Rosanna Spera

Prototypes of Newly Conceived Inorganic and Biological Sensors for Health and Environmental Applications

This paper describes the optimal implementation of three newly conceived sensors for both health and environmental applications, utilizing a wide range of detection methods and complex nanocomposites. The first one is inorganic and based on matrices of calcium oxide, the second is based on protein arrays and a third one is based on Langmuir-Blodgett laccase multi-layers. Special attention was paid to detecting substances significant to the environment (such as carbon dioxide) and medicine (drug administration, cancer diagnosis and prognosis) by means of amperometric, quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies. The resulting three implemented nanosensors are described here along with proofs of principle and their corresponding applications.

Conductometric monitoring of protein-protein interactions.

Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.

Gene expression in the cell cycle of human T-lymphocytes: II. Experimental determination by DNASER technology

Human lymphocytes gene expression before and after PHA stimulation is monitored by DNASER technology, a novel bioinstrumentation entirely constructed in our laboratories as previously reported. The validity of the DNASER measurements is confirmed by standard fluorescence microscopy equipped with CCD. The human lymphocytes gene expression here experimentally probed using commercially available DNA microarrays such as Human Starter, appears compatible both with independent bioinformatic prediction and with existing experimental data, pointing to MYC as the key gene in the G0–G1 transition induced by PHA in resting lymphocytes. It does not escape our notice that in cell biology and cancer research DNASER technology based on microarray constructed with few leader genes identified from bioinformatics represents a meaningful cost‐effective route alternative to massive frequently misleading molecular genomics. J. Cell. Biochem. 97: 1151–1159, 2006.

Atomic Force Microscopy and Anodic Porous Allumina of Nucleic Acid Programmable Protein Arrays

The methodological aspects are here presented for the NAPPA (Nucleic Acid Programmable Protein Arrays) characterization by atomic force microscopy and anodic porous alumina. Anodic Porous Alumina represents also an advanced on chip laboratory for gene expression contained in an engineered plasmid vector. The results obtained with CdK2, CDKN1A, p53 and Jun test genes expressed on NAPPA and the future developments are discussed in terms of our pertinent and recent Patents and of their possibility to overcome some limitations of present fluorescence detection in probing protein-protein interaction in both basic sciences and clinical studies.

SpADS and SNAP-NAPPA Microarrays towards Biomarkers Identification in Humans: Background Subtraction in Mass Spectrometry with E.coli Cell Free Expression System

We present a useful approach towards for biomarkers identification in an innovative self-assembling protein microarray based on “Nucleic Acid Programmable Protein Array” (NAPPA) and SNAP tag coupled to E.coli cell free expression system. This approach prove capable to resolve the “background” problem associated to the above label free detection system for the identification of proteins and of protein-protein interaction in humans that could become used in clinical practice.

NAPPA-Based Nanobiosensors for the Detection of Proteins and of Protein-Protein Interactions Relevant to Cancer

In this manuscript, we report some proofs-of-principle and preliminary applications and results regarding the analytical quantification of protein expression of some genes biologically and clinically related to cancer. Experiments have been carried out coupling Nucleic Acid Programmable Protein Array (NAPPA) with a recently improved nanogravimetric apparatus which exploits the quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies. The selected proteins are BRIP1, JUN and ATF2. Clinically relevant implications for cancer are envisaged and discussed, as well as future perspectives and developments.

Mass Spectrometry and Florescence Analysis of Snap-Nappa Arrays Expressed Using E. coli Cell_Free Expression System

We present the analysis of an innovative kind of self-assembling protein microarray, the “Nucleic Acid Programmable Protein Array” (NAPPA), express with the SNAP tag in E.coli coupled self free expression system. The goal is to develop a standardize procedure to analyze the protein protein interaction occurred on NAPPA array combining label free Mass Spectrometry (MS) and fluorescence technology for protein microarray. We employ in the process “Protein synthesis Using Recombinant Elements” (PURE) system. For the first time an improved version of NAPPA, that allows for functional proteins to be synthesized in situ with a SNAP tag directly from printed cDNAs just in time for assay, has been expressed with a novel cell-free transcription/translation system reconstituted from the purified components necessary for Escherichia coli translation the PURE system – and analyzed both in fluorescence and in a label free manner by four different mass spectrometers, namely three Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), a Voyager, a Bru ker Autoflex and a Bruker Ultraflex, and Liquid Chromatography-Electrospray Ionization Mass Spectrometry (LC-ESI-MS/MS). Due to the high complexity of the system, an ad hoc bioinformatic tool has been needed to develop for their successful analysis. The contemporary fluorescence analysis of NAPPA, expressed by means of PURE system, has been performed to confirm the improved characterization of this new NAPPA-SNAP system. complement to labeling methods [7,8]. Among all these techniques, that usually cannot reveal the identity of interaction proteins, MS is powerful to provide chemical and structural information that is difficult to obtain through other means. Combining MS and other surface techniques could offer new dimensions in protein analysis [5,8]. The integration of microarrays with MS has generated a powerful new tool to deal with the problems in protein analysis and identification area. The most successful example is the ProteinChip® System of Ciphergen Biosystems Inc. The design of the ProteinChip® array was originally derived from chromatography and is divided into two groups according to its surface characteristics; the array reader is a SELDI-TOF-MS instrument equipped with a pulsed UV nitrogen laser source [8]. Also Nedelkov et al. [9] coupled BIACORE with MS to demonstrate its feasibility in detecting multiple protein-protein interactions.In our research we employed two different mass spectrometry (MS) techniques, the Matrix Assisted Laser Desorption Ionization Time-ofFlight (MALDI-TOF) MS and Liquid Chromatography-Electrospray Ionization MS (LC-ESI-MS) (Figure 1). In a previous research we carried out a feasibility study of MALDIJournal of Nanomedicine & Nanotechnology J o u r n a l o f N an om edicine & Natechn o l o g y ISSN: 2157-7439 Citation: Nicolini C, et al. (2013) Mass Spectrometry and Florescence Analysis of Snap-Nappa Arrays Expressed Using E. coli Cell_Free Expression System. J Nanomed Nanotechnol 4: 181. doi:10.4172/2157-7439.1000181

Analysis of gene expression on anodic porous alumina microarrays

This paper investigates the application of anodic porous alumina as an advancement on chip laboratory for gene expressions. The surface was prepared by a suitable electrolytic process to obtain a regular distribution of deep micrometric holes and printed bypen robot tips under standard conditions. The gene expression within the Nucleic Acid Programmable Protein Array (NAPPA) is realized in a confined environment of 16 spots, containing circular DNA plasmids expressed using rabbit reticulocyte lysate. Authors demonstrated the usefulness of APA in withholding the protein expression by detecting with a CCD microscope the photoluminescence signal emitted from the complex secondary antibody anchored to Cy3 and confined in the pores. Friction experiments proved the mechanical resistance under external stresses by the robot tip pens printing. So far, no attempts have been made to directly compare APA with any other surface/substrate; the rationale for pursuing APA as a potential surface coating is that it provides advantages over the simple functionalization of a glass slide, overcoming concerns about printing and its ability to generate viable arrays.

Matrices for Sensors from Inorganic, Organic, and Biological Nanocomposites

Matrices and sensors resulting from inorganic, organic and biological nanocomposites are presented in this overview. The term nanocomposite designates a solid combination of a matrix and of nanodimensional phases differing in properties from the matrix due to dissimilarities in structure and chemistry. The nanoocomposites chosen for a wide variety of health and environment sensors consist of Anodic Porous Allumina and P450scc, Carbon nanotubes and Conductive Polymers, Langmuir Blodgett Films of Lipases, Laccases, Cytochromes and Rhodopsins, Three-dimensional Nanoporous Materials and Nucleic Acid Programmable Protein Arrays.

Groel crystal growth and characterization

Single crystals of ribosomal proteins obtained for the first time by Langmuir-Blodgett (LB) nanotemplate confirm earlier findings (Pechkova et al., 2008), pointing to a new generation of bionanomaterials of unique structure-function relationship. The ribosomal protein phage GroEL was overexpressed in E. coli. Since these proteins samples have some difficulties by classical vapour diffusion method to yield optimal diffraction quality and order (GroEL), the LB nanotemplate method has been applied and compared to the classical method. With the thin film nanotemplate method large phage GroEL crystals appeared in few days and were subsequently characterized by MALDI-TOF Mass Spectroscopy and by a very preliminary X-ray diffraction.