This paper describes the optimisation and the analytical performances of an enzyme-based electrochemical genosensor, developed using disposable oligonucleotide-modified screen-printed gold ...electrodes.
The immobilisation of a thiol-tethered probe was qualitatively investigated by means of faradic impedance spectroscopy. Impedance spectra confirmed that the thiol moiety unambiguously drives the immobilisation of the oligonucleotide probe. Furthermore, both probe surface densities and hybridisation efficiencies were quantified through chronocoulometric measurements.
Electrochemical transduction of the hybridisation process was also performed by means of faradic impedance spectroscopy, after coupling of a streptavidin–alkaline phosphatase conjugate and bio-catalysed precipitation of an insoluble and insulating product onto the sensing interface. Chronocoulometric results allowed discussion of the magnitude of hybridisation signals in terms of probe surface densities and their corresponding hybridisation efficiency. The genosensor response varied linearly (
r
2 = 0.9998) with the oligonucleotide target concentration over three orders of magnitude, between 12
pmol/L and 12
nmol/L. The estimated detection limit was 1.2
pmol/L (i.e., 7.2 × 10
6 target molecules in 10
μL of sample solution).
The analytical usefulness of the impedimetric genosensor was finally demonstrated analysing amplified samples obtained from the pBI121 plasmid and soy and maize powders containing 1 and 5% of genetically modified product. Sensing of such unmodified amplicons was achieved via sandwich hybridisation with a biotinylated signaling probe. The electrochemical enzyme-amplified assay allowed unambiguous identification of all genetically modified samples, while no significant non-specific signal was detected in the case of all negative controls.
This study elucidated the hybridization behavior of surface-bound oligonucleotides to their longer PCR-amplified targets. The screen-printed gold surface of disposable electrodes was the platform ...onto which thiol-tethered oligonucleotides (21-mer) were immobilized by chemisorption. As a model case, approximately 600-bp amplicons were studied. Surface hybridization was monitored by means of an enzyme-linked assay with electrochemical detection. Use of different surface-tethered probe sequences over a wide range of surface densities was explored to achieve the highest duplex yield. Both the surface coverage by the probe and its relative position on the target strand were found to control the efficiency of capture of the target sequence. Interfacial hybridization occurred with the highest efficiency for a probe coverage of approximately 2.9 x 10(12) molecules/cm2 and when the 3' end of the amplicon was involved. An unusual (bell-shaped) response/amplicon concentration profile was additionally found. It was hypothesised that when the amount of solution-phase target is relatively high, random collisions make reannealing of the approximately 600-bp strands favored over formation of the surface-tethered probe-amplicon complex. This paper also describes a strategy to enhance the sensitivity of enzyme-linked hybridization assays. Such a strategy relies on formation, around the long target sequence, of dendritic-like structures, which could offer multiple anchoring points for the enzyme conjugate. The results shown in this work might have great significance for the practical application of hybridization to oligonucleotide chips.
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.
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.
An electrochemical genosensor for the detection of specific sequences of DNA has been developed using disposable screen-printed gold electrodes.
Screen-printed gold electrodes were firstly modified ...with a mixed monolayer of a 25-mer thiol-tethered DNA probe and a spacer thiol, 6-mercapto-1-hexanol (MCH). The DNA probe sequence was internal to the sequence of the 35S promoter, which sequence is inserted in the genome of GMOs regulating the transgene expression.
An enzyme-amplified detection scheme, based on the coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the electroinactive α-naphthyl phosphate to α-naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry.
The assay was, firstly, characterised using synthetic oligonucleotides. Relevant parameters, such as the probe concentration and the immobilisation time, the use of the MCH and different enzymatic conjugates, were investigated and optimised. The genosensor response was found to be linearly related to the target concentration between 0 and 25
nmol/L; the detection limit was 0.25
nmol/L.
The analytical procedure was then applied for the detection of the 35S promoter sequence, which was amplified from the pBI121 plasmid by polymerase chain reaction (PCR). Hybridisation conditions (i.e., hybridisation buffer and hybridisation time) were further optimised. The selectivity of the assay was confirmed using biotinylated non-complementary amplicons and PCR blanks. The results showed that the genosensor enabled sensitive (detection limit: 1
nmol/L) and specific detection of GMO-related sequences, thus providing a useful tool for the screening analysis of bioengineered food samples.
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.
Background:
The objective of this work was to determine the clinical accuracy of GlucoMen®Day, a new microdialysis-based continuous glucose monitoring system (CGMS) from A. Menarini Diagnostics ...(Florence, Italy). Accuracy evaluation was performed using continuous glucose-error grid analysis (CG-EGA), as recommended by the Performance Metrics for Continuous Interstitial Glucose Monitoring; Approved Guideline (POCT05-A).
Methods:
Two independent clinical trials were carried out on patients with types 1 and 2 diabetes mellitus, the glycemic levels of whom were monitored in an in-home setting for 100-hour periods. A new multiparametric algorithm was developed and used to compensate in real-time the GlucoMen®Day signal.
The time lag between continuous glucose monitoring (CGM) and reference data was first estimated using the Poincaré plot method. The entire set of CGM/reference data pairs was then evaluated following the CG-EGA criteria, which allowed an estimation of the combined point and rate accuracy stratified by glycemic ranges.
Results:
With an estimated time lag of 11 minutes, the linear regression analysis of the CGM/reference glucose values yielded r = 0.92. The mean absolute error (MAE) was 11.4 mg/dl. The calculated mean absolute rate deviation (MARD) was 0.63 mg/dl/min. The data points falling within the A+B zones of CG-EGA were 100% in hypoglycemia, 95.7% in euglycemia, and 95.2% in hyperglycemia.
Conclusions:
The GlucoMen®Day system provided reliable, real-time measurement of subcutaneous glucose levels in patients with diabetes for up to 100 hours. The device showed the ability to follow rapid glycemic excursions and detect severe hypoglycemic events accurately. Its accuracy parameters fitted the criteria of the state-of-the-art consensus guideline for CGMS, with highly consistent results from two independent studies.
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 α-naphthyl phosphate solution. Differential pulse voltammetry was finally used to detect the α-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.
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.
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.
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.
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.
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.
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.
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 system's output.
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.
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