FAST slides: a novel surface for microarrays

We have evaluated FAST slides, a glass slide with a microporous polymeric surface that is a suitable substrate for microarray technology. The surface is a nitrocellulose-based polymer that binds DNA and proteins in a noncovalent but irreversible manner. FAST slides are compatible with robotic systems currently used to create microarrays and can easily accommodate volumes of 0.03-2 nL/spot. Our data indicate that FAST slides have a much higher binding capacity for DNA and better spot-to-spot consistency than traditional poly-lysine-coated slides. In addition, FAST slides are well suited for fluorescent detection because of their relatively low light scatter and efficient retention of arrayed DNA. These properties translate into fluorescent sensitivity comparable to modified glass surfaces. FAST slides are also ideal for arraying proteins, making them the only substrate of their kind currently available for microarray applications.     

Development of renewable surface biosensors to meet industrial needs for measurement of glucose in fruit juices.

Most biosensors reported to date have been prepared, studied and used under laboratory conditions. The feasibility of a very great number of biosensors seems to be demonstrated and their characteristics, very often, established as corresponding to the demands of the modern analysis. The operational stability of the biosensors, according to authors, is almost always acceptable. The long term storage, with analytical quality conservation that is necessary to commercialise products, has rarely been studied. The stability of biosensors has to remain not only during the fabrication step or their subsequent utilisation, but also throughout the whole commercial shelf-life of the sensor, from producer to end user, through wholesaler and/or retailer. We developed the manufacturing processes, on a large scale, of renewable surface electrodes modified with enzymes such as oxidoreductases. The process consisting of several steps is described and the analytical behaviours of resulting biosensors is studied and correlated with the effects of different constraints applied during the fabrication process.     

Novel 3-dimensional dendrimer platform for glycolipid microarray

Glycolipids are important biological molecules that modulate cellular recognitions and pathogen adhesions. In this paper, we report a sensitive glycolipid microarray for non-covalently immobilizing glycolipids on a microarray substrate and we perform a set of immunoassays to explore glycolipid-protein interactions. This substrate utilizes a three-dimensional hydrazide-functionalized dendrimer monolayer attached onto a microscopic glass surface, which possesses the characteristics to adsorb glycoliplids non-covalently and facilitates multivalent attributes on the substrate surface. In the proof-of-concept experiments, gangliosides such as GM1, FucGM1, GM3, GD1b, GT1b, and GQ1b, and a lipoarabinomannan were tested on the substrate and interrogated with toxins and antibodies. The resulting glycolipid microarrays exhibited hypersensitivity and specificity for detection of glycolipid-protein interactions. In particular, a robust and specific binding of a pentameric cholera toxin B subunit to the GM1 glycolipid spotted on the array has demonstrated its superiority in sensitivity and specificity. In addition, this glycolipid microarray substrate was used to detect lipoarabinomannan in buffer within a limit-of-detection of 125 ng/mL. Furthermore, Mycobacterium tuberculosis (Mtb) Lipoarabinomannan was tested in human urine specimens on this platform, which can effectively identify urine samples either infected or not infected with Mtb. The results of this work suggest the possibility of using this glycolipid microarray platform to fabricate glycoconjugate microarrays, which includes free glycans and glycolipids and potential application in detection of pathogen and toxin.     

Technique of surface modification of a cell-adhesion-resistant hydrogel by a cell-adhesion-available inorganic microarray

A microtransfer technique for micropattern fabrication using a dithiol macromolecular linker is suggested by transferring a conventionally photolithography-prepared gold microarray on a hard inorganic substrate to a polymeric substrate. The linker was synthesized by end-capping a poly(ethylene glycol) (PEG) chain by the thiol groups. The efficiency of this technique is demonstrated by the transfer of gold microdots from glass to a cell-adhesion-resistant PEG hydrogel, which was formed by polymerizing PEG diacrylate macromers. The stability and biocompatibility of the resulting polymeric-inorganic hybrid material and cell-adhesion contrast of the patterned surface is confirmed by preliminary cell experiments.     

Next generation non-vacuum, maskless, low temperature nanoparticle ink laser digital direct metal patterning for a large area flexible electronics

Flexible electronics opened a new class of future electronics. The foldable, light and durable nature of flexible electronics allows vast flexibility in applications such as display, energy devices and mobile electronics. Even though conventional electronics fabrication methods are well developed for rigid substrates, direct application or slight modification of conventional processes for flexible electronics fabrication cannot work. The future flexible electronics fabrication requires totally new low-temperature process development optimized for flexible substrate and it should be based on new material too. Here we present a simple approach to developing a flexible electronics fabrication without using conventional vacuum deposition and photolithography. We found that direct metal patterning based on laser-induced local melting of metal nanoparticle ink is a promising low-temperature alternative to vacuum deposition- and photolithography-based conventional metal patterning processes. The "digital" nature of the proposed direct metal patterning process removes the need for expensive photomask and allows easy design modification and short turnaround time. This new process can be extremely useful for current small-volume, large-variety manufacturing paradigms. Besides, simple, scalable, fast and low-temperature processes can lead to cost-effective fabrication methods on a large-area polymer substrate. The developed process was successfully applied to demonstrate high-quality Ag patterning (2.1 μΩ·cm) and high-performance flexible organic field effect transistor arrays.     

Electron transfer mediator micro-biosensor fabrication by organic plasma process

We propose a new strategy for constructing a mediator-type biosensor as a Bio-MicroElectroMechanical Systems (BioMEMS) application. A vinylferrocene plasma-polymerized film (PPF) was deposited directly onto the surface of an electrode under dry conditions. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly crosslinked network structure. This technique, capable of polymeric deposition of any kind of monomer, can also serve the purpose of anti-fouling coating, or layer-to-layer interface creation. With a subsequent plasma process, additional polymeric layer of hydrophilic acetonitrile was superimposed onto the existing vinylferrocene-PPF surface to offer crucial features such that the wettability could be adjusted for a better electron transfer, and amino functional groups could be attached to immobilize a large amount of enzyme. Based upon this scheme, the device fabrication could be designed in a manner that the whole procedure was made up of dry wafer-handling processes, which is compatible with mass production. A prototype device was fabricated to have an array of needle-shaped amperometric micro-biosensors. The resultant thin polymer layer carried a large number of the mediator molecules, accomplishing a lower overpotential (+410 mV) and a rapid response time (<5s). Stressing the advantages of the plasma polymerization process together with some additional features accomplished in our device fabrication, we would discuss new possibilities in the field of BioMEMS.     

Arraying glycomics: a novel bi-functional spacer for one-step microscale derivatization of free reducing glycans

Glycan array development is limited by the complexity of efficiently generating derivatives of free reducing glycans with primary amines or other functional groups. A novel bi-functional spacer with selective reactivity toward the free glycan and a second functionality, a primary amine, was synthesized. We demonstrated an efficient one-step derivatization of various glycans including naturally isolated N-glycans, O-glycans, milk oligosaccharides, and bacterial polysaccharides in microgram scale. No protecting group manipulations or activation of the anomeric center was required. To demonstrate its utility for glycan microarray fabrication, we compared glycans with different amine-spacers for incorporation onto an amine-reactive glass surface. Our study results revealed that glycans conjugated with this bi-functional linker were effectively printed and detected with various lectins and antibodies.     

A high-performance glucose biosensor based on monomolecular layer of glucose oxidase covalently immobilised on indium-tin oxide surface

In this paper, a mediatorless amperometric glucose biosensor based on direct covalent immobilisation of monomolecular layer of glucose oxidase (GOx) on a semiconducting indium-tin oxide (ITO) is demonstrated. The abundance of surface hydroxyl functional group of ITO allows it to be used as a suitable platform for direct covalent immobilisation of the enzyme for sensor architecture. The anodic current corresponding to electrochemical oxidation of the enzymatic product, hydrogen peroxide, at a sputtered Pt electrode at 0.500 V (vs. SCE) was obtained as the sensor signal. It was found that the biosensor based on the direct immobilisation scheme shows a fast biosensor response, minimum interference from other common metabolic species and ease of biosensor miniaturisation. A linear range of 0-10 mM of glucose was demonstrated, which exhibits a high sensitivity as far as performance per immobilised GOx molecule is concerned. A detection limit as low as 0.05 mM and long-term stability were observed. Even more important, the biosensor design allows fabrication through a dry process. These characteristics make it possible to achieve mass production of biosensor compatible with the current electronic integrated circuit manufacturing technologies.     

Versatile characterization of thiol-functionalized printed metal electrodes on flexible substrates for cheap diagnostic applications

Background

Cheap, reliable, point-of-care diagnostics is a necessity for the growing and aging population of the world. Paper substrate and printing method, combined together, are the cheapest possible method for generating high-volume diagnostic sensor platforms. Electrical transduction tools also minimize the cost and enhance the simplicity of the devices.

Methods

Standard surface characterization techniques, namely contact angle measurements, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to analyze the growth of the organic thiol layers on top of the printed metal electrodes on paper substrates. The results were compared with those obtained by impedimetric electrical characterization method.

Results

This article reports the fabrication and characterization of printed metal electrodes and their functionalization by organic layers on paper and plastic substrates for biosensing and diagnostic applications. Impedimetric measurement is proposed as a simple, yet elegant, method of characterization of the organic layer growth.

Conclusions

Very good correlation was observed between the results of organic layer growth from different measurement methods, justifying the use of paper as a substrate, printing as a method for fabricating metal and organic layers and impedance as a suitable measurement method for hand-held diagnostic devices.

General significance

This result paves the way for the fabrication of more advanced bio-recognition layers for bio-affinity sensors using a printing technology that is compatible with flexible and cheap paper substrates. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.     

DNA probe attachment on plastic surfaces and microfluidic hybridization array channel devices with sample oscillation

DNA probe immobilization on plastic surfaces and device assembly are both critical to the fabrication of microfluidic hybridization array channel (MHAC) devices. Three oligonucleotide (oligo) probe immobilization procedures were investigated for attaching oligo probes on four different types of plastic surfaces (polystyrene, polycarbonate, poly(methylmethacrylate), and polypropylene). These procedures are the Surmodics procedure, the cetyltrimethylammonium bromide (CTAB) procedure, and the Reacti-Bind procedure. To determine the optimal plastic substrate and attachment chemistry for array fabrication, we investigated plastic hydrophobicity, intrinsic fluorescence, and oligo attachment efficiency. The Reacti-Bind procedure is least effective for attaching oligo probes in the microarray format. The CTAB procedure performs well enough to use in array fabrication, and the concentration of CTAB has a significant effect on oligo immobilization efficiency. We also found that use of amine-modified oligo probes resulted in better immobilization efficiency than use of unmodified oligos with the CTAB procedure. The oligo probe immobilization on plastic surfaces by the Surmodics procedure is the most effective with regard to probe spot quality and hybridization sensitivity. A DNA hybridization assay on such a device results in a limit of detection of 12pM. Utilizing a CO(2) IR laser machining and adhesive layer approach, we have developed an improved procedure for realizing a DNA microarray inside a microfluidic channel. This device fabrication procedure allows for more feasible spot placement in the channel and reduced sample adsorption by adhesive tapes used in the fabrication procedure. We also demonstrated improved hybridization kinetics and increased detection sensitivity in MHAC devices by implementing sample oscillation inside the channel. A limit of detection of 5pM has been achieved in MHAC devices with sample oscillation.     

Design and fabrication development of a micro flow heated channel with measurements of the inside micro-scale flow and heat transfer process

The current work provides a design and fabrication technique for a micro channel system that can provide a uniform heat flux boundary condition on the channel wall and a well insulation on the wall to prevent heat loss from the channel to the outside ambient. Therefore, detailed micro-scale flow and heat transfer process and information along the channel can be studied. Semiconductor sensor material was selected to fabricate both the heaters and the arrays of temperature sensors on a silicon substrate. These heaters and sensors were then moved to a low thermal conductivity epoxy-glass substrate for fabrication of the channel. Design consideration and fabrication techniques involved in this processes will be discussed. A final measurement for the validation of the heaters and the sensors fabricated and a study of the flow friction behavior and the heat transfer coefficient distributions inside the micro channel will be presented. The local Nusselt number distrubution inside the micro channel is reported the first time in the open literature.     

Direct protein detection with a nano-interdigitated array gate MOSFET

A new protein sensor is demonstrated by replacing the gate of a metal oxide semiconductor field effect transistor (MOSFET) with a nano-interdigitated array (nIDA). The sensor is able to detect the binding reaction of a typical antibody Ixodes ricinus immunosuppressor (anti-Iris) protein at a concentration lower than 1 ng/ml. The sensor exhibits a high selectivity and reproducible specific detection. We provide a simple model that describes the behavior of the sensor and explains the origin of its high sensitivity. The simulated and experimental results indicate that the drain current of nIDA-gate MOSFET sensor is significantly increased with the successive binding of the thiol layer, Iris and anti-Iris protein layers. It is found that the sensor detection limit can be improved by well optimizing the geometrical parameters of nIDA-gate MOSFET. This nanobiosensor, with real-time and label-free capabilities, can easily be used for the detection of other proteins, DNA, virus and cancer markers. Moreover, an on-chip associated electronics nearby the sensor can be integrated since its fabrication is compatible with complementary metal oxide semiconductor (CMOS) technology.     

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.     

DNA hybridization sensor based on pentacene thin film transistor

A DNA hybridization sensor using pentacene thin film transistors (TFTs) is an excellent candidate for disposable sensor applications due to their low-cost fabrication process and fast detection. We fabricated pentacene TFTs on glass substrate for the sensing of DNA hybridization. The ss-DNA (polyA/polyT) or ds-DNA (polyA/polyT hybrid) were immobilized directly on the surface of the pentacene, producing a dramatic change in the electrical properties of the devices. The electrical characteristics of devices were studied as a function of DNA immobilization, single-stranded vs. double-stranded DNA, DNA length and concentration. The TFT device was further tested for detection of λ-phage genomic DNA using probe hybridization. Based on these results, we propose that a "label-free" detection technique for DNA hybridization is possible through direct measurement of electrical properties of DNA-immobilized pentacene TFTs.     

Lithographic techniques and surface chemistries for the fabrication of PEG-passivated protein microarrays

This article presents a new technique to fabricate patterns of functional molecules surrounded by a coating of the inert poly(ethylene glycol) (PEG) on glass slides for applications in protein microarray technology. The chief advantages of this technique are that it is based entirely on standard lithography processes, makes use of glass slides employing surface chemistries that are standard in the microarray community, and has the potential to massively scale up the density of microarray spots. It is shown that proteins and antibodies can be made to self-assemble on the functional patterns in a microarray format, with the PEG coating acting as an effective passivating agent to prevent non-specific protein adsorption. Various standard surface chemistries such as aldehyde, epoxy and amine are explored for the functional layer, and it is conclusively demonstrated that only an amine-terminated surface satisfies all the process constraints imposed by the lithography process sequence. The effectiveness of this microarray technology is demonstrated by patterning fluorescent streptavidin and a fluorescent secondary antibody using the well-known and highly specific interaction between biotin and streptavidin.     

Measuring adsorption of a hydrophobic probe with a surface plasmon resonance sensor to monitor conformational changes in immobilized proteins

Conformational changes of proteins immobilized on solid matrices were observed by measuring the adsorption of Triton X-100 (TX), a nonionic detergent, as a hydrophobic probe with BIACORE, a biosensor that utilizes the phenomenon of surface plasmon resonance (SPR). Two kinds of proteins, alpha-glucosidase and lysozyme, were covalently attached to dextran matrices on the sensor surface in the flow cell and then exposed to various concentrations of TX solution. We measured SPR signal changes derived from adsorption of TX to the immobilized proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller (BET) equation. The results demonstrated that monolayer adsorption capacity is proportional to the amount of immobilized proteins. Further, the unfolding process of immobilized proteins on the sensor surface induced by guanidine hydrochloride was investigated by monitoring SPR signal increases due to the adsorption of TX to the exposed hydrophobic region of the protein. Results strongly suggested that the increase in the SPR signal reflected the formation of the agglutinative unfolded state. We expect our measuring method using the SPR sensor and TX adsorption will be a novel tool to provide conformational information regarding various proteins on solid matrices.     

Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging

We developed a peptide microarray based on surface plasmon resonance (SPR) imaging for monitoring protein kinase activities in cell lysates. The substrate peptides of kinases were tethered to the microarray surface modified with a self-assembled monolayer of an alkanethiol with triethylene glycol terminus to create a low nonspecific binding surface. The phosphorylation of the substrate peptides immobilized on the surface was detected with the following phosphate specific binders by amplifying SPR signals: anti-phosphotyrosine antibody for tyrosine kinases and Phos-tag biotin (a phosphate-specific ligand with biotin tag) for serine/threonine kinases. Using the microarray, 9 kinds of protein kinases were evaluated as a pattern of phosphorylation of 26 kinds of substrate peptides. The pattern was unique for each protein kinase. The microarray could be used to evaluate the inhibitory activities of kinase inhibitors. The microarray was applied successfully for kinase activity monitoring of cell lysates. The chemical stimuli responsive activity changes of protein kinases in cell lysates could also be monitored by the peptide microarray. Thus, the peptide microarray based on SPR imaging would be applicable to cell-based drug discovery, diagnosis using tissue lysates, and biochemical studies to reveal signal transduction pathways.     

Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells

We describe here the development of a carbohydrate-based microarray to extend the scope of biomedical research on carbohydrate-mediated molecular recognition and anti-infection responses. We have demonstrated that microbial polysaccharides can be immobilized on a surface-modified glass slide without chemical conjugation. With this procedure, a large repertoire of microbial antigens (approximately 20,000 spots) can be patterned on a single micro-glass slide, reaching the capacity to include most common pathogens. Glycoconjugates of different structural characteristics are shown here to be applicable for microarray fabrication, extending the repertoires of diversity and complexity of carbohydrate microarrays. The printed microarrays can be air-dried and stably stored at room temperature for long periods of time. In addition, the system is highly sensitive, allowing simultaneous detection of a broad spectrum of antibody specificities with as little as a few microliters of serum specimen. Finally, the potential of carbohydrate microarrays is demonstrated by the discovery of previously undescribed cellular markers, Dex-Ids.     

Anchoring of unmodified oligonucleotides on a chitosan-based array: applications to genotyping and gene expression

Oligonucleotide arrays are one of the most used technologies in genotyping and gene expression experiments. Here, a previously developed chitosan-based platform is characterized in its binding of unmodified oligonucleotides, increasing the cost effectiveness of the microarray production process. The unmodified oligonucleotides on the activated chitosan surface showed the same or better performances than amino-modified probes. Moreover, we show applications in genotyping (a ligation detection reaction experiment on cyanobacteria) and in gene expression (on peach RNA samples). The platform was demonstrated to be reliable, robust, and reproducible for immobilizing unmodified oligonucleotides in high quality oligonucleotide array fabrication.