[Show abstract][Hide abstract] ABSTRACT: We report a versatile particle-based route to dense arrays of parallel submicron pores with high aspect ratio in silicon, and explore the application of these arrays in sensors, optics, and polymer micropatterning. Polystyrene (PS) spheres are convectively assembled on gold-coated silicon wafers and sputter-etched, resulting in well-defined gold disc arrays with excellent long-range order. The gold discs act as catalysts in Metal-Assisted Chemical Etching (MACE), yielding uniform pores with straight walls, flat bottoms and high aspect ratio. The resulting pore arrays can be used as robust antireflective surfaces, in biosensing applications, and as templates for polymer replica molding.
[Show abstract][Hide abstract] ABSTRACT: Immunosensors are devices that exploit immobilized antibodies to promote the binding of specific analytes related to diseases of medical importance, such as cancer or cardiac dysfunctions. Label-free immunosensors have an important role, due to their simplicity and fast read-out. Here, the proof of concept for an immunosensor based on a 2-D photonic crystal silicon nitride membrane is presented. The device has been fabricated by means of a well-tuned nanofabrication protocol, achieving a high-quality photonic pattern on a large-area membrane (1 mm × 1 mm), and it has been tested for the detection of interleukin-6, getting protein detection at pg/mL concentrations.
[Show abstract][Hide abstract] ABSTRACT: The use of carbon nanotubes (CNTs) as building blocks in the design of electrochemical biosensors has been attracting attention over the last few years, mainly due to their high electrical conductivity and large surface area. Here, we present two approaches based on tailored single-walled CNTs (SWCNTs) architectures to develop immunosensors for the bacteriophage MS2, a virus often detected in sewage-impacted water supplies. In the first approach, SWCNTs were used in the bottom-up design of sensors as antibody immobilization support. Carboxy-functionalised SWCNTs were covalently tethered onto gold electrodes via carbodiimide coupling to cysteamine-modified gold electrodes. These SWCNTs were hydrazide functionalized by electrochemical grafting of diazonium salts. Site-oriented immobilization of antibodies was then carried out through hydrazone bond formation. Results showed microarray electrode behavior, greatly improving the signal-to-noise ratio. Excellent sensitivity and limit of detection (9.3pfu/mL and 9.8pfu/mL in buffer and in river water, respectively) were achieved, due to the combination of the SWCNTs' ability to promote electron transfer reactions with electroactive species at low overpotentials and their high surface-to-volume ratio providing a favorable environment to immobilize biomolecules. In the second approach, SWCNTs were decorated with iron oxide nanoparticles. Diazonium salts were electrochemically grafted on iron-oxide-nanoparticle-decorated SWCNTs to functionalize them with hydrazide groups that facilitate site-directed immobilization of antibodies via hydrazone coupling. These magnetic immunocarriers facilitated MS2 separation and concentration on an electrode surface. This approach minimized non-specific adsorptions and matrix effects and allowed low limits of detection (12pfu/mL and 39pfu/mL in buffer and in river water, respectively) that could be further decreased by incubating the magnetic immunocarriers with larger volumes of sample. Significantly, both approaches permitted the detection of MS2 to levels regularly encountered in sewage-impacted environments.
[Show abstract][Hide abstract] ABSTRACT: In this work, protein phosphatase inhibition assays (PPIAs) have been used to evaluate the performance of recombinant PP1 and recombinant and wild-type PP2As. The enzymes have been compared using microcystins-LR (MC-LR) as a model cyanotoxin. Whereas PP2ARec provides a limit of detection (LOD) of 3.1 μg/L, PP1Rec and PP2AWild provide LODs of 0.6 and 0.5 μg/L, respectively, lower than the guideline value proposed by the World Health Organization (1 μg/L). The inhibitory potencies of seven MC variants (-LR, -RR, -dmLR, -YR, -LY, -LW and -LF) have been evaluated, resulting on 50 % inhibition coefficient (IC50) values ranging from 1.4 to 359.3 μg/L depending on the MC variant and the PP. The PPIAs have been applied to the determination of MC equivalent contents in a natural cyanobacterial bloom and an artificially contaminated sample, with multi-MC profiles. The inhibition equivalency factors (IEFs) have been applied to the individual MC quantifications determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the estimated MC-LR equivalent content has been compared to PPIA results. PPIAs have demonstrated to be applicable as MC screening tools for environmental applications and to protect human and animal health.
Environmental Science and Pollution Research 09/2014; 21(18):10652. DOI:10.1007/s11356-014-3065-7 · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper reports the development of three peptide modified sensors in which glutathione (GSH) and its fragments Cys-Gly and γ-Glu-Cys were immobilized respectively through aryl diazonium electrochemical grafting onto the surface of graphite-epoxy composite electrodes (GEC), and used for the simultaneous determination of Cd(II), Pb(II) and Zn(II). The concentration interval ranged from 0.1 to 1.5 μmol L-1 for each metal, and the technique used was differential pulse adsorptive stripping voltammetry. This study aimed to the comparison of the information provided by one single modified electrode at both fixed and multiple pH values (pH 6.8, 7.5 and 8.2) for the simultaneous determination of the three metals, with those supplied by the three-sensor array at multiple pH values. For the processing of the voltammograms, the fast Fourier transform was selected as the preprocessing tool for data compression coupled with an artificial neural network for the modeling of the obtained responses.
[Show abstract][Hide abstract] ABSTRACT: The progress in medical field has provided fast remedies for the most of diseases affecting today's society. Now, the next step is to propose a fast way of diagnosis, avoiding long waiting time and expensive laboratory assays. Thanks to the recent research findings and progress in nano-technological fabrication techniques, new biosensors have been developed with the purpose to introduce portable and user-friendly instruments for medical analysis aimed to improve life quality and lower healthcare costs. In this work, we propose a proof-of-concept of an immunosensor based on a two-dimensional silicon nitride photonic crystal membrane able to efficiently detect the concentration of Interleukin-6 in a buffer solution.
Photonics Conference, 2014 Third Mediterranean, Trani; 05/2014
[Show abstract][Hide abstract] ABSTRACT: A novel sensing strategy for electrochemical aptamer-based sensors is presented. Nucleic acid aptamers are considered alternatives to antibodies. However, some of their intrinsic properties, such as that they can undergo conformational changes during the binding of the target, can be used to design novel sensing strategies. Unlike other electrochemical "signal off" aptamer-based sensors, we report a strategy based on enzymatic inhibition. Our approach shows the feasibility to detect small molecules based on the aptamer conformational change induced by the target that leads to the inhibition of the enzyme used as label. Additionally, we prove the ability to regenerate the function of the aptasensor by simply applying a short potential pulse. As a proof-of-concept, the widely used aptamer for ochratoxin A (OTA) has been selected as a model. After self-assembling short oligonucleotides onto a gold electrode, complementary to the 3' end of the aptamer, hybridisation of the aptamer takes place. To investigate the mechanism induced by the OTA-binding, Surface Plasmon Resonance assays were performed, which confirmed the conformational switch of the aptamer rather than the aptamer displacement by dehybridisation from the DNA-modified sensor surface. The electrochemical sensor can successfully detect OTA in wine at the limits stipulated by the European Commission. Given its sensitivity, rapid and easy detection and regeneration, it can be envisaged as screening tool for OTA detection. Moreover, this sensing strategy has the potential to be applied to other aptamer-based biochemical assays for the detection of small molecules in the fields of food safety, environmental monitoring and medical diagnostics.
[Show abstract][Hide abstract] ABSTRACT: Appropriate site-directed chemistry is essential to maximise the performance of immunosensors. We present two new functionalisation strategies that preserve proper folding and binding potential of antibodies by forcing their oriented immobilisation. Both strategies are based on the formation of hydrazone bonds between aldehyde groups on the Fc moieties of periodate-oxidised antibodies and hydrazide groups on functionalised gold electrodes. Those hydrazide groups are introduced by electrografting of diazonium salts or by self-assembly of mono- and dithiolated hydrazide linkers, resulting in films with tailored functional groups and, thus, antibody distribution and spacing. Their barrier properties and permeability towards electroactive species are evaluated. To demonstrate the potential of these new functionalisation strategies, detection of bacteriophage MS2 is performed through either a direct assay using electrochemical impedance spectroscopy (EIS) or through a sandwich assay using differential pulse voltammetry (DPV). Diazonium and monothiolated self-assembled monolayers-modified electrodes enable the detection of less than 1 plaque forming unit (pfu)/mL in a direct EIS assay. However, non-specific adsorption renders measurements in river water samples difficult. In contrast, sandwich-assays on electrodes with electrografted diazonium salts and monothiolated self-assembled monolayers do not show significant matrix effects using river water samples, but the limits of detection are 108 times higher than those of the direct assay. Best results are achieved for immunosensors based on mixed monolayers of hydrazide and hydroxyl diothiolated linkers (15 pfu/mL). These new functionalisation techniques are facile to implement. They afford the possibility to tune surface composition and tailor the electrochemical properties of electrochemical sensors. These advantages should translate into broad interest in this type of surface chemistry for biosensor development.
[Show abstract][Hide abstract] ABSTRACT: We report a new label-free colorimetric aptasensor based on DNAzyme-aptamer conjugate for rapid and high-throughput detection of Ochratoxin A (OTA, a possible human carcinogen, group 2B) in wine. Two oligonucleotides were designed for this detection. One is N1 for biorecognition, which includes two adjacent sequences: the OTA-specific aptamer sequence and the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The other is a blocking DNA (B2), which is partially complementary to a part of the OTA aptamer and partially complementary to a part of the DNAzyme. The existence of OTA reduces the hybridization between N1 and B2. Thus, the activity of the non-hybridized DNAzyme is linearly correlated with the concentration of OTA up to 30nM with a limit of detection of 4nM (3σ). Meanwhile, a double liquid-liquid extraction (LLE) method is accordingly developed to purify OTA from wine. Compared with the existing HPLC-FD or immunoassay methods, the proposed strategy presents the most appropriate balance between accuracy and facility, resulting in a considerable improvement of real-time quality control, and thereby, preventing chronic poisoning caused by OTA contained red wine.
[Show abstract][Hide abstract] ABSTRACT: Biosensors are devices able to reduce costs and time in biological assays. They are efficient and useful tools also in biochemical assays, providing high performance in sensitivity and selectivity of measurements. In this work, we developed a label-free biosensor based on a two-dimensional photonic crystal patterned on a large area silicon nitride membrane that operates in the visible spectrum for the detection of proteins. The biosensor has been preliminarily tested in solutions of different refractive index, showing as expected a shift of the resonance in the reflection spectrum.
Photonics Technologies, 2014 Fotonica AEIT Italian Conference on, Napoli; 05/2013
[Show abstract][Hide abstract] ABSTRACT: The presence of enterohemorrhagic Escherichia coli bacteria in food can cause serious foodborne disease outbreaks. Early detection and identification of these pathogens is extremely important for public health and safety. Here we present a highly sensitive label-free immunosensor for the detection of pathogenic E. coli O157:H7. Anti-E. coli antibodies were covalently immobilised onto gold electrodes via a self-assembled monolayer (SAM) of mercaptohexadecanoic acid and the pathogenic bacteria were detected by electrochemical impedance spectroscopy (EIS). Surface Plasmon Resonance (SPR) was used to monitor the antibody immobilisation protocol and antibody patterned surfaces were used to demonstrate the specificity of the antibody coated surfaces against the pathogenic bacteria. The immunosensor showed a very low limit of detection (2CFU/mL) and a large linear range (3×10-3×10(4)CFU/mL). Finally, the selectivity of the sensor was demonstrated and no significant adsorption of Salmonella typhimurium was observed.
[Show abstract][Hide abstract] ABSTRACT: Ochratoxin A (OTA) is a mycotoxin found in a wide range of food and feedstuffs. Intake of OTA-contaminated food causes health concern due to the harmful effects reported on humans and animals. Much effort is currently devoted to set up and optimise highly sensitive and accurate methods of OTA analysis. This work describes the comparison of fluorescence-based immunosensing strategies for the analysis of OTA. First, an indirect competitive fluoroimmunoassay was designed and optimised. The assay enabled the quantification of the toxin at the levels set by the European legislation. Then, a flow-immunoassay based on kinetic exclusion measurements was developed. It showed the theoretical lowest limit of detection enabled by the affinity of the anti-OTA antibody (IC(80)=12ngL(-1) in the assay solution). Wine and cereal samples were analysed using the optimised flow system. No significant matrix effects were observed after simple pre-treatment of wine and OTA extraction from corn-flakes samples. This simple and highly sensitive automated biosensing-system allows OTA quantification in food and beverages. It is envisaged as a powerful tool for rapid and reliable toxin screening.
[Show abstract][Hide abstract] ABSTRACT: This article gives an overview of the biosensors for the analysis of mycotoxins, marine toxins and cyanobacterial toxins, describing in depth the electrochemical biosensors that incorporate nanobiotechnological concepts. Firstly, it presents tailor-designed biomolecules, such as recombinant enzymes, recombinant antibody fragments and aptamers as novel biorecognition elements in biosensors. It also reviews the use of metallic nanoparticles (NPs) and carbon nanotubes (CNTs) aiming at improving the electrochemical transduction strategies. Finally, the exploitation of magnetic particles (MPs) as immobilisation carriers in flow-systems and the development of arrays are also described. The incorporation of these nanobiotechnological concepts provides with electrochemical biosensors with superior analytical performance in terms of specificity, sensitivity, stability and analysis time.
The Analyst 03/2012; 137(5):1055-67. DOI:10.1039/c2an15736e · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report an aptasensor for biosensing of Ochratoxin A (OTA) using aptamer-DNAzyme hairpin as biorecognition element. The structure of this engineered nucleic acid includes the horseradish peroxidase (HRP)-mimicking DNAzyme and the OTA specific aptamer sequences. A blocking tail captures a part of these sequences in the stem region of the hairpin. In the presence of OTA, the hairpin is opened due to the formation of the aptamer-analyte complex. As a result, self-assembly of the active HRP-mimicking DNAzyme occurs. The activity of this DNAzyme is linearly correlated with OTA concentration up to 10 nM, showing a limit of detection of 2.5 nM.
[Show abstract][Hide abstract] ABSTRACT: Ochratoxin A (OTA) is one of the most important mycotoxins because of its high toxicity to both humans and animals and its occurrence in a number of basic foods and agro-products. The need to develop high-performing methods for OTA analysis able to improve the traditional ones is evident. In this work, through in vitro SELEX (Systematic Evolution of Ligands by EXponential enrichment) two aptamers, designated H8 and H12 were produced that bind with nanomolar affinity with Ochratoxin A (OTA). Two strategies were investigated by using an indirect and a direct competitive Enzyme-Linked Aptamer Assay (ELAA) and were compared to the classical competitive Enzyme-Linked Immunosorbent Assay (ELISA) for the determination of OTA in spiked red wine samples. The limit of detection attained (1 ng/mL), the midpoint value obtained (5 ng/mL) and the analysis time needed (125 min) for the real sample analysis validate the direct competitive ELAA as useful screening tool for routine use in the control of OTA level in wine.
Food Control 05/2011; 22(5-22):737-743. DOI:10.1016/j.foodcont.2010.11.005 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The valuable properties of aptamers, such as specificity, sensitivity, stability, cost-effectiveness and design flexibility,
have favoured their use as biorecognition elements in biosensor development. These synthetic affinity probes can be developed
for almost any target molecule, covering a wide range of applications in fields such as clinical diagnosis and therapy, environmental
monitoring and food control. The combination of aptamers with high-performance electrochemical transducers, with their inherent
high sensitivities, fast response times and simple equipment, has already provided several electrochemical aptamer-based sensors.
Moreover, the small size and versatility of aptamers allow efficient immobilisations in high-density monolayers, an important
feature towards miniaturisation and integration of compact electrochemical devices. This review describes the state-of-the-art
of electrochemical aptamer-based sensors, entering into the details of the different strategies and types of electrochemical
transduction and also considering their advantages when applied to the analysis of complex matrices.
KeywordsAptamer-Biosensor-Electrochemical detection-Aptabeacon-Redox label
[Show abstract][Hide abstract] ABSTRACT: Secondary metabolites are chemical compounds that are not directly involved in the normal growth, development or reproduction of organisms. Due to the toxicity shown by some of these compounds, their presence can represent a threat to human health. Reliable detection systems able to control their presence are required, as a tool to ensure public health. This chapter offers an overview of different techniques developed for the detection of toxic secondary metabolites, taking ochratoxin A and microcystins as two representative examples. While ochratoxin A is a mycotoxin produced by several species of fungi, microcystins are cyanotoxins released by certain strains of cyanobacteria. Biosensor-based strategies are emphasized as powerful screening tools.
Advances in Experimental Medicine and Biology 01/2010; 698:282-92. DOI:10.1007/978-1-4419-7347-4_21 · 1.96 Impact Factor