevulinic acid (ALA) was administered as the photosensitizer in rat cerebellum. An amperometric sensor, selective to NO, was designed and tested both in vitro and in vivo during PDT. ALA-mediated PDT resulted in rapid generation of NO, starting as early as the application of light on the tissue. Simultaneous amperometric recordings have been carried out for 5 min during PDT. The progressive increase in NO concentration peaked at 1.10 min and then the response current began to decrease until it reached a plateau at around 70% of its peak value. This study, for the first time, electrochemically demonstrates the generation of NO during PDT. Rapid and stable responses obtained by the experimental setup confirmed that this method could be used as an online monitoring system for PDT-mediated apoptosis.     

Theoretical and experimental comparisons of gene expression indexes for oligonucleotide arrays

MOTIVATION: Oligonucleotide expression arrays exhibit systematic and reproducible variation produced by the multiple distinct probes used to represent a gene. Recently, a gene expression index has been proposed that explicitly models probe effects, and provides improved fits of hybridization intensity for arrays containing perfect match (PM) and mismatch (MM) probe pairs. RESULTS: Here we use a combination of analytical arguments and empirical data to show directly that the estimates provided by model-based expression indexes are superior to those provided by commercial software. The improvement is greatest for genes in which probe effects vary substantially, and modeling the PM and MM intensities separately is superior to using the PM-MM differences. To empirically compare expression indexes, we designed a mixing experiment involving three groups of human fibroblast cells (serum starved, serum stimulated, and a 50:50 mixture of starved/stimulated), with six replicate HuGeneFL arrays in each group. Careful spiking of control genes provides evidence that 88-98% of the genes on the array are detectably transcribed, and that the model-based estimates can accurately detect the presence versus absence of a gene. The use of extensive replication from single RNA sources enables exploration of the technical variability of the array.     

The efficiencies of the component steps of oxidative phosphorylation. II. Experimental determination of the efficiencies in mitochondria and examination of the equivalence of membrane potential and pH gradient in phosphorylation

In the accompanying article (T.E. Gunter and B.D. Jensen, 1986 Arch. Biochem. Biophys. 248, 289-304), a method is described for measuring the efficiencies of individual steps of the process of oxidative phosphorylation. The results of applying this method to the case of state 3 phosphorylation in rat liver mitochondria are reported here. The rate of energy use (or power use) at the gradient generation, leakage, and phosphorylation steps are reported as efficiencies and energy use factors in tabular form. The limits of the degrees of coupling of the gradient generation and phosphorylation steps are also determined and under the current conditions of measurement these degrees of coupling are found to be quite close to unity. The data can be used to show that the only sets of the stoichiometric parameters noH (the charge/2e- ratio in this case from succinate to oxygen), nPH (the H+/ATP ratio), and nTH (number of protons translocated during substrate-product transport) which are simultaneously consistent with both the laws of thermodynamics and with the current data are 8, 3, 1, and 6, 3, 0. The The efficiency of the phosphorylation step which is independent of noH and nTH averages 80% for the control data analyzed. If noH is 8 (succinate to oxygen), the average value of the efficiency of generation of the electrochemical proton gradient is approximately 91 percent. Since very little power (energy) would then be left over to be coupled in parallel to phosphorylation through some other means of coupling, this would place the electrochemical proton gradient in the direct path of power flow and identify it as "an" intermediate in the process. This would suggest that any other intermediate should be considered as being "in series" with the electrochemical proton gradient. The agents butyrate and propionate have been employed to permit investigation over a range of pH gradient and membrane potential. Both butyrate and propionate decrease the efficiency of generation of the electrochemical proton gradient and increase proton leakage. In addition, butyrate activates electron transport whereas propionate inhibits it. By using butyrate to modify the values of pH gradient and membrane potential, it can be shown that the ratio of the efficiency with which the pH gradient is used in phosphorylation to that with which the membrane potential is used is 1.08 +/- 0.38.     

Electrochemical Gating of Tricarboxylic Acid Cycle in Electricity-Producing Bacterial Cells of Shewanella

Energy-conversion systems mediated by bacterial metabolism have recently attracted much attention, and therefore, demands for tuning of bacterial metabolism are increasing. It is widely recognized that intracellular redox atmosphere which is generally tuned by dissolved oxygen concentration or by appropriate selection of an electron acceptor for respiration is one of the important factors determining the bacterial metabolism. In general, electrochemical approaches are valuable for regulation of redox-active objects. However, the intracellular redox conditions are extremely difficult to control electrochemically because of the presence of insulative phospholipid bilayer membranes. In the present work, the limitation can be overcome by use of the bacterial genus Shewanella , which consists of species that are able to respire via cytochromes abundantly expressed in their outer-membrane with solid-state electron acceptors, including anodes. The electrochemical characterization and the gene expression analysis revealed that the activity of tricarboxylic acid (TCA) cycle in Shewanella cells can be reversibly gated simply by changing the anode potential. Importantly, our present results for Shewanella cells cultured in an electrochemical system under poised potential conditions showed the opposite relationship between the current and electron acceptor energy level, and indicate that this unique behavior originates from deactivation of the TCA cycle in the (over-)oxidative region. Our result obtained in this study is the first demonstration of the electrochemical gating of TCA cycle of living cells. And we believe that our findings will contribute to a deeper understanding of redox-dependent regulation systems in living cells, in which the intracellular redox atmosphere is a critical factor determining the regulation of various metabolic and genetic processes.     

An Interface, Allowing a Commercial Paper-Tape Reader to be used in the Continuous Programming of Growth-Room Temperatures

A circuit is described which forms a link between a commerciallyavailable tape reader and the temperature-control circuit ofa plant growth-room. Successive temperature adjustments areat 5 min intervals and the resulting programming system, subjectto the limitations of the room control circuit, has a possibletemperature span of 49·5 °C with a resolution of0·5 °C. The paper tapes used may be (i) derived fromexisting data for the simulation of field conditions, (ii) outputfrom a computer program to produce a temperature curve whichobeys a mathematical law, or (iii) manually punched. The tapesmay provide a continuously changing regime for a successionof days or, for diurnal repetition, may be spliced to form aloop. The system provides the means of studying plant responseto fluctuating regimes of temperature and other environmentalfactors.