Several techniques for detecting chemical drug precursors have been developed in the last decade. Most of them are able to identify molecules at very low concentration under lab conditions. Other commercial devices are able to detect a fixed number and type of target substances based on a single detection technique providing an absence of flexibility with respect to target compounds. The construction of compact and easy to use detection systems providing screening for a large number of compounds being able to discriminate them with low false alarm rate and high probability of detection is still an open concern. Under CUSTOM project, funded by the European Commission within the FP7, a stand-alone portable sensing device based on multiple techniques is being developed. One of these techniques is based on the LED induced fluorescence polarization to detect Ephedrine and Benzyl Methyl Keton (BMK) as a first approach. This technique is highly selective with respect to the target compounds due to the generation of properly engineered fluorescent proteins which are able to bind the target analytes, as it happens in an “immune-type reaction”. This paper deals with the advances in the design, construction and validation of the LED induced fluorescence sensor to detect BMK analytes. This sensor includes an analysis module based on high performance LED and PMT detector, a fluidic system to dose suitable quantities of reagents and some printed circuit boards, all of them fixed in a small structure (167mm × 193mm × 228mm) with the capability of working as a stand-alone application.
Alberto Secchi, Anna Maria Fiorello, Massimiliano Dispenza, Sabato D'Auria, Antonio Varriale, Alessandro Ulrici, Renato Seeber, Juho Uotila, Vincenzo Venditto, Paolo Ciambelli, Juan Carlos Antolín, Francesco Colao, Tom Kuusela, Ilkka Tittonen, Päivi Sievilä, Grégory Maisons
A large number of techniques for drug precursors chemical sensing has been developed in the latest decades. These techniques are able to screen and identify specific molecules even at very low concentration in lab environment, nevertheless the objective to build up a system which proves to be easy to use, compact, able to provide screening over a large number of compounds and discriminate them with low false alarm rate (FA) and high probability of detection (POD) is still an open issue. The project CUSTOM, funded by the European Commission within the FP7, deals with stand alone portable sensing apparatus based on multiple techniques, integrated in a complex system with a complimentary approach. The objective of the project is to achieve an optimum trade-off between opposite requirements: compactness, simplicity, low cost, sensitivity, low false alarm rate and selectivity. The final goal is the realization of an optical sensing platform able to detect traces of drug precursors compounds, such as ephedrine, safrole, acetic anhydride and the Benzyl Methyl Keton (BMK). This is reached by implementing two main sensing techniques: the fluorescence enhanced by the use of specially developed Organic macro-molecules, and a spectroscopic technique in Mid-IR optical range. The fluorescence is highly selectivewith respect to the target compounds, because it is based on properly engineered fluorescent proteins which are able to bind the target analytes, as it happens in an 'immune-type' reaction. The spectroscopic technique is based on the Photo-Acoustic effect, enhanced by the use of a widely Tunable Quantum Cascade Laser. Finally, the sensing platform is equipped with an air sampling system including a pre-concentrator module based on a sorption desorption cycles of a syndiotactic polystyrene polymer.
Using commercially available organic fluorophores, the current applications of Förster (fluorescence) resonance energy transfer (FRET) are limited to about 80 Å. However, many essential activities in cells are spatially and/or temporally dependent on the assembly/disassembly of transient complexes consisting of large-size macromolecules that are frequently separated by distances greater than 100 Å. Expanding the accessible range for FRET to 150 Å would open up many cellular interactions to fluorescence and fluorescence-lifetime imaging. Here, we demonstrate that the use of multiple randomly distributed acceptors on proteins/antibodies, rather than the use of a single localized acceptor, makes it possible to significantly enhance FRET and detect interactions between the donor fluorophore and the acceptor-labeled protein at distances greater than 100 Å. A simple theoretical model for spherical bodies that have been randomly labeled with acceptors has been developed. To test the theoretical predictions of this system, we carried out FRET studies using a 30-mer oligonucleotide-avidin system that was labeled with the acceptors DyLight649 or Dylight750. The opposite 5′-end of the oligonucleotide was labeled with the Alexa568 donor. We observed significantly enhanced energy transfer due to presence of multiple acceptors on the avidin protein. The results and simulation indicate that use of a nanosized body that has been randomly labeled with multiple acceptors allows FRET measurements to be extended to over 150 Å when using commercially available probes and established protein-labeling protocols.
Antonio Varriale, Maria Staiano, Maria Strianese, Vincenzo Marzullo, Giuseppe Ruggiero, Alberto Secchi, Massimiliano Dispenza, Anna Maria Fiorello, Sabato D'Auria
In this work we describe the synthesis of a new ephedrine derivative with a carbon linker featuring an amino reactive
group, and its conjugation to the glutamine binding protein (GlnBP) from E. coli as a carrier protein for the production of
polyclonal antibodies in rabbits against ephedrine.
Proof-of-principle results that an efficient SPR-based indirect competitive immunoassay for the detection and
quantification of ephedrine are presented. The detection limit of this assay was found to be about 33ng/ml.
Glucose sensing and odorant molecules sensing are used as a models to explore the advantages and problems deriving
from the use of either enzymes or odorant-binding proteins to develop stable optical biosensors. We report on a novel
approach to address the problem of substrate consumption by sensors based on enzymes, namely the utilization of apoenzymes
as non-active forms of the protein which are still able to bind the substrate/ligand. We also show studies in
which the isolation of an odorant-binding protein from the nose of the dog is used as non-consuming analyte probe for
the realization of an integrated optical sensor.
The interaction between an analyte and a biological recognition system is normally detected in biosensors by the
transducer element which converts the molecular event into a measurable effect, such as an electrical or optical signal.
Porous silicon microstructures have unique optical and morphological properties that can be exploited in biosensing. The
large specific surface area (even greater than 500 m2/cm3) and the resonant optical response allow detecting the effect of
a change in refractive index of liquid solutions, which interact with the porous matrix, with very high sensitivity.
Moreover, the porous silicon surface can be chemically modified to link the bioprobe which recognize the target
analytes, in order to enhance the selectivity and specificity of the sensor device. The molecular probe we used was
purified by an extremophile organism, Thermococcus litoralis: the protein is very stable in a wide range of temperatures
even if with different behavior respect to the interaction with the ligand.
KEYWORDS: Proteins, Picosecond phenomena, Glucose, Luminescence, Scanning electron microscopy, Silicon, Nanostructures, Lab on a chip, Electron beams, Molecules
A protein microarray has been realized on a porous silicon (PS) chip by means of electron beam irradiation using a
standard SEM equipped with a nanopattern generator system. Two proteins have been used to generate the array: the
glucose-binding protein (GBP) and the glutamine-binding protein (GlnBP), both isolated from Escherichia coli.
The proteins functionality have been tested by means of a competitive assay.
The odorant-binding proteins (OBPs) are abundant low-molecular weight soluble proteins, which are secreted by the olfactory epithelium in the nasal mucus of vertebrates. These protein reversibly bind odorants with dissociation constants in the micromolar range. For this reason, they are good candidates as biological elements in the development of biosensors. Vertebrate OBPs belong to the lipocalin superfamily. Even if the members of this superfamily display low sequence similarity, all of them show a conserved folding pattern, that is an 8-stranded β-barrel flanked by an α-helix at the C-terminal end of the protein chain. The β-barrel defines a central apolar cavity, called calyx, whose role is to bind and transport hydrophobic odorant molecules. The detection of hazard exposure is becoming a priority in the third millennium, and OBPs are good candidates for detecting traces of explosive molecules in different environments such as luggage's storage rooms and public places. In this context, the measurement of refractive index of odor-binding protein in absence and in presence of odorant molecules have been performed in order to assess its usefulness as a probe for detection of hazardous agents. The work is instrumental to explore the possibility to realize a biosensor where the concentration of searched for substances is analyzed as a variation of the protein refractive index by means of suitable optoelectronic devices.
We developed a new method of glucose sensing using inactive forms of glucose oxidase from Aspergillus niger and glucose dehydrogenase from the thermophilic microorganism Thermoplasma acidophilum. Glucose oxidase was rendered inactive by removal of the FAD cofactor. The resulting apo- glucose oxidase still binds glucose as observed from a decrease in its intrinsic tryptophan fluorescence. 8- Anilino-1-naphthalene sulfonic acid (ANS) was found to bind spontaneously to apo-glucose oxidase as seen from an enhancement of the ANS fluorescence. The steady state intensity of the bound ANS decreased 25% upon binding of glucose, and the mean lifetime of the bound ANS decreased about 40%. These spectral changes occurred with a midpoint from 10 to 20 mM glucose, which is comparable to the Ko of holo-glucose oxidase. These results suggest that apo- glucose oxidase can be used as a reversible non-consuming sensor for glucose.
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