Improved DNA microarray detection sensitivity through immobilization of preformed in solution streptavidin/biotinylated oligonucleotide conjugates

A novel immobilization approach involving binding of preformed streptavidin/biotinylated oligonucleotide conjugates onto surfaces coated with biotinylated bovine serum albumin is presented. Microarrays prepared according to the proposed method were compared, in terms of detection sensitivity and specificity, with other immobilization schemes employing coupling of biotinylated oligonucleotides onto directly adsorbed surface streptavidin, or sequential coupling of streptavidin and biotinylated oligonucleotides onto a layer of adsorbed biotinylated bovine serum albumin. A comparison was performed employing biotinylated oligonucleotides corresponding to wild- and mutant-type sequences of seven single point mutations of the BRCA1 gene. With respect to the other immobilization protocols, the proposed oligonucleotide immobilization approach offered the highest hybridization signals (at least 5 times higher) and permitted more elaborative washings, thus providing considerably higher discrimination between complimentary and non-complementary DNA sequences for all mutations tested. In addition, the hybridization kinetics were significantly enhanced compared to two other immobilization protocols, permitting PCR sample analysis in less than 40. min. Thus, the proposed oligonucleotide immobilization approach offered improved detection sensitivity and discrimination ability along with considerably reduced analysis time, and it is expected to find wide application in DNA mutation detection. © 2015 Elsevier B.V

Data on step-by-step atomic force microscopy monitoring of changes occurring in single melanoma cells undergoing ToF SIMS specialized sample preparation protocol

Data included in this article are associated with the research article entitled ‘Protocol of single cells preparation for time-of-flight secondary ion mass spectrometry’ (Bobrowska et al., 2016 in press) [1]. This data file contains topography images of single melanoma cells recorded using atomic force microscopy (AFM). Single cells cultured on glass surface were subjected to the proposed sample preparation protocol applied to prepare biological samples for time-of-flight secondary ion mass spectrometry (ToF SIMS) measurements. AFM images were collected step-by-step for the single cell, after each step of the proposed preparation protocol. It consists of four main parts: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying. In total 13 steps are required, starting from imaging of a living cell in a culture medium and ending up at images of a dried cell in the air. The protocol was applied to melanoma cells from two cell lines, namely, WM115 melanoma cells originated from primary melanoma site and WM266-4 ones being the metastasis of WM115 cells to skin. Keywords: ToF SIMS, Single cells preparation, AFM imagin

Effects of Polythiophene Surface Structure on Adsorption and Conformation of Bovine Serum Albumin: A Multivariate and Multitechnique Study

Protein interactions with surfaces of promising conducting polymers are critical for development of bioapplications. Surfaces of spin-cast and postbaked poly­(3-alkylthiophenes), regiorandom P3BT, and regioregular RP3HT are examined prior to and after adsorption of model protein, bovine serum albumin, with time-of-flight secondary ion mass spectrometry, atomic force microscopy, and X-ray photoelectron spectroscopy. The multivariate method of principal component analysis applied to ToF-SIMS data maximizes information on subtle differences in surface chemistry: PCA reveals alkyl side chains and conjugated backbones, exposed for RP3HT and P3BT, respectively. Phase imaging AFM shows semicrystalline microstructure of RP3HT and amorphous morphology of P3BT films. A cellular-like pattern of proteins adsorbed on RP3HT develops with coverage to more uniform overlayer, observed always on P3BT. The amount of adsorbed protein, determined by XPS as a function of BSA concentration (up to 10 mg/mL), is ∼21% lower for RP3HT than P3BT (up to 1.1 mg/m²). Although PCA differentiates protein from polythiophene, relative protein surface composition evaluated from ToF-SIMS saturates rather than increases with amount of adsorbed BSA from XPS. This reflects ToF-SIMS sensitivity to outermost layer of proteins, enabling multivariate analysis of protein conformation or orientation. PCA distinguishes between amino acids characteristic for external regions of BSA adsorbed to P3BT and RP3HT. These amino acids are identified for P3BT and RP3HT as hydrophilic and hydrophobic, respectively, by relative hydrophobicity of amino acid side chains. Alternative identification with BSA domains fails, pointing to substrate-induced changes in conformation and degree of denaturation rather than orientation of adsorbed protein

Structures in Multicomponent Polymer Films: Their Formation, Observation and Applications in Electronics and Biotechnology

Several strategies to form multicomponent films of functional polymers, with micron, submicron and nanometer structures, intended for plastic electronics and biotechnology are presented. These approaches are based on film deposition from polymer solution onto a rotating substrate (spin-casting), a method implemented already on manufacturing lines. Film structures are determined with compositional (nanometer) depth profiling and (submicron) imaging modes of dynamic secondary ion mass spectrometry, near-field scanning optical microscopy (with submicron resolution) and scanning probe microscopy (revealing nanometer features). Self-organization of spin-cast polymer mixtures is discussed in detail, since it offers a one-step process to deposit and align simultaneously domains, rich in different polymers, forming various device elements: (i) Surface segregation drives self-stratification of nanometer lamellae for solar cells and anisotropic conductors. (ii) Cohesion energy density controls morphological transition from lamellar (optimal for encapsulated transistors) to lateral structures (suggested for light emitting diodes with variable color). (iii) Selective adhesion to substrate microtemplates, patterned chemically, orders lateral structures for plastic circuitries. (iv) Submicron imprints of water droplets (breath figures) decorate selectively micron-sized domains, and can be used in devices with hierarchic structure. In addition, selective protein adsorption to regular polymer micropatterns, formed with soft lithography after spin-casting, suggests applications in protein chip technology. An approach to reduce lateral blend film structures to submicron scale is also presented, based on (annealed) films of multicomponent nanoparticles

Structures in Multicomponent Polymer Films: Their Formation, Observation and Applications in Electronics and Biotechnology

Several strategies to form multicomponent films of functional polymers, with micron, submicron and nanometer structures, intended for plastic electronics and biotechnology are presented. These approaches are based on film deposition from polymer solution onto a rotating substrate (spin-casting), a method implemented already on manufacturing lines. Film structures are determined with compositional (nanometer) depth profiling and (submicron) imaging modes of dynamic secondary ion mass spectrometry, near-field scanning optical microscopy (with submicron resolution) and scanning probe microscopy (revealing nanometer features). Self-organization of spin-cast polymer mixtures is discussed in detail, since it offers a one-step process to deposit and align simultaneously domains, rich in different polymers, forming various device elements: (i) Surface segregation drives self-stratification of nanometer lamellae for solar cells and anisotropic conductors. (ii) Cohesion energy density controls morphological transition from lamellar (optimal for encapsulated transistors) to lateral structures (suggested for light emitting diodes with variable color). (iii) Selective adhesion to substrate microtemplates, patterned chemically, orders lateral structures for plastic circuitries. (iv) Submicron imprints of water droplets (breath figures) decorate selectively micron-sized domains, and can be used in devices with hierarchic structure. In addition, selective protein adsorption to regular polymer micropatterns, formed with soft lithography after spin-casting, suggests applications in protein chip technology. An approach to reduce lateral blend film structures to submicron scale is also presented, based on (annealed) films of multicomponent nanoparticles

Direct covalent biomolecule immobilization on plasma-nanotextured chemically stable substrates

A new method for direct covalent immobilization of protein molecules (including antibodies) on organic polymers with plasma-induced random micronanoscale topography and stable-in-time chemical functionality is presented. This is achieved using a short (1–5 min) plasma etching and simultaneous micronanotexturing process, followed by a fast thermal annealing step, which induces accelerated hydrophobic recovery while preserving important chemical functionality created by the plasma. Surface-bound biomolecules resist harsh washing with sodium dodecyl sulfate and other detergents even at elevated temperatures, losing less than 40% of the biomolecules bound even at the harshest washing conditions. X-ray photoelectron spectroscopy, secondary-ion mass spectrometry, and electron paramagnetic resonance are used to unveil the chemical modification of the plasma-treated and stabilized surfaces. The nanotextured and chemically stabilized surfaces are used as substrates for the development of immunochemical assays for the sensitive detection of C-reactive protein and salmonella lipopolysaccharides through immobilization of the respective analyte-specific antibodies onto them. Such substrates are stable for a period of 1 year with ambient storage