Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay.
ABSTRACT: DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems.
Project description:Transparent surfaces within microfluidic devices are essential for accurate quantification of chemical, biological, and mechanical interactions. Here, we report how to create low-cost, rapid 3D-printed microfluidic devices that are optically free from artifacts and have transparent surfaces suitable for visualizing a variety of fluid phenomenon. The methodology described here can be used for creating high-pressure microfluidic systems (significantly higher than PDMS-glass bonding). We develop methods for annealing Poly-Lactic Acid (PLA) microfluidic devices demonstrating heat resistance typically not achievable with other plastic materials. We show DNA melting and subsequent fluorescent imaging analysis, opening the door to other high-temperature applications. The FDM techniques demonstrated here allow for fabrication of microfluidic devices for precise visualization of interfacial dynamics, whether mixing between two laminar streams or droplet tracking. In addition to these characterizations, we include a printer troubleshooting guide and printing recipes for device fabrication to facilitate FDM printing for microfluidic device development.
Project description:Plastics are one of the most commonly used materials for fabricating microfluidic devices. While various methods exist for fabricating plastic microdevices, hot embossing offers several unique advantages including high throughput, excellent compatibility with most thermoplastics and low start-up costs. However, hot embossing requires metal or silicon molds that are fabricated using CNC milling or microfabrication techniques which are time consuming, expensive and required skilled technicians. Here, we demonstrate for the first time the fabrication of plastic microchannels using 3D printed metal molds. Through optimization of the powder composition and processing parameters, we were able to generate stainless steel molds with superior material properties (density and surface finish) than previously reported 3D printed metal parts. Molds were used to fabricate poly(methyl methacrylate) (PMMA) replicas which exhibited good feature integrity and replication quality. Microchannels fabricated using these replicas exhibited leak-free operation and comparable flow performance as those fabricated from CNC milled molds. The speed and simplicity of this approach can greatly facilitate the development (i.e. prototyping) and manufacture of plastic microfluidic devices for research and commercial applications.
Project description:Microarrays of RNA aptamers are fabricated in a one-step, multiplexed enzymatic synthesis on gold thin films in a microfluidic format and then employed in the detection of protein biomarkers with surface plasmon resonance imaging (SPRI) measurements. Single-stranded RNA (ssRNA) oligonucleotides are transcribed on-chip from double-stranded DNA (dsDNA) templates attached to microarray elements (denoted as generator elements) by the surface transcription reaction of T7 RNA polymerase. As they are synthesized, the ssRNA oligonucleotides diffuse in the microfluidic channel and are quickly captured by hybridization adsorption onto adjacent single-stranded DNA (ssDNA) microarray elements (denoted as detector elements) that contain a sequence complementary to 5'-end of the ssRNA. The RNA aptamers attached to these detector elements are subsequently used in SPRI measurements for the bioaffinity detection of protein biomarkers. The microfluidic generator-detector element format permits the simultaneous fabrication of multiple ssRNA oligonucleotides with different capture sequences that can hybridize simultaneously to distinct detector elements and thus create a multiplexed aptamer microarray. In an initial set of demonstration experiments, SPRI measurements are used to monitor the bioaffinity adsorption of human thrombin (hTh) and vascular endothelial growth factor (VEGF) proteins onto RNA aptamer microarrays fabricated in situ with this on-chip RNA polymerase synthesis methodology. Additional SPRI measurements of the hydrolysis and desorption of the surface-bound ssRNA aptamers with a surface RNase H are used to verify the capture of ssRNA with RNA-DNA surface hybridization onto the detector elements. The on-chip RNA synthesis described here is an elegant, one-step multiplexed methodology for the rapid and contamination-free fabrication of RNA aptamer microarrays for protein biosensing with SPRI.
Project description:Direct and fast (10s of seconds) deposition of flame-made, high surface-area aerosol films on polymers and polymeric microfluidic devices is demonstrated. Uniform TiO2 nanoparticle films were deposited on cooled Poly(methyl methacrylate) (PMMA) substrates by combustion of titanium(IV) isopropoxide (TTIP) - xylene solution sprays. Films were mechanically stabilized by in-situ annealing with a xylene spray flame. Plasma-etched microfluidic chromatography columns, comprising parallel microchannels were also coated with such nanoparticle films without any microchannel deformation. These microcolumns were successfully used in metal-oxide affinity chromatography (MOAC) to selectively trap phosphopeptides on these high surface-area nanostructured films. The chips had a high capacity retaining 1.2 ?g of standard phosphopeptide. A new extremely fast method is developed for MOAC microchip stationary phase fabrication with applications in proteomics.
Project description:We report a novel method for wafer level, high throughput optical chemical sensor patterning, with precise control of the sensor volume and capability of producing arbitrary microscale patterns. Monomeric oxygen (O(2)) and pH optical probes were polymerized with 2-hydroxyethyl methacrylate (HEMA) and acrylamide (AM) to form spin-coatable and further crosslinkable polymers. A micro-patterning method based on micro-fabrication techniques (photolithography, wet chemical process and reactive ion etch) was developed to miniaturize the sensor film onto glass substrates in arbitrary sizes and shapes. The sensitivity of fabricated micro-patterns was characterized under various oxygen concentrations and pH values. The process for spatially integration of two sensors (Oxygen and pH) on the same substrate surface was also developed, and preliminary fabrication and characterization results were presented. To the best of our knowledge, it is the first time that poly (2-hydroxylethyl methacrylate)-co-poly (acrylamide) (PHEMA-co-PAM)-based sensors had been patterned and integrated at the wafer level with micron scale precision control using microfabrication techniques. The developed methods can provide a feasible way to miniaturize and integrate the optical chemical sensor system and can be applied to any lab-on-a-chip system, especially the biological micro-systems requiring optical sensing of single or multiple analytes.
Project description:In this paper, we study characteristics of polymers (methylcellulose, hypromellose ((hydroxypropyl)methyl cellulose), poly(vinylpyrrolidone), and poly(vinyl alcohol)) with different chemical structures for microchip electrophoresis of non-denatured protein samples in a plastic microchip made of poly(methyl methacrylate) (PMMA). Coating efficiency of these polymers for controlling protein adsorption onto the channel surface of the plastic microchip, wettability of the PMMA surface, and electroosmotic flow in the PMMA microchannels in the presence of these polymers were compared. Also relative electrophoretic mobility of protein samples in solutions of these polymers was studied. We showed that when using low polymer concentrations (lower than the polymer entanglement point) where the sieving effect is substantially negligible, the interaction of the samples with the polymer affected the electrophoretic mobility of the samples. This effect can be used for achieving better resolution in microchip electrophoresis of protein samples.
Project description:Herein is described the fabrication and use of a plastic multilayer 3-channel microfluidic fixture. Multilayer devices were produced by laser machining of plastic polymethylmethacrylate and polyethyleneterapthalate laminates by ablation. The fixture consisted of an array of nine individually addressable gold or gold/ITO working electrodes, and a resistive platinum heating element. Laser machining of both the fluidic pathways in the plastic laminates, and the stencil masks used for thermal evaporation to form electrode regions on the plastic laminates, enabled rapid and inexpensive implementation of design changes. Electrochemiluminescence reactions in the fixture were achieved and monitored through ITO electrodes. Electroaddressable aryl diazonium chemistry was employed to selectively pattern gold electrodes for electrochemical multianalyte DNA detection from double stranded DNA (dsDNA) samples. Electrochemical detection of dsDNA was achieved by melting of dsDNA molecules in solution with the integrated heater, allowing detection of DNA sequences specific to breast and colorectal cancers with a non-specific binding control. Following detection, the array surface could be renewed via high temperature (95 °C) stripping using the integrated heating element. This versatile and simple method for prototyping devices shows potential for further development of highly integrated, multi-functional bioanalytical devices.
Project description:Valorisation of the urban plastic waste in high-quality recyclates is an imperative challenge in the new paradigm of the circular economy. In this scenario, a key role in the improvement of the recycling process is exerted by the optimization of waste sorting. In spite of the enormous developments achieved in the field of automated sorting systems, the quest for the reduction of cross-contamination of incompatible polymers as well as a rapid and punctual sorting of the unmatched polymers has not been sufficiently developed. In this paper, we demonstrate that a miniaturized handheld near-infrared (NIR) spectrometer can be used to successfully fingerprint and classify different plastic polymers. The investigated urban plastic waste comprised polyethylene (PE), polypropylene (PP), poly(vinyl chloride) (PVC), poly(ethylene terephthalate) (PET), and poly(styrene) (PS), collected directly in a recycling plastic waste plant, without any kind of sample washing or treatment. The application of unsupervised and supervised chemometric tools such as principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) on the NIR dataset resulted in a complete classification of the polymer classes. In addition, several kinds of PET (clear, blue, coloured, opaque, and boxes) were correctly classified as PET class, and PE samples with different branching degrees were properly separated.
Project description:Compared to conventional bench-top instruments, microfluidic devices possess advantageous characteristics including great portability potential, reduced analysis time (minutes), and relatively inexpensive production, putting them on the forefront of modern analytical chemistry. Fabrication of these devices, however, often involves polymeric materials with less-than-ideal surface properties, specific instrumentation, and cumbersome fabrication procedures. In order to overcome such drawbacks, a new hybrid platform is proposed. The platform is centered on the use of 5 interconnecting microfluidic components that serve as the injector or reservoirs. These plastic units are interconnected using standard capillary tubing, enabling in-channel detection by a wide variety of standard techniques, including capacitively-coupled contactless conductivity detection (C(4)D). Due to the minimum impact on the separation efficiency, the plastic microfluidic components used for the experiments discussed herein were fabricated using an inexpensive engraving tool and standard Plexiglas. The presented approach (named 5(2)-platform) offers a previously unseen versatility: enabling the assembly of the platform within minutes using capillary tubing that differs in length, diameter, or material. The advantages of the proposed design are demonstrated by performing the analysis of inorganic cations by capillary electrophoresis on soil samples from the Atacama Desert.
Project description:DNA microarrays have gained wide use in biomedical research by simultaneously monitoring the expression levels of a large number of genes. The successful implementation of DNA microarray technologies requires the development of methods and techniques for the fabrication of microarrays, the selection of probes to represent genes, the quantification of hybridization, and data analysis. In this paper, we concentrate on probes that are either spotted or synthesized on the glass slides through several aspects: sources of probes, the criteria for selecting probes, tools available for probe selections, and probes used in commercial microarray chips. We then provide a detailed review of one type of DNA microarray: Affymetrix GeneChips, discuss the need to re-annotate probes, review different methods for regrouping probes into probe sets, and compare various redefinitions through public available datasets.