Maria Minunni

Surface plasmon resonance imaging for affinity-based biosensors

SPR imaging (SPRi) is at the forefront of optical label-free and real-time detection. It offers the possibility of monitoring hundreds of biological interactions simultaneously and from the binding profiles, allows the estimation of the kinetic parameters of the interactions between the immobilised probes and the ligands in solution. We review the current state of development of SPRi technology and its application including commercially available SPRi instruments. Attention is also given to surface chemistries for biochip functionalisation and suitable approaches to improve sensitivity.

New trends in affinity sensing: aptamers for ligand binding

Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and amplification. This review described the in vitro process to obtain aptamers (SELEX). It mentions the main characteristics of these molecules (i.e. affinity, specificity and stability). Moreover, it discusses advantages over antibodies. It reports potential applications of aptamers in analytical and diagnostic assays as biocomponents of biosensors (aptasensors) and allosteric ribozymes (aptazymes).

Electrochemical and piezoelectric DNA biosensors for hybridisation detection

DNA biosensors (or genosensors) are analytical devices that result from the integration of a sequence-specific probe and a signal transducer. Among other techniques, electrochemical and piezoelectric methods have recently emerged as the most attractive due to their simplicity, low instrumentation costs, possibility for real-time and label-free detection and generally high sensitivity. Focusing on the most recent activity of worldwide researchers, the aim of the present review is to give the readers a critical overview of some important aspects that contribute in creating successful genosensing devices. Advantages and disadvantages of different sensing materials, probe immobilisation chemistries, hybridisation conditions, transducing principles and amplification strategies will be discussed in detail. Dedicated sections will also address the issues of probe design and real samples pre-treatment. Special emphasis will be finally given to those protocols that, being implemented into an array format, are already penetrating the molecular diagnostics market.

Quartz crystal microbalance (QCM) affinity biosensor for genetically modified organisms (GMOs) detection.

A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.

Surface modifications for the development of piezoimmunosensors.

Four different techniques for the immobilisation of proteins onto the gold electrode of a piezoelectric quartz crystal were investigated. The examined techniques were adsorption, avidin-biotin binding and two different types of covalent binding on self-assembled monolayers (SAM), dithiobis(succinimidylpropionate) (DSP) and a dextran modified thiol monolayer. The reaction of the immobilised proteins (bovine serum albumin (BSA) and anti-human IgG) with their specific antibodies, anti-BSA and hIgG (50 and 200 micrograms/ml) were studied using a quartz crystal microbalance and then compared. Many cycles of measurements were performed on the same crystal regenerating the gold surface with a solution of glycine.HCl, 100 mM, pH 2.1. The interactions of the immobilised reagents with non-specific antibodies were also studied. The adsorption protocol was the quickest, but did not allow regeneration with glycine.HCl. Thiol-dextran coated surfaces did not show any detectable response to non-specific reagents, but needed a very long and complicated protocol. DSP and avidin-biotin coating procedures were easy and not too long. They seemed to have the best characteristics of reproducibility among different crystals and possibility of regeneration of the coated surface, but the percentage of non-specific binding was high.

Detection of Pesticide in Drinking Water Using Real-Time Biospecific Interaction Analysis (BIA)

Abstract It is reported an application of a biosensor based technology employing Surface Plasmon Resonance (SPR) for studies of molecular interaction in real time. An inhibition immunoassay for the detection of the herbicide atrazine has been developed. The biospecific interface is a sensor chip to which an atrazine derivative has been covalently bound. Monoclonal antibodies (Mabs) against atrazine are mixed with the sample containing herbicide, then the solution reacted with the biospecific interface. As the interaction between free antibodies and the immobilized derivative of atrazine bound to the surface proceeds, the SPR response changes inversely related to the atrazine concentration. A detection limit of 0.05 ppb of atrazine in water can be reached; the concentration range is 0–1 ppb, the analysis time is 15 minutes. After each measure the sensor chip surface can be regenerated. The analysis has been performed with standard solutions (buffer) in distilled and tap water.

The Quartz Crystal Microbalance as Biosensor. A Status Report on Its Future

Abstract A report on the status of the quartz crystal microbalance (QCM) technique for applications in biochemistry, food, environmental and clinical analysis is presented. With the advent of pre-coated crystals, used in a flow cell with the microprocessor controlled PZ 106 Immunobiosensor System, assays can be performed with the QCM in a manner equivalent to the SPR (surface plasmon resonance) technique both in sensitivity and selectivity. ∗Humboldt Fellow on Leave from the University of New Orleans, Louisiana 70148 (USA).

Surface plasmon resonance biosensor for genetically modified organisms detection

The development of a surface plasmon resonance (SPR) affinity biosensor based on DNA hybridisation is described. This biosensor has been applied to genetically modified organisms (GMOs) detection. Single stranded DNA (ssDNA) probes were immobilised on the sensor chip of an SPR device and the hybridisation between the immobilised probe and the complementary sequence (target) was monitored. The probe sequences were internal to the sequence of 35S promoter and NOS terminator which are inserted sequences in the genome of GMO regulating the transgene expression. The system has been optimised using synthetic oligonucleotides, then applied to real samples analysis. Samples, containing the transgenic target sequences, were amplified by polymerase chain reaction (PCR) and then detected with the SPR biosensor.

A quartz crystal microbalance displacement assay for Listeria monocytogenes

Abstract A novel, piezoelectric, quartz crystal microbalance (QCM) biosensor has been developed for the detection of Listeria monocytogenes . The Listeria was immobilized on the gold surface of the crystals using different immobilization approaches. Using a liquid flow cell, it was possible to directly monitor the antigen (Ag)-antibody (Ab) binding without the use of a label, i.e. enzymes, radioactive compounds, etc. The calibration curve was prepared using a displacement assay with a response range from 2.5 × 10 5 to 2.5 × 10 7 cells crystal . The assay was also performed in milk which was injected with the non-specific antigen Serratia or Listeria . The method is as sensitive as ELISA and results can be obtained in less than 15 min.

An optimized digestion method coupled to electrochemical sensor for the determination of Cd, Cu, Pb and Hg in fish by square wave anodic stripping voltammetry

An optimized digestion method coupled to electrochemical detection to monitor lead, copper, cadmium and mercury in fish tissues was developed. Square wave anodic stripping voltammetry (SWASV) coupled to disposable screen-printed electrodes (SPEs) was employed as fast and sensitive electroanalytical method for heavy metals detection. Different approaches in digestion protocols were assessed. The study was focused on Atlantic hake fillets because of their wide diffusion in the human nutrition. Best results were obtained by digesting fish tissue with hydrogen peroxide/hydrochloric acid mixture coupled to solid phase (SP) purification of the digested material. This combined treatment allowed quantitative extraction from fish tissue (muscle) of the target analytes, with fast execution times, high sensitivity and avoiding organic residues eventually affecting electrochemical measurements. Finally, the method has been validated with reference standard materials such as dogfish muscle (DORM-2) and mussel tissues (NIST 2977).