Oxygen plasma treated interactive polycarbonate DNA microarraying platform.

A novel DNA microarrying platform based on oxygen plasma activation of polycarbonate surface of compact disks (DVD) is presented. Carboxylic acid groups are generated in few seconds on polycarbonate in an efficient, fast, and clean way. Following this surface activation strategy, amino-modified oligonucleotide probes were covalently attached, reaching an immobilization density of 2 pmol cm(-2). Atomic force microscopy imaging revealed the nondestructive character of this treatment when applied for short times, allowing for disk scanning in standard DVD drives. DNA assays performed on oxygen plasma treated disks resulted very efficient with maximum hybridization yield of 93% and reaching a low limit of detection (200 pM) for perfect match synthetic oligonucleotide targets when reading the disk with a standard drive as detector. The approach was also evaluated by scoring single nucleotide polymorphisms with a discrimination ratio of 12.8. As proof of concept, the oxygen plasma treated interactive polycarbonate DNA microarraying platform was applied to the detection of PCR products of Salmonella spp., reaching a detection limit of 2 nM that corresponds to a DNA concentration of only 1 c.f.u./mL. The results confirm the suitability of the microarray platform for analysis of biological samples with high sensitivity.

Direct hapten-linked multiplexed immunoassays on polycarbonate surface.

Direct hapten-linked multiplexed immunoassay is developed on the polycarbonate surface of standard Digital Versatile Discs (DVDs) for six compounds of environmental concern, as proof of concept. Carboxylated haptens are directly linked to the aminated polycarbonate surface through carbodiimide/succinimide coupling. The modified DVD surface maintained its physical and optical properties. Multiplexed assay reached detection limits down to 0.1 µg/L for chlorpyrifos, 2,4,5-trichlorophenoxypropionic acid, sulfathiazole and sulfasalazine and down to 1.0 µg/L for fenthion and malathion. This approach presents advantages such as the improvement in sensitivity in comparison to protein-hapten conjugate format for all the studied analytes and the absence of cross-interference effects, allowing high throughput multianalysis on the same surface. Also, a comparison of the performance of two sensing strategies indicated that DVD disc and drive approach turned out in a simpler mode, the assays being more reproducible and with higher signal to noise ratios.

Determination of microcystins in river waters using microsensor arrays on disk.

The development of simple, accurate, and rapid multisample analytical methodologies to find out critical targets in waters is highly demanded. Optical microsensor arrays to determine microcystins in river waters are developed on the polycarbonate side of compact discs. The working principle of the sensors relied on an indirect competitive microimmunoassay, where free microcystin LR (MC-LR) competes with immobilized conjugate for specific monoclonal antibody. The results of the immunoreaction are detected with a DVD drive, showing the readouts in minutes. The method reached a sensitivity (IC(50)) for MC-LR of 1.04 µg/L and a linear response in the range 0.12-2.00 µg/L, allowing its determination below the upper limit proposed by the World Health Organization in drinking water. The developed analytical approach shows simplicity, good sensitivity, high throughput capability, and rapidity (37 min) in field use. The optimized assay showed also high congener reactivity to MC-LY (144%), MC-LA (125%), MC-LF (119%), MC-LW (102%), MC-YR (83%), and nodularin (94%). Furthermore, the suitability of the disk biosensor to quantify MC-LR was successfully evaluated analyzing river water samples, obtaining excellent recoveries (78-113%). Precoated discs are stable for at least seven weeks without loosing their analytical performances. Also, the portability of the analytical system permits on-site analysis and quantification, saving time and other resources. To our knowledge, this is the only work where a portable, easy-to-use, array based system has been developed for on-site microcystin quantification and applied to simultaneously analyze 42 samples plus the calibration curve, reaching microgram per liter sensitivity.

Simple replication methods for producing nanoslits in thermoplastics and the transport dynamics of double-stranded DNA through these slits.

Mixed-scale nano- and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2-7 µm in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass. The device also contained a microchannel network that was hot embossed into the polymer substrate using a metal molding tool prepared via HPMM. The mixed-scale device could also be used as a master to produce a polymer stamp, which was made from polydimethylsiloxane, PDMS, and used to generate the mixed-scale fluidic network in a single step. Thermal fusion bonding of the cover plate to the substrate at a temperature below their respective T(g) was accomplished by oxygen plasma treatment of both the substrate and cover plate, which significantly reduced thermally induced structural deformation during assembly: ∼6% for PMMA and ∼9% for COC nanoslits. The electrokinetic transport properties of double-stranded DNA (dsDNA) through the polymeric nanoslits (PMMA and COC) were carried out. In these polymer devices, the dsDNA demonstrated a field-dependent electrophoretic mobility with intermittent transport dynamics. DNA mobilities were found to be 8.2 ± 0.7 × 10(-4) cm(2) V(-1) s(-1) and 7.6 ± 0.6 × 10(-4) cm(2) V(-1) s(-1) for PMMA and COC, respectively, at a field strength of 25 V cm(-1). The extension factors for λ-DNA were 0.46 in PMMA and 0.53 in COC for the nanoslits (2-6% standard deviation).

Development of hapten-linked microimmunoassays on polycarbonate discs.

An amino-modified polycarbonate surface of compact discs is used to link haptens covalently and directly as an alternative to the classic protein-hapten conjugate adsorption coating strategy employed in immunoassays. The modified surface maintains its physical and optical properties, and a standard disk drive can then read the assay results. Advantages are evaluated, such as the use of a broader spectrum of coupling media including organic solvents that are inappropriate for proteins but necessary for some water-insoluble haptens and the bypassing of the synthesis and purification for protein conjugates. As proof of concept, competitive microimmunoassays were developed for chlorpyrifos, atrazine, and 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP), in microarray format, obtaining detection limits of 37.2, 8.1, and 76 ng/L, respectively. The sensitivity was 1 order of magnitude better than that obtained for all the studied systems using hapten-protein conjugates adsorbed on polystyrene enzyme-linked immunosorbent assay (ELISA) plates and polycarbonate surfaces. Further, the influence of hapten structure and presentation on molecular recognition pattern is discussed. To our knowledge, this is the first time that microarray and compact disc technologies converge with this particular hapten immobilization mode. The great potential of the approach is demonstrated through the high-throughput capability of the disc in a range of analytical applications, as well as the inherent advantages of compact disc reading technology.

Analytical prospect of compact disk technology in immunosensing.

A sensitive and versatile methodology involving recordable compact disks as molecular screening surfaces and a standard optical CD/DVD drive as detector, is reported. Quantitative immunoanalysis, in microarray format, of a cancer marker (alpha-fetoprotein, AFP) and a selective herbicide (atrazine) on four types of audio-video disc was conducted. Enzyme or gold nanoparticle-labeled antibodies were used as tracers, forming a precipitate on the sensing disk surface. The principle of disk reading is based on capture of analog signals with the disk drive that were proportional to the darkness of the immunoreaction product. Detection limits for AFP (8.0 microg L(-1)) and for atrazine (0.04 microg L(-1)) were under the threshold needed to detect nonseminomatous testicular cancer, and below the maximum E.U. residue limit for drinking water, respectively. The described methodology improves the previous developments using CDs and highlights the enormous potential of immunoassay methods using standard audio-video disk surfaces in combination with the CD/DVD drive for clinical analysis, drug discovery, or high-throughput multiresidue screening applications.

Use of polystyrene spin-coated compact discs for microimmunoassaying.

The analytical potential of polystyrene (PS) spin-coated modified compact discs (CDs) surface as platforms for the development of microarray immunoassays is presented. The surface maintained the optical characteristics of compact discs, obtaining a transparent and smooth film polymer of 70 nm thickness, the track being read (lambda 780 nm) without errors in a commercial CD reader/writer. The analytical capability of the methodology was demonstrated through an analysis of a neurotoxic compound (2560 spots per disc), reaching 0.08 microg L(-1) as limit of detection. These figures demonstrate the enormous potential of using PS spin-coated compact discs in combination with CD players as an easy-to-operate and portable device to develop lab-on-a-disc analytical applications.