Andrea Medeiros Salgado

The evolution and developments of immunosensors for health and environmental monitoring: problems and perspectives

Abstract - This paper is an overview of the recent developments in immunosensors, which have attracted considerable attention. Immunosensors can play an important role in the improvement of public health by providing applications for which rapid detection, high sensitivity, and specificity are important, in areas such as clinical chemistry, food quality, and environmental monitoring. This review focuses on the current research in immunoassay methods based on electrochemical detection for the analysis of environmental samples or medical diagnostic methods with emphasis on recent advances, challenges and trends. Technological aspects in the development of immunosensors such as kinetics of biomolecular interaction, techniques of immobilization, simplification of assay procedures, immunointeration and catalytic studies and system miniaturization are presented Keywords : Immunoassay; Protein; Immunosensor; Bioengineering approach. INTRODUCTION Since the first biosensor of Clark & Lyons (Clark, 1992) aiming to detect glucose levels in serum samples, several analytes have been the aim of detection by the development of many biosensors - analytical devices that include a biologically sensitive element firmly immobilized or integrated into a physical transducer. Biosensors are one of the most promising lines in the production of analytical devices and monitoring. There is no doubt that the practical use in the medical area, in the food industry and in the monitoring of toxic substances in the environment has greatly stimulated the research and development of biosensors. These researches were stimulated mainly by the demands of clinical analyses and medical diagnoses; in particular, for the fast analysis of clinical preparations, for continuous monitoring

Development of an amperometric biosensor for phenol detection.

With the aim of searching for an in situ method for monitoring phenol, Agaricus bisporus tissue with tyrosine activity was used as a biocomponent and an oxygen electrode used as a transducer to develop a biosensor. The experimental methodology investigated the relation between dissolved oxygen and phenol concentration using a standard solution. Biosensor calibration was evaluated by studying reaction time and tissue amount necessary to promote a reliable response and to minimize errors. The influence of air saturation of the sample and washing of the electrode was also investigated. Results showed that 5 g of mushroom tissue with a 1 min reaction time promoted the best biosensor response within a phenol concentration range of 5–10 ppm. Washing of the electrode did not change the performance of the analysis; however, initial air saturation caused less variation amongst the samples.

Analysis of methane biodegradation by Methylosinus trichosporium OB3b

A oxidacao microbiologica de metano na atmosfera e realizada por bacterias metanotroficas, que o utilizam como fonte unica de carbono e energia. O objetivo deste trabalho consistiu na investigacao das melhores condicoes de biodegradacao do metano por bacterias metanotroficas Methylosinus trichosporium OB3b, que o oxidam a dioxido de carbono, para o emprego destes microrganismos em metodos de monitoracao para metano. Os resultados obtidos mostraram que M.trichosporium OB3b foi capaz de degradar o metano de forma mais eficaz partindo-se de uma concentracao inicial de microrganismos de 0.0700 g.L-1, a uma temperatura de 30oC, pH igual a 6.5 e empregando-se 1.79 mmol de metano. Nestas mesmas condicoes, nao houve crescimento bacteriano quando foram empregados 2.69 mmol de metano. A taxa especifica de crescimento do microrganismo, o fator de conversao, a eficiencia e a produtividade volumetrica para as condicoes otimizadas de biodegradacao foram, respectivamente, 0.0324 h-1, 0.6830 gcelulas/gCH4, 73.73% e 2.7732.10-3 gcelulas/L.h. O produto final da degradacao microbiologica do metano, o dioxido de carbono, foi quantificado atraves do emprego de um eletrodo comercial, e, atraves desta medida, foi calculado o percentual de conversao de metano em dioxido de carbono.

Development of a sucrose enzymatic biosensor

An enzymatic biosensor for sucrose determination was developed for on-line and continuous monitoring of sucrose concentration. The sensor was adapted to two different measurement schemes, one continuous and another injection sampling lines. The sensor adapted with the injection sampling line presented a linear measurement range of 5–20 g sucrose/1, good reproducibility, and a high versatility permitting the substitution of the immobilized enzymes when their activity decreased.

Development and Application of an Integrated System for Monitoring Ethanol Content of Fuels

An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-aminophenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05–1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyses/h, and relative SD of 3.5%. A new system was designed consisting of a microreactor with a 0.91-mL internal volume filled with alcohol oxidase immobilized on glass beads and an addition of free peroxidase, adapted in an FIA line, for continued reuse. This integrated biosensor-FIA system is being used for quality control of biofuels, gasohol, and hydrated ethanol. The FIA system integrated with the microreactor showed a calibration curve in the range of 0.05–1.5 g of ethanol/L, and good results were obtained compared with the ethanol content measured by high-performance liquid chromatography and gas chromatography standard methods.

A Sequential Enzymatic Microreactor System for Ethanol Detection of Gasohol Mixtures

A sequential enzymatic double microreactor system with dilution line was developed for quantifying ethanol from gasohol mixtures, using a colorimetric detection method, as a new proposal to the single micro reactor system used in previous work. Alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on glass beads, one in each microreactor, were used with phenol and 4-aminophenazone and the red-colored product was detected with a spectrophotometer at 555 nm. Good results were obtained with the immobilization technique used for both AOD and HRP enzymes, with best retention efficiencies of 95.3 +/- 2.3% and 63.2 +/- 7.0%, respectively. The two microreactors were used to analyze extracted ethanol from gasohol blends in the range 1-30 % v/v (10.0-238.9 g ethanol/L), with and without an on-line dilution sampling line. A calibration curve was obtained in the range 0.0034-0.087 g ethanol/L working with the on-line dilution integrated to the biosensor FIA system proposed. The diluted sample concentrations were also determined by gas chromatography (GC) and high-pressure liquid chromatography (HPLC) methods and the results compared with the proposed sequential system measurements. The effect of the number of analysis performed with the same system was also investigated.

Agaricus bisporus as a source of tyrosinase for phenol detection for future biosensor development

Phenols are toxic compounds that are present in several industrial wastewaters, so their quantification has great environmental importance. In order to permit an analytical methodology for in situ monitoring, this work aims to study the application of Agaricus bisporus tissue as a source of tyrosinase and the optimum reaction conditions for the development of a phenol biosensor. Such an enzyme is a polyphenol oxidase that transforms many different phenolic compounds into quinones. Experiments with fungi tissue were performed to evaluate different sizes of tissue (0.5, 1.0 and 1.5 cm), different temperatures (23.5°C to 60°C), and different pH values (6, 7 and 8) to quantify analytically phenol content. Amongst the tested conditions, those that had presented larger efficiency in phenol oxidation were attained with the fungal tissue size of 1 cm, at pH 8.0, in the temperature range from 35°C to 45°C.

Benzene as a Chemical Hazard in Processed Foods.

This paper presents a literature review on benzene in foods, including toxicological aspects, occurrence, formation mechanisms, and mitigation measures and analyzes data reporting benzene levels in foods. Benzene is recognized by the IARC (International Agency for Research on Cancer) as carcinogenic to humans, and its presence in foods has been attributed to various potential sources: packaging, storage environment, contaminated drinking water, cooking processes, irradiation processes, and degradation of food preservatives such as benzoates. Since there are no specific limits for benzene levels in beverages and food in general studies have adopted references for drinking water in a range from 1–10 ppb. The presence of benzene has been reported in various food/beverage substances with soft drinks often reported in the literature. Although the analyses reported low levels of benzene in most of the samples studied, some exceeded permissible limits. The available data on dietary exposure to benzene is minimal from the viewpoint of public health. Often benzene levels were low as to be considered negligible and not a consumer health risk, but there is still a need of more studies for a better understanding of their effects on human health through the ingestion of contaminated food.This paper presents a literature review on benzene in foods, including toxicological aspects, occurrence, formation mechanisms, and mitigation measures and analyzes data reporting benzene levels in foods. Benzene is recognized by the IARC (International Agency for Research on Cancer) as carcinogenic to humans, and its presence in foods has been attributed to various potential sources: packaging, storage environment, contaminated drinking water, cooking processes, irradiation processes, and degradation of food preservatives such as benzoates. Since there are no specific limits for benzene levels in beverages and food in general studies have adopted references for drinking water in a range from 1–10 ppb. The presence of benzene has been reported in various food/beverage substances with soft drinks often reported in the literature. Although the analyses reported low levels of benzene in most of the samples studied, some exceeded permissible limits. The available data on dietary exposure to benzene is minimal from the viewpoint of public health. Often benzene levels were low as to be considered negligible and not a consumer health risk, but there is still a need of more studies for a better understanding of their effects on human health through the ingestion of contaminated food.

Biosensors for Contaminants Monitoring in Food and Environment for Human and Environmental Health

Environmental security is one of the fundamental requirements of our well-being. However, it still remains a major global challenge, on account of the increasing number of potentially harmful pollutants (chemical compounds, toxins and pathogens) discharges into the environ‐ ment [1]. In this context, the detection and monitoring of environmental pollutants in soil, water and air is very important in the overall safety and security of humans, other animals and plants.

Model based soft-sensor for on-line determination of substrate

A software sensor for on-line determination of substrate was developed based on a model for fed-batch alcoholic fermentation process and on-line measured signals of ethanol, biomass, and feed flow. The ethanol and biomass signals were obtained using a colorimetric biosensor and an optical sensor developed in previous works that permitted determination of ethanol at a concentration of 0–40 g/L and biomass of 0–60 g/L. The volume in the fermentor could be continuously calculated using the total measured signal of the feed flow. The results obtained show that the model used is adequate for the proposed software sensor and determines continuously the substrate concentration with efficiency and security during the fermentation process.