Sonia Centi

Different approaches for the detection of thrombin by an electrochemical aptamer-based assay coupled to magnetic beads.

Different assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection of thrombin by using a thrombin specific aptamer. By using the thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin have been developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of the enzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the electrochemical transduction with the innovative and advantageous use of magnetic beads. The assays based on the immobilisation of the protein were not successful since no binding was recorded between thrombin and its aptamer. With the direct competitive assay, when the aptamer was immobilised onto the magnetic beads, a detection limit of 430nM for thrombin was achieved. A lower detection limit for the protein (175nM) was instead obtained by detecting the product of the enzymatic reaction catalysed by thrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45nM of thrombin demonstrating the best analytical performances. With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances of the best assay configuration has been demonstrated.

Chitosan Films Doped with Gold Nanorods as Laser-Activatable Hybrid Bioadhesives

Chitosan is a linear chain polysaccharide comprising glucosamine and N -acetyl glucosamine residues joined together by β -1,4-glycosidic bonds. It is derived as an acids-soluble material by deacetylation of chitin, which is commonly found in the outer skeleton of crustaceans and in the cell walls of fungi. A variety of fundamental properties such as excellent biocompatibility and biodegradability make chitosan a very attractive material for biomedical applications including wound dressing, tissue engineering and drug delivery. [ 1 , 2 ] The peculiar tendency of chitosan to form films with high mechanical strength, good elasticity and rather slow biodegradation is among the principal reasons why this material is gaining a primary role in medical technology. [ 2–4 ] Examples of applications of chitosan films are the localized chemotherapy based on the local delivery and sustained release of antineoplastic agents after cancer removal [ 5 ] and the tissue repair by closing chronic, accidental, and surgical wounds. [ 4 , 6 ] In order to enable these applications, the chitosan films need to be secured to the target, which may be achieved by suturing. [ 7 ] However, in many cases, suturing may be hardly feasible such as for poorly accessible or delicate body regions (e.g., intracranial, myocardial, etc.). In addition metals and synthetic materials in use for implantation can interfere with the functional rehabilitation of the site due to foreign-body reactions, causing infl ammation and abnormal wound healing. [ 8 ]

Gold nanorods as new nanochromophores for photothermal therapies

Results and perspectives on the biomedical exploitation of gold nanorods with plasmon resonances in the near infrared window are reported. The authors describe experimental studies of laser-activated nanoparticles in the direct welding of connective tissues, which may become a valuable technology in biomedicine. In particular, colloidal gold nanorods excited by diode laser radiation at 810 nm were used to mediate functional photothermal effects and weld eyes lens capsules and arteries. The preparation of biopolymeric matrices including gold nanorods is also described, as well as preliminary tests for their application in the closure of wounds in vessels and tendons. Finally, the use of these nanoparticles for future applications in the diagnosis, imaging and therapy of cancer is discussed.

Improvement of analytical performances of a disposable electrochemical immunosensor by using magnetic beads

A comparison of two electrochemical immunosensing strategies for PCBs detection, based on the use of two different solid phases, is here discussed. In both cases, carbon-based screen-printed electrodes (SPEs) are used as transducers in a direct competitive immunoassay scheme, where PCBs in solution compete with the tracer PCB28-alkaline phosphatase (AP) labeled for antibodies immobilized onto the solid-phase. In the standard format (called EI strategy), SPEs are both the solid-phase for immunoassay and electrochemical transducers: in this case the immunochemical reaction occurs onto the working electrode. Finally, the enzymatic substrate is added and an electroactive product is generated and detected by electrochemical measurement. In order to improve the performances of the system, a new approach (called EMI strategy) is developed by using functionalized magnetic beads as solid phase for the competitive assay; only after the immunosensing step they are captured by a magnet onto the working surface of the SPE for the electrochemical detection. Experimental results evidenced that the configuration based on the use of separate surfaces for immunoassay and for electrochemical detection gave the best results in terms of sensitivity and speed of the analysis. The improvement of analytical performances of the immunosensor based on EMI strategy was also demonstrated by the analysis of some spiked samples.

Strategies for electrochemical detection in immunochemistry

In this review, the current status of research in electrochemical immunosensors is considered. We primarily focus on label-free and enzyme-labeled immunosensors, and the analytical capabilities of these devices are discussed. Moreover, the use of magnetic beads as new materials for immunosensors coupled with electrochemical sensing is also described, together with the application of new molecules such as aptamers as specific biorecognition elements. Examples of the applicability of these devices in solving various analytical problems in clinical, environmental and food fields are reported. Finally, the prospects for the further development of immunosensor technologies are shown.

Size dependent biological profiles of PEGylated gold nanorods

The perspective of introducing plasmonic particles for applications in biomedical optics is receiving much interest. However, their translation into clinical practices is delayed by various factors, which include a poor definition of their biological interactions. Here, we describe the preparation and the biological profiles of gold nanorods belonging to five different size classes with average effective radii between ∼5 and 20 nm and coated with polyethylene glycol (PEG). All these particles exhibit decent stability in the presence of representative proteins, low cytotoxicity and satisfactory compatibility with intravenous administration, in terms of their interference with blood tissue. However, the suspension begins to become unstable after a few days of exposure to blood proteins. Moreover, the cytotoxicity is a little worse for smaller particles, probably because their purification is more critical, while undesirable interactions with the mononuclear phagocyte system are minimal in the intermediate size range. Overall, these findings hold implications of practical relevance and suggest that PEGylated gold nanorods may be a versatile platform for a variety of biomedical applications.

In vitro assessment of antibody-conjugated gold nanorods for systemic injections

BackgroundThe interest for gold nanorods in biomedical optics is driven by their intense absorbance of near infrared light, their biocompatibility and their potential to reach tumors after systemic administration. Examples of applications include the photoacoustic imaging and the photothermal ablation of cancer. In spite of great current efforts, the selective delivery of gold nanorods to tumors through the bloodstream remains a formidable challenge. Their bio-conjugation with targeting units, and in particular with antibodies, is perceived as a hopeful solution, but the complexity of living organisms complicates the identification of possible obstacles along the way to tumors.ResultsHere, we present a new model of gold nanorods conjugated with anti-cancer antigen 125 (CA125) antibodies, which exhibit high specificity for ovarian cancer cells. We implement a battery of tests in vitro, in order to simulate major nuisances and predict the feasibility of these particles for intravenous injections. We show that parameters like the competition of free CA125 in the bloodstream, which could saturate the probe before arriving at the tumors, the matrix effect and the interference with erythrocytes and phagocytes are uncritical.ConclusionsAlthough some deterioration is detectable, anti-CA125-conjugated gold nanorods retain their functional features after interaction with blood tissue and so represent a powerful candidate to hit ovarian cancer cells.

Detection of Polychlorinated Biphenyls (PCBs) in Milk using a Disposable Immunomagnetic Electrochemical Sensor

Abstract PCBs are among the most persistent and widely distributed pollutants in the global ecosystem and therefore it is of great importance for both human and environmental health to develop practical analytical systems to detect them. In this context, this work presents the application of an electrochemical immunosensor based on Screen‐Printed Carbon‐based Electrodes as transducers with antibody‐coated magnetic microparticles as a solid phase. The immunoassay was based on direct competition between the analyte present in the samples and an alkaline phosphatase‐labeled tracer. The product of the enzymatic reaction between AP and its substrate (α‐naphthyl phosphate) was detected using differential pulse voltammetry. Sample extraction and clean‐up was achieved using Solid Phase Extraction. Detection of low concentrations PCB was achieved and the method was shown to be applicable to both skimmed and whole milk. SPE was shown to improve the analysis.

Quantitative measurement of scattering and extinction spectra of nanoparticles by darkfield microscopy

We demonstrate a versatile concept for the quasi simultaneous and quantitative measurement of light extinction and scattering cross section spectra of nanoparticles in a darkfield microscope. We validate this method by the analysis of an aqueous suspension of gold nanorods and comparison with both numerical simulations and standard spectrophotometry measurements. Our approach holds the promise to allow one to map the principal optical properties of nanoparticles in a biological sample with μm spatial resolution, which is an issue of particular relevance for applications in biomedical optics such as photothermolysis and laser hyperthermia.

Detection of biomarkers for inflammatory diseases by an electrochemical immunoassay: The case of neopterin

An electrochemical immunoassay for neopterin was developed using recently produced specific antibodies immobilized to protein A-coated magnetic beads in combination with differential pulse voltammetry and screen-printed array of electrodes. Neopterin-alkaline phosphatase conjugate was used as label in a competitive assay format. Multiplexed analysis of neopterin was demonstrated by replacing the traditional ELISA with electrochemical detection and the traditional plastic wells with screen-printed array of electrodes. The optimized electrochemical method, based on polyclonal antibodies, reached a limit of detection of 0.008 ng/mL with an average RSD %=10. Serum samples collected from patients with sepsis, healthy volunteers and other patients without a confirmed clinical diagnosis were also analyzed. The obtained results, compared with those of a commercial ELISA kit, had a significant correlation, showing the possibility to distinguish among the serum samples from ill or healthy subjects.