A Förster Resonance Energy Transfer (FRET)-Based Immune Assay for the Detection of Microcystin-LR in Drinking Water.

Cyanobacteria bloom is the term used to describe an abnormal and rapid growth of cyanobacteria in aquatic ecosystems such as lakes, rivers, and oceans as a consequence of anthropic factors, ecosystem degradation, or climate change. Cyanobacteria belonging to the genera Microcystis, Anabaena, Planktothrix, and Nostoc produce and release toxins called microcystins (MCs) into the water. MCs can have severe effects on human and animal health following their ingestion and inhalation. The MC structure is composed of a constant region (composed of five amino acid residues) and a variable region (composed of two amino acid residues). When the MC variable region is composed of arginine and leucine, it is named MC-LR. The most-common methods used to detect the presence of MC-LR in water are chromatographic-based methods (HPLC, LC/MS, GC/MS) and immunological-based methods (ELISA). In this work, we developed a new competitive Förster resonance energy transfer (FRET) assay to detect the presence of traces of MC-LR in water. Monoclonal antibody anti-MC-LR and MC-LR conjugated with bovine serum albumin (BSA) were labeled with the near-infrared fluorophores CF568 and CF647, respectively. Steady-state fluorescence measurements were performed to investigate the energy transfer process between anti-MC-LR 568 and MC-LR BSA 647 upon their interaction. Since the presence of unlabeled MC-LR competes with the labeled one, a lower efficiency of FRET process can be observed in the presence of an increasing amount of unlabeled MC-LR. The limit of detection (LoD) of the FRET assay is found to be 0.245 nM (0.245 µg/L). This value is lower than the provisional limit established by the World Health Organization (WHO) for quantifying the presence of MC-LR in drinking water.

A Surface-Enhanced Raman Spectroscopy-Based Biosensor for the Detection of Biological Macromolecules: The Case of the Lipopolysaccharide Endotoxin Molecules.

The development of sensitive methods for the detection of endotoxin molecules, such as lipopolysaccharides (LPS), is essential for food safety and health control. Conventional analytical methods used for LPS detection are based on the pyrogen test, plating and culture-based methods, and the limulus amoebocyte lysate method (LAL). Alternatively, the development of reliable biosensors for LPS detection would be highly desirable to solve some critical issues, such as high cost and a long turnaround time. In this work, we present a label-free Surface-Enhanced Raman Spectroscopy (SERS)-based method for LPS detection in its free form. The proposed method combines the benefits of plasmonic enhancement with the selectivity provided by a specific anti-lipid A antibody (Ab). A high-enhancing nanostructured silver substrate was coated with Ab. The presence of LPS was quantitatively monitored by analyzing the changes in the Ab spectra obtained in the absence and presence of LPS. A limit of detection (LOD) and quantification (LOQ) of 12 ng/mL and 41 ng/mL were estimated, respectively. Importantly, the proposed technology could be easily expanded for the determination of other biological macromolecules.

An Impedance-Based Immunosensor for the Detection of Ovalbumin in White Wine.

Food allergies are an exceptional response of the immune system caused by the ingestion of specific foods. The main foods responsible for allergic reactions are milk, eggs, seafood, soy, peanuts, tree nuts, wheat, and their derived products. Chicken egg ovalbumin (OVA), a common allergen molecule, is often used for the clarification process of wine. Traces of OVA remain in the wine during the fining process, and they can cause significant allergic reactions in sensitive consumers. Consequently, the European Food Safety Authority (EFSA) and the American Food and Drug Administration (FDA) have shown the risks for allergic people to assume allergenic foods and food ingredients, including eggs. Commonly, OVA detection requires sophisticated and time-consuming analytical techniques. Intending to develop a faster assay, we designed a proof-of-concept non-Faradaic impedimetric immunosensor for monitoring the presence of OVA in wine. Polyclonal antibodies anti-OVA were covalently immobilised onto an 11-mercaptoundecanoic-acid (11-MUA)-modified gold surface. The developed immunosensor was able to detect OVA in diluted white wine without the need for an external probe or any pre-treatment step with a sensitivity of 0.20 µg/mL, complying with the limit established by the resolution OIV/COMEX 502-2012 for the quantification of allergens in wine.

An Impedimetric Biosensor for Detection of Volatile Organic Compounds in Food.

The demand for a wide choice of food that is safe and palatable increases every day. Consumers do not accept off-flavors that have atypical odors resulting from internal deterioration or contamination by substances alien to the food. Odor response depends on the volatile organic compounds (VOCs), and their detection can provide information about food quality. Gas chromatography/mass spectrometry is the most powerful method available for the detection of VOC. However, it is laborious, costly, and requires the presence of a trained operator. To develop a faster analytic tool, we designed a non-Faradaic impedimetric biosensor for monitoring the presence of VOCs involved in food spoilage. The biosensor is based on the use of the pig odorant-binding protein (pOBP) as the molecular recognition element. We evaluated the affinity of pOBP for three different volatile organic compounds (1-octen-3-ol, trans-2-hexen-1-ol, and hexanal) related to food spoilage. We developed an electrochemical biosensor conducting impedimetric measurements in liquid and air samples. The impedance changes allowed us to detect each VOC sample at a minimum concentration of 0.1 µM.

SPR-Based Detection of ASF Virus in Cells.

African swine fever (ASF) is one of the most dangerous hemorrhagic infectious diseases that affect domestic and wild pigs. Currently, neither a vaccine nor effective treatments are available for this disease. As regards the degree of virulence, ASFV strains can be divided into high, moderate, or low virulence. The main detection methods are based on the use of the polymerase chain reaction (PCR). In order to prevent an uncontrolled spread of ASF, new on-site techniques that can enable the identification of an early-stage disease are needed. We have developed a specific immunological SPR-based assay for ASFV antigen detection directly in liquid samples. The developed assay allows us to detect the presence of ASFV at the dose of 103 HAD50/mL.

The Porcine Odorant-Binding Protein as a Probe for an Impedenziometric-Based Detection of Benzene in the Environment.

Odorant-binding proteins (OBPs) are a group of small and soluble proteins present in both vertebrates and insects. They have a high level of structural stability and bind to a large spectrum of odorant molecules. In the environmental field, benzene is the most dangerous compound among the class of pollutants named BTEX (benzene, toluene, ethylbenzene, and xylene). It has several effects on human health and, consequently, it appears to be important to monitor its presence in the environment. Commonly, its detection requires the use of very sophisticated and time-consuming analytical techniques (GC-MS, etc.) as well as the presence of specialized personnel. Here, we present the application of an odorant-binding protein (pOBP) isolated from pigs as a molecular recognition element (MRE) for a low-energy impedenziometric biosensor for outdoor and real-time benzene detection. The obtained results show that the biosensor can detect the presence of 64 pM (5 µg/m3) benzene, the limit value of exposure for human health set by the European Directive 2008/50/EC.

Photonic Label-Free Biosensors for Fast and Multiplex Detection of Swine Viral Diseases.

In this paper we present the development of photonic integrated circuit (PIC) biosensors for the label-free detection of six emerging and endemic swine viruses, namely: African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PPRSV), Porcine Parvovirus (PPV), Porcine Circovirus 2 (PCV2), and Swine Influenza Virus A (SIV). The optical biosensors are based on evanescent wave technology and, in particular, on Resonant Rings (RRs) fabricated in silicon nitride. The novel biosensors were packaged in an integrated sensing cartridge that included a microfluidic channel for buffer/sample delivery and an optical fiber array for the optical operation of the PICs. Antibodies were used as molecular recognition elements (MREs) and were selected based on western blotting and ELISA experiments to ensure the high sensitivity and specificity of the novel sensors. MREs were immobilized on RR surfaces to capture viral antigens. Antibody-antigen interactions were transduced via the RRs to a measurable resonant shift. Cell culture supernatants for all of the targeted viruses were used to validate the biosensors. Resonant shift responses were dose-dependent. The results were obtained within the framework of the SWINOSTICS project, contributing to cover the need of the novel diagnostic tools to tackle swine viral diseases.

Characterization of Two NMN Deamidase Mutants as Possible Probes for an NMN Biosensor.

Nicotinamide mononucleotide (NMN) is a key intermediate in the nicotinamide adenine dinucleotide (NAD+) biosynthesis. Its supplementation has demonstrated beneficial effects on several diseases. The aim of this study was to characterize NMN deamidase (PncC) inactive mutants to use as possible molecular recognition elements (MREs) for an NMN-specific biosensor. Thermal stability assays and steady-state fluorescence spectroscopy measurements were used to study the binding of NMN and related metabolites (NaMN, Na, Nam, NR, NAD, NADP, and NaAD) to the PncC mutated variants. In particular, the S29A PncC and K61Q PncC variant forms were selected since they still preserve the ability to bind NMN in the micromolar range, but they are not able to catalyze the enzymatic reaction. While S29A PncC shows a similar affinity also for NaMN (the product of the PncC catalyzed reaction), K61Q PncC does not interact significantly with it. Thus, PncC K61Q mutant seems to be a promising candidate to use as specific probe for an NMN biosensor.

Emergent Biosensing Technologies Based on Fluorescence Spectroscopy and Surface Plasmon Resonance.

The purpose of this work is to provide an exhaustive overview of the emerging biosensor technologies for the detection of analytes of interest for food, environment, security, and health. Over the years, biosensors have acquired increasing importance in a wide range of applications due to synergistic studies of various scientific disciplines, determining their great commercial potential and revealing how nanotechnology and biotechnology can be strictly connected. In the present scenario, biosensors have increased their detection limit and sensitivity unthinkable until a few years ago. The most widely used biosensors are optical-based devices such as surface plasmon resonance (SPR)-based biosensors and fluorescence-based biosensors. Here, we will review them by highlighting how the progress in their design and development could impact our daily life.

A fluorescence immunoassay for a rapid detection of Listeria monocytogenes on working surfaces.

Listeria monocytogenes is a foodborne pathogen responsible for human listeriosis. The increasing incidence of listeriosis induced governments and food manufacturing enterprises to act to diminish the problem. Several methods for the detection of Listeria monocytogenes in food industries were developed. However, they are time-consuming and require the use of specialized equipment. To reduce the detection time of Listeria monocytogenes in food, in this work we developed a fluorescence sandwich immunoassay based on the use of an innovative chitosan-cellulose nanocrystal (CNC) membrane that improves the antigen capture during bacterial growth. The combined use of CNC film for the capture of p60 protein-specific antigen together with the use of fluorescence detection reduced the time of analysis from 24 to 12 h with a limit of detection (LOD) of the assay of 102 CFU/mL (2 Log). In addition, the use of monoclonal anti-PepD covalently immobilized to a CNC membrane assured a high specificity of the assay. Interestingly, the obtained results show no cross-reactivity with the five most diffused pathogen bacteria strains tested.

Structural features of the glutamate-binding protein from Corynebacterium glutamicum.

L-glutamate (Glu) is the major excitatory transmitter in mammalian brain. Inadequate concentration of Glu in the brain correlates to mood disorder. In industry, Glu is used as a flavour enhancer in food and in foodstuff processing. A high concentration of Glu has several effects on human health such as hypersensitive effects, headache and stomach pain. The presence of Glu in food can be detected by different analytical methods based on chromatography, or capillary electrophoresis or amperometric techniques. We have isolated and characterized a glutamate-binding protein (GluB) from the Gram-positive bacteria Corynebacterium glutamicum. Together with GluC protein, GluD protein and the cytoplasmic protein GluA, GluB permits the transport of Glu in/out of cell. In this study, we have investigated the binding features of GluB as well as the effect of temperature on its structure both in the absence and in the presence of Glu. The results have showed that GluB has a high affinity and selectivity versus Glu (nanomolar range) and the presence of the ligand induces a higher thermal stability of the protein structure.

Fluorescence polarization assay to detect the presence of traces of ciprofloxacin.

Detection of ciprofloxacin residues in milk by sensitive and rapid methods is of great interest due to its use in the treatment of dairy livestock health. Current analytical approaches to antibiotics detection, are laboratory-based methods and they are time-consuming and require trained personnel. To cope this problem, we propose an assay, based on fluorescence polarization principle, able to detect the presence of ciprofloxacin in diluted milk sample without any pre-treatment. The proposed method is based on the use of ciprofloxacin-protein conjugate labeled with near infrared fluorescence dye, which upon binding to specific antibody causes an increase of the fluorescence polarization emission signal. The developed assay allows for the detection of ciprofloxacin at a concentration of 1ppb, which represents an amount lower than the maximum residual limit (MRL) of ciprofloxacin in milk, as set by the European Union regulation (100 ppb).

Design and Development of Photonic Biosensors for Swine Viral Diseases Detection.

In this paper we introduce a field diagnostic device based on the combination of advanced bio-sensing and photonics technologies, to tackle emerging and endemic viruses causing swine epidemics, and consequently significant economic damage in farms. The device is based on the use of microring resonators fabricated in silicon nitride with CMOS compatible techniques. In the paper, the designed and fabricated photonic integrated circuit (PIC) sensors are presented and characterized, showing an optimized performance in terms of optical losses (30 dB per ring) and extinction ration for ring resonances (15 dB). Furthermore, the results of an experiment for porcine circovirus 2 (PCV2) detection by using the developed biosensors are presented. Positive detection for different virus concentrations has been obtained. The device is currently under development in the framework of the EU Commission co-funded project SWINOSTICS.

Detection of naphthalene in sea-water by a label-free plasmonic optical fiber biosensor.

In this study we developed an optical fiber biosensor able to detect the presence of naphthalene in sea-water. With this aim, we designed and produced an antibody specific for the naphthalene molecule. The capability of the antibody to bind to naphthalene was characterized by ELISA tests. A surface plasmon resonance (SPR) sensor platform was realized by sputtering a gold layer on a modified plastic optical fiber (POF). The gold surface was derivatizated and functionalized with the produced antibody by using the EDC/NHS amino-coupling immobilization protocol. The obtained results indicated that the POF-biosensor is able to sense the presence of naphthalene in a sea-water solution. The limit of detection (LOD) value was calculated to be 0.76 ng/mL, a value lower than the maximum residue limit value of naphthalene (0.13 µg/mL) referred as the water environmental quality standards (EQS). In addition, to the high sensitivity of the assay, it is remarkable to point out the possibility to monitor the presence of naphthalene in a real sea water solution by exploiting a simple experimental setup with a remote sensing capability offered by the POF-biosensor.

Setting up and exploitation of a nano/technological platform for the evaluation of HMGA1b protein in peripheral blood of cancer patients.

Even if cancer specific biomarkers are present in peripheral blood of cancer patients, it is very difficult to detect them with conventional technology because of their low concentration. A potential cancer biomarker is the HMGA1b protein, whose overexpression is a feature of several human malignant neoplasias. By taking advantage of the surface plasmon resonance (SPR) phenomenon, we realized a specific nano/technology-based assay for cancer detection. More in details, anti-HMGA1b monoclonal antibodies, whose affinity was previously defined by ELISA, were immobilized onto metallic surfaces to develop a direct SPR-based assay. After having analyzed blood samples from colorectal cancer patients and healthy people for the presence of HMGA1b, we observed a 2-fold increase of the HMGA1b levels in the blood of cancer patients with respect to the healthy control people. We conclude that the set-up technology might allow to detect a tumoral mass through the evaluation of HMGA1b protein blood levels.

The porcine odorant-binding protein as molecular probe for benzene detection.

In recent years, air pollution has been a subject of great scientific and public interests for the strong impact on human health. Air pollution is due to the presence in the atmosphere of polluting substances, such as carbon monoxide, sulfur and nitrogen oxides, particulates and volatile organic compounds (VOCs), derived predominantly from various combustion processes. Benzene is a VOC belonging to group-I carcinogens with a toxicity widely demonstrated. The emission limit values and the daily exposure time to benzene (TLV-TWA) are 5µg/m3 (0.00157 ppm) and 1.6mg/m3 (0.5 ppm), respectively. Currently, expensive and time-consuming analytical methods are used for detection of benzene. These methods require to perform a few preliminary steps such as sampling, and matrices pre-treatments. In addition, it is also needed the support of specialized personnel. Recently, single-walled carbon nanotube (SWNTs) gas sensors with a limit detection (LOD) of 20 ppm were developed for benzene detection. Other innovative bioassay, called bio-report systems, were proposed. They use a whole cell (Pseudomona putida or Escherichia coli) as molecular recognition element and exhibit a LOD of about 10 µM. Here, we report on the design of a highly sensitive fluorescence assay for monitoring atmospheric level of benzene. For this purpose, we used as molecular recognition element the porcine odorant-binding protein (pOBP). 1-Aminoanthracene was selected as extrinsic fluorescence probe for designing a competitive fluorescence resonance energy transfer (FRET) assay for benzene detection. The detection limit of our assay was 3.9µg/m3, a value lower than the actual emission limit value of benzene as regulated by European law.

Cloning and bacterial expression systems for recombinant human heparanase production: Substrate specificity investigation by docking of a putative heparanase substrate.

Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed of a subunit of 8 kDa and another of 50 kDa. The two protein subunits are noncovalently associated. The cloning and expression of the two protein subunits in Escherichia coli and their subsequent purification to homogeneity under native conditions result in the production of an active HPSE enzyme. The substrate specificity of the HPSE was studied by docking of a putative substrate that is a designed oligosaccharide with the minimum recognition backbone, with the additional 2-N-sulfate and 6-O-sulfate groups at the nonreducing GlcN and a fluorogenic tag at the reducing extremity GlcN. To develop a quantitative fluorescence assay with this substrate would be extremely useful in studies on HPSE, as the HPSE cleavage of fluorogenic tag would result in a measurable response.

Enzymes as Sensors.

Over the last few decades the development of new technologies, the fabrication of new materials, and the introduction of nanotechnologies created new trends in a series of advances that produced innovations in biological sensing devices with a wide range of application from health, security, defense, food, and medicine, to the environment. Specificity, low cost, rapidity, sensitivity, and multiplicity are some of the reasons for their growth, and their commercial success is expected to increase in the next future. Biosensors are devices in which the recognition part of the target molecule is accomplished by biological macromolecules such as proteins, enzymes, antibodies, aptamers, etc. These biomolecules are able to bind to the target molecules with high selectivity and specificity. The interaction between the target molecule and the specific biomolecule is reflected as a change of the biomolecule structural features. The extent of this change is strictly related to the biosensor response. Fluorescence spectroscopy, due to its sensitivity, is often used as the principal technique to monitor biological interactions, and thus the biosensor response as well. Both the intrinsic ultraviolet fluorescence of protein, arising from aromatic amino acids (tryptophan, tyrosine, and phenylalanine), and extrinsic fluorescent labels emitting in the visible region of the spectrum together allow for very flexible transduction of the analyte recognition, suitable for many different applications. This chapter focuses special attention on enzymes as practically unmatched recognition elements for biosensors and emphasizes the potential advantages of customized biosensor devices using apo- or holo forms of enzymes also isolated from thermophile sources.

On the possibility of ephedrine detection: time-resolved fluorescence resonance energy transfer (FRET)-based approach.

Ephedrine is one of the main precursor compounds used in the illegal production of amphetamines and related drugs. Actually, conventional analytical methods such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and gas chromatography-mass spectrometry (GC-MS) are used for the detection of ephedrine; sadly, these methods require qualified personnel and are time-consuming and expensive. In order to overcome these problems, in recent years, different methods have been developed based on the surface plasmon resonance (SPR) and electrochemical method. In this work, we present a simple, rapid, and effective method to detect the presence of ephedrine in solution, based on competitive fluorescence resonance energy transfer (FRET) assay. The antibody anti-ephedrine and ephedrine derivative were produced and labeled respectively, with two different fluorescent probes (donor and acceptor). The change in FRET signal intensity between donor and acceptor ephedrine compounds gives the possibility of detecting ephedrine traces of at least 0.81 ± 0.04 ppm (LOD). Graphical abstract A new Time-resolved Fluorescence Resonance Energy Transfer (FRET) assay for ephedrine detection.

A Fluorescence Polarization Assay To Detect Steroid Hormone Traces in Milk.

Steroids are a class of hormones improperly used in livestock as growth-promoting agents. Due to their high risk for human health, the European Union (EU) has strictly forbidden the administration of all natural and synthetic steroid hormones to food-producing animals, and the development of new rapid detection methods are greatly encouraged. This work reports a novel fluorescence polarization assay, ready to use, capable of detecting 17ß-estradiol directly in milk samples with a low limit of detection of <10 pmol. It is based on the coupling of monospecific antibodies against 17ß-estradiol and fluorophores, capable of modulating the fluorescence polarization emission on the basis of the specific binding of antibodies to fluorescence-labeled 17ß-estradiol derivative. The successful detection of 17ß-estradiol has disclosed the development of an efficient method, easily extensible to any food matrix and having the potential to become a milestone in food quality and safety.