Diego Voccia

Strategies for the development of an electrochemical bioassay for TNF-alpha detection by using a non-immunoglobulin bioreceptor.

TNF-α is an inflammatory cytokine produced by the immune system. Serum TNF-α level is elevated in some pathological states such as septic shock, graft rejection, HIV infection, neurodegenerative diseases, rheumatoid arthritis and cancer. Detecting trace amount of TNF-α is, also, very important for the understanding of tumor biological processes. Detection of this key biomarker is commonly achieved by use of ELISA or cytofluorimetric based methods. In this study the traditional optical detection was replaced by differential pulse voltammetry (DPV) and an affinity molecule, produced by evolutionary approaches, has been tested as capture bioreceptor. This molecule, namely a combinatorial non-immunoglobulin protein (Affibody®) interacts with TNF-α selectively and was here tested in a sandwich assay format. Moreover magnetic beads were used as support for bioreceptor immobilization and screen printed carbon electrodes were used as transducers. TNF-α calibration curve was performed, obtaining the detection limit of 38pg/mL, the quantification range of 76-5000pg/mL and RSD%=7. Preliminary results of serum samples analysis were also reported.

Direct determination of small RNAs using a biotinylated polythiophene impedimetric genosensor

Herein, direct determination of small RNAs is described using a functional-polymer modified genosensor. The analytical strategy adopted involves deposition by electropolymerization of biotinylated polythiophene films on the surface of miniaturized, disposable, gold screen-printed electrodes, followed by the layer-by-layer deposition of streptavidin, and then biotynilated capture probes. A small RNA (miR-221) target was determined via the impedimetric measurement of the hybridization event in a label-free and PCR-free approach. Under optimized conditions, the limit of detection (LOD) was 0.7 pM miR-221 (15% RSD). The genosensor was applied for determination of miR-221 in total RNA extracted from human lung and breast cancer cell lines, discriminating between the cancer-positive and -negative cells, without any amplification step, in less than 2h.

Electrochemical, Electrochemiluminescence, and Photoelectrochemical Aptamer-Based Nanostructured Sensors for Biomarker Analysis

Aptamer-based sensors have been intensively investigated as potential analytical tools in clinical analysis providing the desired portability, fast response, sensitivity, and specificity, in addition to lower cost and simplicity versus conventional methods. The aim of this review, without pretending to be exhaustive, is to give the readers an overview of recent important achievements about electrochemical, electrochemiluminescence, and photoelectrochemical aptasensors for the protein biomarker determination, mainly cancer related biomarkers, by selected recent publications. Special emphasis is placed on nanostructured-based aptasensors, which show a substantial improvement of the analytical performances.

Electrochemical Liposome-Based Biosensors for Nucleic Acid Detection

MicroRNAs (miRNAs) are intensely studied as candidates for diagnostic and prognostic clinical biomarkers. Faradic impedance spectroscopy (EIS) and differential pulse voltammetry (DPV), coupled to disposable gold electrodes and enzyme amplification of the hybridization event, were used for the development of biosensors for the detection of miRNAs. Biotin-labeled liposomes were employed as nanointerfaces that amplify the primary miRNA-sensing events by their association to the probe–/DNA–miRNA–analyte complex generated onto the transducer.

Electrochemical and Photoelectrochemical Biosensors for Biomarker Detection

A rapid and accurate medical diagnosis is essential in order to determine the health status of a patient. Nowadays, most of the clinical analyses are performed in specialized laboratory, which required specific instrumentation and trained personal, resulting in an increase of analysis costs and time. In this context, biosensors represent ideal tools capable to provide a specific and fast response together with low cost, easy use and portable size features. This work attempts to provide a review of the research progresses of electrochemical and photoelectrochemical biosensor platforms in clinical applications that have been published in recent years. Special emphasis will be devoted to discuss examples for breast cancer biomarker detection, because breast cancer, is considered the leading cause of cancer-related deaths worldwide in women, representing 15% of all cancer related amongst women, with a 6% mortality rate (based on overall cancer deaths). The manuscript is focused on aptamer-based biosensors, because, due to their stability and their relatively low cost, they have been successfully applied in many biosensor formats for breast cancer biomarker detection.

Different enzyme-based strategies for the development of disposable electrochemical biosensors: Application to environmental pollutant monitoring

The increased concerns with the toxic effects of chemicals have led to the necessity of monitoring pollution levels into the different environmental compartments. Thus, in order to fulfill the increasing request of rapid analysis of environmental samples, electrochemical biosensors have been proposed as reliable, fast and inexpensive devices for the analysis of pollutants. Biosensors combine the selectivity of the biomolecular reactions with operational simplicity. Enzymes have found widespread use as sensing elements in catalytic electrochemical biosensors. Enzymes are also used in affinity biosensors, as label of the biorecognition event. The successful use of enzymes is essentially due to their specificity and to their ability to convert single biorecognition events into a multitude of detectable molecules. In this work, the use of enzymes for the development of both catalytic and affinity biosensors for the detection of environmental pollutants is described. In particular, different strategies for the development of catalytic, esterase-based biosensors for the detection of organophosphorous and carbamate pesticides, recently proposed by the authors lab, will be revised. Moreover, preliminary results regarding the development of an emerging class of environmental pollutants, i.e. PBDEs, by means of an affinity biosensor will be also presented.

Different strategies for the detection of bioagents using electrochemical and photoelectrochemical genosensors

In recent years various kinds of biosensors for the detection of pathogens have been developed. A genosensor consists in the immobilization, onto the surface of a chosen transducer, of an oligonucleotide with a specific base sequence called capture probe. The complementary sequence (the analytical target, i.e. a specific sequence of the DNA/RNA of the pathogen) present in the sample is recognized and captured by the probe through the hybridization reaction. The evaluation of the extent of the hybridization allows one to confirm whether the sample contains the complementary sequence of the probe or not. Electrochemical transducers have received considerable attention in connection with the detection of DNA hybridization. Moreover, recently, with the emergence of novel photoelectrochemically active species and new detection schemes, photoelectrochemistry has resulted in substantial progress in its analytical performance for biosensing applications. In this paper, some examples of electrochemical genosensors for multiplexed pathogen detection are shown. Moreover, the preliminary experiments towards the development of a photoelectrochemical genosensor using a TiO2 – nanocrystal-modified ITO electrode are discussed.

Label-Free Impedimetric Determination of miRNA Using Biotinylated Conducting Polymer Modified Carbon Electrodes

Preliminary results are reported on label-free miRNA determination with faradaic electrochemical impedance spectroscopy (EIS) measurements on glassy carbon electrodes (GCEs) and screen-printed carbon electrodes (SPCEs). These electrodes were coated with films of functionalized conducting polymers. For that, bis(2,2′-bithien-5-yl)-(4-hydroxyphenyl)methane biotin ester was used as the functional monomer. This monomer was potentiodynamically electropolymerized to result in deposition of thin polymer films on the electrodes. Next, streptavidin, and then the biotinylated DNA capture probe were immobilized via streptavidin–biotin interactions. Detectability of the resulting biosensor with respect to the complementary miRNA was well below 100 pM.

Electrochemical Biosensors for miRNA Detection

MicroRNAs (miRNAs) are intensely studied as candidates for diagnostic and prognostic biomarkers. They are naturally occurring small RNAs (approximately 22 nucleotides in length) that act as regulators of protein translation. Because many diseases are caused by the misregulated activity of proteins, miRNAs have been implicated in a number of diseases including a broad range of cancers, heart disease, and immunological and neurological diseases. A great deal of effort, therefore, has been devoted to developing analytical methods for miRNA analysis. The consideration when selecting existing or designing new methods for miRNA analysis includes sensitivity and multiplexing capability without PCR. In this chapter, novel electrochemical strategies for miRNA detection and quantification will be reviewed.