Optical Sensor for Real-Time pH Monitoring in Human Tissue.

This article reports on a fiber-based ratiometric optical pH sensor for use in real-time and continuous in vivo pH monitoring in human tissue. Stable hybrid sol-gel-based pH sensing material is deposited on a highly flexible plastic optical fiber tip and integrated with excitation and detection electronics. The sensor is extensively tested in a laboratory environment before it is applied in vivo in a human model. The pH sensor performance in the laboratory environment outperforms the state-of-the-art reported in the current literature. It exhibits the highest sensitivity in the physiological pH range, resolution of 0.0013 pH units, excellent sensor to sensor reproducibility, long-term stability, short response time of <2 min, and drift of 0.003 pH units per 22 h. The sensor also exhibits promising performance in in vitro whole blood samples. In addition, human evaluations conducted under this project demonstrate successful short-term deployment of this sensor in vivo.

In situ generation of plasmonic cavities for high sensitivity fluorophore and biomolecule detection.

Plasmonic cavities are widely studied for the large amplifications in fluorophore emission intensity that they can achieve. Exploiting these properties for biological sensing applications requires strategies to selectively insert the target antigen into a resonant cavity, which are often of similar size or smaller than the target molecule. Here we demonstrate that using relatively simple solution processing, cavity structures can be grown at the stochastic locations where antigen binding takes place, which yields large enhancements in fluorophore emission intensities and over an order of magnitude improvement in bioassay response. The fluorescence amplification generated by the in situ growth of plasmonic structures is sufficiently large to enable both single antigen and single low-quantum efficiency fluorophore detection. The simplicity of the process demonstrated negates the requirement for complex surfaces or geometries and is readily adaptable for a wide range of diagnostic applications.

Controlled surface plasmon enhanced fluorescence from 1D gold gratings via azimuth rotations.

We demonstrate a method to maximise the fluorescence enhancement from a dye using gold coated diffraction gratings. Rotations about the azimuth provides a convenient approach to maximise the coupling between the grating and excitation source while achieving enhancements comparable to traditional optical configurations where the grating and in plane light vectors are parallel. This approach yields a 30 fold enhancement in the fluorescence signal over metal free substrates, while opening up the range of possible orientations and configurations suitable for fluorescence enhancement applications.

Cyanine5-doped silica nanoparticles as ultra-bright immunospecific labels for model circulating tumour cells in flow cytometry and microscopy.

In this work, ultra-bright fluorescent silica nanoparticles (NPs) labels have been shown to selectively bind to a model circulating tumour cell (CTC) line, MCF-7, a metastatic breast cancer by targeting epithelial cellular adhesion molecule (EpCAM) present on the MCF-7 cell membrane. Silica NPs approximately 40nm in diameter were doped with different concentrations of Cyanine5 dye molecules, using the reverse microemulsion method. The NPs were two orders of magnitude brighter than Cyanine5 free dye and the measured fluorescence intensity matched a homo-Förster Resonance Energy Transfer model. NPs were conjugated with anti-EpCAM antibody to the NP surface for immunospecific targeting. In flow cytometry experiments the NPs were twice as bright as two commercial anti-EpCAM red fluorophore conjugates, APC and AlexaFluor®647. This increase is achieved while keeping non-specific binding low as established in control tests with a non-metastatic cancer cell line (HeLa). The NPs were also immunospecific in fluorescence microscopy experiments performed at room temperature on non-fixed cells. Confocal microscopy was used to confirm the NPs were located on the surface of the cells, matching with the location of the EpCAM marker. These NPs labels have excellent potential in biomedical diagnostics, particularly when high signal to noise and good photostability are needed, for example, in point-of-care testing.

Baking Powder Actuated Centrifugo-Pneumatic Valving for Automation of Multi-Step Bioassays.

We report a new flow control method for centrifugal microfluidic systems; CO2 is released from on-board stored baking powder upon contact with an ancillary liquid. The elevated pressure generated drives the sample into a dead-end pneumatic chamber sealed by a dissolvable film (DF). This liquid incursion wets and dissolves the DF, thus opening the valve. The activation pressure of the DF valve can be tuned by the geometry of the channel upstream of the DF membrane. Through pneumatic coupling with properly dimensioned disc architecture, we established serial cascading of valves, even at a constant spin rate. Similarly, we demonstrate sequential actuation of valves by dividing the disc into a number of distinct pneumatic chambers (separated by DF membranes). Opening these DFs, typically through arrival of a liquid to that location on a disc, permits pressurization of these chambers. This barrier-based scheme provides robust and strictly ordered valve actuation, which is demonstrated by the automation of a multi-step/multi-reagent DNA-based hybridization assay.

Investigating the colloidal stability of fluorescent silica nanoparticles under isotonic conditions for biomedical applications.

Fluorescent silica nanoparticle (NP) labels are of great interest in biomedical diagnostics, however, when used in bioassays under physiological conditions they rapidly agglomerate and precipitate from solution leading to high levels of non-specific binding. In this work, using size and zeta-potential data obtained from Dynamic and Electrophoretic Light Scattering analysis, the improvement in colloidal stability of silica NPs under physiological conditions was correlated with an increase in the concentration of three additives: (1) a protein, bovine serum albumin (BSA); (2) a neutral surfactant, Tween 20®; and (3) a charged surfactant, sodium dodecyl sulfate (SDS). The number of BSA molecules present in the NP corona at each concentration was calculated using UV-Vis spectroscopy and a bicinchoninic acid protein assay (BCA). The optimal concentration of each additive was also effective in stabilizing antibody labeled fluorescent nanoparticles (αNPs) under physiological conditions. Using a fourth additive, trehalose, the colloidal stability of αNPs after freeze-drying and long-term storage also significantly improved. Both as-prepared and freeze-dried αNPs were tested in a standard fluorescence immunoassay for the detection of human IgG. The as-prepared assay showed a higher sensitivity at low concentration and a lower limit of detection when compared to a free dye assay. Assays performed with freeze dried αNPs after 4 and 22 days also showed good reproducibility.

High efficiency ring-lens supercritical angle fluorescence (SAF) detection for optimum bioassay performance.

We present a polymer biochip with embedded optics which allows the detection of supercritical angle fluorescence (SAF) without losses due to total internal reflection within the substrate. The chip design comprises structured spherical and aspherical optical elements on the bottom, while the top is chemically functionalized for direct binding of biomolecules. Furthermore, this design facilitates integration in lab-on-a-chip systems with appropriate microfluidics. In the confocal optical setup an ellipsoidal mirror is used for collection of SAF light above the critical angle of the water-polymer interface, which is detected by a photon-counting detector. The work presented here represents a proof of concept for performing sensitive and rapid point-of-care testing, using this low-cost, robust and disposable optical biochip platform. The performance of the platform was validated using direct binding DNA and human IgG assays which yielded low limits of detection 10 pM for DNA and 10 pg/ml for human IgG.

A chemical quenching- and physical blocking-based method to minimize process-mediated aggregation of antibody-crosslinked nanoparticles for imaging application.

Critical limitation of nanoparticles (NP) is their aggregation after functionalisation and antibody cross-linking. We analysed the cause of this aggregation with respect to functionalities (carboxyls and amines) on the NP surface. We have devised a low cost novel method to reduce such aggregations during protein cross-linking and validated it by probing the platelet surface with platelet surface-specific anti-CD41 antibody conjugated NPs.

At-line bioprocess monitoring by immunoassay with rotationally controlled serial siphoning and integrated supercritical angle fluorescence optics.

In this paper we report a centrifugal microfluidic "lab-on-a-disc" system for at-line monitoring of human immunoglobulin G (hIgG) in a typical bioprocess environment. The novelty of this device is the combination of a heterogeneous sandwich immunoassay on a serial siphon-enabled microfluidic disc with automated sequential reagent delivery and surface-confined supercritical angle fluorescence (SAF)-based detection. The device, which is compact, easy-to-use and inexpensive, enables rapid detection of hIgG from a bioprocess sample. This was achieved with, an injection moulded SAF lens that was functionalized with aminopropyltriethoxysilane (APTES) using plasma enhanced chemical vapour deposition (PECVD) for the immobilization of protein A, and a hybrid integration with a microfluidic disc substrate. Advanced flow control, including the time-sequenced release of on-board liquid reagents, was implemented by serial siphoning with ancillary capillary stops. The concentration of surfactant in each assay reagent was optimized to ensure proper functioning of the siphon-based flow control. The entire automated microfluidic assay process is completed in less than 30 min. The developed prototype system was used to accurately measure industrial bioprocess samples that contained 10 mg mL(-1) of hIgG.

Dendrimer driven self-assembly of SPR active silver-gold nanohybrids.

A fourth generation PAMAM dendrimer has been successfully employed for the development of a single step synthesis strategy for self-assembled Ag-Au nanohybrid structures. The surface plasmon resonance properties and the degree of self-assembly of the nanohybrid are strongly correlated with the stoichiometry of the metals which gives rise to enhanced plasmonic properties. The enhanced plasmonic response of the nanohybrids is modeled and is validated experimentally in a model HRP (horseradish peroxidise) bioassay carried out on an SPR-based biochip platform.

Optimization of size, morphology and colloidal stability of fluorescein dye-doped silica NPs for application in immunoassays.

Fluorescent nanoparticle (NP) labels are of great interest for point-of-care medical diagnostics where high fluorescence signals combined with low limits of detection are required. In this work, hydrophilic and hydrophobic fluorescein dye derivatives were covalently doped into silica NPs. The NPs were prepared in a range of sizes from 16 to 80 nm using both ternary and quaternary microemulsion methods where the diameter varied linearly with changes in the water to surfactant ratio. The morphology and colloidal stability of the NPs were characterised using transmission electron microscopy and photon correlation spectroscopy; NPs doped with hydrophobic fluorescein dye were significantly smaller and more polydispersed. Optical properties including absorption, fluorescence and quantum efficiency were also determined. Representative NPs from each microemulsion method (ternary, Ø = 25 nm and quaternary, Ø = 80 nm) were tested as labels in a fluorescence based immunoassay for the detection of human IgG and human chorionic gonadotropin. Both sets of nanoparticle assays showed lower limits of detection and better coefficients of variance than a free dye label with good day to day reproducibility. The optimal surface coverage of detection antibody was also found to depend on the size of the nanoparticle.

Synthesis and characterization of model silica-gold core-shell nanohybrid systems to demonstrate plasmonic enhancement of fluorescence.

In this work, gold-silica plasmonic nanohybrids have been synthesized as model systems which enable tuning of dye fluorescence enhancement/quenching interactions. For each system, a dye-doped silica core is surrounded by a 15 nm spacer region, which in turn is surrounded by gold nanoparticles (GNPs). The GNPs are either covalently conjugated via mercapto silanization to the spacer or encapsulated in a separate external silica shell. The intermediate spacer region can be either dye doped or left undoped to enable quenching and plasmonic enhancement effects respectively. The study indicates that there is a larger enhancement effect when GNPs are encapsulated in the outer shell compared to the system of external conjugation. This is due to the environmental shielding provided by shell encapsulation compared to the exposure of the GNPs to the solvent environment for the externally conjugated system. The fluorescence signal enhancement of the nanohybrid systems was evaluated using a standard HRP-anti-HRP fluorescence based assay platform.

Inhibition of neuroblastoma tumor growth by targeted delivery of microRNA-34a using anti-disialoganglioside GD2 coated nanoparticles.

BACKGROUND: Neuroblastoma is one of the most challenging malignancies of childhood, being associated with the highest death rate in paediatric oncology, underlining the need for novel therapeutic approaches. Typically, patients with high risk disease undergo an initial remission in response to treatment, followed by disease recurrence that has become refractory to further treatment. Here, we demonstrate the first silica nanoparticle-based targeted delivery of a tumor suppressive, pro-apoptotic microRNA, miR-34a, to neuroblastoma tumors in a murine orthotopic xenograft model. These tumors express high levels of the cell surface antigen disialoganglioside GD2 (GD(2)), providing a target for tumor-specific delivery. PRINCIPAL FINDINGS: Nanoparticles encapsulating miR-34a and conjugated to a GD(2) antibody facilitated tumor-specific delivery following systemic administration into tumor bearing mice, resulted in significantly decreased tumor growth, increased apoptosis and a reduction in vascularisation. We further demonstrate a novel, multi-step molecular mechanism by which miR-34a leads to increased levels of the tissue inhibitor metallopeptidase 2 precursor (TIMP2) protein, accounting for the highly reduced vascularisation noted in miR-34a-treated tumors. SIGNIFICANCE: These novel findings highlight the potential of anti-GD(2)-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD(2)-expressing tumors, and as a basic discovery tool for elucidating biological effects of novel miRNAs on tumor growth.

Synthesis and characterization of a Noble metal Enhanced Optical Nanohybrid (NEON): a high brightness detection platform based on a dye-doped silica nanoparticle.

A highly bright and photostable, fluorescent nanohybrid particle is presented which consists of gold nanoparticles (GNPs) embedded in dye-doped silica in a core-shell configuration. The dye used is the near-infrared emitting 4,5-benzo-5'-(iodoacetaminomethyl)-1',3,3,3',3'-pentamethyl-1-(4-sulfobutyl) indodicarbo cyanine. The nanohybrid architecture comprises a GNP core which is separated from a layer of dye molecules by a 15 nm buffer layer and has an outer protective, undoped silica shell. Using this architecture, a brightness factor of 550 has been achieved compared to the free dye. This hybrid system, referred to as Noble metal Enhanced Optical Nanohybrid (NEON) in this paper, is the first nanohybrid construct to our knowledge which demonstrates such tunable fluorescence property. NEON has enhanced photostability compared to the free dye and compared to a control particle without GNPs. Furthermore, the NEON particle, when used as a fluorescent label in a model bioassay, shows improved performance over assays using a conventional single dye molecule label.

Label-free optical characterization methods for detecting amine silanization-driven gold nanoparticle self-assembly.

Fluorescence lifetime correlation spectroscopy (FLCS) is presented as a single-step label-free detection method for probing the amine silanization-driven spontaneous 3D self-assembly of freestanding gold nanoparticles (GNPs) in solution. Unlike the conventional methods of studying self-assembly, for example, UV-vis spectroscopy and electron microscopy, FLCS utilizes the intrinsic gold fluorescence. The significance of this approach is to amalgamate the measurement of optical and hydrodynamic size properties simultaneously to achieve a more coherent description of the self-assembly pathway. GNP self-assembly has two-stage kinetics. Electrostatic interaction drives the initial amine silanization, and this is followed by siloxane bond formation between hydrolyzed ethoxy groups of GNP-attached APTES, resulting in the formation of micrometer-sized superstructures. The self-assembly has resulted in a 5-fold increase in the fluorescence lifetime (FL), and the FLCS study has shown an 8- to 10-fold increase in the diffusion coefficient using the pure diffusion model. This result is consistent with the transmission electron microscopy (TEM) observation, which shows a few hundred fold increase in the diameter due to assembly formation by the GNPs.

Dextran-coated silica nanoparticles for calcium-sensing.

This article describes the synthesis and characterisation of fluorescent composite nanoparticles consisting of a silica core and a dextran shell. The silica core contains a rhodamine-based reference dye, which allows ratiometric measurements and the dextran shell is labelled with the Ca(2+)-sensitive dye Fluo-4. The nanoparticles have an average hydrodynamic diameter of 95 nm, good colloidal stability and show a 2.9-fold increase in fluorescence intensity upon binding to Ca(2+) ions. The apparent dissociation constant of K'(d) ≈ 520 nM is well suited for measurements in the physiological range.

Signal enhancement of surface plasmon-coupled emission (SPCE) with the evanescent field of surface plasmons on a bimetallic paraboloid biochip.

We present a novel approach to the enhancement of surface plasmon-coupled emission (SPCE) using surface plasmon excitation in a bimetal (Ag/Au) layer and we validate the enhancement by presenting the results of a model human IgG immunoassay. Theoretical calculations using Fresnel's equations have been carried out to determine the optimum bimetallic composition and the resulting electric field enhancement. Signal enhancement of SPCE was confirmed using a range of bimetallic layers which were deposited on the surface of a high collection efficiency polymer array biochip and subsequently immobilized with Alexa Fluor 647 labeled anti-human IgG. The bimetallic film of Ag/Au (36/10nm) was determined as an optimum substrate for maximum SPCE signal which was a compromise between the long-term stability of the metal layer and the optimized evanescent field enhancement. An enhanced dose-dependent response was also demonstrated which was ∼3 times greater than that detected with a pure gold layer. A human IgG immunoassay showed a dose-dependent response yielding a limit of detection of 1pg/ml by the 3σ rule. The improved performance of the bimetal layer compared to that of an assay carried out on a pure gold layer is attributed to the enhanced evanescent field intensity of surface plasmons in the bimetal combination which excites more fluorescence hence producing an enhanced SPCE signal. This result demonstrates the potential of the SPCE-based array biochips as a sensitive and high-throughput analysis platform for biomolecular interactions.

Novel multiparametric approach to elucidate the surface amine-silanization reaction profile on fluorescent silica nanoparticles.

This Article addresses the important issue of the characterization of surface functional groups for optical bioassay applications. We use a model system consisting of spherical dye-doped silica nanoparticles (NPs) that have been functionalized with amine groups whereby the encapsulated cyanine-based near-infrared dye fluorescence acts as a probe of the NP surface environment. This facilitates the identification of the optimum deposition parameters for the formation of a stable ordered amine monolayer and also elucidates the functionalization profile of the amine-silanization process. Specifically, we use a novel approach where the techniques of fluorescence correlation spectroscopy (FCS) and fluorescence lifetime measurement (FL) are used in conjunction with the more conventional analytical techniques of zeta potential measurement and Fourier transfer infrared spectroscopy (FTIR). The dynamics of the ordering of the amine layer in different stages of the reaction have been characterized by FTIR, FL, and FCS. The results indicate an optimum reaction time for the formation of a stable amine layer, which is optimized for further biomolecular conjugation, whereas extended reaction times lead to a disordered cross-linked layer. The results have been validated using an immunoglobulin (IgG) plate-based direct binding assay where the maximum number of IgG-conjugated aminated NPs were captured by immobilized anti-IgG antibodies for the NP sample corresponding to the optimized amine-silanization condition. Importantly, these results point to the potential of FCS and FL as useful analytical tools in diverse fields such as characterization of surface functionalization.

Ratiometric fluorescence-based dissolved carbon dioxide sensor for use in environmental monitoring applications.

The focus of this work is on the development and characterisation of a fluorescence-based ratiometric sol-gel-derived dissolved carbon dioxide (dCO(2)) sensor for use in environmental monitoring applications. Fluorescence-based dCO(2) sensors are attractive as they facilitate the development of portable and low-cost systems that can be easily deployed outside the laboratory environment. The sensor developed for this work exploits a pH fluorescent dye 1-hydroxypyrene-3,6,8-trisulfonic acid, ion-paired with cetyltrimethylammonium bromide (HPTS-IP), which has been entrapped in a hybrid sol-gel-based matrix derived from n-propyltriethoxysilane along with the liphophilic organic base. The sensor spot deposited on a cover slip has been interrogated with a robust, ratiometric optical probe that combines effective fluorescence excitation and detection and thus facilitates the production of a highly sensitive sensor system using low-cost optoelectronic components. The probe design involves the use of dual-LED excitation in order to facilitate ratiometric operation and uses a silicon PIN photodiode. HPTS-IP exhibits two pH-dependent changes in excitation bands, which allows for dual excitation ratiometric detection as an indirect measure of the dCO(2). Such measurements are insensitive to changes in dye concentration, leaching and photobleaching of the fluorophore and instrument fluctuations unlike unreferenced fluorescence intensity measurements. The performance of the sensor system is characterised by a high degree of repeatability, reversibility and stability. Calculated limit of detection for the sensor was 35 ppb. The sensor probe was used to monitor dCO(2) levels in a laboratory-based aquatic habitat, and the expected diurnal pattern was clearly visible. The influence of temperature, biofouling and photobleaching on sensor performance has been also investigated.