Microarray-based Functional Nanoproteomics for an Industrial Approach to Cancer. II Mass Spectrometry and Nanoconductimetry

Claudio Nicolini, Nicola Luigi Bragazzi and Eugenia Pechkova
 

Abstract

Using the New England BioLabs (NEBL) SNAP-based Genes Expression in conjunction with our “sub-micron arrays” (Anodic Porous Allumina and/ or Kapton based Nanopores), we exploit our proprietary microarrays scanner (DNASER, DNA analyzer) and Label Free Nanotechnologies to carry out the following tasks:
1) Construction of SNAP-based Genes Nanoarrays, using gold surface coated for 10 minutes with 2% solution of 3-Aminopropyltriethoxysilane (APTES) in acetone, rinsed in acetone and dried with filtered air. Full length complementary DNAs (cDNAs) for onco-suppressor 53 (p53), Cyclin-dependent kinase 2 (CDK2), SH2 (Src Homology 2) domain of the proto-oncogene tyrosine-protein kinase (Src) and tyrosine-protein phosphatase non-receptor type 11 (PTPN11) were amplified and cloned. Printing mix was prepared with 0.66 μg/μl DNA capture reagent BG-PEG-NH2 for the one-step synthesis of SNAP-tag substrates from esters on labels or surfaces;
2) Determination of Protein-Protein Interaction for the chosen cancer following the identification of leader genes (or hub genes, investigated with theoretical ab initio bioinformatics analysis using in-house software and algorithms, and then experimentally confirmed via DNASER). These genes are expressed by PURE (Protein synthesis Using Recombinant Elements) Express in spots less than 1 micron size piezo-microdispensed and then characterized via Label Free proprietary Autoflex Mass Spectrometry (MS) integrated with ad hoc software, namely the Spectrum Analyzer and Data Set manager (SpADS) and a proprietary Quartz Crystal Micro-balance with Dissipation factor monitoring (QCM_D) Nanoconductimetry, enabling to describe properties such as changes in frequency and conductance, viscoelasticity and dissipation factor. Solutions without DNA were prepared (called Master Mix, MM), as negative controls, in printing mix. Negative controls were prepared with a varying concentration range of SNAP capture reagent. As a positive control (for fluorescence analysis) mouse IgG or rabbit IgG (Pierce, IL, USA) were added in a printing mix instead of DNA.

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