The potential of molecular imprinting as a biosensing devices for monitoring the CEA cancer biomarker

6th Graduate Student Symposium on Molecular Imprinting6th Graduate Student Symposium on Molecular Imprinting, in Medway School of Pharmacy, Kent, UK, August 27-28, 2015Colorectal cancer is the third most common type of cancer and the major cause of the death throughout the world. Widely known, carcinoembryonic antigen (CEA) is an important tumour marker responsible for clinical diagnosis of 95% of all colon tumors1. The discovery of novel non-invasive biomarkers, as CEA, and its fast determination at low cost is presently required, to enable its use over wide screening programs and applications in point-of-care context, and, thus, its monitoring quite early. As a novel approach, this work proposes a novel support with molecular imprinted polymer (MIP) for CEA cancer biomarker based on carbon ink matrix linked by sol-gel chemistry on top of conductive glass covered by fluorine-doped tin oxide (FTO glass). In brief, the electrical biosensor was tailored on top of a disposable conductive glass electrode, following a bottom-up approach. The several stages of this process included the chemical modification of a homemade carbon ink layer and the assembly of a MIP or non-imprinted polymer (NIP) layer. The analytical performance of the obtained devices was followed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Chemical modifications of the surface were characterized using Fourier Transform Infrared (FTIR), and Raman spectroscopy with confocal microscopy. Overall, the MIP/FTO glass-based device displayed linear responses to CEA in EIS assays from 2.5×10-3 µg.mL-1 to 1.25 µg.mL-1 in PBS buffer, with detection limits of 2.5×10-3 µg.mL-1. Successful detection of CEA was, also, achieved in spiked samples of fetal bovine serum. In conclusion, the devices developed are a promising tool for the monitoring of CEA in a point-of-care applications, due to its detection capability below the normal physiological levels expected for this cancer biomarker, simplicity of manufacture, low-cost and good sensitivity and selectivity

Synthesis of an antibody-like material for the detection of Albumin

6th Graduate Student Symposium on Molecular Imprinting6th Graduate Student Symposium on Molecular Imprinting, Medway School of Pharmacy, Kent, 27-28 de Agosto 2015A novel molecularly imprinted polymer (MIP) is presented for the detection of Albumin, currently a biomarker of several diseases. The material acted as an antibody for Albumin and was obtained through a bulk imprinting approach, by electropolymerizing Eriochrome blackT (EBT) around the target protein

Development of paper-based color test-strip for drug detection in aquatic environment: Application to oxytetracycline

The wide use of antibiotics in aquaculture has led to the emergence of resistant microbial species. It should be avoided/minimized by controlling the amount of drug employed in fish farming. For this purpose, the present work proposes test-strip papers aiming at the detection/semi-quantitative determination of organic drugs by visual comparison of color changes, in a similar analytical procedure to that of pH monitoring by universal pH paper. This is done by establishing suitable chemical changes upon cellulose, attributing the paper the ability to react with the organic drug and to produce a color change. Quantitative data is also enabled by taking a picture and applying a suitable mathematical treatment to the color coordinates given by the HSL system used by windows. As proof of concept, this approach was applied to oxytetracycline (OXY), one of the antibiotics frequently used in aquaculture. A bottom-up modification of paper was established, starting by the reaction of the glucose moieties on the paper with 3-triethoxysilylpropylamine (APTES). The so-formed amine layer allowed binding to a metal ion by coordination chemistry, while the metal ion reacted after with the drug to produce a colored compound. The most suitable metals to carry out such modification were selected by bulk studies, and the several stages of the paper modification were optimized to produce an intense color change against the concentration of the drug. The paper strips were applied to the analysis of spiked environmental water, allowing a quantitative determination for OXY concentrations as low as 30 ng/mL. In general, this work provided a simple, method to screen and discriminate tetracycline drugs, in aquaculture, being a promising tool for local, quick and cheap monitoring of drugs

Backside-surface imprinting as a new strategy to generate specific plastic antibody materials

A backside protein-surface imprinting process is presented herein as a novel way to generate specific synthetic antibody materials. The template is covalently bonded to a carboxylated-PVC supporting film previously cast on gold, let to interact with charged monomers and surrounded next by another thick polymer. This polymer is then covalently attached to a transducing element and the backside of this structure (supporting film plus template) is removed as a regular “tape”. The new sensing layer is exposed after the full template removal, showing a high density of re-binding positions, as evidenced by SEM. To ensure that the templates have been efficiently removed, this re-binding layer was cleaned further with a proteolytic enzyme and solution washout. The final material was named MAPS, as in the back-side reading of SPAM, because it acts as a back-side imprinting of this recent approach. It was able to generate, for the first time, a specific response to a complex biomolecule from a synthetic material. Non-imprinted materials (NIMs) were also produced as blank and were used as a control of the imprinting process. All chemical modifications were followed by electrochemical techniques. This was done on a supporting film and transducing element of both MAPS and NIM. Only the MAPS-based device responded to oxLDL and the sensing layer was insensitive to other serum proteins, such as myoglobin and haemoglobin. Linear behaviour between log(C, μg mL−1) versus charged tranfer resistance (RCT, Ω) was observed by electrochemical impedance spectroscopy (EIS). Calibrations made in Fetal Calf Serum (FCS) were linear from 2.5 to 12.5 μg mL−1 (RCT = 946.12 × log C + 1590.7) with an R-squared of 0.9966. Overall, these were promising results towards the design of materials acting close to the natural antibodies and applied to practical use of clinical interest

New and low cost plastic membrane electrode with low detection limits for sulfadimethoxine determination in aquaculture waters

Sulfadimethoxine (SDM) is one of the drugs, often used in the aquaculture sector to prevent the spread of disease in freshwater fish aquaculture. Its spread through the soil and surface water can contribute to an increase in bacterial resistance. It is therefore important to control this product in the environment. This work proposes a simple and low-cost potentiometric device to monitor the levels of SDM in aquaculture waters, thus avoiding its unnecessary release throughout the environment. The device combines a micropipette tip with a PVC membrane selective to SDM, prepared from an appropriate cocktail, and an inner reference solution. The membrane includes 1% of a porphyrin derivative acting as ionophore and a small amount of a lipophilic cationic additive (corresponding to 0.2% in molar ratio). The composition of the inner solution was optimized with regard to the kind and/or concentration of primary ion, chelating agent and/or a specific interfering charged species, in different concentration ranges. Electrodes constructed with inner reference solutions of 1 × 10−8 mol/L SDM and 1 × 10−4 mol/L chromate ion showed the best analytical features. Near-Nernstian response was obtained with slopes of −54.1 mV/decade, an extraordinary detection limit of 7.5 ng/mL (2.4 × 10−8 mol/L) when compared with other electrodes of the same type. The reproducibility, stability and response time are good and even better than those obtained by liquid contact ISEs. Recovery values of 98.9% were obtained from the analysis of aquaculture water samples

Determination of tartaric acid in wines by FIA with tubular tartrate-selective electrodes

A flow injection analysis (FIA) system comprising a tartrate- (TAT) selective electrode has been developed for determination of tartaric acid in wines. Several electrodes constructed for this purpose had a PVC membrane with a complex of quaternary ammonium and TAT as anion exchanger, a phenol derivative as additive, and a more or less polar mediator solvent. Characterization of the electrodes showed behavior was best for membranes with o-nitrophenyl octyl ether as solvent. On injection of 500 μL into a phosphate buffer carrier (pH = 3.1; ionic strength 10–2 mol/L) flowing at 3 mL/min, the slope was 58.06 ± 0.6 with a lower limit of linear range of 5.0 × 10–4 mol/L TAT and R2 = 0.9989. The interference of several species, e.g. chloride, bromide, iodide, nitrate, gallic acid, tannin, sucrose, glucose, fructose, acetate, and citrate, was evaluated in terms of potentiometric selectivity coefficients. The Hofmeister series was followed for inorganic species and the most interfering organic ion was citrate. When red and white wines were analyzed and the results compared with those from an independent method they were found to be accurate, with relative standard deviations below 5.0%

New modified electrochemical conductive paper support for BSA detection

Chemical sensors and biosensors are widely used to detect various kinds of protein target biomolecules. Molecularly Imprinted Polymers (MIPs) have raised great interest in this area, because these act as antibody-like recognition materials, with high affinity to the template molecule. Compared to natural antibodies, these are also of lower cost and higher stability. There are different types of supports used to carry MIP materials, mostly of these made of gold, favourably assembled on a Screen Printed Electrode (SPE) strategy. For this work a new kind of support for the sensing layer was developed: conductive paper. This support was made by modifying first cellulose paper with paraffin wax (to make it waterproof), and casting a carbon-ink on it afterwards, to turn it conductive. The SPAM approach previously reported in1 was employed herein to assemble to MIP sensing material on the conductive paper. The selected charged monomers were (vinylbenzyl) trimethlammonium chloride (positive charge) or vinylbenzoic acid (negative charge), used to generate binding positions with single-type charge (positive or negative). The non-specific binding area of the MIP layer was assembled by chronoamperometry-assisted polymerization (at 1 V, for 60, 120 or 180 seconds) of vinylbenzoate, cross-linked with ethylene glycol vinyl ether. The BSA biomolecules lying within the polymeric matrix were removed by Proteinase K action. All preparation stages of the MIP assembly were followed by FTIR, Raman spectroscopy and, electrochemical analysis. In general, the best results were obtained for longer polymerization times and positively charged binding sites (which was consistent with a negatively-charged protein under physiological pH, as BSA). Linear responses against BSA concentration ranged from 0.005 to 100 mg/mL, in PBS buffer standard solutions. The sensor was further calibrated in standard solutions that were prepared in synthetic or real urine, and the analytical response became more sensitive and stable. Compared to the literature, the detection capability of the developed device is better than most of the reported electrodes. Overall, the simplicity, low cost and good analytical performance of the BSA SPE device, prepared with positively charged binding positions, seems a suitable approach for practical application in clinical context. Further studies with real samples are required, as well as gathering with electronic-supporting devices to allow on-site readings

Emerging (Bio)Sensing Technology for Assessing and Monitoring Freshwater Contamination - Methods and Applications

Ecological Water Quality - Water Treatment and ReuseWater is life and its preservation is not only a moral obligation but also a legal requirement. By 2030, global demands will exceed more than 40 % the existing resources and more than a third of the world's population will have to deal with water shortages (European Environmental Agency [EEA], 2010). Climate change effects on water resources will not help. Efforts are being made throughout Europe towards a reduced and efficient water use and prevention of any further deterioration of the quality of water (Eurostat, European Comission [EC], 2010). The Water Framework Directive (EC, 2000) lays down provisions for monitoring, assessing and classifying water quality. Supporting this, the Drinking Water sets standards for 48 microbiological and chemical parameters that must be monitored and tested regularly (EC, 1998). The Bathing Water Directive also sets concentration limits for microbiological pollutants in inland and coastal bathing waters (EC, 2006), addressing risks from algae and cyanobacteria contamination and faecal contamination, requiring immediate action, including the provision of information to the public, to prevent exposure. With these directives, among others, the European Union [EU] expects to offer its citizens, by 2015, fresh and coastal waters of good quality

Chlormequat selective electrodes: construction, evaluation and application at FIA systems

A flow injection analysis (FIA) system having a chlormequat selective electrode is proposed. Several electrodes with poly(vinyl chloride) based membranes were constructed for this purpose. Comparative characterization suggestedthe use of membrane with chlormequat tetraphenylborate and dibutylphthalate. On a single-line FIA set-up, operating with 1x10-2 mol L-1 ionic strength and 6.3 pH, calibration curves presented slopes of 53.6±0.4mV decade-1 within 5.0x10-6 and1.0x10-3 mol L-1, andsquaredcorrelation coefficients >0.9953. The detection limit was 2.2x10-6 mol L-1 and the repeatability equal to ±0.68mV (0.7%). A dual-channel FIA manifold was therefore constructed, enabling automatic attainment of previous ionic strength andpH conditions and thus eliminating sample preparation steps. Slopes of 45.5±0.2mV decade -1 along a concentration range of 8.0x10-6 to 1.0x10-3 mol L-1 with a repeatability ±0.4mV (0.69%) were obtained. Analyses of real samples were performed, and recovery gave results ranging from 96.6 to 101.1%

Sol-gel biomimetic material designed to target CEA cancer biomarker

1st ASPIC International CongressPoster J3, presented at 1st ASPIC International Congress, in Fundação Calouste Gulbenkian, Lisbon, Portugal, November 25-26, 2014.Introduction Carcinoembryonic antigen (CEA) is an important tumor marker responsible for clinical diagnosis of over 95% of all colon tumors, 50% of breast tumors, as well as tumors of the lung cancer or ovarian carcinoma [1]. The detection of CEA levels in biological samples plays an important role in the pre-diagnosis evaluation and in the follow-up examination during therapy stage [2]. The most common tool for the analysis of CEA in hospitals and clinical laboratories relies on ELISA-based procedures using antibodies as capturing probe. The overall principal offers the selectivity and sensitivity out coming from the use of antibodies, but it could be further improved by assembling the biosensors over a receptor platform and establishing a label-free measure by electrical impedance spectroscopy (EIS). Thus, the present work proposes the development of an immunosensor for CEA. Materials and Methods Electrochemical signals were measured in a Methrom Autolab potentiostat/galvanostat (Autolab PGSTAT302N) interfaced to a computer and controlled by NOVA 1.9 software. The chemical modification of the surface of the conductive glass was characterized by Raman spectroscopy with confocal microscopy (Thermo Scientific). The immunosensor was assembled by modifying conductive glass (with ITO) with an amino silane compound (APTES), activating the antibody via carbodiimide chemistry (EDAC/NHS) and binding the antibody to the amine surface over the ITO glass. The performance of the imunosensor was evaluated by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SVW). Results and discussion The immunosensor made with an optimized composition displayed linear behavior against CEA concentration by EIS and SWV techniques. The corresponding linear ranges were 0.502-1.5 and 0.252-1.5ng/mL, with detection limits of 0.417 and 0.043 ng/mL, respectively. Overall, the obtained device may be potential method to apply for screening CEA in point-of-care due to the simplicity of fabrication, short time response, low cost and good sensitivity when compared to other analytical techniques, such as ELISA assays