Comparison between a Direct-Flow SPR Immunosensor for Ampicillin and a Competitive Conventional Amperometric Device: Analytical Features and Possible Applications to Real Samples.

In this research, we developed a direct-flow surface plasmon resonance (SPR) immunosensor for ampicillin to perform direct, simple, and fast measurements of this important antibiotic. In order to better evaluate the performance, it was compared with a conventional amperometric immunosensor, working with a competitive format with the aim of finding out experimental real advantages and disadvantages of two respective methods. Results showed that certain analytical features of the new SPR immunodevice, such as the lower limit of detection (LOD) value and the width of the linear range, are poorer than those of a conventional amperometric immunosensor, which adversely affects the application to samples such as natural waters. On the other hand, the SPR immunosensor was more selective to ampicillin, and measurements were more easily and quickly attained compared to those performed with the conventional competitive immunosensor.

Bioethanol in Biofuels Checked by an Amperometric Organic Phase Enzyme Electrode (OPEE) Working in "Substrate Antagonism" Format.

The bioethanol content of two samples of biofuels was determined directly, after simple dilution in decane, by means of an amperometric catalase enzyme biosensor working in the organic phase, based on substrate antagonisms format. The results were good from the point of view of accuracy, and satisfactory for what concerns the recovery test by the standard addition method. Limit of detection (LOD) was on the order of 2.5 × 10(-5) M.

A new surface plasmon resonance immunosensor for triazine pesticide determination in bovine milk: a comparison with conventional amperometric and screen-printed immunodevices.

A detailed comparison was made of the analytical features of a new Surface Plasmon Resonance (SPR) immunodevice for triazine pesticide determination with those of two other amperometric (conventional and screen-printed) immunosensors and the advantages and disadvantages of the SPR method were thoroughly investigated. For conventional amperometric and screen-printed devices, "competitive" assays were used; conversely, the SPR transduction technique allowed a "direct" measurement format to be used. As far as the main analytical data are concerned, the SPR method does not seem to offer substantial advantages. Nevertheless the measurement time is much shorter and the measurement itself much easier to perform. Lastly several applications and recovery tests were carried out on bovine milk samples, before and after spiking, to check for triazine pesticides in the samples, obtaining satisfactory results.

Simple and suitable immunosensor for ß-lactam antibiotics analysis in real matrixes: milk, serum, urine.

The anti-penicillin G was conjugated to avidin-peroxidase and biotin to obtain immunogen and competitor which were then used to develop a competitive immunosensor assay for the detection of penicillin G and other ß-lactam antibiotics, with Kaff values of the order of 10(8) M(-1). The new immunosensor appears to afford a number of advantages in terms of sensitivity, possibility of "in situ" analysis, but especially of simplicity and lower costs, compared with other existing devices, or different chemical instrumental methods reported in the literature and used for the analysis of ß-lactam compounds. Satisfactory results were found in the analysis of real matrixes and good recoveries were obtained by applying the standard addition method to spiked milk, urine, serum and drug samples. The new device uses an amperometric electrode for hydrogen peroxide as transducer, the BSA-penicillin G immobilized on polymeric membrane overlapping the amperometric transducer and the peroxidase enzyme as marker. It proved to be highly sensitive, inexpensive and easily reproducible; LOD was of the order of 10(-11)M. Lastly, the new immunosensor displayed low selectivity versus the entire class of ß-lactam antibiotics and higher selectivity toward other classes of non-ß-lactam antibiotics.

Determination of traces of several pesticides in sunflower oil using organic phase immuno electrodes (OPIEs).

Testing for traces of different pesticides (triazinic, organophosphates and chlorurates), present in hydrophobic matrices such as sunflower oil was checked using new immunosensors working in organic solvent mixtures (OPIEs). The competitive process took place in an n-hexane-chloroform 75% (V/V) mixture, while the subsequent final enzymatic measurement was performed in decane using tert-butylhydroperoxide as substrate of the enzymatic reaction. A Clark electrode was used as transducer and peroxidase enzyme as marker. A linear response of between about 10 nM and 4 µM was usually obtained in the presence of sunflower oil. Immunosensors show satisfactory selectivity and precision and recovery tests carried out on commercial sunflower oil samples gave excellent results. Lastly, theoretical confirmation of the possibility that immunosensors can act positively in organic solvent mixtures was discussed on the basis of Hill׳s coefficient values.

Agent orange herbicides, organophosphate and triazinic pesticides analysis in olive oil and industrial oil mill waste effluents using new organic phase immunosensors.

New immunosensors working in organic solvent mixtures (OPIEs) for the analysis of traces of different pesticides (triazinic, organophosphates and chlorurates) present in hydrophobic matrices such as olive oil were developed and tested. A Clark electrode was used as transducer and peroxidase enzyme as marker. The competitive process took place in a chloroform-hexane 50% (V/V) mixture, while the subsequent enzymatic final measurement was performed in decane and using tert-butylhydroperoxide as substrate of the enzymatic reaction. A linear response of between about 10nM and 5.0µM was usually obtained in the presence of olive oil. Recovery tests were carried out in commercial or artisanal extra virgin olive oil. Traces of pesticides were also checked in the oily matrix, in pomace and mill wastewaters from an industrial oil mill. Immunosensors show good selectivity and satisfactory precision and recovery tests performed in olive oil gave excellent results.

Comparison of three immunosensor methods (surface plasmon resonance, screen-printed and classical amperometric immunosensors) for immunoglobulin G determination in human serum and animal or powdered milks.

Within the framework of research carried out by our team aimed at developing new immunological methods to determine proteins such as immunoglobulins G in different biological matrixes, for instance, serum and milk, tests were performed on several immunosensors based on different transducer types, i.e. amperometric (classical or screen-printed) electrodes for hydrogen peroxide. Lastly the feasibility of constructing immunosensors based on surface plasmon resonance (SPR) was investigated. "Competitive" immunological procedures were used in the first two cases. Conversely, the surface plasmon resonance transduction technique allowed a "direct" measurement. Applications were performed on human serum, powdered milks for babies and particularly on several animal milks, in the case of buffalo milk seeking a routine control method to identify possible inflammatory affections in the animals.

Reducing the pollutant load of olive mill wastewater by photocatalytic membranes and monitoring the process using both tyrosinase biosensor and COD test.

Photocatalytic technique had already been employed in the treatment of olive mill wastewater (OMW) using the photocatalysis in suspension. The coupling of photocatalytic and membrane techniques should result in a very powerful process bringing great innovation to OMW depollution. Despite the potential advantages using these hybrid photoreactors, research on the combined use of photocatalysis and membranes has so far not been sufficiently developed. The present paper describes a study to assess the photocatalytic efficacy of a new ceramic membrane containing titanium dioxide, irradiated by UV light, used to abate the pollutant load of OMW. Good results were obtained (more than 90% of the phenol content was removed and the COD decrease was of the order of 46-51% in 24 h) particularly using the ceramic membrane compared with those offered by analogous catalytic membranes made of metallic or polymeric materials.

Synthesis and biological evaluation of 3,7-diazabicyclo[4.3.0]nonan-8-ones as potential nootropic and analgesic drugs.

A series of cis and trans 3,7-diazabicyclo[4.3.0]nonan-8-ones has been synthesized and tested for their ability to revert scopolamine-induced amnesia in the mouse passive-avoidance test. The racemates of the most potent compounds 4 and 7 were separated and tested, but no enantioselectivity was found for the nootropic activity. Compounds 4 and 7 and their enantiomers displayed interesting antihyperalgesic activity in two models of neuropathic pain (streptozotocin-induced and oxalilplatin-induced neuropathy) in comparison with pregabalin.

Design, synthesis, and preliminary biological evaluation of new isoform-selective f-current blockers.

New I(f) blockers have been designed and tested on HEK293 cells stably expressing the HCN1, HCN2, and HCN4 channels to find compounds able to discriminate among the channel isoforms. Among the synthesized compounds, the cis-butene derivative (R)-5 shows some preference for HCN2 while the pseudodimeric product (R)-6 shows selectivity for HCN1. These compounds can be important pharmacological tools to study the channels in native tissues and may be useful to design safe drugs.

Further development of lactoferrin immunosensor (part III).

The last year we fabricated some new immunosensors for the analysis of lactoferrin protein. In the present research the immunological and analytical characteristics of the immunosensor method have been extensively investigated. The study was therefore extended to cover the ability of the analyte and the corresponding antibody to produce the immunocomplex. A rough estimation of the K(aff) value was obtained at the midpoint of the Langmuir curve, where K(aff)=1/IC(50). The K(aff) value was found to be of the order of 10(6)M(-1). In addition we attempted in the present study to reduce the excessively long time required for each measurement, due to the fact that, in the previous researches the measurement procedure used was the classic "competition" ELISA type, employing an ad hoc enzymatic marker. One possible way of reducing measurement time was found to be to perform a kind of completely innovative "direct" measurement developed by us, which still uses an enzymatic marker and an amperometric transducer, as in the previous competition method.

Synthesis, affinity profile and functional activity of potent chiral muscarinic antagonists with a pyrrolidinylfuran structure.

Starting from the structure of previously studied muscarinic agonists, characterized by a pyrrolidinylfuran scaffold, a new series of muscarinic antagonists was synthesized by substituting the 5-position of the furane cycle with bulky hydrophobic groups. Both tertiary amines and the corresponding iodomethyl derivatives were obtained and studied. All the new compounds show high affinity toward cloned human muscarinic M(1)-M(5) receptors expressed in Chinese hamster ovary (CHO) cells and behave as competitive antagonists on classical models of muscarinic receptors. The diastereoisomeric mixture of the highest affinity compound of the series was resolved into the four optical isomers by chiral HPLC. The relative and absolute configuration of the obtained compounds was established by means of a combined strategy based on X-ray crystallography and chiroptical techniques. Although generally fairly potent, the compounds showed only modest subtype selectivity, with the exception of 2a and 6a, which in functional assays presented clear-cut selectivity for the muscarinic receptors present in rabbit vas deferens.

Design, synthesis and nootropic activity of new analogues of sunifiram and sapunifiram, two potent cognition-enhancers.

A series of amides and sulfonamides, structurally related to DM235 (sunifiram) and MN19 (sapunifiram), derived by ring expansion or contraction, or by inversion of the exocyclic amide function, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency than piracetam, and with a potency similar to the parent compounds.

[35S]GTP gamma S binding studies of amphiphilic drugs-activated Gi proteins: a caveat.

This paper documents a serious problem met during the testing of Gi protein-activating properties of a new series of synthetic compounds by measuring the induced binding of [(35)S]GTPgammaS to different subtypes of Gi protein. The problem arose from the strong affinity between [(35)S]GTPgammaS and the tested compounds, that are characterized by several (2-4) positive charges and high lipophilicity. Apparently, such affinity yields insoluble, labelled complexes that, also in the absence of Gi protein, are retained on the filters and give rise to false positive results.

Determination of lactoferrin and immunoglobulin g in animal milks by new immunosensors.

Two different immunosensors, recently developed for the determination of antibacterial proteins (lactoferrin and immunoglobulin G) in buffalo milk and in other commercial animal milks samples, were used in the present study. The aim was to propose these immunosensor methods for routine control of important diet products, such as cow and goat milks, and in particular buffalo milk. To this end we employed two different kinds of immunosensors: one for the analysis of immunoglobulin G (IgG), the other was a new amperometric immunosensor for lactoferrin analysis. Lactoferrin and IgG immunosensors were also used for the determination of lactoferrin and immunoglobulin G in buffalo milk on different days of lactation.

Design, synthesis and preliminary pharmacological evaluation of new analogues of DM232 (unifiram) and DM235 (sunifiram) as cognition modulators.

A series of amides, structurally related to DM232 (unifiram) and DM235 (sunifiram), characterized by a 1,2,3,4-tetrahydropyrazino[2,1-a]isoindol-6(2H)-one, 1,4-diamino-cyclohexane or 1,4-diaminobenzene ring, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency than piracetam, while some cyclohexane derivatives are endowed with amnesia inducing properties.

Synthesis and pharmacological characterization of chiral pyrrolidinylfuran derivatives: the discovery of new functionally selective muscarinic agonists.

Building on the previously and successfully applied hypothesis that stereochemical complication in the proximity of the critical cationic head of a cholinergic agonist would result in subtype selective compounds, we synthesized a series of chiral derivatives of furmethide and 5-methylfurmethide, with the aim of obtaining compounds that are useful for treating diseases derived from cholinergic receptor dysfunctions and/or useful for further characterizing subtypes of cholinergic receptors. Unlike their parent compounds, the new molecules lack nicotinic activity, being pure muscarinic ligands. While binding studies on the five cloned human muscarinic receptors showed no subtype selectivity, functional assays revealed that some of the molecules of the series are potent M 2 selective partial agonists with interesting pharmacological profiles.

New immunosensor for Lactoferrin determination in human milk and several pharmaceutical dairy milk products recommended for the unweaned diet.

Thorough research was carried out on Lactoferrin immunosensor development. Furthermore, two different competitive procedures were used for Lactoferrin determination, in which either the antigen (Lactoferrin) or the antibody (anti-Lactoferrin) was, respectively, conjugated with horseradish peroxidase enzyme using a biotinylation process. The biotinylation of Lactoferrin and the subsequently used competition procedure for the immunosensor measurement were to get ready. Three different kinds of immunosensors were implemented, in all cases using the peroxidase enzyme as marker and hydrogen peroxide as substrate, but alternatively using as transducers one of the following sensors: (i) an amperometric electrode for H2O2, (ii) a Clark electrode and (iii) an iodide electrode. After optimizing the "competitive" measurement procedures and the transducer, the new Lactoferrin immunosensor was used for the determination of Lactoferrin content in human milk and in different types of dried milks or other dairy products, specifically produced and sold in chemist's shops to feed unweaned children in the first few months of life.

Immunoglobulin G Determination in Human Serum and Milk Using an Immunosensor of New Conception Fitted with an Enzyme Probe as Transducer.

To completely overcome the problem of the presence of urea in the serum, which can be the cause (especially at low immunoglobulin G concentrations) of a small but non negligible interference in the enzyme reaction of the enzymatic marker, when the measurement was performed by a potentiometric immunosensor that we constructed and characterized in previous work, and which used urease as marker, we have now constructed an entirely different and highly innovative immunosensor. This new device uses the enzyme alkaline phosphatase as marker, sodium phenylphosphate as substrate but above all, a tyrosinase biosensor obtained by coupling a Clark type gas diffusion amperometric electrode and the tyrosinase enzyme, immobilized in a cellulose triacetate membrane, as transducer. After optimizing the 'competitive' measurement procedures, the new immunosensor was used to determine both HIgG and the anti-HIgG, with a limit of detection (LOD) of the order of 3x10-11 M. Clearly this highly innovative construction geometry makes the immunosensor extremely selective. This makes it possible to determine immunoglobulin G both in human serum and milk without the slightest interference by any urea present in these biological matrixes.

Design, synthesis and preliminary pharmacological evaluation of new piperidine and piperazine derivatives as cognition-enhancers.

A series of 2-oxopiperazine, 4-aminomethyl-, 3-amino- and 3-aminomethylpiperidine analogues of DM235 (sunifiram) and MN19 (sapunifiram), two previously reported potent cognition-enhancers, have been synthesized and tested in the mouse passive-avoidance test. The compounds display minimal effective doses in the range 0.3-10mg/kg. Although the new substances do not show improved activity when compared to the parent compounds, some useful information has been obtained to understand structure-activity relationships. In addition, the 3-aminopiperidine moiety appears to be a promising scaffold to synthesize new drugs endowed with cognition-enhancing activity.