Multiplex enzymatic synthesis of DNA with single-base resolution.

Enzymatic DNA synthesis (EDS) is a promising benchtop and user-friendly method of nucleic acid synthesis that, instead of solvents and phosphoramidites, uses mild aqueous conditions and enzymes. For applications such as protein engineering and spatial transcriptomics that require either oligo pools or arrays with high sequence diversity, the EDS method needs to be adapted and certain steps in the synthesis process spatially decoupled. Here, we have used a synthesis cycle comprising a first step of site-specific silicon microelectromechanical system inkjet dispensing of terminal deoxynucleotidyl transferase enzyme and 3' blocked nucleotide, and a second step of bulk slide washing to remove the 3' blocking group.

An Aqueous Analog MAC Machine.

Using ions in aqueous milieu for signal processing, like in biological circuits, may potentially lead to a bioinspired information processing platform. Studies, however, have focused on individual ionic diodes and transistors rather than circuits comprising many such devices. Here a 16 × 16 array of new ionic transistors is developed in an aqueous quinone solution.

CMOS electrochemical pH localizer-imager.

pH controls a large repertoire of chemical and biochemical processes in water. Densely arrayed pH microenvironments would parallelize these processes, enabling their high-throughput studies and applications. However, pH localization, let alone its arrayed realization, remains challenging because of fast diffusion of protons in water.

Antibody recognition of a human chorionic gonadotropin epitope (hCGbeta66-80) depends on local structure retained in the free peptide.

Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where it is routinely detected and quantified by specific immunoassays. Intelligent epitope selection is essential to achieving the required assay performance. We present binding affinity measurements demonstrating that a typical β3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCGβ(66-80) and the closely related luteinizing hormone (LH) fragment LHβ(86-100), which differ only by a single amino acid residue.

The rational development of molecularly imprinted polymer-based sensors for protein detection.

The detection of specific proteins as biomarkers of disease, health status, environmental monitoring, food quality, control of fermenters and civil defence purposes means that biosensors for these targets will become increasingly more important. Among the technologies used for building specific recognition properties, molecularly imprinted polymers (MIPs) are attracting much attention. In this critical review we describe many methods used for imprinting recognition for protein targets in polymers and their incorporation with a number of transducer platforms with the aim of identifying the most promising approaches for the preparation of MIP-based protein sensors (277 references).

MiS-MALDI: microgel-selected detection of protein biomarkers by MALDI-ToF mass spectrometry.

Intensified efforts to decipher the origin of disease at the molecular level stimulate the emergence of more efficient proteomic technologies. To complement this, attempts are being made to identify new predictive biomarkers for building more reliable biomarker patterns. As biomarker research gathers pace an immediate interest becomes focused on platforms, which although based on mainstream approaches, are more amenable to specialist tasks.

Polymer- and colloid-mediated bioassays, sensors and diagnostics.

Synthetic polymers and colloids are increasingly being exploited in bioassays to help measure gene expression, sequence genomes, monitor metabolic disorders and detect the presence of disease. This can be attributed to their potential to reduce reaction scales, improve throughput, lower costs and improve the sensitivity, selectivity, stability and reproducibility of assays. This review highlights the newest application areas, including some of the strategies employed, as well as major technical challenges and future opportunities.

Accurate whole human genome sequencing using reversible terminator chemistry.

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost.

Measurement of glucose in blood with a phenylboronic acid optical sensor.

Background: Current methods of glucose monitoring rely predominantly on enzymes such as glucose oxidase for detection. Phenylboronic acid receptors have been proposed as alternative glucose binders. A unique property of these molecules is their ability to bind glucose in a fully reversible covalent manner that facilitates direct continuous measurements.

pH-sensitive holograms for continuous monitoring in plasma.

Conventional electrochemical methods of determining the pH of body fluids have drawbacks. Newer optical methods offer the promise of miniaturisation and continuous in vivo measurements without drift. This report examines the ability of a holographic sensor based on a thin-film, biocompatible hydrogel (approximately 10 microm) of poly(2-hydroxyethyl methacrylate) and ionisable 2-(dimethylaminoethyl) methacrylate to accurately measure the pH of blood plasma ex vivo.

Continuous blood glucose monitoring with a thin-film optical sensor.

Background: We recently described a holographic optical sensor with improved selectivity for glucose over fructose that was based on a thin-film polymer hydrogel containing phenylboronic acid receptors. The aim of the present work was to measure glucose in human blood plasma as opposed to simple buffers and track changes in concentration at a rate mimicking glucose changes in vivo.

Methods: We used holographic sensors containing acrylamide, N,N'-methylenebisacrylamide, 3-acrylamidophenylboronic acid, and (3-acrylamidopropyl)trimethylammonium chloride to measure 7 human blood plasma samples at different glucose concentrations (3-33 mmol/L) in static mode.

Selective holographic detection of glucose using tertiary amines.

Introducing tertiary amine monomers into holographic sensors containing phenylboronic acids gives greatly improved selectivity for glucose.

Crosslinking of phenylboronic acid receptors as a means of glucose selective holographic detection.

A holographic sensor for the detection of glucose has been developed that is based on a hydrogel film containing phenylboronic acid receptors. Changes to the replay wavelength of the hologram were used to characterise the swelling and de-swelling behaviour of the hydrogel matrix upon receptor-ligand binding. The effect of introducing a fixed positive charge into the polymer matrix by modification of the hydrogel with a quaternary amine group (3-acrylamidopropyl)trimethylammonium chloride (ATMA), was investigated for a range of sugars and the alpha-hydroxy acid, lactate, at physiological pH.

Poly(butyl methacrylate-g-methoxypoly(ethylene glycol)) and poly(methyl methacrylate-g-methoxypoly(ethylene glycol)) graft copolymers: preparation and aqueous solution properties.

A series of water-soluble, amphiphilic graft copolymers has been prepared by free-radical copolymerization of methoxypoly(ethylene glycol) macromonomers, with either methyl methacrylate or butyl methacrylate as the comonomers, in water/ethanol solvent mixtures. Lower molecular weight copolymers were obtained by increasing the concentration of the initiator, azobisisobutyronitrile (AIBN), used in the polymerization reaction. However, the route used also led to the formation of significant quantities of tetramethylsuccinodinitrile, a toxic byproduct resulting from the cage reaction of AIBN.

Polystyrene nanoparticles based on poly(butyl methacrylate-g-methoxypoly(ethylene glycol)) and poly(methyl methacrylate-g-methoxypoly(ethylene glycol)) graft copolymers.

The solubilization of styrene by poly(butyl methacrylate-g-methoxypoly(ethylene glycol)) and poly(methyl methacrylate-g-methoxypoly(ethylene glycol)) graft copolymers has been examined. From turbidity measurements the solubility limit of the monomer in the micelles was obtained and the distribution coefficients were evaluated. Dynamic light scattering revealed that below the solubility limit, solubilization leads to a slight increase in micelle size, while above the solubility limit, there is a dramatic increase in particle size and turbidity as oil-in-water emulsions are formed through coalescence of monomer-swollen micelles.

Physicochemical perspectives on DNA microarray and biosensor technologies.

Detection and sequence-identification of nucleic acid molecules is often performed by binding, or hybridization, of specimen "target" strands to immobilized, complementary "probe" strands. A familiar example is provided by DNA microarrays used to carry out thousands of solid-phase hybridization reactions simultaneously to determine gene expression patterns or to identify genotypes. The underlying molecular process, namely sequence-specific recognition between complementary probe and target molecules, is fairly well understood in bulk solution.

Reaction of N-phenyl maleimide with aminosilane monolayers.

Reaction of N-phenyl maleimide (NPM) with silica surfaces modified with a self-assembled monolayer of (aminopropyl)triethoxysilane (APTES) was investigated using infrared spectroscopy (FTIR), elemental analysis, and titration assays. This reaction is of interest as a test case for using amine-maleimide coupling for immobilization of biomolecules. Addition of NPM to surface APTES residues was consistently sub-stoichiometric, with typical yields of about 75% on monolayers with a coverage of 1.