Bioconjugation of enzymes and proteins on multifunctional and nanostructured solid supports for biomolecular interactions monitoring

Biosensors are interesting tools in various biotechnological fields, because of their versatility in fields such as diagnostics, development of biomedical devices and monitoring of pollutants in drinking water. Biosensors are constituted by an optical (or electrical, mechanical, etc.) transducer, which constitutes the sensitive element, and a biomolecular probe, which gives specificity to the device, because it is able to selectively bind target molecules. Both elements are the subject of this thesis, that studies the properties of different materials and characterization techniques for biosensing devices development. Nanostructured materials have physical and chemical properties arising interesting peculiarities (such as photoluminescence, reflectivity, etc.) that make them the fundamental building blocks for the development of the next generation of tools and devices for biotechnological applications. In particular, the following two types of nanostructured material were studied: • porous silicon (PSi); • gold nanoparticles (AuNPs). Further item of the present thesis is the bioconjugation of molecular bioprobes. It is well known that there are several methods of bioconjugation useful in the development of nano-bio-systems for sensor applications. Self-assembling materials, found in nature, are extremely interesting as functional interfaces between the artificial transducers and the living world. These interfaces do not require any chemical procedure, nor special equipment for their deposition. Along with the self-assembling materials, essential is the study of the use of aminosilanes and crosslinker. Moreover, it is well known that an optimization study of bioprobes immobilization is a hot topic in biosensors study. Therefore, objectives of this PhD project are: • Study of spotting conditions of bioconjugation onto nanostructured materials by two strategies: o immobilization of biomolecules by covalent approach (using aminosilane and crosslinker); o immobilization by non-covalent approach due to self-assembling of proteins; • Development of biosensors for biomedical and environmental applications. Molecular interactions monitored by label-free optical techniques performed onto different nanostructured materials found application in both biomedical and environmental monitoring fields. Interesting devices were finally obtained: • glucose interaction monitoring with an affinity constant of about 40mM; • PSA-Anti/PSA interaction monitoring with a sensitivity of 37±2 cm-1/mgL-1; • reversible device for lead ions detection in aqueous environment were developed, with a LOD of 2ppb (5 times lower then World Health Organization legal limit for drinking water); • naked eye assay for arsenic ions speciation (also at 85μM)

Modified denatured lysozyme effectively solubilizes fullerene c60 nanoparticles in water

Fullerenes, allotropic forms of carbon, have very interesting pharmacological effects and engineering applications. However, a very low solubility both in organic solvents and water hinders their use. Fullerene C60, the most studied among fullerenes, can be dissolved in water only in the form of nanoparticles of variable dimensions and limited stability. Here the effect on the production of C60 nanoparticles by a native and denatured hen egg white lysozyme, a highly basic protein, has been systematically studied. In order to obtain a denatured, yet soluble, lysozyme derivative, the four disulfides of the native protein were reduced and exposed cysteines were alkylated by 3-bromopropylamine, thus introducing eight additional positive charges. The C60 solubilizing properties of the modified denatured lysozyme proved to be superior to those of the native protein, allowing the preparation of biocompatible highly homogeneous and stable C60 nanoparticles using lower amounts of protein, as demonstrated by dynamic light scattering, transmission electron microscopy and atomic force microscopy studies. This lysozyme derivative could represent an effective tool for the solubilization of other carbon allotropes

Peptide functionalization of silicon for detection and classification of prostatic cells

The development of simple, rapid, and low costmethods for early detection, identification, andmeasurement ofmultiple biomarkers remains a challenge to improve diagnosis, treatment monitoring, and prognosis of cancer. Biosensing technology, combining the properties of biological systems with functional advanced materials, guarantees rapid, reproducible, and highly sensitive cell detection. In this study, we developed silicon-based biochips for prostate cancer PC3 cells detection by using cytokeratin 8/18 and Urotensin Receptor (UTR) as markers in order to obtain a biochip-based diagnostic system. Spectroscopic ellipsometry and fluorescence microscopy were used to characterize surface homogeneity and chemical properties. Cell detection was investigated by optical microscopy.Moreover, synthetic fluorescently labeled peptides were prepared and used for developing faster and lowercost identification assay compared with classic ELISA immunoassay. Results showed an effective immobilization of PC3 cells on silicon surface and the specific recognition of these cells by fluorescent Urotensin II (4-11). In conclusion, this strategy could be really useful as diagnostic system for prostate cancer

Nanogravimetric and Optical Characterizations of Thrombin Interaction with a Self-Assembled Thiolated Aptamer

Efficient biorecognition of thrombin (TB), a serine protease with crucial role in physiological and pathological blood coagulation, is a hot topic in medical diagnostics. In this work, we investigate the ability of synthetic thrombin aptamer (TBA), immobilized on a gold substrate, to bind thrombin by two different label-free techniques: the quartz crystal microbalance (QCM) and the spectroscopic ellipsometry (SE). By QCM characterization in the range from 20 to 110 nM, we demonstrate high specificity of TBA-TB interaction and determine affinity constant (Kd) of 17.7 ± 0.3 nM, system sensitivity of 0.42 ± 0.03 Hz nM-1, and limit of detection (LOD) of 240 ± 20 pM. The interaction between TBA and TB is also investigated by SE, an all-optical method, by quantifying the thickness increase of the TBA film assembled on gold substrate. AFM characterization of TBA and TB molecules deposited on flat silicon surface is also supplied

The Lung Screen Uptake Trial (LSUT): protocol for a randomised controlled demonstration lung cancer screening pilot testing a targeted invitation strategy for high risk and ‘hard-to-reach’ patients

Background Participation in low-dose CT (LDCT) lung cancer screening offered in the trial context has been poor, especially among smokers from socioeconomically deprived backgrounds; a group for whom the risk-benefit ratio is improved due to their high risk of lung cancer. Attracting high risk participants is essential to the success and equity of any future screening programme. This study will investigate whether the observed low and biased uptake of screening can be improved using a targeted invitation strategy. Methods/design A randomised controlled trial design will be used to test whether targeted invitation materials are effective at improving engagement with an offer of lung cancer screening for high risk candidates. Two thousand patients aged 60–75 and recorded as a smoker within the last five years by their GP, will be identified from primary care records and individually randomised to receive either intervention invitation materials (which take a targeted, stepped and low burden approach to information provision prior to the appointment) or control invitation materials. The primary outcome is uptake of a nurse-led ‘lung health check’ hospital appointment, during which patients will be offered a spirometry test, an exhaled carbon monoxide (CO) reading, and an LDCT if eligible. Initial data on demographics (i.e. age, sex, ethnicity, deprivation score) and smoking status will be collected in primary care and analysed to explore differences between attenders and non-attenders with respect to invitation group. Those who attend the lung health check will have further data on smoking collected during their appointment (including pack-year history, nicotine dependence and confidence to quit). Secondary outcomes will include willingness to be screened, uptake of LDCT and measures of informed decision-making to ensure the latter is not compromised by either invitation strategy. Discussion If effective at improving informed uptake of screening and reducing bias in participation, this invitation strategy could be adopted by local screening pilots or a national programme. Trial registration This study was registered with the ISRCTN (International Standard Registered Clinical/soCial sTudy Number : ISRCTN21774741) on the 23rd September 2015 and the NIH ClinicalTrials.gov database (NCT0255810) on the 22nd September 2015

A Biological Global Positioning System: Considerations for Tracking Stem Cell Behaviors in the Whole Body

Many recent research studies have proposed stem cell therapy as a treatment for cancer, spinal cord injuries, brain damage, cardiovascular disease, and other conditions. Some of these experimental therapies have been tested in small animals and, in rare cases, in humans. Medical researchers anticipate extensive clinical applications of stem cell therapy in the future. The lack of basic knowledge concerning basic stem cell biology-survival, migration, differentiation, integration in a real time manner when transplanted into damaged CNS remains an absolute bottleneck for attempt to design stem cell therapies for CNS diseases. A major challenge to the development of clinical applied stem cell therapy in medical practice remains the lack of efficient stem cell tracking methods. As a result, the fate of the vast majority of stem cells transplanted in the human central nervous system (CNS), particularly in the detrimental effects, remains unknown. The paucity of knowledge concerning basic stem cell biology—survival, migration, differentiation, integration in real-time when transplanted into damaged CNS remains a bottleneck in the attempt to design stem cell therapies for CNS diseases. Even though excellent histological techniques remain as the gold standard, no good in vivo techniques are currently available to assess the transplanted graft for migration, differentiation, or survival. To address these issues, herein we propose strategies to investigate the lineage fate determination of derived human embryonic stem cells (hESC) transplanted in vivo into the CNS. Here, we describe a comprehensive biological Global Positioning System (bGPS) to track transplanted stem cells. But, first, we review, four currently used standard methods for tracking stem cells in vivo: magnetic resonance imaging (MRI), bioluminescence imaging (BLI), positron emission tomography (PET) imaging and fluorescence imaging (FLI) with quantum dots. We summarize these modalities and propose criteria that can be employed to rank the practical usefulness for specific applications. Based on the results of this review, we argue that additional qualities are still needed to advance these modalities toward clinical applications. We then discuss an ideal procedure for labeling and tracking stem cells in vivo, finally, we present a novel imaging system based on our experiments

Arsenate reductase from Thermus thermophilus conjugated to polyethylene glycol-stabilized gold nanospheres allow trace sensing and speciation of arsenic ions.

Water sources pollution by arsenic ions is a serious environmental problem all around the world. Arsenate reductase enzyme (TtArsC) from Thermus thermophilus extremophile bacterium, naturally binds arsenic ions, As(V) and As (III), in aqueous solutions. In this research, TtArsC enzyme adsorption onto hybrid polyethylene glycol-stabilized gold nanoparticles (AuNPs) was studied at different pH values as an innovative nanobiosystem for metal concentration monitoring. Characterizations were performed by UV/Vis and circular dichroism spectroscopies, TEM images and in terms of surface charge changes. The molecular interaction between arsenic ions and the TtArsC-AuNPs nanobiosystem was also monitored at all pH values considered by UV/Vis spectroscopy. Tests performed revealed high sensitivities and limits of detection equal to 10 ± 3 M−12 and 7.7 ± 0.3 M−12 for As(III) and As(V), respectively

Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein

A fusion protein designed in order to combine the fluorescence emission of the Green Fluorescent Protein (GFP) with the adhesion ability of the class I hydrophobin Vmh2 was heterologously produced in the yeast Pichia pastoris. The Vmh2-GFP fusion protein has proven to be a smart and effective tool for the study of Vmh2 self-assembling. Since the two proteins were linked by the specific cutting site of the thrombin, the fusion protein was used as the active biological element in the realization of a thrombin biosensor. When the thrombin present in the target solution specifically hydrolyzed its cleavage sequence, a consequent decrease in the fluorescence intensity of the sample could be observed. The Vmh2-GFP based assay allowed quantification of thrombin in solution with a detection limit of 2.27aM. The specificity of the assay with respect to other proteases and proteins granted the measurement of thrombin added to healthy human plasma with same high sensitivity and a limit of detection of 2.3aM. Further advantages of the developed biosensor are the simplicity of its design and preparation, and the low requirements in terms of samples, reagents and time

Hybrid hydrophobin/gold nanoparticles: Synthesis and characterization of new synthetic probes for biological applications

We report a simple and original method to synthesize gold nanoparticles in which a fungal protein, the hydrophobin Vmh2 from Pleurotus ostratus, mixed to cetyltetrammonium bromide (CTAB) has been used as additional component in a one-step synthesis, leading to shell-like hybrid protein-metal nanoparticles (NPs). The nanoparticles have been characterized by ultra-violet/visible and infrared spectroscopies, and also by electron microscopy imaging. The results of these analytical techniques highlight nanometric sized, stable, hybrid complexes of about 10 nm, with a micelles-like hydrophobins rearrangement