Philip J. R. Roche

A Camera Phone Localised Surface Plasmon Biosensing Platform towards Low-Cost Label-Free Diagnostic Testing

Developmental work towards a camera phone diagnostic platform applying localized surface plasmon resonance (LSPR) label-free sensing is presented. The application of spherical gold nanoparticles and nanorods are considered and assessed against ease of application, sensitivity, and practicality for a sensor for the detection of CCL2 (chemokine ligand 2). The sensitivity of the platform is compared with that of a commercial UV/Vis spectrometer. The sensitivity of the camera phone platform is found to be 30% less than that of the commercial system for an equivalent incubation time, but approaches that of the commercial system as incubation time increases. This suggests that the application of LSPR sensing on a portable camera phone devices may be a highly effective label-free approach for point-of-care use as a low-cost diagnostic sensing tool in environments where dedicated equipment is not available.

Design and analysis of a spectro-angular surface plasmon resonance biosensor operating in the visible spectrum

Surface plasmon resonance (SPR) sensing is one of the most widely used methods to implement biosensing due to its sensitivity and capacity for label-free detection. Whilst most commercial SPR sensors operate in the angular regime, it has recently been shown that an increase in sensitivity and a greater robustness against noise can be achieved by measuring the reflectivity when varying both the angle and wavelength simultaneously, in a so-called spectro-angular SPR biosensor. A single value decomposition method is used to project the two-dimensional spectro-angular reflection signal onto a basis set and allow the image obtained from an unknown refractive index sample to be compared very accurately with a pre-calculated reference set. Herein we demonstrate that a previously reported system operated in the near infra-red has a lower detection limit when operating in the visible spectrum due to the improved spatial resolution and numerical precision of the image sensor. The SPR biosensor presented here has an experimental detection limit of 9.8 × 10(-7) refractive index unit. To validate the system as a biosensor, we also performed the detection of synthetic RNA from pathogenic Legionella pneumophila with the developed biosensing platform.

Analysis of surface plasmon spectro-angular reflectance spectrum: real-time measurement, resolution limits, and applications to biosensing

A surface plasmon biosensing technique based on real-time measurement of the spectro-angular reflectance spectrum of a gold surface is presented. A significant improvement in refractive index resolution and drift compensation has been achieved for the spectro-angular technique to demonstrate a biosensing platform that is, in addition, applicable to plasmonic bandgap measurements. Instrumental improvements are detailed and constants for the model bovine serum albumin (BSA):oxacillin bioassay are presented.

Enhancement of luminescent quenching based oxygen sensing by gold nanoparticles: comparison between luminophore:matrix:nanoparticle thin films on glass and gold coated substrates

For weak luminescence, quenching of insensitive luminophores by proximity to a gold film improves signal to noise by suppression of background luminescence of Ru(4,7-diphenyl-1,10-anthroline) 3 Cl 2 . Initially it was expected that the effects of gold film quenching and nanoparticle enhanced luminescence could be combined to give a summative improvement, but the increase caused by the nanoparticles generates a larger signal to noise ratio and greater sensitivity of those luminophores to the dynamic quenching by gaseous oxygen. Impressive detection limits were achieved on gold coated glass and plain glass, where detection limit was 0.05% and 0.004% and sensitivity 0.02 and 0.05%, respectively.

Controlling optical properties and surface morphology of dry etched porous silicon

This PDF file contains the errata for “JNP Vol. 5 Issue 01 Paper 3592487” for JNP Vol. 5 Issue 01

A rapid diagnostic method for E. coli serogroups responsible for gastro-intestinal diseases using loop-mediated isothermal amplification

A rapid diagnostic method for Escherichia coli serogroup identification was developed, employing the loop-mediated isothermal amplification reaction (LAMP). Identifying the serogroup responsible for infection is accomplished using primers that are specific to sequences that are exclusive to the particular serogroup. Previous work has primarily focused on detecting one specific sequence or gene as a method of detection of a pathogenic entity. The novel approach involves using primers specific to various genes to determine which are present as a method of identification of the unknown pathogen rather than simple detection. The experiments conducted involved running the LAMP reaction and detecting amplification using gel electrophoresis, fluorescence, and localized surface plasmon resonance. The results obtained demonstrate that the LAMP reaction is efficient, and specific in that it only amplifies target DNA, and that it requires minimal instrumentation in comparison to various other nucleic acid amplification methods. This assays principle and instrumentation is suited to resource-limited environments and when mobility is required for testing.

Dynamical thermal effects in InGaAsP microtubes at telecom wavelengths

We report on the observation of a dynamical thermal effect in InGaAsP microtubes at telecom wavelengths. The microtubes are fabricated by releasing a strained semiconductor bilayer and are picked up by abruptly tapered optical fibers for subsequent coupling with adiabatically tapered optical fibers. As a result of absorption by InAs quantum dots embedded in the tube structure, these microtubes show dynamical thermal effects at wavelengths around 1525 nm and 1578 nm, while they are passive at longer wavelengths near 1634 nm. The photon absorption induced thermal effect is visualized by generating a pair of microbottles. The dynamical thermal effect can be avoided or exploited for passive or active applications by utilizing appropriate resonance wavelengths.

Efficient Homology Directed Repair by Cas9:DNA Localization and Cationic Polymeric Transfection in Mammalian Cells

Homology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox. Summary Precision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.Homology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining repair. In order to obtain high HDR frequency in mammalian cells, we delivered donor DNA to a DSB site by engineering a Cas9 protein fused to a high-affinity monoavidin domain to accept biotinylated DNA donors. In addition, we used the cationic polymer, polyethylenimine, to efficiently deliver our Cas9-DNA donor complex into the nucleus, thereby avoiding drawbacks such as cytotoxicity and limited in vivo translation associated with the more commonly used nucleofection technique. Combining these strategies led to an improvement in HDR rates of up to 90% on several test loci (CXCR4, EMX1, TLR). Our approach offers a cost effective, simple and broadly applicable editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.

Demonstration of a reusable plasmonic polymer microarray sensing platform

High throughput plasmonic sensors are a popular research field, standard surface plasmon resonance (SPR) instruments can achieve high throughput only in imaging configuration. This leads to consideration of pattern substrates and isolated nanoparticle arrays, both of which have some disadvantages. Spot functionalisation relies upon mask or pin printing to accomplish density, and this increase the complexity of use and standard operating procedures. Both patterned and nanoparticle arrays assay platforms are also commonly single use, unlike some SPR imaging and multi channel angular sensing SPR approaches. The microarray format proposed here is intended for multiple usages and regenerated, with a simple optical readout method. A plasmonic polymer of exquisite refractive index sensitivity and incorporate glass-like physical and mechanical stability provides the sensing element to the platform. Further, the standard sol-gel chemistry is well understood and amenable to easy covalent functionalisation as well as matrix methods such as nitrocellulose for biomolecule fictionalization. Two forms of polymer templating have been developed. For spots greater than 700μm a double side tape method can be applied and for sub 700μm patterned SU-8 and 100nm Aluminum reflective layer allow greater spot resolution. Proof of concept through refractive index sensing is demonstrated.

Enhancement in sensitivity and detection of luminescent quenching based oxygen sensing by gold nanoparticles

The field of plasmonics has shown a great promise in the enhancement of luminescence detection. Here, a simple method to enhance oxygen detection by quenching of Ru[(4,7-diphenyl-1,10-anthroline)3]2+ (or Ru[dpp]2+) in a sol-gel matrix by localized surface plasmon resonance (LSPR) of gold nanoparticles (AuNP) is presented. In the experiments, AuNP (10 ± 1.5 nm diameter) were added to a sol that was prepared by hydrolysis of trimethoxysilane, octyltrimethoxysilane and ethanol in the presence of Ru[dpp]2+ luminophore. The resulting sol of the mixture was spincoated on glass and allowed to age in the dark for one week to form the sol-gel film. A control sample was also prepared using the procedure, except that AuNP was not added to the sol. The resulting AuNP embedded sol-gel shows 8.3 times improvement in the baseline (0% O2) intensity (I0) over the control. Moreover, there is a dramatic improvement in the sensitivity from 0.0011 per % O2 in the control to 0.059 per % O2 with AuNP, for O2 level below 15%. Signal to noise ratio also improved, thus leading to a 100-fold improvement in the detection limit. Using phaseluminometry, it was determined that there is a reduction in the luminescence lifetime when AuNP is added to the sol-gel matrix. This reduction in the lifetime can be explained by the near-field interaction between the luminophores and the AuNP.