Protein immobilisation on micro/nanostructures fabricated by laser microablation.

The performance of biomedical microdevices requires the accurate control of the biomolecule concentration on the surface, as well as the preservation of their bioactivity. This desideratum is even more critical for proteins, which present a significant propensity for surface-induced denaturation, and for microarrays, which require high multiplexing. We have previously proposed a method for protein immobilisation on micro/nanostructures fabricated via laser ablation of a thin metal layer deposited on a transparent polymer. This study investigates the relationship between the properties of the micro/nanostructured surface, i.e., topography and physico-chemistry, and protein immobilisation, for five, molecularly different proteins, i.e., lysozyme, myoglobin, α-chymotrypsin, human serum albumin, and human immunoglobulin. Protein immobilisation on microstructures has been characterised using quantitative fluorescence measurements and atomic force microscopy. It has been found that the sub-micrometer-level, combinatorial nature of the microstructure translates in a 3-10-fold amplification of protein adsorption, as compared to flat, chemically homogenous polymeric surfaces. This amplification is more pronounced for smaller proteins, as they can capitalize better on the newly created surface and variability of the nano-environments.

Staleya guttiformis attachment on poly(tert-butylmethacrylate) polymeric surfaces.

The attachment behaviour of Staleya guttiformis DSM 11458(T) on poly(tert-butyl methacrylate) (P(tBMA)) polymeric surfaces has been studied. The electrostatic charge of the S. guttiformis cell surface (measured as zeta potential via microelectrophoresis) was -43.18 mV. S. guttiformis cells appeared weakly hydrophilic as the water contact angle measured on lawns of bacterial cells was found to be 55+/-4.9 degrees. It was found that while attaching on P(tBMA) surfaces, S. guttiformis cells produced extracellular polymeric substances (EPS) as observed from atomic force microscopy (AFM) and scanning electron microscopy (SEM) analysis. The AFM high resolution imaging revealed the nano-topography of the 'free' (the EPS that is produced by the bacterial cells, but no longer directly attached to the cells) EPS associated on the cell surface and also found on P(tBMA) surface. The 'free' EPS exhibited granular structure with lateral dimensions of 30-50 nm and a vertical nano-roughness of 7-10nm. Another type of the EPS secreted by S. guttiformis cells appeared as a hydrogel substance, presumably polysaccharide that formed a biopolymer network that facilitated bacterial attachment.

ATP level variations in heterotrophic bacteria during attachment on hydrophilic and hydrophobic surfaces.

A survey of the extracellular ATP levels of 86 heterotrophic bacteria showed that gram-negative bacteria of the genera Sulfitobacter, Staleya, and Marinobacter secreted elevated amounts of extracellular ATP, ranging from 6.0 to 9.8 pM ATP/colony forming unit (cfu), and that gram-positive bacteria of the genera Kocuria and Planococcus secreted up to 4.1 pM ATP/cfu. Variations in the levels of extracellular and intracellular ATP-dependent luminescence were monitored in living cells of Sulfitobacter mediterraneus ATCC 700856T and Planococcus maritimus F 90 during 48 h of attachment on hydrophobic (poly[tert-butyl methacrylate], PtBMA) and hydrophilic (mica) surfaces. The bacteria responded to different polymeric surfaces by producing either intracellular or extracellular ATP. The level of intracellular ATP in S. mediterraneus ATCC 700856T attached to either surface was as high as 50-55 pM ATP/cfu, while in P. maritimus F 90 it was 120 and 250 pM ATP/cfu on PtBMA and mica, respectively. S. mediterraneus ATCC 700856T generated about 20 and 50 pM of extracellular ATP/cfu on PtBMA and mica, respectively, while the amount generated by P. maritimus F 90 was about the same for both surfaces, 6 pM ATP/cfu. The levels of extracellular ATP generated by S. mediterraneus during attachment on PtBMA and mica were two to five times higher than those detected during the initial screening. High-resolution atomic force microscopy imaging revealed a potentially interesting correlation between the porous cell-surface of certain alpha- and gamma-proteobacteria and their ability to secrete high amounts of ATP.

ATP level variations in heterotrophic bacteria during attachment on hydrophilic and hydrophobic surfaces / Variaciones en los niveles de ATP en bacterias heterotrofas durante la adherencia a superficies hidrofílicas e hidrofóbicas

A survey of the extracellular ATP levels of 86 heterotrophic bacteria showed that gram-negative bacteria of the genera Sulfitobacter, Staleya, and Marinobacter secreted elevated amounts of extracellular ATP, ranging from 6.0 to 9.8 pM ATP/colony forming unit (cfu), and that gram-positive bacteria of the genera Kocuria and Planococcus secreted up to 4.1 pM ATP/cfu. Variations in the levels of extracellular and intracellular ATP-dependent luminescence were monitored in living cells of Sulfitobacter mediterraneus ATCC 700856T and Planococcus maritimus F 90 during 48 h of attachment on hydrophobic (poly[tert-butyl methacrylate], PtBMA) and hydrophilic (mica) surfaces. The bacteria responded to different polymeric surfaces by producing either intracellular or extracellular ATP. The level of intracellular ATP in S. mediterraneus ATCC 700856T attached to either surface was as high as 50-55 pM ATP/cfu, while in P. maritimus F 90 it was 120 and 250 pM ATP/cfu on PtBMA and mica, respectively. S. mediterraneus ATCC 700856T generated about 20 and 50 pM of extracellular ATP/cfu on PtBMA and mica, respectively, while the amount generated by P. maritimus F 90 was about the same for both surfaces, 6 pM ATP/cfu. The levels of extracellular ATP generated by S. mediterraneus during attachment on PtBMA and mica were two to five times higher than those detected during the initial screening. High-resolution atomic force microscopy imaging revealed a potentially interesting correlation between the porous cell-surface of certain a- and g-proteobacteria and their ability to secrete high amounts of ATP (AU)

Un estudio de los niveles del ATP extracelular de 86 bacterias heterotrofas mostró que las bacterias gram-negativas de los géneros Sulfitobacter, Staleya y Marinobacter secretaron grandes cantidades de ATP extracelular 6,0 a 9,8 pM ATP/unidad formadora de colonia (cfu) y las bacterias gram-positivas de los géneros Kocuria y Planococcus secretaban cantidades de hasta 4,1 pM ATP/cfu. Las variaciones de los niveles extracelulares e intracelulares de luminiscencia dependiente de ATP fueron controlados en células vivas de Sulfitobacter mediterraneus ATCC 700856T y Planococcus maritimus F 90 durante 48 h de adherencia a superficies hidrofóbicas (poli[tert-butil metacrilato], PtBMA) e hidrofílicas (mica). Las bacterias respondieron a las diferentes superficies produciendo ATP intracelular o extracelular. El nivel de ATP intracelular en S. mediterraneus ATCC 700856T llegó hasta 50-55 pM ATP/cfu en ambas superficies, mientras que para P. maritimus F 90 fue de 120 pM ATP/cfu en PtBMA y de 250 pM ATP/cfu en mica. El ATP extracelular generado por las células de S. mediterraneus ATCC 700856T se situó entre 20 pM ATP/cfu en PtBMA y 50 pM ATP/cfu en mica, mientras que para P. maritimus F 90 era casi igual en ambas superficies, 6 pM ATP/cfu. Los niveles detectados de ATP extracelular generados por S. mediterraneus durante la adherencia a PtBMA y a mica fueron de 2 a 5 veces los detectados en el cribado inicial. Las imágenes obtenidas mediante microscopia de fuerza atómica de alta resolución pusieron de manifiesto una correlación de posible interés entre la superficie celular porosa de ciertas a- y g-proteobacterias y la capacidad de secretar grandes cantidades de ATP (AU)

A comparative study between the adsorption and covalent binding of human immunoglobulin and lysozyme on surface-modified poly(tert-butyl methacrylate).

The adsorption and covalent immobilization of human immunoglobulin (HIgG) and lysozyme (LYZ) on surface-modified poly(tert-butyl methacrylate) PtBMA films have been evaluated using x-ray photoelectron spectroscopy (XPS), ellipsometry and atomic force microscopy (AFM). Surface modification of PtBMA (UV irradiation) afforded surfaces suitable for both the physical and covalent attachment of proteins. The XPS and ellipsometry results showed good correlation in terms of variable-dense/thickness protein layer formation between physisorbed and covalently bound proteins. The amount of physisorbed HIgG ranged from 23.0 +/- 1.6 ng mm(2) on PtBMA, with corresponding film thicknesses 17.0 +/- 1.2 nm. Covalent immobilization mediated through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) coupling chemistry, afforded 5.6-8 ng mm(2) of HIgG with a corresponding thickness of 5.9 +/- 0.6 nm on PtBMA. The attachment of LYZ to modified PtBMA surface was similarly translated, where adsorption yielded up to 15 ng mm(2), while covalent immobilization afforded typically 7-8 ng mm(2). The thickness of the adsorbed LYZ protein layer was 11.0 +/- 3.2 nm (PtBMA), suggesting the greater portion of protein adsorbs on surface-modified PtBMA.

Poly(L-lysine)-mediated immobilisation of oligonucleotides on carboxy-rich polymer surfaces.

The immobilisation efficiency of the complexes of oligonucleotide/poly(L-lysine) on two polymeric carboxy-rich surfaces, i.e. poly(styrene/maleic acid) (PSMA) and poly(styrene/maleic anhydride) (PSMAA), has been investigated using X-ray photoelectron spectroscopy, atomic force microscopy (AFM) and fluorescence-based measurements of DNA attachment. A molecularly thin layer of either electrostatically or covalently (via amide bond) bound poly(L-lysine) allows the 'switching' from COOH-based to NH(2)-based surface functionality. The results indicate that approximately 54-57% and 55-62% of the applied oligonucleotides bind to polymeric surfaces via the route of electrostatic adsorption of poly(L-lysine) and covalent bonding of poly(L-lysine), respectively. This system can be applied conveniently for the detection of nucleic acids in both disposable and reusable biosensors.

Detection of coccoid forms of Sulfitobacter mediterraneus using atomic force microscopy.

The adhesion of the marine alpha-Proteobacteria Sulfitobacter pontiacus, Sulfitobacter mediterraneus, Sulfitobacter brevis, and Staleya guttiformis to a poly(tert-butyl methacrylate) (PtBMA) polymeric surface generates unusual cell morphological peculiarities following attachment. While the type strains S. pontiacus and S. brevis failed to attach to PtBMA, the vegetative cells of type strain S. mediterraneus underwent morphological conversion into coccoid forms during the attachment over an incubation period of 24-72 h. Type strain St. guttiformis cells formed a multilayered biofilm on the PtBMA surface, presumably facilitated by bacterial production of extracellular polysaccharides. The attachment behavior and fine structure of these coccoid forms have been described using atomic force microscopy. The impact of polymeric surfaces of defined hydrophobicity on the formation of coccoid bodies is discussed.