Molecularly-Imprinted Materials for Potentiometric Transduction: Application to the Antibiotic Enrofloxacin

Enrofloxacin (ENR) is an antimicrobial used both in humans and in food producing species. Its control is required in farmed species and their surroundings in order to reduce the prevalence of antibiotic resistant bacteria. Thus, a new biomimetic sensor enrofloxacin is presented. An artificial host was imprinted in specific polymers. These were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. The potentiometric sensors exhibited a near-Nernstian response. Slopes expressing mV/Δlog([ENR]/M) varied within 48–63. The detection limits ranged from 0.28 to 1.01 µg mL−1. Sensors were independent from the pH of test solutions within 4–7. Good selectivity was observed toward potassium, calcium, barium, magnesium, glycine, ascorbic acid, creatinine, norfloxacin, ciprofloxacin, and tetracycline. In flowing media, the biomimetic sensors presented good reproducibility (RSD of ± 0.7%), fast response, good sensitivity (47 mV/Δlog([ENR]/M), wide linear range (1.0 × 10−5–1.0 × 10−3 M), low detection limit (0.9 µg mL−1), and a stable baseline for a 5 × 10−2 M acetate buffer (pH 4.7) carrier. The sensors were used to analyze fish samples. The method offered the advantages of simplicity, accuracy, and automation feasibility. The sensing membrane may contribute to the development of small devices allowing in vivo measurements of enrofloxacin or parent-drugs.

“Green Electrodes” Modified with Au Nanoparticles Synthesized in Glycerol, as Electrochemical Nitrite Sensor

A new environmentally friendly Au nanoparticles (Au NPs) synthesis in glycerol by using ultraviolet irradiation and without extra-added stabilizers is described. The synthesis proposed in this work may impact on the non-polluting production of noble nanoparticles with simple chemicals normally found in standard laboratories. These Au NPs were used to modify a carbon paste electrode (CPE) without having to separate them from the reaction medium. This green electrode was used as an electrochemical sensor for the nitrite detection in water. At the optimum conditions the green sensor presented a linear response in the 2.0×10−7–1.5×10−5 M concentration range, a good detection sensitivity (0.268 A L mol−1), and a low detection limit of 2.0×10−7 M of nitrite. The proposed modified green CPE was used to determine nitrite in tap water samples.

Determinação rápida de hidroquinona usando análise por injeção em batelada (BIA) com detecção amperométrica

A fast analytical method for determination of hydroquinone in pharmaceutical formulations employing batch injection analysis (BIA) with amperometric detection using a boron-doped diamond electrode is described. The supporting electrolyte was a 0.1 mol L-1 H2SO4 solution (the single reagent used for analysis). The method showed good repeatability (RSD of 0.45%, n=20), wide linear range (from 10 to 2000 µmol L-1, R=0.9999), low detection limit (0.016 µmol L-1) and satisfactory recovery values (91-96%). Accuracy of the method was evaluated by comparative analyses using high-performance liquid-chromatography. The ability to replace the electronic pipette by disposable syringes (injection procedure) in BIA systems was also shown.

Synthesis of isotope-labelled methoxypyrazine compounds as internal standards and quantitative determination of aroma methoxypyrazines in water and wines by solid-phase extraction with isotope dilution-GC-MS /

An efficient way of synthesizing the deuterium labelled analogues of three methoxypyrazine compounds: 2-d3-methoxy-3-isopropylpyrazine, 2-d3-methoxy-3- isobutylpyrazine, and 2-d3-methoxy-3-secbutylpyrazine, has been developed. To confirm that the deuterium labels had been incorporated into the expected positions in the molecules synthesized, the relevant characterization by NMR, HRMS and GC/MS analysis was conducted. Another part of this work involved quantitative determination of methoxypyrazines in water and wines. Solid-phase extraction (SPE) proved to be a suitable means for the sample separation and concentration prior to GC/MS analysis.Such factors as the presence of ethanol, salt, and acid have been investigated which can influence the recovery by SPE for the pyrazines from the water matrix. Significantly, in this work comparatively simple fractional distillation was attempted to replace the conventional steam distillation for pre-concentrating a sample with a relatively large volume prior to SPE. Finally, a real wine sample spiked with the relevant isotope-labelled methoxypyrazines was quantitatively analyzed, revealing that the wine with 10 beetles per litre contained 138 ppt of 2-methoxy-3-isopropylpyrazine. Interestingly, we have also found that 2-methoxy-3-secbutylpyrazine exhibits an extremely low detection limit in GC/MS analysis compared with the detection limit of the other two methoxypyrazines: 2- methoxy-3-isopropylpyrazine and 2-methoxy-3-isobutylpyrazine.

Quantification of N-acetylcysteine in pharmaceuticals using cobalt phthalocyanine modified graphite electrodes

Flow injection analysis (FIA) with amperometric detection was employed for the quantification of N-acetylcysteine (NAC) in pharmaceutical formulations, utilizing an ordinary pyrolytic graphite (OPG) electrode modified with cobalt phthalocyanine (CoPc). Cyclic voltammetry was used in preliminary studies to establish the best conditions for NAC analysis. In FIA-amperometric experiments the OPG-CoPc electrode exhibited sharp and reproducible current peaks over a wide linear working range (5.0 x 10(-5)-1.0 x 10(-3) mol L(-1)) in 0.1 mol L(-1) NaOH solution. High sensitivity (130 mA mol(-1) cm(2)) and a low detection limit (9.0 x 10(-7) mol L(-1)) were achieved using the sensor. The repeatability (R.S.D.%) for 13 successive flow injections of a solution containing 5.0 x 10(-4) mol L(-1) NAC was 1.1%. The new procedure was applied in analyses of commercial pharmaceutical products and the results were in excellent agreement with those obtained using the official titrimetric method. The proposed amperometric method is highly suitable for quality control analyses of NAC in pharmaceuticals since it is rapid, precise and requires much less work than the recommended titrimetric method. (C) 2010 Elsevier B.V. All rights reserved.

Aplicabilidade da voltametria de pulso diferencial na quantificação de ácido fólico e ácido oxálico: um método comparativo

The development of more selective and sensitive analytical methods is of great importance in different areas of knowledge, covering, for example, food, biotechnological, environmental and pharmaceutical sectors. The study aimed to employ the technique electroanalytical differential pulse voltammetry (DPV) as an innovative and promising alternative for identification and quantification of organic compounds. The organic compounds were investigated in this study oxalic acid (OA) and folic acid (FA). The electrochemical oxidation of oxalic acid has been extensively studied as a model reaction in the boundary between the organic and inorganic electrochemistry. Since the AF, an essential vitamin for cell multiplication in all tissues, which is essential for DNA synthesis. The AF has been investigated using analytical techniques, liquid chromatography and molecular absorption spectrophotometry. The results obtained during the experimental procedure indicated that the process of electrochemical oxidation of oxalic acid is strongly dependent on the nature of the anode material and the oxidation mechanism, which affects their detection. Efficient removal was observed in Ti/PbO2 anodes, graphite, BDD and Pt 90, 85, 80 and 78% respectively. It was also shown that the DPV employing glassy carbon electrode offers a fast, simple, reliable and economical way to determine the AO during the process of electrochemical oxidation. Furthermore, electroanalytical methods are more expensive than commonly used chromatographic analysis and other instrumental methods involving toxic reagents and higher cost. Compared with the classical method of titration and DPV could be a good fit, confidence intervals and detection limits confirming the applicability of electroanalytical technique for monitoring the degradation of oxalic acid. For the study of AF was investigated the electrocatalytic activity of the carbon paste electrode for identification and quantification in pharmaceutical formulations by applying the DPV. The results obtained during the experimental procedure showed an irreversible oxidation peak at 9.1 V characteristic of FA. The carbon paste sensor showed low detection limit of 5.683×10−8 mol L-1 reducing matrix effects. The spectrophotometric analysis showed lower concentrations of HF compared with those obtained by HPLC and DPV. The levels of AF were obtained according to the methodology proposed by the Brazilian Pharmacopoeia. The electroanalytical method (DPV) proposed is cheaper than GC analysis commonly used by the pharmaceutical industry. The results demonstrated the potential of these electroanalytical techniques for future applications in environmental, chemical and biological sensors

Determinação de chumbo por análise voltamétrica de pulso diferencial por redissolução anódica em regiões de falhas geológicas com emissão de radônio e filhos de radônio no município de Presidente Prudente

Pós-graduação em Química - IBILCE

Caracterização e aplicação analítica de eletrodos modificados com sistemas porfirínicos supramoleculares

Estudos com eletrodos modificados foram conduzidos utilizando dois sistemas porfirínicos supramoleculares diferentes. O primeiro foi baseado na modificação de eletrodo de carbono vítreo com uma porfirina de níquel tetrarrutenada, [NiIITPyP{RuII(bipy)2Cl}4]4+. A modificação do eletrodo foi realizada por meio de sucessivos ciclos voltamétricos em meio alcalino (pH 13), gerando um eletrodo com característica similar a eletrodos modificados com α-Ni(OH)2. A caracterização química do filme formado foi realizada através das técnicas de voltametria cíclica, ressonância paramagnética eletrônica, espectroscopia eletrônica por reflectância e espectroscopia Raman com ensaio espectro-eletroquímico. Os resultados sugerem a formação de um polímero de coordenação, [µ-O2-NiIITPyP{RuII(bipy)2Cl}4]n, composto por subunidades porfirínicas ligadas entre si por pontes µ-peroxo axialmente coordenadas aos átomos de níquel (Ni-O-O-Ni). O crescimento do filme apresentou dependência da alcalinidade do meio pela formação do precursor octaédrico [Ni(OH)2TRPyP]2+ em solução, pela coordenação de OH- nas posições axiais do átomo de níquel. O processo de eletropolimerização indicou a participação de radical hidroxil, gerado por oxidação eletrocatalítica da água nos sítios periféricos da porfirina contendo o complexo de rutênio. O mesmo eletrodo foi aplicado como sensor eletroquímico para análise amperométrica de ácido fólico em comprimidos farmacêuticos. O sensor foi associado a um sistema de Batch Injection Analysis (BIA) alcançando considerável rapidez e baixo limite de detecção. Para as análises das amostras também foi proposto um método para a remoção da lactose, que agia como interferente. O segundo estudo envolveu a modificação de eletrodos de carbono vítreo com diferentes hemoglobinas, naturais (HbA0, HbA2 e HbS) e sintéticas (Hb-PEG5K2, αα-Hb-PEG5K2 e BT-PEG5K4), para a avaliação da eficiência na redução eletrocatalítica de nitrito mediada por FeI-heme. Os filmes foram produzidos pela mistura de soluções das hemoglobinas com brometo de didodecildimetiltrimetilamônio (DDAB), aplicados nas superfícies com consecutiva evaporação, formando filmes estáveis. Os valores de potencial redox para os processos do grupo heme e a sua associação com a disponibilidade do grupo na proteína foram avaliados por voltametria cíclica. Os valores das constantes de velocidade, k, para redução de nitrito foram obtidos por cronoamperometria em -1,1 V (vs Ag/AgCl(KCl 3M)) que foram utilizados para estudo comparativo entre as espécies sintéticas para eventual aplicação clínica.

Optical fibre sensors with applications in gas and biological sensing

This thesis describes the study of various grating based optical fibre sensors for applications in refractive index sensing. The sensitivity of these sensors has been studied and in some cases enhanced using novel techniques. The major areas of development are as follows. The sensitivity of long period gratings (LPGs) to surrounding medium refractive index (SRI) for various periods was investigated. The most sensitive period of LPG was found to be around 160 µm and this was due to the core mode coupling to a single cladding mode but phase matching at two wavelength locations, creating two attenuation peaks, close to the waveguide dispersion turning point. Large angle tilted fibre gratings (TFGs) have similar behaviour to LPGs, in that they couple to the co-propagating cladding modes. The tilted structure of the index modulation within the core of the fibre gives rise to a polarisation dependency, differing the large angle TFG from a LPG. Since the large angle TFG couple to the cladding mode they are SRI sensitive, the sensitivity to SRI can be further increased through cladding etching using HF acid. The thinning of the cladding layer caused a reordering of the cladding modes and shifted to more SRI sensitive cladding modes as the investigation discovered. In a SRI range of 1.36 to 1.40 a sensitivity of 506.9 nm/URI was achieved for the etched large angle TFG, which is greater than the dual resonance LPG. UV inscribed LPGs were coated with sol-gel materials with high RIs. The high RI of the coating caused an increase in cladding mode effective index which in turn caused an increase in the LPG sensitivity to SRI. LPGs of various periods of LPG were coated with sol-gel TiO2 and the optimal thickness was found to vary for each period. By coating of the already highly SRI sensitive 160µm period LPG (which is a dual resonance) with a sol-gel TiO2, the SRI sensitivity was further increased with a peak value of 1458 nm/URI, which was an almost 3 fold increase compared to the uncoated LPG. LPGs were also inscribed using a femtosecond laser which produced a highly focused index change which was no uniform throughout the core of the optical fibre. The inscription technique gave rise to a large polarisation sensitivity and the ability to couple to multiple azimuthal cladding mode sets, not seen with uniform UV inscribed gratings. Through coupling of the core mode to multiple sets of cladding modes, attenuation peaks with opposite wavelength shifts for increasing SRI was observed. Through combining this opposite wavelength shifts, a SRI sensitivity was achieved greater than any single observed attenuations peak. The maximum SRI achieved was 1680 nm/URI for a femtosecond inscribed LPG of period 400 µm. Three different types of surface plasmon resonance (SPR) sensors with a multilayer metal top coating were investigated in D shape optical fibre. The sensors could be separated into two types, utilized a pre UV inscribed tilted Bragg grating and the other employed a post UV exposure to generate surface relief grating structure. This surface perturbation aided the out coupling of light from the core but also changed the sensing mechanism from SPR to localised surface plasmon resonance (LSPR). This greatly increased the SRI sensitivity, compared to the SPR sensors; with the gold coated top layer surface relief sensor producing the largest SRI sensitivity of 2111.5nm/URI was achieved. While, the platinum and silver coated top layer surface relief sensors also gave high SRI sensitivities but also the ability to produce resonances in air (not previously seen with the SPR sensors). These properties were employed in two applications. The silver and platinum surface relief devices were used as gas sensors and were shown to be capable of detecting the minute RI change of different gases. The calculated maximum sensitivities produced were 1882.1dB/URI and 1493.5nm/URI for silver and platinum, respectively. Using a DFB laser and power meter a cheap alternative approach was investigated which showed the ability of the sensors to distinguish between different gases and flow rates of those gases. The gold surface relief sensor was coated in a with a bio compound called an aptamer and it was able to detect various concentrations of a biological compound called Thrombin, ranging from 1mM to as low as 10fM. A solution of 2M NaCl was found to give the best stripping results for Thrombin from the aptamer and showed the reusability of the sensor. The association and disassociation constants were calculated to be 1.0638×106Ms-1 and 0.2482s-1, respectively, showing the high affinity of the Aptamer to thrombin. This supports existing working stating that aptamers could be alternative to enzymes for chemical detection and also helps to explain the low detection limit of the gold surface relief sensor.

Generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-PCR and nonfluorescent low-density microarray

A nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.

Low Bone Mass In Human Immunodeficiency Virus-infected Climacteric Women Receiving Antiretroviral Therapy: Prevalence And Associated Factors.

Low bone mineral density (BMD) has been found in human immunodeficiency virus (HIV)-infected patients; however, data on associated factors remain unclear, specifically in middle-aged women. This study aims to evaluate factors associated with low BMD in HIV-positive women. In this cross-sectional study, a questionnaire was administered to 206 HIV-positive women aged 40 to 60 years who were receiving outpatient care. Clinical features, laboratory test results, and BMD were assessed. Yates and Pearson χ(2) tests and Poisson multiple regression analysis were performed. The median age of women was 47.7 years; 75% had nadir CD4 T-cell counts higher than 200, and 77.8% had viral loads below the detection limit. There was no association between low BMD at the proximal femur and lumbar spine (L1-L4) and risk factors associated with HIV infection and highly active antiretroviral therapy. Poisson multiple regression analysis showed that the only factor associated with low BMD at the proximal femur and lumbar spine was postmenopause status. Low BMD is present in more than one third of this population sample, in which most women are using highly active antiretroviral therapy and have a well-controlled disease. The main associated factor is related to estrogen deprivation. The present data support periodic BMD assessments in HIV-infected patients and highlight the need to implement comprehensive menopausal care for these women to prevent bone loss.

Detection Of Explosives On The Surface Of Banknotes By Raman Hyperspectral Imaging And Independent Component Analysis.

The aim of this study was to develop a methodology using Raman hyperspectral imaging and chemometric methods for identification of pre- and post-blast explosive residues on banknote surfaces. The explosives studied were of military, commercial and propellant uses. After the acquisition of the hyperspectral imaging, independent component analysis (ICA) was applied to extract the pure spectra and the distribution of the corresponding image constituents. The performance of the methodology was evaluated by the explained variance and the lack of fit of the models, by comparing the ICA recovered spectra with the reference spectra using correlation coefficients and by the presence of rotational ambiguity in the ICA solutions. The methodology was applied to forensic samples to solve an automated teller machine explosion case. Independent component analysis proved to be a suitable method of resolving curves, achieving equivalent performance with the multivariate curve resolution with alternating least squares (MCR-ALS) method. At low concentrations, MCR-ALS presents some limitations, as it did not provide the correct solution. The detection limit of the methodology presented in this study was 50μgcm(-2).

Sensitive detection of nitric oxide using seeded parametric four-wave mixing

A sensitive near-resonant four-wave mixing technique based on two-photon parametric four-wave mixing has been developed. Seeded parametric four-wave mixing requires only a single laser as an additional phase matched seeder field is generated via parametric four-wave mixing of the pump beam in a high gain cell. The seeder field travels collinearly with the pump beam providing efficient nondegenerate four-wave mixing in a second medium. This simple arrangement facilitates the detection of complex molecular spectra by simply scanning the pump laser. Seeded parametric four-wave mixing is demonstrated in both a low pressure cell and an air/acetylene flame with detection of the two-photon C (2) Pi(upsilon'=0)<--X (2) Pi(upsilon =0) spectrum of nitric oxide. From the cell data a detection limit of 10(12) molecules/cm(3) is established. A theoretical model of seeded parametric four-wave mixing is developed from existing parametric four-wave mixing theory. The addition of the seeder field significantly modifies the parametric four-wave mixing behaviour such that in the small signal regime, the signal intensity can readily be made to scale as the cube of the laser pump power while the density dependence follows a more familiar square law dependence, In general, we find excellent agreement between theory and experiment. Limitations to the process result from an ac Stark shift of the two-photon resonance in the high pressure seeder cell caused by the generation of a strong seeder field, as well as a reduction in phase matching efficiency due to the presence of certain buffer species. Various optimizations are suggested which should overcome these limitations, providing even greater detection sensitivity. (C) 1998 American Institute of Physics, [S0021-9606(98)01014-9].

Oriented Tailoring of Plastic Antibodies for Prostate Specific Antigen and Application of the Imprinted Material as Ionophore in Potentiometric Detection

Prostate Specific Antigen (PSA) is the biomarker of choice for screening prostate cancer throughout the population, with PSA values above 10 ng/mL pointing out a high probability of associated cancer1. According to the most recent World Health Organization (WHO) data, prostate cancer is the commonest form of cancer in men in Europe2. Early detection of prostate cancer is thus very important and is currently made by screening PSA in men over 45 years old, combined with other alterations in serum and urine parameters. PSA is a glycoprotein with a molecular mass of approximately 32 kDa consisting of one polypeptide chain, which is produced by the secretory epithelium of human prostate. Currently, the standard methods available for PSA screening are immunoassays like Enzyme-Linked Immunoabsorbent Assay (ELISA). These methods are highly sensitive and specific for the detection of PSA, but they require expensive laboratory facilities and high qualify personal resources. Other highly sensitive and specific methods for the detection of PSA have also become available and are in its majority immunobiosensors1,3-5, relying on antibodies. Less expensive methods producing quicker responses are thus needed, which may be achieved by synthesizing artificial antibodies by means of molecular imprinting techniques. These should also be coupled to simple and low cost devices, such as those of the potentiometric kind, one approach that has been proven successful6. Potentiometric sensors offer the advantage of selectivity and portability for use in point-of-care and have been widely recognized as potential analytical tools in this field. The inherent method is simple, precise, accurate and inexpensive regarding reagent consumption and equipment involved. Thus, this work proposes a new plastic antibody for PSA, designed over the surface of graphene layers extracted from graphite. Charged monomers were used to enable an oriented tailoring of the PSA rebinding sites. Uncharged monomers were used as control. These materials were used as ionophores in conventional solid-contact graphite electrodes. The obtained results showed that the imprinted materials displayed a selective response to PSA. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8X10-11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed smaller sensitivity, with average slopes of -24.8 mV/decade. The best sensors were successfully applied to the analysis of serum samples, with percentage recoveries of 106.5% and relatives errors of 6.5%.

Label-free human chorionic gonadotropin detection at picogram levels using oriented antibodies bound to graphene screen-printed electrodes

Human chorionic gonadotropin (hCG) is a key diagnostic marker of pregnancy and an important biomarker for cancers in the prostate, ovaries and bladder and therefore of great importance in diagnosis. For this purpose, a new immunosensor of screen-printed electrodes (SPEs) is presented here. The device was fabricated by introducing a polyaniline (PANI) conductive layer, via in situ electropolymerization of aniline, onto a screen-printed graphene support. The PANI-coated graphene acts as the working electrode of a three terminal electrochemical sensor. The working electrode is functionalised with anti-hCG, by means of a simple process that enabled oriented antibody binding to the PANI layer. The antibody was attached to PANI following activation of the –COOH group at the Fc terminal. Functionalisation of the electrode was analysed and optimized using Electrochemical Impedance Spectroscopy (EIS). Chemical modification of the surface was characterised using Fourier transform infrared, and Raman spectroscopy with confocal microscopy. The graphene–SPE–PANI devices displayed linear responses to hCG in EIS assays from 0.001 to 50 ng mL−1 in real urine, with a detection limit of 0.286 pg mL−1. High selectivity was observed with respect to the presence of the constituent components of urine (urea, creatinine, magnesium chloride, calcium chloride, sodium dihydrogen phosphate, ammonium chloride, potassium sulphate and sodium chloride) at their normal levels, with a negligible sensor response to these chemicals. Successful detection of hCG was also achieved in spiked samples of real urine from a pregnant woman. The immunosensor developed is a promising tool for point-of-care detection of hCG, due to its excellent detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.