Bartolomeo Della Ventura

Detection of Parathion Pesticide by Quartz Crystal Microbalance Functionalized with UV-Activated Antibodies

Photonic immobilization technique (PIT) has been used to develop an immunosensor for the detection of parathion. An antibody solution has been activated by breaking the disulfide bridge in the triad Trp/Cys-Cys through absorption of ultrashort UV laser pulses. The free thiol groups so produced interact with gold lamina making the antibody oriented upside, that is, with its variable parts exposed to the environment, thereby greatly increasing the detection efficiency. PIT has been applied to anchor polyclonal antiparathion antibodies to the gold electrode of a Quartz Crystal Microbalance (QCM) giving rise to very high detection sensitivity once the parathion is made heavier by complexion with BSA (bovine serum albumin), this latter step only required by the mass based transducer used in this case. The comparison of the sensor response with irradiated antibodies against different analytes shows that the high degree of antibody specificity is not affected by PIT nor is it by the complexion of parathion with BSA. These results pave the way to important applications in biosensing, since the widespread occurrence of the Trp/Cys-Cys residues triads in proteins make our procedure very general and effective to detect light analytes.

Light assisted antibody immobilization for bio-sensing

Ultrashort UV pulses at 258 nm with repetition rate of 10 kHz have been used to irradiate buffer solution of antibody. The tryptophan residues strongly absorb this radiation thus becoming capable to disrupt the disulfide bridges located next to them. Due to their high reactivity the opened bridges can anchor a gold plate more efficiently than other sites of the macromolecule giving rise to preferential orientations of the variable part of the antibody. UV irradiation has been applied to anchor antiIgG antibody to the electrode of a Quartz Crystal Microbalance (QCM) that lends itself as a sensor, the antibody acting as the bio-receptor. An increase of the QCM sensitivity and of the linear range has been measured when the antibody is irradiated with UV laser pulses. The photo-induced reactions leading to disulfide bridge breakage have been analyzed by means of a chemical assay that confirms our explanation. The control of disulfide bridges by UV light paves the way to important applications for sensing purpose since cysteine in combination with tryptophan can act as a hook to link refractory bio-receptors to surfaces.

Glucose Sensing by Time-Resolved Fluorescence of Sol-Gel Immobilized Glucose Oxidase

A monolithic silica gel matrix with entrapped glucose oxidase (GOD) was constructed as a bioactive element in an optical biosensor for glucose determination. Intrinsic fluorescence of free and immobilised GOD was investigated in the visible range in presence of different glucose concentrations by time-resolved spectroscopy with time-correlated single-photon counting detector. A three-exponential model was used for analysing the fluorescence transients. Fractional intensities and mean lifetime were shown to be sensitive to the enzymatic reaction and were used for obtaining calibration curve for glucose concentration determination. The sensing system proposed achieved high resolution (up to 0.17 mM) glucose determination with a detection range from 0.4 mM to 5 mM.

A simple MALDI plate functionalization by Vmh2 hydrophobin for serial multi-enzymatic protein digestions.

The development of efficient and rapid methods for the identification with high sequence coverage of proteins is one of the most important goals of proteomic strategies today. The on-plate digestion of proteins is a very attractive approach, due to the possibility of coupling immobilized-enzymatic digestion with direct matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) analysis. The crucial step in the development of on-plate immobilization is however the functionalization of the solid surface. Fungal self-assembling proteins, the hydrophobins, are able to efficiently functionalize surfaces. We have recently shown that such modified plates are able to absorb either peptides or proteins and are amenable to MALDI-TOF-MS analysis. In this paper, the hydrophobin-coated MALDI sample plates were exploited as a lab-on-plate for noncovalent immobilization of enzymes commonly used in protein identification/characterization, such as trypsin, V8 protease, PNGaseF, and alkaline phosphatase. Rapid and efficient on-plate reactions were performed to achieve high sequence coverage of model proteins, particularly when performing multiple enzyme digestions. The possibility of exploiting this direct on-plate MALDI-TOF/TOF analysis has been investigated on model proteins and, as proof of concept, on entire whey milk proteome.

Effective antibodies immobilization and functionalized nanoparticles in a quartz-crystal microbalance-based immunosensor for the detection of parathion

Background Biosensor-based detection provides a rapid and low-cost alternative to conventional analytical methods for revealing the presence of the contaminants in water as well as solid matrices. Although important to be detected, small analytes (few hundreds of Daltons) are an issue in biosensing since the signal they induce in the transducer, and specifically in a Quartz-Crystal Microbalance, is undetectable. A pesticide like parathion (M = 292 Da) is a typical example of contaminant for which a signal amplification procedure is desirable. Methods/Findings The ballasting of the analyte by gold nanoparticles has been already applied to heavy target as proteins or bacteria to improve the limit of detection. In this paper, we extend the application of such a method to small analytes by showing that once the working surface of a Quartz-Crystal Microbalance (QCM) has been properly functionalized, a limit of detection lower than 1 ppb is reached for parathion. The effective surface functionalization is achieved by immobilizing antibodies upright oriented on the QCM gold surface by a simple photochemical technique (Photonic Immobilization Technique, PIT) based on the UV irradiation of the antibodies, whereas a simple protocol provided by the manufacturer is applied to functionalize the gold nanoparticles. Thus, in a non-competitive approach, the small analyte is made detectable by weighing it down through a “sandwich protocol” with a second antibody tethered to heavy gold nanoparticles. The immunosensor has been proved to be effective against the parathion while showing no cross reaction when a mixture of compounds very similar to parathion is analyzed. Conclusion/Significance The immunosensor described in this paper can be easily applied to any small molecule for which polyclonal antibodies are available since both the functionalization procedure of the QCM probe surface and gold nanoparticle can be applied to any IgG, thereby making our device of general application in terms of target analyte.

Femtosecond UV-laser pulses to unveil protein–protein interactions in living cells

A hallmark to decipher bioprocesses is to characterize protein–protein interactions in living cells. To do this, the development of innovative methodologies, which do not alter proteins and their natural environment, is particularly needed. Here, we report a method (LUCK, Laser UV Cross-linKing) to in vivo cross-link proteins by UV-laser irradiation of living cells. Upon irradiation of HeLa cells under controlled conditions, cross-linked products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected, whose yield was found to be a linear function of the total irradiation energy. We demonstrated that stable dimers of GAPDH were formed through intersubunit cross-linking, as also observed when the pure protein was irradiated by UV-laser in vitro. We proposed a defined patch of aromatic residues located at the enzyme subunit interface as the cross-linking sites involved in dimer formation. Hence, by this technique, UV-laser is able to photofix protein surfaces that come in direct contact. Due to the ultra-short time scale of UV-laser-induced cross-linking, this technique could be extended to weld even transient protein interactions in their native context.

Photophysics and Photochemistry of a DNA–Protein Cross-Linking Model: A Synergistic Approach Combining Experiments and Theory

The photophysical and photochemical properties of 5-benzyluracil and 5,6-benzyluracil, the latter produced by photocyclization of the former through irradiation with femtosecond UV laser pulses, are investigated both experimentally and theoretically. The absorption spectra of the two molecules are compared, and the principal electronic transitions involved are discussed, with particular emphasis on the perturbation induced on the two chromophore species (uracil and benzene) by their proximity. The photoproduct formation for different irradiation times was verified with high-performance liquid chromatography and nuclear magnetic resonance measurements. The steady-state fluorescence demonstrates that the fluorescence is a distinctive physical observable for detection and selective identification of 5- and 5,6-benzyluracil. The principal electronic decay paths of the two molecules, obtained through TDDFT calculations, explain the features observed in the emission spectra and the photoreactivity of 5-benzyluracil. The order of magnitude of the lifetime of the excited states is derived with steady-state fluorescence anisotropy measurements and rationalized with the help of the computational findings. Finally, the spectroscopic data collected are used to derive the photocyclization and fluorescence quantum yields. In obtaining a global picture of the photophysical and photochemical properties of the two molecules, our findings demonstrates that the use of 5-benzyluracil as a model system to study the proximity relations of a DNA base with a close-lying aromatic amino acid is valid at a local level since the main characteristics of the decay processes from the excited states of the uracil/thymine molecules remain almost unchanged in 5-benzyluracil, the main perturbation arising from the presence of the close-lying aromatic group.

Flexible immunosensor for the detection of salivary α-amylase in body fluids

The development of a portable testing device for detecting Human Salivary α-Amylase (HSA) is very timely since such an enzyme is a valuable biomarker for diagnosing many diseases and monitoring the human stress. We show that an easy-to-use and robust device like the Quartz-Crystal Microbalance (QCM) can be a suitable platform for HSA sensing with a limit of detection of 1µg/mL (77 U/L). The functionalization of the gold surface is realized by the Photochemical Immobilization Technique (PIT), a powerful and simple method based on an appropriate UV-activation of antibodies. The resulting QCM-based immunosensor allows one to detect HSA in saliva by simple dilution and one-step protocol, whereas the measurement of HSA content in body fluids like urine and serum could be carried out by introducing an additional step consisting of analyte ballasting through the formation of sandwich complexes, which pushes the limit of detection to less than 10 U/L. The validation of the one-step protocol with a standard laboratory method like Phadebas test demonstrates the reliability of the proposed immunosensors, which can be applied to the amylase concentration in body fluids like blood serum and urine for which the physiological level is above 20 U/L.

Label-Free Detection of Gliadin in Food by Quartz Crystal Microbalance-Based Immunosensor

Gluten is a protein composite found in wheat and related grains including barley, rye, oat, and all their species and hybrids. Gluten matrix is a biomolecular network of gliadins and glutenins that contribute to the texture of pastries, breads, and pasta. Gliadins are mainly responsible for celiac disease, one of the most widespread food-related pathologies in Western world. In view of the importance of gliadin proteins, by combining the quartz crystal microbalance technology, a cheap and robust piezoelectric transducer, with the so-called photonic immobilization technique, an effective surface functionalization method that provides spatially oriented antibodies on gold substrates, we realized a sensitive and reliable biosensor for quantifying these analytes extracted from real samples in a very short time. The resulting immunosensor has a limit of detection of about 4 ppm and, more remarkably, shows excellent sensitivity in the range 7.5-15 ppm. This feature makes our device reliable and effective for practical applications since it is able to keep low the influence of false positives.

The tumor necrosis factor g1022G>A polymorphism is associated with resistance to tuberculosis in water buffalo (Bubalus bubalis)

Marina Papaianni*, Gianfranco Cosenza*, Giorgia Borriello, Giorgio Galiero, Fernando Grasso*, Bartolomeo Della Ventura, Marco Iannaccone* and Rosanna Capparelli* *Department of Agriculture, University of Naples Federico II, via Universit a 100, 80055 Portici, Napoli, Italy; IZS Mezzogiorno, Via della Salute 2, 80055 Portici, Naples, Italy; CNISM and Dipartimento di Fisica, University of Naples Federico II, Via Cintia 26, Naples 80126, Italy