Enzymes Immobilized In Langmuir-Blodgett films: Why determining the surface properties in Langmuir monolayer is important?

In this review we discuss about the immobilization of enzymes in Langmuir-Blodgett films in order to determine the catalytic properties of these biomacromolecules when adsorbed on solid supports. Usually, the conformation of enzymes depends on the environmental conditions imposed to them, including the chemical composition of the matrix, and the morphology and thickness of the film. In this review, we show an outline of manuscripts that report the immobilization of enzymes as LB films since the 1980's, and also some examples of how the surface properties of the floating monolayer prepared previously to the transfer to the solid support are important to determine the efficiency of the resulting device.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Univ Fed Sao Paulo, Inst Environm Chem & Pharmaceut Sci, Rua Sao Nicolau 210, BR-09913030 Diadema, SP, BrazilUniv Fed Sao Paulo, Inst Environm Chem & Pharmaceut Sci, Rua Sao Nicolau 210, BR-09913030 Diadema, SP, BrazilFAPESP: 2015/10851-0Web of Scienc

Enzymes immobilized in Langmuir-Blodgett films: Why determining the surface properties in Langmuir monolayer is important?

ABSTRACT In this review we discuss about the immobilization of enzymes in Langmuir-Blodgett films in order to determine the catalytic properties of these biomacromolecules when adsorbed on solid supports. Usually, the conformation of enzymes depends on the environmental conditions imposed to them, including the chemical composition of the matrix, and the morphology and thickness of the film. In this review, we show an outline of manuscripts that report the immobilization of enzymes as LB films since the 1980’s, and also some examples of how the surface properties of the floating monolayer prepared previously to the transfer to the solid support are important to determine the efficiency of the resulting device

The design, development and application of novel, screen-printed amperometric glutamate biosensors

The aim of the studies presented in this thesis was to develop a screen-printed electrochemical biosensor for the measurement of glutamate and to apply this device to the determination of the analyte in food, serum and toxicity studies.Chapter 1 serves as an introduction to both the physiological significance of glutamate and the fundamental principles underpinning the electrochemical techniques used throughout this thesis.Chapter 2 is a review chapter, separated into two main sections. The first section details glutamate biosensors fabricated with glutamate oxidase (GluOx), the second section details biosensors fabricated with glutamate dehydrogenase (GLDH). The immobilization techniques, ease of fabrication and sample preparation techniques employed are compared. Biosensor characteristics such as sensitivity, limit of detection and linear range are summarised within a table.The studies described in Chapter 3 focus on the development of a non-reagentless glutamate biosensor. A Meldola’s Blue screen-printed carbon electrode (MB-SPCE) was employed as the base transducer. The biosensor was constructed by drop coating the biopolymer chitosan (CHIT) and GLDH onto the surface of the MB-SPCE. For this study, NAD+ was present in free solution. Meldola’s Blue served as the electrocatalyst, whereby NADH produced by the GLDH/NAD+ reaction, was electrocatalytically oxidised at a low operating potential (+0.1V (vs. Ag/AgCl)). The applied potential, temperature, pH and concentration of the co-factors required for the biosensor operation were optimised in this study. The assay exhibited a linear range of 12.5 µM to 150 µM, limit of detection of 1.5 µM, response time of 2s and a sensitivity of 0.44 nA/ µM. The optimised biosensor was subsequently applied to the determination of endogenous and fortified concentrations of glutamate in both serum and food samples (OXO cubes). The serum was fortified with and the resulting mean recovery was 96% with a CV of 3.3% (n = 6). For the food sample, an unfiltered beef OXO cube was analysed for monosodium glutamate (MSG) content. The endogenous content of MSG was 125.43 mg/g, with a CV of 8.98% (n = 6). The solution was fortified with 100mM of glutamate and a resulting mean recovery of 91% with a CV of 6.39% (n = 6) was determined.In Chapter 4, the glutamate biosensor was further developed in order to produce a reagentless device whereby the cofactor NAD+ and GLDH were immobilized on to the surface of the electrode utilising CHIT. The reagentless device was developed in order to monitor glutamate release from human liver carcinoma cells (HepG2) as a result of cell toxicity from exposure to paracetamol. The biosensor was miniaturised in the form of a microband biosensor, whereby one dimension of the electrode is of micrometre size and the other millimetre size. Micro bands exhibit unique diffusion properties in comparison to conventional sized electrodes. Calibration studies were carried out with an applied potential of +0.1V (vs. Ag/AgCl) using both phosphate buffer and cell media. In phosphate buffer the following microband biosensor characteristics were determined: linear range; 25 - 125µM, sensitivity; 0.0636 nA/µM and a theoretical limit of detection of 1.20µM. In cell media; linear range; 25 – 150 µM, sensitivity; 0.128 nA/µM and a theoretical limit of detection of 4.2µM. As the HepG2 cells were grown in an incubator at a fixed temperature and pH, studies were carried out at pH 7, 37ºC, in a 5% CO2 atmosphere. The miniaturised biosensor was applied to the determination of glutamate and the quantification was done by standard addition in cell media after 24 hours exposure to various concentrations of paracetamol. The average endogenous concentrations for glutamate released from the HepG2 cells was 52.07µM (CoV: 13.74%, n = 3), 93.30µM (CoV: 18.41%, n = 3) and 177.14µM (CoV: 14.54% n = 3) for 1mM, 5mM, 10mM doses of paracetamol respectively. The microband biosensor was also applied to the real time monitoring of glutamate over 8 hours. The standard deviations for the final current generated after eight hours are as follows; 1mM (coefficient of variation (CoV): 3.3%), 5mM (CoV: 9.056%) and 10mM (CoV: 13.18%). The study showed that the magnitudes of the steady state currents increased in proportion to the concentration of added paracetamol. The study also demonstrated the possibility of applying microband biosensors, over extended time periods, for toxicity studies; there is no significant removal of analyte owning to the small biosensor dimensions.Chapter 5 describes the development of a reagentless conventional sized glutamate biosensor whereby the cofactor NAD+ and GLDH were immobilized using a combination of multi-walled carbon nanotubes (MWCNT), CHIT and additional water based MB in a layer-by-layer fashion. The MWCNT/CHIT/MB combination facilitates electron transfer to the surface of working electrode. The MWCNT/CHIT also entraps GLDH and the NAD+ on the surface of the electrode. The pH, temperature, optimum applied potential, concentrations of NAD+, CHIT and the addition of water-based MB were optimised. The electrocatalyst MB allowed a operating potential of +0.1V (vs. Ag/AgCl) to be utilised. The biosensor was examined with standard glutamate solutions and the following biosensor characteristics were determined; linear range; 7 - 105µM, LOD; 3 µM, sensitivity; 0.39 nA/µM, response time 20-30s. A food sample was analysed for MSG and found to contain 90.56 mg/g with a CV of 7.52% (n = 5). The reagentless biosensor was also applied to the determination of glutamate in serum. The endogenous concentration was found to be 1.44mM (n = 5), CV: 8.54%. The recovery of glutamate in fortified serum was 104% (n = 5), CV of 2.91%. The results indicate that the new biosensor holds promise for food and biomedical studies

Toward realizing power scalable and energy proportional high-speed wireline links

Growing computational demand and proliferation of cloud computing has placed high-speed serial links at the center stage. Due to saturating energy efficiency improvements over the last five years, increasing the data throughput comes at the cost of power consumption. Conventionally, serial link power can be reduced by optimizing individual building blocks such as output drivers, receiver, or clock generation and distribution. However, this approach yields very limited efficiency improvement. This dissertation takes an alternative approach toward reducing the serial link power. Instead of optimizing the power of individual building blocks, power of the entire serial link is reduced by exploiting serial link usage by the applications. It has been demonstrated that serial links in servers are underutilized. On average, they are used only 15% of the time, i.e. these links are idle for approximately 85% of the time. Conventional links consume power during idle periods to maintain synchronization between the transmitter and the receiver. However, by powering-off the link when idle and powering it back when needed, power consumption of the serial link can be scaled proportionally to its utilization. This approach of rapid power state transitioning is known as the rapid-on/off approach. For the rapid-on/off to be effective, ideally the power-on time, off-state power, and power state transition energy must all be close to zero. However, in practice, it is very difficult to achieve these ideal conditions. Work presented in this dissertation addresses these challenges. When this research work was started (2011-12), there were only a couple of research papers available in the area of rapid-on/off links. Systematic study or design of a rapid power state transitioning in serial links was not available in the literature. Since rapid-on/off with nanoseconds granularity is not a standard in any wireline communication, even the popular test equipment does not support testing any such feature, neither any formal measurement methodology was available. All these circumstances made the beginning difficult. However, these challenges provided a unique opportunity to explore new architectural techniques and identify trade-offs. The key contributions of this dissertation are as follows. The first and foremost contribution is understanding the underlying limitations of saturating energy efficiency improvements in serial links and why there is a compelling need to find alternative ways to reduce the serial link power. The second contribution is to identify potential power saving techniques and evaluate the challenges they pose and the opportunities they present. The third contribution is the design of a 5Gb/s transmitter with a rapid-on/off feature. The transmitter achieves rapid-on/off capability in voltage mode output driver by using a fast-digital regulator, and in the clock multiplier by accurate frequency pre-setting and periodic reference insertion. To ease timing requirements, an improved edge replacement logic circuit for the clock multiplier is proposed. Mathematical modeling of power-on time as a function of various circuit parameters is also discussed. The proposed transmitter demonstrates energy proportional operation over wide variations of link utilization, and is, therefore, suitable for energy efficient links. Fabricated in 90nm CMOS technology, the voltage mode driver, and the clock multiplier achieve power-on-time of only 2ns and 10ns, respectively. This dissertation highlights key trade-off in the clock multiplier architecture, to achieve fast power-on-lock capability at the cost of jitter performance. The fourth contribution is the design of a 7GHz rapid-on/off LC-PLL based clock multi- plier. The phase locked loop (PLL) based multiplier was developed to overcome the limita- tions of the MDLL based approach. Proposed temperature compensated LC-PLL achieves power-on-lock in 1ns. The fifth and biggest contribution of this dissertation is the design of a 7Gb/s embedded clock transceiver, which achieves rapid-on/off capability in LC-PLL, current-mode transmit- ter and receiver. It was the first reported design of a complete transceiver, with an embedded clock architecture, having rapid-on/off capability. Background phase calibration technique in PLL and CDR phase calibration logic in the receiver enable instantaneous lock on power-on. The proposed transceiver demonstrates power scalability with a wide range of link utiliza- tion and, therefore, helps in improving overall system efficiency. Fabricated in 65nm CMOS technology, the 7Gb/s transceiver achieves power-on-lock in less than 20ns. The transceiver achieves power scaling by 44x (63.7mW-to-1.43mW) and energy efficiency degradation by only 2.2x (9.1pJ/bit-to-20.5pJ/bit), when the effective data rate (link utilization) changes by 100x (7Gb/s-to-70Mb/s). The sixth and final contribution is the design of a temperature sensor to compensate the frequency drifts due to temperature variations, during long power-off periods, in the fast power-on-lock LC-PLL. The proposed self-referenced VCO-based temperature sensor is designed with all digital logic gates and achieves low supply sensitivity. This sensor is suitable for integration in processor and DRAM environments. The proposed sensor works on the principle of directly converting temperature information to frequency and finally to digital bits. A novel sensing technique is proposed in which temperature information is acquired by creating a threshold voltage difference between the transistors used in the oscillators. Reduced supply sensitivity is achieved by employing junction capacitance, and the overhead of voltage regulators and an external ideal reference frequency is avoided. The effect of VCO phase noise on the sensor resolution is mathematically evaluated. Fabricated in the 65nm CMOS process, the prototype can operate with a supply ranging from 0.85V to 1.1V, and it achieves a supply sensitivity of 0.034oC/mV and an inaccuracy of ±0.9oC and ±2.3oC from 0-100oC after 2-point calibration, with and without static nonlinearity correction, respectively. It achieves a resolution of 0.3oC, resolution FoM of 0.3(nJ/conv)res2 , and measurement (conversion) time of 6.5μs

Electrophysiological study of dopamine neurons in the dorsal raphe nucleus & ventrolateral periaqueductal grey

The midbrain dopamine system plays a fundamental conserved role in regulating behaviour, and its dysfunction is associated with several neuropsychiatric disorders including addiction, schizophrenia, and Parkinson’s Disease. Midbrain dopamine neurons display considerable heterogeneity in their neurochemical, electrophysiological, and functional properties, and project to many cortical and subcortical structures. The majority of these neurons reside within the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA), but a less well-known, and understudied, dopamine population are housed within the dorsal raphe nucleus (DRN) and ventrolateral periaqueductal grey (vlPAG). These neurons provide the majority of the dopaminergic input to the central extended amygdala and have been implicated in the sleep-wake cycle and mediating the effects of opiates. However, their electrophysiological properties have not been examined. I have studied these neurons in mice using immunohistochemistry and electrophysiology in an acute brain slice preparation ex vivo. I found that they display similar properties to some VTA dopamine neurons, and noted co-expression of vasoactive intestinal peptide in a subset of this population. A characteristic feature of VTA dopamine neurons is the potentiation observed at glutamatergic synapses following a single dose of addictive drug or an acute stress experience. I investigated whether DRN/vlPAG dopamine neurons would show cocaine-induced plasticity and found that glutamatergic synapses were potentiated not only by a single dose of cocaine, but also by acute social isolation. This potentiation was associated with a change in AMPAR transmission, which is similar to that observed following cocaine in the VTA. I also investigated the hypothesis that this plasticity is a result of acute anxiety using behavioural analysis and administration of anxiolytic compounds. These findings suggest another form of salient stimulus which can induce plasticity in dopamine neurons, and have relevance to several neuropsychiatric diseases including those modelled by social isolation

Bovine Science

Bovine Science - Challenges and Advances presents up-to-date knowledge of bovine health, covering both introductory topics and more advanced concepts. Chapters cover such topics as new techniques in bovine science and development, health and risk factors and diagnosis of disease in bovines, and production and reproductive technologies and advancements