Plasma nanotextured polymeric lab-on-a-chip for highly efficient bacteria capture and lysis.

We describe the design, fabrication, and successful demonstration of a sample preparation module comprising bacteria cell capture and thermal lysis on-chip with potential applications in food sample pathogen analysis. Plasma nanotexturing of the polymeric substrate allows increase of the surface area of the chip and the antibody binding capacity. Three different anti-Salmonella antibodies were directly and covalently linked to plasma treated chips without any additional linker chemistry or other treatment. Then, the Ab-modified chips were tested for their capacity to bind bacteria in the concentration range of 10(2)-10(8) cells per mL; the module exhibited 100% efficiency in Salmonella enterica serovar Typhimurium bacteria capture for cell suspensions below 10(5) cells per mL (10(4) cells injected with a 100 µL sample volume) and efficiency higher than 50% for 10(7) cells per mL. Moreover, thermal lysis achieved on-chip from as low as 10 captured cells was demonstrated and shown to compare well with off-chip lysis. Excellent selectivity (over 1 : 300) was obtained in a sample containing, in addition to S. Typhimurium and E. coli bacteria.

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.

Plasma-assisted nanoscale protein patterning on Si substrates via colloidal lithography.

Selective immobilization of proteins in well-defined patterns on substrates has recently attracted considerable attention as an enabling technology for applications ranging from biosensors and BioMEMS to tissue engineering. In this work, a method is reported for low-cost, large scale and high throughput, selective immobilization of proteins on nanopatterned Si, based on colloidal lithography and plasma processing to define the areas (<300 nm) where proteins are selectively immobilized. A close-packed monolayer of PS microparticles is deposited on oxidized Si and, either after microparticle size reduction or alternatively after metal deposition through the PS close-packed monolayer, is used as etching mask to define SiO2 nanoislands (on Si). C4F8 plasma was used to selectively etch and modify the SiO2 nanoislands while depositing a fluorocarbon layer on the Si surface. The plasma-treated surfaces were chemically characterized in terms of functional group identification through XPS analysis and reaction with specific molecules. Highly selective protein immobilization mainly through physical adsorption on SiO2 nanoislands and not on surrounding Si was observed after C4F8 plasma-induced chemical modification of the substrate. The thickness of the immobilized protein monolayer was estimated by means of AFM image analysis. The method reported herein constitutes a cost-efficient route toward rapid, large surface, and high-density patterning of biomolecules on solid supports that can be easily applied in BioMEMS or microanalytical systems.

High-capacity and high-intensity DNA microarray spots using oxygen-plasma nanotextured polystyrene slides.

Commercially available polystyrene (PS) slides were plasma nanotextured (nano-roughened) through treatment in oxygen plasma discharges to create substrates with increased surface area for microarray applications. Conditions of plasma treatment were determined for maximum and uniform oligonucleotide immobilization on these nanotextured PS slides. Oligonucleotides were immobilized onto the surface in the form of biotinylated oligonucleotide/streptavidin conjugates to take advantage of increased protein binding capacity of the substrate. It was found that the amount of oligonucleotides that could be immobilized was increased up to ten times on plasma treated as compared with untreated slides. The sensitivity of detection of labelled hybridized probes was improved by a factor of 20. Optimized nanotextured PS slides were subsequently used to develop a microarray for the detection of three deleterious BRCA1 gene mutations by immobilizing oligonucleotides corresponding to wild and mutant-type sequences. The microarray developed on the nanotextured PS slides provided higher specific hybridization signal and discrimination ratios as compared with flat untreated PS slides.

Plasma nanotextured PMMA surfaces for protein arrays: increased protein binding and enhanced detection sensitivity.

Poly(methyl methacrylate) (PMMA) substrates were nanotextured through treatment in oxygen plasma to create substrates with increased surface area for protein microarray applications. Conditions of plasma treatment were found for maximum uniform protein adsorption on these nanotextured PMMA surfaces. Similar results were obtained using both a high-density plasma (HDP) and a low-density reactive ion etcher (RIE), suggesting independence from the plasma reactor type. The protein binding was evaluated by studying the adsorption of two model proteins, namely, biotinylated bovine serum albumin (b-BSA) and rabbit gamma-globulins (RgG). The immobilization of these proteins onto the surfaces was quantitatively determined through reaction with fluorescently labeled binding molecules. It was found that the adsorption of both proteins was increased up to 6-fold with plasma treatment compared to untreated surfaces and up to 4-fold compared to epoxy-coated glass slides. The sensitivity of detection was improved by 2 orders of magnitude. Moreover, highly homogeneous protein spots were created on optimized plasma-nanotextured surfaces through deposition with an automated microarray spotter, revealing the potential of plasma-nanotextured surfaces as protein microarray substrates.

Mechanisms of oxygen plasma nanotexturing of organic polymer surfaces: from stable super hydrophilic to super hydrophobic surfaces.

Plasma processing is used to fabricate super hydrophilic or super hydrophobic polymeric surfaces by means of O2 plasma etching of two organic polymers, namely, poly(methyl methacrylate) (PMMA) and poly(ether ether ketone) (PEEK); a C4F8 plasma deposition follows O2 plasma etching, if surface hydrophobization is desired. We demonstrate high aspect ratio pillars with height ranging from 16 nm to several micrometers depending on the processing time, and contact angle (CA) close to 0 degrees after O2-plasma treatment or CA of 153 degrees (with CA hysteresis lower than 5 degrees) after fluorocarbon deposition. Super hydrophobic surfaces are robust and stable in time; in addition, aging of super hydrophilic surfaces is significantly retarded because of the beneficial effect of the nanotextured topography. The mechanisms responsible for the plasma-induced PMMA and PEEK surface nanotexturing are unveiled through intelligent experiments involving intentional modification of the reactor wall material and X-ray photoelectron spectroscopy, which is also used to study the surface chemical modification in the plasma. We prove that control of plasma nanotexture can be achieved by carefully choosing the reactor wall material.