James E. Noble

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).

QUANTITATION OF PROTEIN

The measurement of protein concentration in an aqueous sample is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Methods are described to provide information on how to analyze protein concentration using UV protein spectroscopy measurements, traditional dye-based absorbance measurements; BCA, Lowry, and Bradford assays and the fluorescent dye-based assays; amine derivatization and detergent partition assays. The observation that no single assay dominates the market is due to specific limitations of certain methods that investigators need to consider before selecting the most appropriate assay for their sample. Many of the dye-based assays have unique chemical mechanisms that are prone to interference from chemicals prevalent in many biological buffer preparations. A discussion of which assays are prone to interference and the selection of alternative methods is included.

Bioconjugation and characterisation of gold colloid-labelled proteins.

Colloidal metal particles, in particular gold, have found many biological applications often as probes in light and electron microscopy, and more recently since the 1980s in membrane-based rapid immunoaffinity tests. The surface plasmon resonance absorbance properties in the visible spectroscopy region of gold colloids make them useful tools in medical devices, as the colloids are directly visible to the naked eye. Despite the relative ease with which gold-protein conjugates can be prepared a major issue is the manufacture of poor-quality and poorly characterised bioconjugates that can result in the under performance of subsequent diagnostic tests. This paper describes the preparation of good-quality conjugates for use in immunoassays by optimising the adsorption of antibodies onto the surface of gold colloids, followed by their subsequent characterisation. The conjugates were characterized for size, aggregation and quality using a range of techniques: UV-visible (UV/Vis) absorption spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). The biological activities of the conjugated products were also assessed using an immunoassay format and electrochemical measurements. By utilising a number of measurement techniques we aimed to gain a better understanding of the extent of particle aggregation, and the resulting stability and activity of the biological molecule on the surfaces of nanoparticles. The tools developed will enable researchers and companies to ensure the sensitivity, quality and reproducibility of batches of nanoparticle bio-conjugates.

Standardisation of cardiac troponin I measurement: past and present

&NA; The laboratory measurement of cardiac troponin (cTn) concentration is a critical tool in the diagnosis of acute myocardial infarction (MI). Current cTnI assays produce different absolute troponin numbers and use different clinical cut‐off values; hence cTnI values cannot be interchanged, with consequent confusion for clinicians. A recent Australian study compared patient results for seven cTnI assays and showed that between‐method variation was approximately 2‐ to 5‐fold. A major reason for poor method agreement is the lack of a suitable common reference material for the calibration of cTnI assays by manufacturers. Purified complexed troponin material lacks adequate commutability for all assays; hence a serum‐based secondary reference material is required for cTnI with value assignment by a higher order reference measurement procedure. There is considerable debate about how best to achieve comparability of results for heterogeneous analytes such as cTnI, whether it should be via the harmonisation or the standardisation process. Whereas harmonisation depends upon consensus value assignment and uses those commercial methods which give the closest agreement at the time, standardisation comes closer to the true value through a reference measurement system that is based upon long‐term calibration traceability. The current paper describes standardisation efforts by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of cTnI (IFCC WG‐TNI) to establish a reference immunoassay measurement procedure for cTnI of a higher order than current commercial immunoassay methods and a commutable secondary reference material for cTnI to which companies can reference their calibration materials.

Rapid duplex immunoassay for wound biomarkers at the point-of-care.

In this study we describe a novel method of sampling and quantifying wound biomarkers for clinical settings. We believe the chosen format will allow rapid assessments of wound healing and provide biomarker evidence-based decision points for treatment of the wound at the time of presentation. The wound monitoring principle uses a proprietary sample collection tool (a thermally reversible hydrogel) to sample and isolate biomarkers within a wound environment without further sample extraction/preparation steps. We show how gel samples can be analysed in a lateral flow assay format utilising fluorescent microspheres with optically discrete emission characteristics and demonstrate quantitative detection of two analytes (duplexing) achieved in a single test line. As a model assay, the chronic wound biomarkers interleukin 6 (IL6) and tumour necrosis factor alpha (TNFα) are used. Limits of detection of 48.5 pg/mL and 55.5 pg/mL respectively in hydrogel samples and 7.15 pg/mL and 10.7 pg/mL respectively in plasma are reported. We believe this is the first literature example of quantitative detection of multiple analytes within a single test line using spectral separation to distinguish the analytes.

A De Novo Virus-Like Topology for Synthetic Virions

A de novo topology of virus-like assembly is reported. The design is a trifaceted coiled-coil peptide helix, which self-assembles into ultrasmall, monodisperse, anionic virus-like shells that encapsulate and transfer both RNA and DNA into human cells. Unlike existing artificial systems, these shells share the same physical characteristics of viruses being anionic, nonaggregating, abundant, hollow, and uniform in size, while effectively mediating gene silencing and transgene expression. These are the smallest virus-like structures reported to date, both synthetic and native, with the ability to adapt and transfer small and large nucleic acids. The design thus offers a promising solution for engineering bespoke artificial viruses with desired functions.

Quantification of Protein Concentration Using UV Absorbance and Coomassie Dyes

The measurement of a solubilized protein concentration in solution is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantification assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Where multiple samples need measurement, and/or the sample volume and concentration is limited, preparations of the Coomassie dye commonly known as the Bradford assay can be used.

Evaluation of standardization capability of current cardiac troponin I assays by a correlation study: results of an IFCC pilot project

Abstract Background: As a part of an International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) project to prepare a commutable reference material for cardiac troponin I (cTnI), a pilot study evaluated current cTnI assays for measurement equivalence and their standardization capability. Methods: cTnI-positive samples collected from 90 patients with suspected acute myocardial infarction were assessed for method comparison by 16 cTnI commercial assays according to predefined testing protocols. Seven serum pools prepared from these samples were also assessed. Results: Each assay was assessed against median cTnI concentrations measured by 16 cTnI assays using Passing-Bablok regression analysis of 79 patient samples with values above each assay’s declared detection limit. We observed a 10-fold difference in cTnI concentrations for lowest to highest measurement results. After mathematical recalibration of assays, the between-assay variation for patient samples reduced on average from 40% to 22% at low cTnI concentration, 37%–20% at medium concentration, and 29%–14% at high concentration. The average reduction for pools was larger at 16%, 13% and 7% for low, medium and high cTnI concentrations, respectively. Overall, assays demonstrated negligible bias after recalibration (y-intercept: –1.4 to 0.3 ng/L); however, a few samples showed substantial positive and/or negative differences for individual cTnI assays. Conclusions: All of the 16 commercial cTnI assays evaluated in the study demonstrated a significantly higher degree of measurement equivalence after mathematical recalibration, indicating that measurement harmonization or standardization would be effective at reducing inter-assay bias. Pooled sera behaved similarly to individual samples in most assays.

Aptamer-mediated detection of thrombin using silver nanoparticle signal enhancement

We present the first assay combining a dual aptamer sandwich format with detection by anodic stripping voltammetry with ionic silver amplification. This assay format lends itself to rapid point-of-care tests, where the use of aptamers could improve the overall stability of the assay. We have used human alpha-thrombin as a model system, and demonstrate a detection limit of 6.09 μg L−1. We present the optimization of the aptamer-silver colloid attachment chemistry and the final assay format to achieve sensitive analyte detection. The use of a sandwich assay coupled with magnetic separation and ionic silver amplification, generates an assay with similar sensitivity than those reported in the literature in a format that can be used in rapid, portable testing regimes.

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. The move away from conventional assay approaches is being driven by a desire to improve our basic understanding of human biology, to diagnose diseases earlier, and to manage healthcare resources more efficiently. These endeavors are important owing to a rising world population and an increasing average life span.