Lucia Mosiello

A biosensor for Escherichia coli based on a potentiometric alternating biosensing (PAB) transducer

Routinary methods for the detection of bacterial contamination in table water are costly and time consuming. In the present paper we describe a biosensor based on an immunoassay test. Escherichia coli cells present in water samples were detected by a complete Potentiometric alternating biosensor (PAB). The PAB system consists of a measuring chamber, acquisition and driving electronics and the appropriate software: the apparatus is computer controlled in order to obtain on-line acquisitions and recording of data. The transducer principle is based on a LAPS (Light Addressable Potentiometrie Sensor) technology which, in our case, reveals the production of NH3 by a urease-E. coli antibody conjugate. The proposed system appears to be very sensitive: concentrations of 10 cells per mL were clearly detected over blank of E. coli free water. No signals were detected from other bacteria, eventually polluting drinking water such as Pseudomonas marina, and also from bacteria such as Klebsiella oxytoca phyletically related to E. coli.

A fibre-optic immunosensor for 2,4-dichlorophenoxyacetic acid detection

A new total internal reflection fluorescence (TIRF) fibre-optic sensor working with the red fluorophore Cy5 and based on a compact, inexpensive solid-state laser source and photodetector is presented. The sensor has been tested with a monoclonal antibody (mAb) developed in our laboratory against the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The perfomances of mAb- and hapten-coated fibres are compared. A detection threshold below 60 pM is achieved with the latter configuration, with mAb as the analyte. An immunoassay for 2,4-D in solution has also been developed and compared with an analogous immunoenzymatic assay (ELISA). The TIRF assay allowed the mAb equilibrium dissociation constant for the liquid-phase antibody-antigen reaction to be calculated.

Laser-Assisted Fabrication of Biomolecular Sensing Microarrays

With the aim of obtaining high density biosensing arrays we use pulsed laser deposition to immobilize functional biomolecules on useful surfaces, and micro- and nanopatterning techniques for fabrication of prototype immunosensing bioarrays. We report biological activity tests demonstrating the functional properties of the immobilized proteins and atomic force microscopy characterization of films of nanometric dimensions. Laser-fabricated immunofluorescent arrays are analyzed to check that the intensity and contrast of the sensing sites allow efficient device fabrication. We have also developed an elementary array of heterogeneous reaction sites and tested its performance by simultaneous incubation with the different specific antigens.

Development of electrochemical aptasensor using dendrimers as an immobilization platform for detection of Aflatoxin B1 in food samples

This project focuses on the development of a DNA-aptamer based biosensor, which is referred to as an aptasensor, that would aid in the detection of mycotoxins in foodstuffs. We developed aptamer based biosensor for detection of Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticus, species of fungi. The characterization of the sensor and detection of aflatoxin B1 was performed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). We tested an electrochemical aptasensor for AFB1 detection through the immobilization of DNA aptamers on the Poly (amido-amine) dendrimers of fourth generation (PAMAM G4) on gold electrode covered by cystamine. The DNA aptamers specific to AFB1 were attached on the PAMAM layer covalently by means of glutaraldehyde. The range of AFB1 concentrations was selected as target interval for further utilization of the biosensor in food samples. The aptasensor has been tested in certificated samples of peanuts as well as in spiked samples of peanuts demonstrating optimal response. The biosensor was also tested in other mycotoxins such as ochratoxin A (OTA) and aflatoxin B2 (AFB2), to investigate non-specific interaction.

Development of a QCM (Quartz Crystal Microbalance) Biosensor to Detection of Mycotoxins

In this study, we have used a direct immunoassay where the simple binding between antigen and an antibody is detected. Immunoassays were performed in a drop system, monitoring the decrease in the frequency of the quartz crystal microbalance device as the mass increases during immunoreaction. The QCM sensor was coated on both sides by gold electrodes; only one side of the crystal (liquid side) was in contact with the solution, the other side (contact side) was always dry. We tested a piezoelectric immunosensor for aflatoxin B1 (AFLA-B1), ochratoxin A (OTA), and fumonisin B1 (FB1) mycotoxin detection through the immobilization of DSP–anti-mycotoxin antibody (AFLA-B1–Ab anti-AFLAB1, OTA-Ab anti-OTA, FB1-Ab anti-FB1) on gold-coated quartz crystals (AT-cut/5 MHz). The DSP (3,3′-dithiodipropionic-acid-di-N-hydroxysuccinimide ester) was used for the covalent attachment of the proteins. The piezoelectric crystal electrodes were pretreated by DSP for 15 min, rinsed with water, and dried in a gentle flow of nitrogen gas. Then the DSP-coated crystals were installed in a sample holder and exposed to the antibody and to the analyte. Frequency and resistance shifts (Δf and ΔR) were measured simultaneously.

Biosensors for Aflatoxins Detection

The availability of rapid and reliable methods for rapid determination of small molecules, as contaminants in food samples, including Aflatoxins, is an increasing need also for human health. In order to monitoring food contaminants, as Mycotoxins (MTXs) Gas Chromatographic (GC) and High Pressure Liquid Chromatography (HPLC) methods are generally utilized, due to their high detection sensibility and selectivity. However, GC and HPLC analyses are time consuming and needs sample pre-treatment or pre-concentration procedures. Immunoassays and biosensors are becoming a recognized alternative or complementary to conventional analytical techniques for the detection of mycotoxins, as Aflatoxins. Recently, biosensors based on the use of monoclonal or polyclonal antibodies have seen a great development in the field of small molecules analytical determination and specifically in the mycotoxins analyses. Among biosensors for mycotoxins monitoring, optical or electrochemical devices for Aflatoxins detection were described by different authors. The present Chapter describes the different biosensors for Aflatoxins developed and utilized in food analysis. The absence of cross-reactivity obtained with most of these biosensor, the possibility of on-line measurement, the absence of sample pre-treatment, can really put it in competition with other conventional systems such as HPLC and ELISA. Chapter describes also main biosensors features and vantages for these innovative devices and various examples of biosensors and reviews some biosensors for Aflatoxins and other mycotoxins detection methods, as microarray. In particular, we will focus our attention on biosensors developed for mycotoxins detection that utilize immunoglobulins or aptamer showing affinity for a correspondent analyte, associated to various transduction elements. Various biosensing platforms will be introduced, including, but not limited to, surface plasmon resonance and quartz microbalance crystals. Examples of biosensors array, as microarray, detecting Aflatoxin and Fumonisin will be also presented. Some of these biosensing devices were developed in our laboratories and the sensing performance of each device will be evaluated and compared in terms of sensitivity and detection limit. Analytical methods used for mycotoxins determination are mainly based on TLC, HPLC or ELISA. Actually biosensor and microsystem technologies are used for different applications including studies of human and veterinary diseases, drug discovery, genetic screening, clinical and food diagnostics. According to these approaches the aim of many authors was to transfer the methods of the immunological assay from microtiter plates into a biosensor format in order to develop a rapid, sensitive and inexpensive method for the detection of

Suspending DNA origami between four gold nanodots

Rectangular DNA origami functionalized with thiols in each of the four corners immobilizes by self-assembly between lithographically patterned gold nanodots on a silicon oxide surface.

High-Sensitive Impedimetric Aptasensor for Detection Ochratoxin A in Food

We report a high sensitive biosensor based on DNA aptamers for detection ochratoxin A (OTA). The thiolated DNA aptamers specific to OTA have been immobilised to a surface of gold electrode. The electrochemical impedance spectroscopy (EIS) at presence of redox probe [Fe(CN)6]−3/−4 has been used for determination of charge transfer resistance, Rct, following addition of OTA. We have shown that Rct increased with increasing OTA in a range of 0.1–100 nM. The sensor revealed high sensitivity (the limit of detection was 0.44 nM), selectivity and was regenerable. The sensor was validated in coffee extract.

A Piezoelectric Quartz Crystal Sensor Applied for Thrombin-Binding Aptamers

The temperature-dependent UV spectroscopy and thickness shear mode acoustic method were applied to study of thermodynamics and binding properties of DNA aptamers sensitive to thrombin depending on the substitution of bases in G-quadruplex. The substitution of thymidines by adenines in TT and TGT loops of G-quadruplex resulted in destabilization of aptamers and in decrease of sensitivity to human thrombin.

A Novel Based Protein Microarray for the Simultaneous Analysis of Activated Caspases

The aim of our work is to develop a new method to study caspases expression in apoptotic cells using an antibody-based protein microarray. Actually we performed the array for two different form of caspase-3 (cleaved and uncleaved), in the next future we plan to enlarge the microarray analysis using caspase-8 and caspase-9 antibodies in order to obtain a new analytical tool to study the expression of these markers in apoptotic cells.