Riccardo Funari

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.

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.

Single Molecule Characterization of UV-Activated Antibodies on Gold by Atomic Force Microscopy

The interaction between proteins and solid surfaces can influence their conformation and therefore also their activity and affinity. These interactions are highly specific for the respective combination of proteins and solids. Consequently, it is desirable to investigate the conformation of proteins on technical surfaces, ideally at single molecule level, and to correlate the results with their activity. This is in particular true for biosensors where the conformation-dependent target affinity of an immobilized receptor determines the sensitivity of the sensor. Here, we investigate for the first time the immobilization and orientation of antibodies (Abs) photoactivated by a photonic immobilization technique (PIT), which has previously demonstrated to enhance binding capabilities of antibody receptors. The photoactivated immunoglobulins are immobilized on ultrasmooth template stripped gold films and investigated by atomic force microscopy (AFM) at the level of individual molecules. The observed protein orientations are compared with results of nonactivated antibodies adsorbed on similar gold films and mica reference samples. We find that the behavior of Abs is similar for mica and gold when the protein are not treated (physisorption), whereas smaller contact area and larger heights are measured when Abs are treated (PIT). This is explained by assuming that the activated antibodies tend to be more upright compared with nonirradiated ones, thereby providing a better exposure of the binding sites. This finding matches the observed enhancement of Abs binding efficiency when PIT is used to functionalize gold surface of QCM-based biosensors.

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.

Simple and Flexible Model for Laser-Driven Antibody–Gold Surface Interactions: Functionalization and Sensing

Interactions between biomolecules and between substrates and biomolecules is a crucial issue in physics and applications to topics such as biotechnology and organic electronics. The efficiency of bio- and mechanical sensors, of organic electronics systems, and of a number of other devices critically depends on how molecules are deposited on a surface so that these acquire specific functions. Here, we tackle this vast problem by developing a coarse grained model of biomolecules having a recognition function, such as antibodies, capable to quantitatively describe in a simple manner essential phenomena: antigen-antibody and antibody substrate interactions. The model is experimentally tested to reproduce the results of a benchmark case, such as (1) gold surface functionalization with antibodies and (2) antibody-antigen immune-recognition function. The agreement between experiments and model prediction is excellent, thus unveiling the mechanism for antibody immobilization onto metals at the nanoscale in various functionalization schemes. These results shed light on the geometrical packing properties of the deposited molecules, and may open the way to a novel coarse-grained based approach to describe other processes where molecular packing is a key issue with applications in a huge number of fields from nano- to biosciences.

Antibody Anchoring on QCM Gold Surfaces by UV Based Strategy

Nowadays there is a strong interest in the development of measurement tools suitable for rapid and low cost analysis. Biosensors face this issue exploiting the intrinsic sensitivity and selectivity of a wide range of biomolecules. One of the main problems in biosensors development is the immobilization of the biological sensitive element onto the sensor surface. In this research topic we applied a novel strategy named Photonic Immobilization Technique (PIT) to anchor antibodies onto a gold electrode of a QCM with their antigen binding sites exposed to the environment (Della Ventura et al., Biomed Opt Express 2:3223–3231, 2011). The antibodies were activated by breaking the disulfide bridge in the triads Trp/Cys-Cys, which are typical of the immunoglobulins (Ioerger et al., Mol Immunol 36:373–386, 1999), through UV irradiation (Neves-Petersen et al., Protein Sci Publ Protein Soc 15:343–351, 2006). The new thiol groups so produced are exploited to anchor in a preferential way the proteins onto a gold surface. Even if the absorption mechanism is of the first order (one-photon absorption) the high UV power required by PIT needs some investigations concerning the most appropriate UV source. We compared femtosecond and nanosecond pulsed laser sources from different prospective, analyzing both their final effects on the biosensors and their effectiveness in protein activation by the Ellman’s assay (Ellman, Arch Biochem Biophys 82:70–77, 1959). Moreover, conformational changes of the antibodies once irradiated were checked through steady state fluorescence (Garidel et al., Biotechnol J 3:1201–1211, 2008), on the other side AFM analysis of the gold surfaces in several irradiation conditions has also been performed. Our results show clear differences between the two laser sources, in particular the irradiation by nanosecond laser pulses seems to lead to a more rapid “photo-bleaching”, as a consequence of the high fluence conveyed by these pulses, making femtosecond rather than nanosecond laser sources more suitable for PIT (Fig. 24.1).

Effective Antibody Anchoring on Gold Plate by Ultra-short UV Pulses

One of the main issues in biosensor research concerns the control of both the amount and the orientation of the bioreceptors. The so-called photonic induced immobilization can be very effective in anchoring antibodies with the variable part preferentially exposed, thus increasing the sensitivity of a Quartz Crystal Microbalance (QCM) immunosensor. In the present paper such a technique is applied to the detection of parathion, a relatively light analyte, demonstrating that the QCM is able to provide a detectable signal only if the photonic induced immobilization is applied, being completely insensitive to parathion when the bioreceptors are left to adhere randomly.

UV-light-assisted functionalization for sensing of light molecules

An antibody immobilization technique based on the formation of thiol groups after UV irradiation of the proteins is shown to be able to orient upside antibodies on a gold electrode of a Quartz Crystal Microbalance (QCM). This greatly affects the aptitude of antibodies in recognizing small antigens thereby increasing the sensitivity of the QCM. The capability of such a procedure to orient antibodies is confirmed by the Atomic Force Microscopy (AFM) of the surface that shows different statistical distributions for the height of the detected peaks, whether the irradiation is performed or not. In particular, the distributions are Gaussian with a standard deviation smaller when irradiated antibodies are used compared to that obtained with no treated antibodies. The standard deviation reduction is explained in terms of higher order induced on the host surface resulting from the trend of irradiated antibodies to be anchored upside on the surface with their antigen binding sites free to catch recognized analytes. As a result the sensitivity of the realized biosensor is increased by even more than one order of magnitude.