Optimisation of distributed feedback laser biosensors

A new integrated optical sensor chip is proposed, based on a modified distributed- feedback (DFB) semiconductor laser. The semiconductor layers of different refractive indices that comprise a laser form the basis of a waveguide sensor, where changes in the refractive index of material at the surface are sensed via changes in the evanescent field of the lasing mode. In DFB lasers, laser oscillation occurs at the Bragg wavelength. Since this is sensitive to the effective refractive index of the optical mode, the emission wavelength is sensitive to the index of a sample on the waveguide surface. Hence, lasers are modelled as planar waveguides and the effective index of the fundamental transverse electric mode is calculated as a function of index and thickness of a thin surface layer using the beam propagation method. We find that an optimised structure has a thin upper cladding layer of ~0.15 mum, which according to this model gives detection limits on test layer index and thickness resolution of 0.1 and 1.57 nm, respectively, a figure which may be further improved using two lasers in an interferometer-type configuration

The coexistence of pressure waves in the operation of quartz-crystal shear-wave sensors

It is demonstrated that an AT-cut quartz crystal driven in the thickness-shear-wave mode and typically used as a sensor to monitor the viscoelastic shear-wave properties of a fluid also produce longitudinal pressure waves. Unlike the shear wave, these waves are capable of long-range propagation through the fluid and of reflection at its boundaries, notably at an outer fluid–air interface. They introduce a component into the measured electrical impedance and resonance frequency shift of the crystal, which reflects the setting up of cyclic pressure-wave resonances in the fluid. This has important implications for the practical employment of these crystal as sensors. Under appropriate conditions, as demonstrated for water and n-octane, it is possible to determine the propagating properties of sound waves in a fluid simultaneously with the viscoelastic shear-wave properties. These experiments are supported by an analysis of the appropriate hydrodynamic equations for waves in the crystal–fluid system, which predicts electrical characteristics in close agreement with those found experimentally

Enhanced selectivity of hydrogel-based molecularly imprinted polymers (HydroMIPs) following buffer conditioning.

We have investigated the effect of buffer solution composition and pH during the preparation, washing and re-loading phases within a family of acrylamide-based molecularly imprinted polymers (MIPs) for bovine haemoglobin (BHb), equine myoglobin (EMb) and bovine catalyse (BCat). We investigated water, phosphate buffer saline (PBS), tris(hydroxymethyl)aminomethane (Tris) buffer and succinate buffer. Throughout the study MIP selectivity was highest for acrylamide, followed by N-hydroxymethylacrylamide, and then N-iso-propylacrylamide MIPs. The selectivity of the MIPs when compared with the NIPs decreased depending on the buffer conditions and pH in the order of Tris>PBS>succinate. The Tris buffer provided optimum imprinting conditions at 50mM and pH 7.4, and MIP selectivities for the imprinting of BHb in polyacrylamide increased from an initial 8:1 to a 128:1 ratio. It was noted that the buffer conditions for the re-loading stage was important for determining MIP selectivity and the buffer conditions for the preparation stage was found to be less critical. We demonstrated that once MIPs are conditioned using Tris or PBS buffers (pH7.4) protein reloading in water should be avoided as negative effects on the MIP's imprinting capability results in low selectivities of 0.8:1. Furthermore, acidifying the pH of the buffer solution below pH 5.9 also has a negative impact on MIP selectivity especially for proteins with high isoelectric points. These buffer conditioning effects have also been successfully demonstrated in terms of MIP efficiency in real biological samples, namely plasma and serum

Coupling dye-integrated polymeric membranes with smartphone detection to classify bacteria

We report the use of a colorimetric plastic-based device to discriminate four pathogenic bacteria: Klebsiella pneumoniae, Proteus vulgaris, Proteus mirabilis and Escherichia coli. The colour changes of the plastic membranes were analysed with RGB values extracted from dye-integrated polymeric membrane images obtained with a smartphone and used as input data for non-supervised pattern recognition methods. © The Royal Society of Chemistry 2015

Use of plastic-based analytical device, smartphone and chemometric tools to discriminate amines

Amine-based volatile compounds released bymicroorganisms offer an alternative diagnostic approach for the identification of foodborne pathogens. Our objective has been to solvent cast cellulose acetate membranes to immobilise dyes and to use the resultant membranes as a plastic device to discriminate between different types of amines (triethylamine, isobutylamine, isopentylamine). The plastic device consisted of an array of membranes with five pH indicators (namely alizarin, bromophenol blue, chlorophenol red, methyl red and thymol blue). To analyse the data using a portable instrument, we used an iPhone (R) to obtain images and to extract red, green and blue colours (RGB) using in-house software before and after contact with each individual amine. All the RGB values extracted for each analyte allowed us to generate a unique colour pattern, which was used as input for non-supervised pattern recognition methods. Based on this analysis, it was possible to clearly discriminate between the amines studied without any misclassification, demonstrating that the device is well-suited for large-scale applications such as non-destructive methods to discriminate amines and, in future, for smart packaging applications in order to give early warning of rotting food that may lead to food poisoning. Additionally, a semi-quantitative analysis was performed and we have demonstrated that it is possible to quantify concentrations of amines down to 1 ppm

Investigation of Polyacrylamide Hydrogel-based Molecularly Imprinted Polymers using Protein Gel Electrophoresis

In conjunction with polyacrylamide gel electrophoresis (PAGE), molecular imprinting methods have been applied to produce a multi-layer mini-slab in order to evaluate how selectively and specifically a hydrogel-based molecularly imprinted polymer (MIP) binds bovine haemoglobin (BHb, ~64.5 kDa). A three-layer mini-slab comprising an upper and lower layer and a MIP, or a non-imprinted control polymer dispersion middle layer has been investigated. The discriminating MIP layer, also based on polyacrylamide, was able to specifically bind BHb molecules in preference to a protein similar in molecular weight such as bovine serum albumin (BSA, ~66 kDa). Protein staining allowed us to visualise the protein retention strength of the MIP layer under the influence of an electric field. This method could be applied to other proteins with implications in effective protein capture, disease diagnostics and protein analysis

Toward Rational Design of Selective Molecularly Imprinted Polymers (MIPs) for Proteins: Computational and Experimental Studies of Acrylamide Based Polymers for Myoglobin

Molecularly imprinted polymers (MIPs) have potential as alternatives to antibodies in the diagnosis and treatment of disease. However, atomistic level knowledge of the prepolymerization process is limited that would facilitate rational design of more efficient MIPs. Accordingly, we have investigated using computation and experiment the protein-monomer binding interactions that may influence the desired specificity. Myoglobin was used as the target protein and five different acrylamide-based monomers considered. Protein binding sites were predicted using SiteMap and binding free energies of monomers at each site calculated using MM-GBSA. Statistical thermodynamic analysis and study of atomistic interactions facilitated rationalization of monomer performance in MIP rebinding studies (% rebind; imprinting factors). CD spectroscopy was used to determine monomer effects on myoglobin secondary structure, with all monomers except the smallest monomer (acrylamide) causing significant changes. A complex interplay between different protein-monomer binding effects and MIP efficacy was observed. Validation of hypotheses for key binding features was achieved by rational selection of two different co-monomer MIP combinations that produced experimental results in agreement with predictions. The co-monomer studies revealed that uniform, non-competitive binding of monomers around a target protein is favourable. This study represents a step towards future rational in silico design of MIPs for proteins

Spectroscopic and quartz crystal microbalance (QCM) characterisation of protein-based MIPs

We have studied acrylamide-based polymers of varying hydrophobicity (acrylamide, AA; N-hydroxymethylacrylamide, NHMA; N-isopropylacrylamide, NiPAm) for their capability of imprinting protein. Rebinding capacities (Q) from spectroscopic studies were highest for bovine haemoglobin (BHb) MIPs based on AA, Q = 4.8 ± 0.21 76 ± 0.5%). When applied to the QCM sensor as thin-film MIPs, NHMA MIPs were found to exhibit best discrimination between MIP and non-imprinted control polymer (NIP) in the order of NiPAm < AA < NHMA. The extent of template removal and rebinding, using both crystal impedance and frequency measurements, demonstrated that 10% (w/v):10% (v/v) sodium dodecyl sulphate:acetic acid (pH 2.8) was efficient at eluting template BHb (with 80 ± 10% removal). Selectivity studies of NHMA BHb-MIPs revealed higher adsorption and selective recognition properties to BHb (64.5 kDa) when compared to non-cognate BSA (66 kDa), myoglobin (Mb, 17.5 kDa), lysozyme (Lyz, 14.7 kDa) thaumatin (Thau, 22 kDa) and trypsin (Tryp, 22.3 kDa). The QCM gave frequency shifts of ∼1500 ± 50 Hz for template BHb rebinding in both AA and NHMA MIPs, whereas AA-based MIPs exhibited an interference signal of ∼2200 ± 50 Hz for non-cognate BSA in comparison to a ∼500 ± 50 Hz shift with NHMA MIPs. Our results show that NHMA-based hydrogel MIP are superior to AA and NIPAM

Developments in nanoparticles for use in biosensors to assess food safety and quality

The following will provide an overview on how advances in nanoparticle technology have contributed towards developing biosensors to screen for safety and quality markers associated with foods. The novel properties of nanoparticles will be described and how such characteristics have been exploited in sensor design will be provided. All the biosensor formats were initially developed for the health care sector to meet the demand for point-of-care diagnostics. As a consequence, research has been directed towards miniaturization thereby reducing the sample volume to nanolitres. However, the needs of the food sector are very different which may ultimately limit commercial application of nanoparticle based nanosensors

Quantification and confocal imaging of protein specific molecularly imprinted polymers

We have employed FITC-albumin as the protein template molecule in an aqueous phase molecular imprinted polymer (HydroMIP) strategy. For the first time, the use of a fluorescently labelled template is reported, with subsequent characterisation of the smart material to show that the HydroMIP possess a significant molecular memory in comparison to that of the nonimprinted control polymer (HydroNIP). The imaging of the FITC-albumin imprinted HydroMIP using confocal microscopy is described, with the in situ removal of imprinted protein displayed in terms of observed changes in the fluorescence of the imprinted polymer, both before and after template elution (using a 10% SDS/10% AcOH (w/v) solution). We also report the imaging of a bovine haemoglobin (BHb) imprinted HydroMIP using two-photon confocal microscopy, and describe the effects of template elution upon protein autofluorescence. The findings further contribute to the understanding of aqueous phase molecular imprinting protocols, and document the use of fluorescence as a useful tool in template labelling/detection and novel imaging strategies