Fausto Lucarelli

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and electrochemical investigation of the surface, are discussed in detail with separate sections devoted to label-free and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples.

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.

Prussian Blue and enzyme bulk-modified screen-printed electrodes for hydrogen peroxide and glucose determination with improved storage and operational stability

The addition to the carbon ink, a major component of a screen-printed electrode (SPE), of an aliquot (10%) of Prussian Blue (PB)-modified glassy carbon (PB-GC) particles, resulted in an interference free “ready to use” amperometric H 2O2 sensor (Vapp =− 50 mV) with a LOD of 3 × 10 −7 mol/l and a sensitivity of 135 mA (mol l −1 cm 2 ). A storage stability of up to 8 months and an operational stability of 3 days has been achieved making these sensors suitable for mass-production. Glucose and lysine biosensors have been assembled immobilizing glucose oxidase (Gox) and lysine oxidase (LyOx) with glutaraldehyde and Nafion ® onto the PB bulk-modified electrodes. A LOD of 4 × 10 −6 mol/l for glucose and 5 × 10 −6 mol/l for lysine with a linear range up to 0.5 and 0.7 mmol/l, respectively, have been observed. A ready to use glucose biosensor was then developed mixing PB-modified glassy carbon (7.5%), carbon ink (87.5%) and glucose oxidase (GOx, 5%) and using the mixture for the printing step of a SPE working electrode. A LOD of 3× 10 −5 mol/l and a linearity range up to 6 × 10 −3 mol/l of glucose have been achieved, together with a storage stability up to 20 weeks (at RT) and an operational stability of 1 day with 11 calibration curves performed.

Electrochemical DNA biosensor as a screening tool for the detection of toxicants in water and wastewater samples.

Applications of a disposable electrochemical DNA biosensor to standard solutions and to real samples are reported. The DNA biosensor is assembled by immobilising the double stranded calf thymus DNA on the surface of a disposable carbon screen-printed electrode. The immobilised ds-DNA interacts with the sample for 2 min; then is washed and immersed in a clean buffer where the analytical signal (the oxidation peak area of the guanine base) is obtained by a square-wave voltammetric scan. The results were compared with some currently used toxicity tests and in particular with a commercial luminescent bacteria test, Toxalert(R)100.

Electrochemical DNA biosensor for analysis of wastewater samples

The application of a disposable electrochemical DNA biosensor to wastewater samples is reported. The DNA biosensor is assembled by immobilising double-stranded calf thymus DNA on the surface of a disposable, carbon screen-printed electrode (SPE). The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal. The presence of compounds with affinity for DNA is measured by their effect on the guanine oxidation. The comparison of the results with a toxicity test based on bioluminescent bacteria has confirmed the applicability of the method to real samples.

Disposable electrochemical DNA-array for PCR amplified detection of hazelnut allergens in foodstuffs

An electrochemical low-density DNA-array has been designed and implemented to be used in combination with polymerase chain reaction (PCR) in order to investigate the presence of hazelnut major allergens (Cor a 1.04, Cor a 1.03) in foodstuff. Unmodified PCR products were captured at the sensor interface via sandwich hybridization with surface-tethered probes and biotinylated signalling probes. The resulting biotinylated hybrids were coupled with a streptavidin-alkaline phosphatase conjugate and then exposed to a alpha-naphthyl phosphate solution. Differential pulse voltammetry was finally used to detect the alpha-naphthol signal. The detection limits for Cor a 1.03 and Cor a 1.04 were 0.3 and 0.1 nmol L(-1), respectively (R.S.D. 10%). The optimized conditions were used to test several commercially available foodstuffs, claiming to contain or not the targeted nuts. The results were compared with those obtained with classical ELISA tests.

Coupling of an indicator-free electrochemical DNA biosensor with polymerase chain reaction for the detection of DNA sequences related to the apolipoprotein E

Abstract This paper describes a disposable indicator-free electrochemical DNA biosensor applied to the detection of apolipoprotein E (apoE) sequences in PCR samples. In the indicator-free assays, the duplex formation was detected by measuring the electrochemical signal of the guanine base of nucleic acids. The biosensor format involved the immobilisation of an inosine-modified (guanine-free) probe onto a screen-printed electrode (SPE) transducer and the detection of the duplex formation in connection with the square-wave voltammetric measurement of the oxidation peak of the guanine of the target sequence. The indicator-free scheme has been characterised using 23-mer oligonucleotides as model: parameters affecting the hybridisation assay such as probe immobilisation conditions, hybridisation time, use of hybridisation accelerators were examined and optimised. The analysis of PCR samples (244xa0bp DNA fragments, obtained by amplification of DNA extracted from human blood) required a further optimisation of the experimental procedure. In particular, a lower steric hyndrance of the probe modified surface was essential to allow an efficient hybridisation of the target DNA fragment. Negative controls have been performed using the PCR blank and amplicons unrelated to the immobilised probe. A 10xa0min hybridisation time allowed a full characterisation of each sample.

GlucoMen Day Continuous Glucose Monitoring System: A Screening for Enzymatic and Electrochemical Interferents

Background: While most of the common drugs with the potential to interfere with continuous glucose monitoring (CGM) systems are accessible over the counter and can be assumed by CGM patients without medical supervision, many other chemicals are frequently used to treat critically ill patients. Continuous glucose monitoring reading accuracy may also be compromised in patients characterized by abnormally high concentrations of physiological interferents. In this article, 22 species selected from endogenous and exogenous chemicals were screened as possible interferents of GlucoMen®Day (GMD), the new microdialysis-based CGM system from A. Menarini Diagnostics. Method: Interference testing was performed according to the EP7-A2 guideline (Clinical and Laboratory Standards Institute 2005). Interference was evaluated at two levels of glucose, with each interferent additionally tested at two concentrations. Furthermore, two configurations of the GMD disposable sensor kit—one designed for subcutaneous application, the other for direct intravascular CGM—were challenged with interferent-spiked serum and blood samples, respectively. Results: With the exception of dopamine (however, at very high, nonphysiological concentrations), no interference was observed for all the tested substances. Interestingly, none of the common electrochemical interferents (including ascorbic acid, acetaminophen, and salicylic acid, which represent the major specificity issue for the competing CGM systems) significantly affected the systems output. Conclusions: These results provide clear insights into the advantages offered by the use of a microdialysis-based CGM system that additionally relies on the detection of hydrogen peroxide at low operating potential. GlucoMen Day may become the CGM system of choice for those patients who require either regular administration of drugs or their glycemia to be tightly controlled in the intensive care unit or similar environments.

Comparative Performance Assessment of Point-of-Care Testing Devices for Measuring Glucose and Ketones at the Patient Bedside

Background: Point-of-care (POC) testing devices for monitoring glucose and ketones can play a key role in the management of dysglycemia in hospitalized diabetes patients. The accuracy of glucose devices can be influenced by biochemical changes that commonly occur in critically ill hospital patients and by the medication prescribed. Little is known about the influence of these factors on ketone POC measurements. The aim of this study was to assess the analytical performance of POC hospital whole-blood glucose and ketone meters and the extent of glucose interference factors on the design and accuracy of ketone results. Methods: StatStrip glucose/ketone, Optium FreeStyle glucose/ketone, and Accu-Chek Performa glucose were also assessed and results compared to a central laboratory reference method. The analytical evaluation was performed according to Clinical and Laboratory Standards Institute (CLSI) protocols for precision, linearity, method comparison, and interference. Results: The interferences assessed included acetoacetate, acetaminophen, ascorbic acid, galactose, maltose, uric acid, and sodium. The accuracies of both Optium ketone and glucose measurements were significantly influenced by varying levels of hematocrit and ascorbic acid. StatStrip ketone and glucose measurements were unaffected by the interferences tested with exception of ascorbic acid, which reduced the higher level ketone value. The accuracy of Accu-Chek glucose measurements was affected by hematocrit, by ascorbic acid, and significantly by galactose. The method correlation assessment indicated differences between the meters in compliance to ISO 15197 and CLSI 12-A3 performance criteria. Conclusions: Combined POC glucose/ketone methods are now available. The use of these devices in a hospital setting requires careful consideration with regard to the selection of instruments not sensitive to hematocrit variation and presence of interfering substances.

Minimizing the Impact of Time Lag Variability on Accuracy Evaluation of Continuous Glucose Monitoring Systems

Background: Despite all commercially available continuous glucose monitoring (CGM) systems being designed to operate in the extracellular interstitial fluid, and even though there is a well-recognized time lag between the interstitial and the venous compartments, the accuracy of the CGM device readings is still evaluated against the glucose concentration in venous blood (VB) samples, thus resulting in a perceived decrease in accuracy. This article explains how different time lag compensation methods (no compensation, compensation with a fixed delay, compensation with a variable delay based on an intercompartmental diffusional model) have an impact on how CGM accuracy is evaluated. Methods: The data set used consisted of 210 CGM/blood glucose data pairs from 18 diabetes subjects (15 type 1 and 3 type 2) selected from a data base collected during two independent clinical trials. All CGM measurements were performed using the GlucoMen®Day CGM system (A. Menarini Diagnostics, Italy), and the reference VB glucose measurements by means of a standard laboratory instrument. For each applied time lag compensation method, the CGM accuracy evaluation was performed as recommended by the POCT05-A consensus guideline. Results: The perceived accuracy of the CGM device significantly improved when applying both the fixed or the variable delay compensation method. However, it is worth noting how the variable delay method, which relies on a closer description of the intercompartmental diffusion processes, provided the best perception of the clinical accuracy of the device. Conclusions: When assessing the accuracy of a CGM system, a crucial step in data analysis is to account for time lag, which enables minimization of the apparent decline in system accuracy.