Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2′-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented methodology favored Ab/Ag affinity and immunodetection of the antigen. The immunosensor design was evaluated by quartz-crystal microbalance with dissipation, atomic force microscopy, electrochemical impedance spectroscopy (EIS) and square-wave voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charge transfer resistance across the electrochemical set-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from glucose, urea and creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label-Free Human Chorionic Gonadotropin Detection

A new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold-nanoparticles over graphene screen-printed electrode (SPE). The antibody was covalently bound to CS via its Fc-terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG-immunosensor displayed linear response against the logarithm-hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

Disposable immunosensor with simple antibody orientation for label-free real-time detection of a cancer biomarker

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2’-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The Anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented proposal favored Ab/Ag affinity. The immunosensor design was evaluated by Quartz-Crystal microbalance with Dissipation, Atomic Force Microscopy, Electrochemical Impedance Spectroscopy (EIS) and Square-Wave Voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charged transfer resistance across the electrochemical sep-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from Glucose, Urea and Creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

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.

Specific label-free and real-time detection of oxidized low density lipoprotein (oxLDL) using an immunosensor with three monoclonal antibodies

Increased levels of plasma oxLDL, which is the oxidized fraction of Low Density Lipoprotein (LDL), are associated with atherosclerosis, an inflammatory disease, and the subsequent development of severe cardiovascular diseases that are today a major cause of death in modern countries. It is therefore important to find a reliable and fast assay to determine oxLDL in serum. A new immunosensor employing three monoclonal antibodies (mAbs) against oxLDL is proposed in this work as a quick and effective way to monitor oxLDL. The oxLDL was first employed to produce anti-oxLDL monoclonal antibodies by hybridoma cells that were previously obtained. The immunosensor was set-up by selfassembling cysteamine (Cyst) on a gold (Au) layer (4 mm diameter) of a disposable screen-printed electrode. Three mAbs were allowed to react with N-hydroxysuccinimide (NHS) and ethyl(dimethylaminopropyl)carbodiimide (EDAC), and subsequently incubated in the Au/Cys. Albumin from bovine serum (BSA) was immobilized further to ensure that other molecules apart from oxLDL could not bind to the electrode surface. All steps were followed by various characterization techniques such as electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The analytical operation of the immunosensor was obtained by incubating the sensing layer of the device in oxLDL for 15 minutes, prior to EIS and SWV. This was done by using standard oxLDL solutions prepared in foetal calf serum, in order to simulate patient's plasma with circulating oxLDL. A sensitive response was observed from 0.5 to 18.0 mg mL 1 . The device was successfully applied to determine the oxLDL fraction in real serum, without prior dilution or necessary chemical treatment. The use of multiple monoclonal antibodies on a biosensing platform seemed to be a successful approach to produce a specific response towards a complex multi-analyte target, correlating well with the level of oxLDL within atherosclerosis disease, in a simple, fast and cheap way.

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins (OMPs) was developed. Two single stranded DNA sequences were tested as recognition elements and compared. The aptamer capture probes were immobilized, with and without 6-mercapto-1-hexanol (MCH) on a gold electrode. Each step of the modification process was characterized by Faradaic impedance spectroscopy (FIS). A linear relationship between the electron-transfer resistance (Ret) and E. coli OMPs concentration was demonstrated in a dynamic detection range of 1 × 10−7–2 × 10−6 M. Moreover, the aptasensor showed selectivity despite the presence of other possible water contaminates and could be regenerated under low pH condition. The developed biosensor shows great potential to be incorporated in a biochip and used for in situ detection of E. coli OMPs in water samples.

Label-free Detection of Microcystin-LR in Waters Using Real-Time Potentiometric Biosensors Based on Single-Walled Carbon Nanotubes Imprinted Polymers

Microcystin-LR (MC-LR) is a dangerous toxin found in environmental waters, quantified by high performance liquid chromatography and/or enzyme-linked immunosorbent assays. Quick, low cost and on-site analysis is thus required to ensure human safety and wide screening programs. This work proposes label-free potentiometric sensors made of solid-contact electrodes coated with a surface imprinted polymer on the surface of Multi-Walled Carbon NanoTubes (CNTs) incorporated in a polyvinyl chloride membrane. The imprinting effect was checked by using non-imprinted materials. The MC-LR sensitive sensors were evaluated, characterized and applied successfully in spiked environmental waters. The presented method offered the advantages of low cost, portability, easy operation and suitability for adaptation to flow methods.

Optimizing potentiometric ionophore and electrode design for environmental on-site control of antibiotic drugs: Application to sulfamethoxazole

Potentiometric sensors are typically unable to carry out on-site monitoring of environmental drug contaminants because of their high limits of detection (LODs). Designing a novel ligand material for the target analyte and managing the composition of the internal reference solution have been the strategies employed here to produce for the first time a potentiometric-based direct reading method for an environmental drug contaminant. This concept has been applied to sulfamethoxazole (SMX), one of the many antibiotics used in aquaculture practices that may occur in environmental waters. The novel ligand has been produced by imprinting SMX on the surface of graphitic carbon nanostructures (CN) < 500 nm. The imprinted carbon nanostructures (ICN) were dispersed in plasticizer and entrapped in a PVC matrix that included (or not) a small amount of a lipophilic additive. The membrane composition was optimized on solid-contact electrodes, allowing near-Nernstian responses down to 5.2 μg/mL and detecting 1.6 μg/mL. The membranes offered good selectivity against most of the ionic compounds in environmental water. The best membrane cocktail was applied on the smaller end of a 1000 μL micropipette tip made of polypropylene. The tip was then filled with inner reference solution containing SMX and chlorate (as interfering compound). The corresponding concentrations were studied for 1 × 10−5 to 1 × 10−10 and 1 × 10−3 to 1 × 10−8 mol/L. The best condition allowed the detection of 5.92 ng/L (or 2.3 × 10−8 mol/L) SMX for a sub-Nernstian slope of −40.3 mV/decade from 5.0 × 10−8 to 2.4 × 10−5 mol/L.

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.

Biomimetic Sensor Potentiometric System for Doxycycline Antibiotic Using a Molecularly Imprinted Polymer as an Artificial Recognition Element

Molecular imprinting is a useful technique for the preparation of functional materials with molecular recognition properties. A Biomimetic Sensor Potentiometric System was developed for assessment of doxycycline (DOX) antibiotic. The molecularly imprinted polymer (MIP) was synthesized by using doxycycline as a template molecule, methacrylic acid (MAA) and/or acrylamide (AA) as a functional monomer and ethylene glycol dimethacrylat (EGDMA) as a cross-linking agent. The sensing elements were fabricated by the inclusion of DOX imprinted polymers in polyvinyl chloride (PVC) matrix. The sensors showed a high selectivity and a sensitive response to the template in aqueous system. Electrochemical evaluation of these sensors under static (batch) mode of operation reveals near-Nernstian response. MIP/MAA membrane sensor was incorporated in flow-through cells and used as detectors for flow injection analysis (FIA) of DOX. The method has the requisite accuracy, sensitivity and precision to assay DOX in tablets and biological fluids.

"Multiplex PCR" identification of the atypical and monophasic Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- in São Paulo State, Brazil: frequency and antibiotic resistance patterns

Salmonella spp. are the etiologic agents of salmonellosis, a worldwide spread zoonoses causing foodborne outbreaks and clinical diseases. By serological identification, Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- accounted for 8.8% of human and 1.6% of nonhuman Salmonella strains isolated in São Paulo State, during 1991-2000. A total of 28.6% of them amplified a fragment corresponding to H:1,2 (flagellar phase two) through PCR analysis and were further assigned as S. Typhimurium. Antimicrobial resistance was detected in 36.3% of the 369 PCR-negative strains tested, including the multiresistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin.

Reconstruction of Large Defect of Foot with Extensive Bone Loss Exclusively Using a Latissimus Dorsi Muscle Free Flap: a Potential New Indication for this Flap

In cases of extensive damage to the foot, with significant bone loss, it is generally accepted that reconstruction must include bone flaps or grafts either in the emergency setting or subsequently. In this report, we describe the case of an 18-year-old student with an avulsion injury of the dorsum of his right foot. Consequently, he lost most of the soft tissue over the dorsum of the foot and the cuboid, navicular, and cuneiform bones. A latissimus dorsi free flap was used to reconstruct the defect. A functional pseudoarthrosis developed between the remaining bones of the foot, and the patient experienced satisfactory foot function after rehabilitation. For this reason, no additional reconstructive procedure was undertaken. This case suggests that it might be adequate to use the latissimus dorsi muscle flap more liberally than previously reported in the reconstruction of extensive defects of the dorsum of the foot, including cases with significant bone loss. This option could avoid the morbidity and inconvenience of a second surgery and the need to harvest a bone flap or graft.

Successful control program to implement the appropriate antibiotic prophylaxis for cesarean section

OBJECTIVE: Describe implementation of a successful program to reduce doses (cefazolin 2 to 1 g) used for antimicrobial prophylaxis. METHODS: Evaluation of an intervention program to reduce prophylactic antimicrobial doses. The intervention included weekly staff discussions, automatic dispensation of 1g-vial of cefazolin by the pharmacy unless expressly requested by surgeon and increase in post-discharge surveillance as a strategy to reassure surgeons of the safety of the reduction. In the pre and post intervention periods, a prospective study of antimicrobial consumption and surgical site infections were measured. RESULTS: There were 5,164 and 5,204 deliveries in 2001-2002 and 2003-2004, respectively; 1,524 (29.5%) and 1,363 (26%) were cesarean sections. There was a 45% decrease in cefazolin vials used on average per cesarean section (2.29 to 1.25). Patients evaluated increased from 16% to 67% and the SSI rates in both periods were 3.34% to 2.42%, respectively. CONCLUSION: An ample intervention, including administrative and educational measures, led to high compliance with dose reduction and saved more than US$4,000 in cefazolin, considered important because government reimbursement in Brazil for cesarean section is $80.

Cell surface hydrophobicity and adherence of a strain of group B streptococci during the post-antibiotic effect of penicillin

The minimum inhibitory concentration and post-antibiotic effects of an antimicrobial agent are parameters to be taken into consideration when determining its dosage schedules. The in vitro post-antibiotic effects on cell surface hydrophobicity and bacterial adherence were examined in one strain of group B streptococci. Exposure of the microorganism for 2 h at 37 °C to 1 x MIC of penicillin induced a PAE of 1.1 h. The cell surface charge of the Streptococcus was altered significantly during the post-antibiotic phase as shown by its ability to bind to xylene: hydrophobicity was decreased. Bacterial adherence to human buccal epithelial cells was also reduced. The results of the present investigation indicate that studies designed to determine therapeutic regimens should evaluate the clinical significance of aspects of bacterial physiology during the post-antibiotic period.

Characterization of an antioxidant surfactant-free topical formulation containing Castanea sativa leaf extract

Context: Inclusion of antioxidants in topical formulations can contribute to minimize oxidative stress in the skin, which has been associated with photoaging, several dermatosis and cancer. Objective: A Castanea sativa leaf extract with established antioxidant activity was incorporated into a semisolid surfactant-free formulation. The objective of this study was to perform a comprehensive characterization of this formulation. Materials and methods: Physical, microbiological and functional stability were evaluated during 6 months storage at 20 °C and 40 °C. Microstructure elucidation (cryo-SEM), in vitro release and in vivo moisturizing effect (Corneometer® CM 825) were also assessed. Results and discussion: Minor changes were observed in the textural and rheological properties of the formulation when stored at 20 °C for 6 months and the antioxidant activity of the plant extract remained constant throughout the storage period. Microbiological quality was confirmed at the end of the study. Under accelerated conditions, higher modifications of the evaluated parameters were observed. Cryo-SEM analysis revealed the presence of oil droplets dispersed into a gelified external phase. The release rate of the antioxidant compounds (610 ± 70 µgh−0.5) followed Higuchi model. A significant in vivo moisturizing effect was demonstrated, that lasted at least 4 h after product’s application. Conclusion: The physical, functional and microbiological stability of the antioxidant formulation was established. Specific storage conditions should be recommended considering the influence of temperature on the stability. A skin hydration effect and good skin tolerance were also found which suggests that this preparation can be useful in the prevention or treatment of oxidative stress-mediated dysfunctions.