Sol-Gel Chemistry in Biosensing Devices of Electrical Transduction: Application to CEA Cancer Biomarker

Sol-gel chemistry allows the immobilization of organic molecules of biological origin on suibtable solid supports, permitting their integration into biosensing devices widening the possibility of local applications. The present work is an application of this principle, where the link between electrical receptor platform and the antibody acting as biorecognition element is made by sol-gel chemistry. The immunosensor design was targeted for carcinoembryonic antigen (CEA), an important biomarker for screening the colorectal cancer, by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SVW). The device displayed linear behavior to CEA in EIS and in SWV assays ranging from 0.50 to 1.5ng/mL, and 0.25 to 1.5ng/mL, respectively. The corresponding detection limits were 0.42 and 0.043 ng/mL. Raman spectroscopy was used to characterize the surface modifications on the conductive platform (FTO glass). Overall, simple sol-gel chemistry was effective at the biosensing design and the presented approach can be a potential method for screening CEA in point-of-care, due to the simplicity of fabrication, short response time and low cost. - See more at: http://www.eurekaselect.com/127192/article#sthash.m1AWhINx.dpuf

Protein-responsive polymers for point-of-care detection of cardiac biomarker

This work describes a novel use for the polymeric film, poly(o-aminophenol) (PAP) that was made responsive to a specific protein. This was achieved through templated electropolymerization of aminophenol (AP) in the presence of protein. The procedure involved adsorbing protein on the electrode surface and thereafter electroploymerizing the aminophenol. Proteins embedded at the outer surface of the polymeric film were digested by proteinase K and then washed away thereby creating vacant sites. The capacity of the template film to specifically rebind protein was tested with myoglobin (Myo), a cardiac biomarker for ischemia. The films acted as biomimetic artificial antibodies and were produced on a gold (Au) screen printed electrode (SPE), as a step towards disposable sensors to enable point-of-care applications. Raman spectroscopy was used to follow the surface modification of the Au-SPE. The ability of the material to rebind Myo was measured by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The devices displayed linear responses to Myo in EIS and SWV assays down to 4.0 and 3.5 μg/mL, respectively, with detection limits of 1.5 and 0.8 μg/mL. Good selectivity was observed in the presence of troponin T (TnT) and creatine kinase (CKMB) in SWV assays, and accurate results were obtained in applications to spiked serum. The sensor described in this work is a potential tool for screening Myo in point-of-care due to the simplicity of fabrication, disposability, short time response, low cost, good sensitivity and selectivity.

Novel biosensing device for point-of-care applications with plastic antibodies grown on Au-Screen Printed Electrodes

A gold screen printed electrode (Au-SPE) was modified by merging Molecular Imprinting and Self-Assembly Monolayer techniques for fast screening cardiac biomarkers in point-of-care (POC). For this purpose, Myoglobin (Myo) was selected as target analyte and its plastic antibody imprinted over a glutaraldehyde (Glu)/cysteamine (Cys) layer on the gold-surface. The imprinting effect was produced by growing a reticulated polymer of acrylamide (AAM) and N,N′-methylenebisacrylamide (NNMBA) around the Myo template, covalently attached to the biosensing surface. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) studies were carried out in all chemical modification steps to confirm the surface changes in the Au-SPE. The analytical features of the resulting biosensor were studied by different electrochemical techniques, including EIS, square wave voltammetry (SWV) and potentiometry. The limits of detection ranged from 0.13 to 8 μg/mL. Only potentiometry assays showed limits of detection including the cut-off Myo levels. Quantitative information was also produced for Myo concentrations ≥0.2 μg/mL. The linear response of the biosensing device showed an anionic slope of ~70 mV per decade molar concentration up to 0.3 μg/mL. The interference of coexisting species was tested and good selectivity was observed. The biosensor was successfully applied to biological fluids.

Smart Plastic Antibody Material (SPAM) tailored on disposable screen printed electrodes for protein recognition: application to Myoglobin detection

This work introduces two major changes to the conventional protocol for designing plastic antibodies: (i) the imprinted sites were created with charged monomers while the surrounding environment was tailored using neutral material; and (ii) the protein was removed from its imprinted site by means of a protease, aiming at preserving the polymeric network of the plastic antibody. To our knowledge, these approaches were never presented before and the resulting material was named here as smart plastic antibody material (SPAM). As proof of concept, SPAM was tailored on top of disposable gold-screen printed electrodes (Au-SPE), following a bottom-up approach, for targeting myoglobin (Myo) in a point-of-care context. The existence of imprinted sites was checked by comparing a SPAM modified surface to a negative control, consisting of similar material where the template was omitted from the procedure and called non-imprinted materials (NIMs). All stages of the creation of the SPAM and NIM on the Au layer were followed by both electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). AFM imaging was also performed to characterize the topography of the surface. There are two major reasons supporting the fact that plastic antibodies were effectively designed by the above approach: (i) they were visualized for the first time by AFM, being present only in the SPAM network; and (ii) only the SPAM material was able to rebind to the target protein and produce a linear electrical response against EIS and square wave voltammetry (SWV) assays, with NIMs showing a similar-to-random behavior. The SPAM/Au-SPE devices displayed linear responses to Myo in EIS and SWV assays down to 3.5 μg/mL and 0.58 μg/mL, respectively, with detection limits of 1.5 and 0.28 μg/mL. SPAM materials also showed negligible interference from troponin T (TnT), bovine serum albumin (BSA) and urea under SWV assays, showing promising results for point-of-care applications when applied to spiked biological fluids.

Sol-gel biomimetic material designed to target CEA cancer biomarker

1st ASPIC International Congress

BIiossensor Enzimático para avaliação da Capacidade Antioxidente

Os radicais livres formam-se naturalmente nos organismos vivos, pois a sua produção/geração está interligada com o processo de produção de energia (respiração), processos inflamatórios (fagocitose), regulação do crescimento celular, sinalização intercelular e síntese de substâncias biológicas relevantes. Estes também podem ser introduzidos por vias exógenas (poluição, radiação, tabaco, alimentação, etc). Os radicais livres têm capacidade de reagir com o material nucleico (ADN e ARN), proteínas e substâncias oxidáveis, causando danos oxidativos responsáveis pelo envelhecimento e originar doenças degenerativas, tais como, o cancro, arteriosclerose, artrite reumatoide, entre outras. De forma a combater os efeitos pejorativos provocados pelos radicais, os organismos vivos desenvolveram complexos sistemas de defesa antioxidante. Estes sistemas são constituídos por antioxidantes endógenos, produzidos pelos seres vivos, tais como enzimas ou por antioxidantes exógenos obtidos por via da alimentação (por exemplo o ácido ascórbico). Neste sentido, um antioxidante tem capacidade de eliminar ou reduzir a propagação da cadeia de geração de radicais livres. Neste trabalho foi desenvolvido um biossensor enzimático para a quantificação da capacidade antioxidante total de matrizes alimentares. A construção deste biossensor consistiu na eletroimobilização da adenina no elétrodo de pasta de carbono (EPC) ou na adsorção física da dA20 na superfície do EPC. O dano oxidativo foi induzido pelo radical hidroxilo gerado pela reação de Fenton. Nesta dissertação, foi estudada a capacidade de alguns antioxidantes em eliminar o efeito pejorativo dos radicais livres e combater a integridade das bases de adenina ou do dA20.Os antioxidantes estudados foram o ácido ascórbico e alguns ácidos fenólicos como o ácido hidroxibenzoico (ácido gálico) e ácidos hidroxicinâmicos (ácido cafeico e ácido cumárico). Estes antioxidantes têm a capacidade de neutralizar o radical hidroxilo e proteger a adenina/dA20 imobilizado na superfície do EPC. O comportamento da Lacase foi estudado na presença do ácido gálico e do ácido ascórbico. Os estudos eletroquímicos foram realizados através da voltametria de onda quadrada (VOQ), sendo que a interação entre a adenina/ou o dA20 imobilizada na superfície do EPC e os radicais livres na ausência e presença de antioxidantes foi avaliada por meio de mudanças no pico anódico produzido pela oxidação da adenina /dA20. Os resultados demonstraram que estes biossensores permitem a avaliação da capacidade antioxidante total em águas aromatizadas.

MnFe2O4@CNT-N as novel electrochemical nanosensor for determination of caffeine, acetaminophen and ascorbic acid

For the first time, a glassy carbon electrode (GCE) modified with novel N-doped carbon nanotubes (CNT-N) functionalized with MnFe2O4 nanoparticles (MnFe2O4@CNT-N) has been prepared and applied for the electrochemical determination of caffeine (CF), acetaminophen (AC) and ascorbic acid (AA). The electrochemical behaviour of CF, AC and AA on the bare GCE, CNT-N/GCE and MnFe2O4@CNT-N/GCE were carefully investigated using cyclic voltammetry (CV) and square-wave voltammetry (SWV). Compared to bare GCE and CNT-N modified electrode, the MnFe2O4@CNT-N modified electrode can remarkably improve the electrocatalytic activity towards the oxidation of CF, AC and AA with an increase in the anodic peak currents of 52%, 50% and 55%, respectively. Also, the SWV anodic peaks of these molecules could be distinguished from each other at the MnFe2O4@CNT-N modified electrode with enhanced oxidation currents. The linear response ranges for the square wave voltammetric determination of CF, AC and AA were 1.0 × 10−6 to 1.1 × 10−3 mol dm−3, 1.0 × 10−6 to 1.0 × 10−3 mol dm−3 and 2.0 × 10−6 to 1.0 × 10−4 mol dm−3 with detection limit (S/N = 3) of 0.83 × 10−6, 0.83 × 10−6 and 1.8 × 10−6 mol dm−3, respectively. The sensitivity values at the MnFe2O4@CNT-N/GCE for the individual determination of AC, AA and CF and in the presence of the other molecules showed that the quantification of AA and CF show no interferences from the other molecules; however, AA and CF interfered in the determination of AC, with the latter molecule showing the strongest interference. Nevertheless, the obtained results show that MnFe2O4@CNT-N composite material acted as an efficient electrochemical sensor towards the selected biomolecules.

Molecularly imprinted sensor for voltammetric detection of norfloxacin

In this work, a norfloxacin selective modified glassy carbon electrode (GCE) based on a molecularly imprinted polymer (MIP) as electrochemical sensor was developed. A suspension of multi-walled carbon nanotubes (MWCNTs) was deposited on the electrode surface. Subsequently, a molecularly imprinted film was prepared by electropolymerization, via cyclic voltammetry of pyrrole (PPy) in the presence of norfloxacin (NFX) as the template molecule. A control electrode (NIP) was also prepared. Scanning electron microscopy (SEM) and cyclic voltammetry in a ferrocyanide solution were performed for morphological and electrochemical characterisation, respectively. Several experimental parameters were studied and optimised. For quantification purposes the MIP/MWCNT/GCE was immersed in NFX solutions for 10 min, and the detection was performed in voltammetric cell by square wave voltammetry. The proposed sensor presented a linear behaviour, between peak current intensity and logarithmic concentration of NFX between 1 × 10−7 and 8 × 10−6 M. The obtained results presented good precision, with a repeatability of 4.3% and reproducibility of 9% and the detection limit was 4.6 × 10−8 M (S/N = 3). The developed sensor displayed good selectivity and operational lifetime, is simple to fabricate and easy to operate and was successfully applied to the analysis of NFX in urine samples.

Estudo eletroquímico do dano oxidativo promovido por RNS e PAH e o efeito protetor de antioxidantes no ADN

No dia-a-dia, os organismos vivos estão sujeitos a vários tipos de agressões de origem endógena e exógena. A produção endógena exagerada de contaminantes que ocorre nos processos metabólicos dos seres vivos está intimamente associada ao aparecimento e desenvolvimento de várias patologias. Por outro lado, e devido às atividades antropogénicas, muitos contaminantes de origem ambiental e alimentar entram por via exógena no organismo dos seres vivos provocando igualmente danos a nível celular. De modo a protegerem-se dos efeitos pejorativos provocados por estes compostos, os organismos vivos desenvolveram mecanismos complexos de defesa antioxidante. Este trabalho consistiu no estudo eletroquímico do dano oxidativo induzido por contaminantes (PAH (hidrocarbonetos aromáticos policíclicos), H2O2, NO• e HClO) e do efeito protetor, ao dano oxidativo, promovido por antioxidantes no material baseado no ADN recorrendo à utilização de um biossensor de bases púricas, adenina-EPC (elétrodo pasta de carbono) e dA20-EPC, utilizando a voltametria de onda quadrada (VOQ) como técnica de deteção. A aplicação da eletroquímica apresenta várias vantagens para a quantificação da capacidade antioxidante total (CAT) pois, permite a redução da quantidade de reagentes e amostra em análise, elimina a etapa de remoção de cor (a cor é um interferente nos métodos óticos) e não requer equipamentos dispendiosos. Foram seguidas diferentes abordagens para a construção dos biossensores. A primeira consistiu na construção de um adenina-EPC em três etapas: i) condicionamento do EPC, ii) eletrodeposição da adenina no EPC e iii) leitura do sinal eletroquímico. Assim, foram otimizados diversos parâmetros: concentração de adenina (150,0 mg/L), potencial de condicionamento (Ec) (+ 1,80 V), potencial de deposição (Ed) (+ 0,40 V), tempo de condicionamento (tc) (180 s) e tempo de deposição (td) (240 s). Foi aplicado o adenina-EPC no estudo do dano oxidativo provocado por PAH (benzo (g,h,i) perileno) e constatou-se que era necessário transformar o benzo (g,h,i) perileno num radical para se possível observar danos oxidativos induzidos no biossensor. A nova estratégia consistiu na construção de um dA20-EPC, através da adsorção física de uma gota de dA20 na superfície do EPC, com posterior secagem e leitura do sinal eletroquímico. Neste procedimento foi otimizada a concentração de dA20 (100,0 mg/L). O dano oxidativo provocado pelo H2O2, NO• e HClO foi estudado sobre o dA20-EPC e verificou-se que os três contaminantes induziam dano oxidativo no dA20-EPC. Confirmou-se a capacidade do ácido ascórbico (AA) em proteger o dA20-EPC do dano oxidativo induzido por H2O2 e NO•. O biossensor desenvolvido (dA20-EPC) foi aplicado na avaliação da CAT de diferentes amostras reais (café, sumo de laranja e água aromatizada de laranja) usando-se como contaminantes o H2O2 e NO•. Todas as amostras analisadas apresentaram ter capacidade antioxidante. Quando se usou o dA20-EPC na presença de H2O2, verificou-se que as amostras de café apresentam valores mais elevados de CAT (1130-1488 mg AAE/L) do que as amostras de bebidas (110 mg AAE/L em água aromatizada e 775 mg AAE/L em sumo). Os valores de CAT obtidos para amostras de sumo e água aromatizada na presença de NO• indicam que a amostra de sumo possui maior teor de CAT (526 mg AAE/L) conforme era esperado, do que a amostra de água aromatizada (172 mg AAE/L).

Processos Oxidativos Avançados para Remediação de Águas Contaminadas por Pesticidas

Este trabalho teve como objectivo inicial o estudo de processos oxidativos avançados de forma a remediar e tratar águas contaminadas por pesticidas. No entanto, ao longo do trabalho experimental, constatou-se que os produtos resultantes da degradação de pesticidas são muitas vezes mais tóxicos do que os compostos que lhes deram origem e que, por isso, degradar um composto nem sempre é o melhor para o ambiente. Assim, neste trabalho, procurou-se estudar o processo de degradação com o objectivo de minimizar o impacto ambiental dos pesticidas na água e no ambiente em geral. A parte experimental deste trabalho foi dividida em duas etapas, sendo que, em ambas, a voltametria de onda quadrada e a espectrofotometria de UV/Vis foram os métodos de análise utilizados, para acompanhar o processo de fotodegradação. Na primeira etapa estudou-se a relação entre a estrutura química dos pesticidas MCPA, MCPP, 2.4-D e Dicloroprop e a sua fotodegradação. Soluções aquosas dos pesticidas enunciados foram submetidas a irradiação UV/vis, com incrementos variáveis de tempo de irradiação. Os resultados obtidos, nesta etapa, permitiram constatar diferenças na percentagem de degradação dos diferentes pesticidas. Dos pesticidas estudados verificou-se uma maior fotodegradação para o MCPA e MCPP seguido do Dicloroprop e finalmente o 2.4-D que se degradou menos. Os dados obtidos sugerem que a fotodegradação destes pesticidas está intimamente ligada com a estrutura das moléculas. A presença de um maior número de grupos cloro ligados ao anel aromático nos pesticidas 2,4-D e Dicloroprop faz com que estes sejam mais estáveis e por isso se degradam menos que o MCPA e o MCPP. Por outro lado, o facto de o 2,4-D apresentar um potencial de oxidação mais elevado do que o Dicloroprop, faz com que este seja mais difícil de degradar, o que justifica a diferença entre os dois. Desta forma, foi possível concluir que a estrutura dos pesticidas condiciona o processo de degradação, como esperado. Na segunda etapa, estudou-se a estabilização dos pesticidas MCPA e MCPP após encapsulação, com 2-hidroxipropil-β-ciclodextrina (HP-β-CD), em água desionizada e em água do rio. Para tal, submeteram-se as soluções aquosas dos pesticidas com e sem ciclodextrina, a irradiação UV/vis, também com incrementos variáveis de tempo. No caso do MCPA verificou-se que, tanto para água desionizada como para água do rio, que este herbicida encapsulado se degrada bastante menos do que o MCPA livre. O encapsulamento permitiu reduzir quase para metade a taxa de fotodegradação. Assim, confirmou-se que a HP-β-CD permite estabilizar este pesticida, tornando-o mais resistente à fotodegradação. Desta forma, originam-se menos produtos de degradação, os quais podem ser mais tóxicos, e reduz-se de o impacto ambiental deste herbicida. Verificou-se também que o MCPA livre se degrada mais em água do rio do que em água desionizada, provavelmente devido à matéria orgânica presente nesta água, que promove o processo de degradação. No que respeita ao MCPP também se constatou que este herbicida se degrada menos encapsulado do que livre, em água desionizada e em água do rio. Neste caso, conseguiu-se reduzir pouco a taxa de fotodegradação, mas, ainda assim se verifica uma estabilização deste pesticida através do encapsulamento. No entanto, tornou-se mais evidente a estabilização do MCPP após encapsulação em água do rio, já que apresenta uma taxa de fotodegradação menor. Este facto demonstra que a HP-β-CD permite estabilizar também este pesticida, tornando-o mais resistente à fotodegradação, e reduzindo seu impacto ambiental.

Iron oxide/gold core/shell nanomagnetic probes and CdS biolabels for amplified electrochemical immunosensing of Salmonella typhimurium

There is an imminent need for rapid methods to detect and determine pathogenic bacteria in food products as alternatives to the laborious and time-consuming culture procedures. In this work, an electrochemical immunoassay using iron/gold core/shell nanoparticles (Fe@Au) conjugated with anti-Salmonella antibodies was developed. The chemical synthesis and functionalization of magnetic and gold-coated magnetic nanoparticles is reported. Fe@Au nanoparticles were functionalized with different self-assembled monolayers and characterized using ultraviolet-visible spectrometry, transmission electron microscopy, and voltammetric techniques. The determination of Salmonella typhimurium, on screen-printed carbon electrodes, was performed by square-wave anodic stripping voltammetry through the use of CdS nanocrystals. The calibration curve was established between 1×101 and 1×106 cells/mL and the limit of detection was 13 cells/mL. The developed method showed that it is possible to determine the bacteria in milk at low concentrations and is suitable for the rapid (less than 1 h) and sensitive detection of S. typhimurium in real samples. Therefore, the developed methodology could contribute to the improvement of the quality control of food samples.

Electrochemical immunosensor for multiplexed detection of food-borne pathogens using nanocrystal bioconjugates and MWCNT screen-printed electrode

Bacterial food poisoning is an ever-present threat that can be prevented with proper care and handling of food products. A disposable electrochemical immunosensor for the simultaneous measurements of common food pathogenic bacteria namely Escherichia coli O157:H7 (E. coli), campylobacter and salmonella were developed. The immunosensor was fabricated by immobilizing the mixture of anti-E. coli, anticampylobacter and anti-salmonella antibodies with a ratio of 1:1:1 on the surface of the multiwall carbon nanotube-polyallylamine modified screen printed electrode (MWCNT-PAH/SPE). Bacteria suspension became attached to the immobilized antibodies when the immunosensor was incubated in liquid samples. The sandwich immunoassay was performed with three antibodies conjugated with specific nanocrystal ( -E. coli-CdS, -campylobacter-PbS and -salmonella-CuS) which has releasable metal ions for electrochemical measurements. The square wave anodic stripping voltammetry (SWASV) was employed to measure released metal ions from bound antibody nanocrystal conjugates. The calibration curves for three selected bacteria were found in the range of 1 × 103 – 5 × 105 cells mL−1 with the limit of detection (LOD) 400 cells mL−1 for salmonella, 400 cells mL−1 for campylobacter and 800 cells mL−1 for E. coli. The precision and sensitivity of this method show the feasibility of multiplexed determination of bacteria in milk samples.

Electrochemical determination of antioxidant capacities in flavored waters by guanine and adenine biosensors

The immobilization and electro-oxidation of guanine and adenine asDNA bases on glassy carbon electrode are evaluated by square wave voltammetric analysis. The influence of electrochemical pretreatments, nature of supporting electrolyte, pH, accumulation time and composition of DNA nucleotides on the immobilization effect and the electrochemical mechanism are discussed. Trace levels of either guanine or adenine can be readily detected following short accumulation time with detection limits of 35 and 40 ngmL−1 for guanine and adenine, respectively. The biosensors of guanine and adenine were employed for the voltammetric detection of antioxidant capacity in flavored water samples. The method relies on monitoring the changes of the intrinsic anodic response of the surface-confined guanine and adenine species, resulting from its interaction with free radicals from Fenton-type reaction in absence and presence of antioxidant. Ascorbic acid was used as standard to evaluate antioxidant capacities of samples. Analytical data was compared with that of FRAP method.

Direct electroanalytical determination of fluvastatin in a pharmaceutical dosage form: batch and flow analysis

The reduction of luvastatin (FLV) at a hanging mercury-drop electrode (HMDE) was studied by square-wave adsorptive-stripping voltammetry (SWAdSV). FLV can be accumulated and reduced at the electrode, with a maximum peak current intensity at a potential of approximately 1.26V vs. AgCl=Ag, in an aqueous electrolyte solution of pH 5.25. The method shows linearity between peak current intensity and FLV concentration between 1.0 10 8 and 2.7 10 6 mol L 1. Limits of detection (LOD) and quantification (LOQ) were found to be 9.9 10 9 mol L 1 and 3.3 10 8 mol L 1, respectively. Furthermore, FLV oxidation at a glassy carbon electrode surface was used for its hydrodynamic monitoring by amperometric detection in a flow-injection system. The amperometric signal was linear with FLV concentration over the range 1.0 10 6 to 1.0 10 5 mol L 1, with an LOD of 2.4 10 7 mol L 1 and an LOQ of 8.0 10 7 mol L 1. A sample rate of 50 injections per hour was achieved. Both methods were validated and showed to be precise and accurate, being satisfactorily applied to the determination of FLV in a commercial pharmaceutical.

Electrochemical determination of citalopram by adsorptive stripping voltammetry–determination in pharmaceutical products

The electrochemical behavior of citalopram was studied by square-wave and square-wave adsorptive-stripping voltammetry (SWAdSV). Citalopram can be reduced and accumulated at a mercury drop electrode, with a maximum peak current intensity being obtained at a potential of approximately -1.25V vs. AgCl/Ag, in an aqueous electrolyte solution of pH 12. A SWAdSV method has been developed for the determination of citalopram in pharmaceutical preparations. The method shows a linear range between 1.0x10-7 and 2.0x10-6 mol L-1 with a limit of detection of 5x10-8 mol L-1 for an accumulation time of 30 s. The precision of the method was evaluated by assessing the repeatability and intermediate precision, achieving good relative standard deviations in all cases (≤2.3%). The proposed method was applied to the determination of citalopram in five pharmaceutical products and the results obtained are in good agreement with the labeled values.