Assessing Different PCR Master Mixes for Ultrarapid DNA Amplification: Important Analytical Parameters.

The basic principles of ultrafast plasmonic PCR have been promulgated in the scientific and technological literature for over a decade. Yet, its everyday diagnostic utility remains unvalidated in pre-clinical and clinical settings. Although the impressive speed of plasmonic PCR reaction is well-documented, implementing this process into a device form compatible with routine diagnostic tasks has been challenging. Here, we show that combining careful system engineering and process control with innovative and specific PCR biochemistry makes it possible to routinely achieve a sensitive and robust "10 min" PCR assay in a compact and lightweight system. The critical analytical parameters of PCR reactions are discussed in the current instrument setting.

Enhancing in vitro photothermal therapy using plasmonic gold nanorod decorated multiwalled carbon nanotubes.

Photothermal therapy (PTT) is a promising approach for cancer treatment that selectively heats malignant cells while sparing healthy cells. Here, the light-to-heat conversion efficiency of multiwalled carbon nanotubes (MWCNTs) within the near-infrared biological transmission window is enhanced by decorating them with plasmonic gold nanorods (GNRs). The results reveal a significant photothermal enhancement of hybrid MWCNTs-GNRs compared to bare MWCNTs, displaying a 4.9 enhancement factor per unit mass. The enhanced plasmonic PTT properties of MWCNTs-GNRs are also investigated in vitro using PC3 prostate cancer cell lines, demonstrating a potent ablation efficiency. These findings advance innovative hybrid plasmonic nanostructures for clinical applications.

Plasmonic and label-free real-time quantitative PCR for point-of-care diagnostics.

In response to the world's medical community's need for accurate and immediate infectious pathogen detection, many researchers have focused on adapting the standard molecular diagnostic method of polymerase chain reaction (PCR) for point-of-care (POC) applications. PCR technology is not without its shortcomings; current platforms can be bulky, slow, and power-intensive. Although there have been some advances in microfluidic PCR devices, a simple-to-operate and fabricate PCR device is still lacking. In the first part of this paper, we introduce a compact plasmonic PCR thermocycler in which fast DNA amplification is derived from efficient photothermal heating of a colloidal reaction mixture containing gold nanorods (AuNRs) using a small-scale vertical-cavity surface-emitting laser (VCSEL). Using this method, we demonstrate 30 cycle-assay time of sub-ten minutes for successful Chlamydia trachomatis DNA amplification in 20 µL total PCR sample volume. In the second part, we report an ultrasensitive real-time amplicon detection strategy which is based on cycle-by-cycle monitoring of 260 nm absorption of the PCR sample. This was accomplished by irradiating the PCR sample using a UV LED and collecting the transmitted optical power with a photodetector. The UV absorption dependency on the nucleotides' structural degree of freedom gives rise to distinctive features in the shape of UV amplification curves for the determination of PCR results, thus circumventing the need for the complicated design of target-specific probes or intercalating fluorophores. This amplicon quantification method has a high detection sensitivity of one DNA copy. This is the first demonstration of a compact plasmonic thermocycler combined with a real-time fluorophore-free quantitative amplicon detection system. The small footprint of our PCR device stems from hardware miniaturization, while abundant sample volume facilitates highly sensitive detection and fluid handling required for in-field sample analysis, thereby making it an excellent candidate for POC molecular diagnostics.

Improved method for estimating adlayer thickness and bulk RI change for gold nanocrescent sensors.

This paper presents a novel method employing the maximum likelihood estimation (MLE) technique alongside a nonlinear sensor response model to improve and extract more quantitative sensing results for localized surface plasmon resonance biosensors. The nonlinear response model treats the sensor response as a nonlinear function of the biomolecular adlayer thickness. This method makes use of the multiple resonance characteristic of nanocrescent structures in order to estimate the adlayer thickness and bulk refractive index (RI) change. Nanoimprint lithography is used here to fabricate the nanostructures. The finite element method (FEM) is used to model the nanocrescents and numerically validate the nonlinear-MLE method. Comparing to the established linear model, the proposed nonlinear-MLE method achieves 75% improvement in the limit of detection based on the estimated adlayer thickness and improves the bulk RI resolution by two orders of magnitude.

Improved self-referenced biosensing with emphasis on multiple-resonance nanorod sensors.

We present a novel approach to improve self-referenced sensing based on multiple-resonance nanorod structures. The method employs the maximum likelihood estimation (MLE) alongside a linear response model (LM), relating the sensor response (shifts in resonance wavelengths) to the changes due to surface binding and bulk refractive index. We also provide a solution to avoid repetitive simulations, that have been previously needed to determine the adlayer thickness sensitivity when measuring biological samples of different refractive indices. The finite element method (FEM) was used to model the nanorod structure, and the nanoimprint lithography was employed to fabricate them. The standard deviation of the results based on the MLE method is lower than that associated with the LM results. The method can be applied to an extended number of resonances to achieve a higher accuracy and precision.

Monitoring of bacterial film formation and its breakdown with an angular-based surface plasmon resonance biosensor.

Bacterial biofilms are a leading cause of infection in health-care settings. Surface plasmon resonance (SPR) biosensors stand as valuable tools not only for the detection of biological entities and the characterisation of biomaterials but also as a suitable means to monitor bacterial film formation. This article reports on a proof-of-concept study for the use of an angular-based SPR biosensor for the monitoring of bacterial cell growth and biofilm formation and removal under the effect of different cleaning agents. The benefit of this custom-made SPR instrument is that it records simultaneously both the critical and resonant angles. This provides unique information on the growth of bacterial cells which is otherwise not obtainable with commonly used intensity-based SPR systems. The results clearly showed that a multilayer biofilm can be formed in 48 hours and the steps involved can be monitored in real-time with the SPR instrument through the measurement of the refractive index change and following the evolution in the shape of the SPR curve. The number, the depth and the sharpness of the reflection ripples varied as the film became thicker. Simulation results confirmed that the number of layers of bacteria affected the number of ripples at the critical angle. Real-time monitoring of the film breakdown with three cleaning agents indicated that bleach solution at 4.5% was the most effective in disrupting the biofilm from the gold sensor. Our overall findings suggest that the SPR biosensor with angular modulation presented in this article can perform real-time monitoring of biofilm formation and has the potential to be used as a platform to test the efficiency of disinfectants.

Real time plasmonic qPCR: how fast is ultra-fast? 30 cycles in 54 seconds.

Polymerase Chain Reaction (PCR) is a critical tool for biological research investigators but recently it also has been making a significant impact in clinical, veterinary and agricultural applications. Plasmonic PCR, which employs the very efficient heat transfer of optically irradiated metallic nanoparticles, is a simple and powerful methodology to drive PCR reactions. The scalability of next generation plasmonic PCR technology will introduce various forms of PCR applications ranging from small footprint portable point of care diagnostic devices to large footprint central laboratory multiplexing devices. In a significant advance, we have introduced a real time plasmonic PCR and explored the ability of ultra-fast cycling compatible with both label-free and fluorescence-based monitoring of amplicon production. Furthermore, plasmonic PCR has been substantially optimized to now deliver a 30 cycle PCR in 54 seconds, with a detectable product. The advances described here will have an immediate impact on the further development of the use of plasmonic PCR playing a critical role in rapid point of care diagnostics.

Projection method for improving signal to noise ratio of localized surface plasmon resonance biosensors.

This paper presents a simple and accurate method (the projection method) to improve the signal to noise ratio of localized surface plasmon resonance (LSPR). The nanostructures presented in the paper can be readily fabricated by nanoimprint lithography. The finite difference time domain method is used to simulate the structures and generate a reference matrix for the method. The results are validated against experimental data and the proposed method is compared against several other recently published signal processing techniques. We also apply the projection method to biotin-streptavidin binding experimental data and determine the limit of detection (LoD). The method improves the signal to noise ratio (SNR) by one order of magnitude, and hence decreases the limit of detection when compared to the direct measurement of the transmission-dip. The projection method outperforms the established methods in terms of accuracy and achieves the best combination of signal to noise ratio and limit of detection.

Surface plasmon resonance biosensor as a tool for the measurement of complex refractive indices.

Optical characterisation of liquids through the measurement of their complex refractive index is critical in environmental monitoring, food industry and medicine. While surface plasmon resonance is widely used for measurement of the real part of the refractive index there have been few studies to date on measurement of complex refractive index with this method. We present a systematic study which highlights the challenges associated with this approach. Instrument design and data analysis techniques are presented together with preliminary experimental results.

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.

Towards more accurate microcavity sensors: maximum likelihood estimation applied to a combination of quality factor and wavelength shifts.

Optical microcavities are widely used for biological and chemical sensing applications. In these devices, a sensing event is estimated by measuring the shift in the resonant wavelength, or in the quality factor of the microcavity. However, all published works to date only use one of these measures to estimate the sensing event. Here, we show that the estimation accuracy of a sensing event can be improved by employing a combination of both the quality factor and the resonant wavelength measurements in a microcavity sensor. We further demonstrate an experimental application of this model by introducing a refractive index change for a microtoroidal cavity sensor immersed in a liquid. By further using the finite element method simulations in conjunction with the estimator model, we show the existence of three distinct measurement regimes as a function of the quality factor of the microcavity. Finally, the estimator model is extended to develop a sensing metric to compare performance of optical or non-optical sensors.

Accurate determination of the quality factor and tunneling distance of axisymmetric resonators for biosensing applications.

Due to ultra high quality factor (10(6) - 10(9)), axisymmetric optical microcavities are popular platforms for biosensing applications. It has been recently demonstrated that a microcavity biosensor can track a biodetection event as a function of its quality factor by using phase shift cavity ring down spectroscopy (PS-CRDS). However, to achieve maximum sensitivity, it is necessary to optimize the microcavity parameters for a given sensing application. Here, we introduce an improved finite element model which allows us to determine the optimized geometry for the PS-CRDS sensor. The improved model not only provides fast and accurate determination of quality factors but also determines the tunneling distance of axisymmetric resonators. The improved model is validated numerically, analytically, and experimentally.

Demonstration of a plasmonic thermocycler for the amplification of human androgen receptor DNA.

A plasmonic heating method for the polymerase chain reaction is demonstrated by the amplification of a section of the human androgen receptor gene. The thermocycler has a simple low-cost design, demonstrates excellent temperature stability and represents the first practical demonstration of plasmonic thermocycling.

Waveguide-coupled drop filters on SOI using quarter-wave shifted sidewalled grating resonators.

We report on the design, fabrication, and demonstration of waveguide coupled channel drop filters at 1550 nm, on a silicon-on-insulator (SOI) substrate. These devices rely on resonant power transfer from a bus waveguide to side-walled Bragg resonators with quarter-wave shifts in the middle. By employing a second mirror resonator, and a tap-off waveguide, reflections along the bus waveguide can be reduced, leading to realization of circulator-free resonance filters. These devices were fabricated on SOI using e-beam lithography and inductively coupled plasma (ICP) etching. Fabricated devices with two coupled cavities are demonstrated to have rejection ratios greater than 20 dB and 3-dB bandwidths of 110 GHz, close to the values predicted by numerical modeling. We also demonstrate power tap-off at resonance of around 16 dB.

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.

Simultaneous measurement of quality factor and wavelength shift by phase shift microcavity ring down spectroscopy.

Optical resonant microcavities with ultra high quality factors are widely used for biosensing. Until now, the primary method of detection has been based upon tracking the resonant wavelength shift as a function of biodetection events. One of the sources of noise in all resonant-wavelength shift measurements is the noise due to intensity fluctuations of the laser source. An alternative approach is to track the change in the quality factor of the optical cavity by using phase shift cavity ring down spectroscopy, a technique which is insensitive to the intensity fluctuations of the laser source. Here, using biotinylated microtoroid resonant cavities, we show simultaneous measurement of the quality factor and the wavelength shift by using phase shift cavity ring down spectroscopy. These measurements were performed for disassociation phase of biotin-streptavidin reaction. We found that the disassociation curves are in good agreement with the previously published results. Hence, we demonstrate not only the application of phase shift cavity ring down spectroscopy to microcavities in the liquid phase but also simultaneous measurement of the quality factor and the wavelength shift for the microcavity biosensors in the application of kinetics measurements.

Selective polarization mode excitation in InGaAs/GaAs microtubes.

We report on selective polarization mode excitation in InGaAs/GaAs rolled-up microtubes. The microtubes are fabricated by selectively releasing a coherently strained InGaAs/GaAs quantum dot layer from its host GaAs substrate. An optical fiber abrupt taper is used to pick up the microtube, while an adiabatically tapered optical fiber is used to couple light into the resonant optical modes of the microtube. By varying the polarization of the light in the adiabatically tapered fiber both transverse electric and transverse magnetic modes are observed in the microtube. We also show that the microtube can be used as a red (0.6 µm) to infrared light (1.5 µm) optical-optical modulator taking advantage of the thermal-optical effect.

Single rolled-up InGaAs/GaAs quantum dot microtubes integrated with silicon-on-insulator waveguides.

We report on single rolled-up microtubes integrated with silicon-on-insulator waveguides. Microtubes with diameters of ~7 µm, wall thicknesses of ~250 nm, and lengths greater than 100 µm are fabricated by selectively releasing a coherently strained InGaAs/GaAs quantum dot layer from the handling GaAs substrate. The microtubes are then transferred from their host substrate to silicon-on-insulator waveguides by an optical fiber abrupt taper. The Q-factor of the waveguide coupled microtube is measured to be 1.5×10(5), the highest recorded for a semiconductor microtube cavity to date. The insertion loss and extinction ratio of the microtube are 1 dB and 34 dB respectively. By pumping the microtube with a 635 nm laser, the resonance wavelength is shifted by 0.7 nm. The integration of InGaAs/GaAs microtubes with silicon-on-insulator waveguides provides a simple, low loss, high extinction passive filter solution in the C+L band communication regime.

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.

Composite superprism photonic crystal demultiplexer: analysis and design.

We present the analysis and design of a superprism-based demultiplexer that employs both group and phase velocity dispersion of the photonic crystal (PhC). Simultaneous diffraction compensation and spatio-angular wavelength channel separation is realized in a slab region that divides the PhC. This avoids the excessive broadening of the beams inside the PhC and enhances the achievable angular dispersion of the conventional superprism topology. As a result, a compact demultiplexer with a relaxed requirement for low divergence input beams is attained. The dynamics of the beams envelops are considered based on the curvature of the band structure. Analysis shows at least 36-fold reduction of the PhC area and much smaller propagation length in slab compared to the preconditioned superprism, based on the same design model. PhC area scales as Δω(-2.5) with Δω being the channel spacing.