Coupling ion channels to receptors for biomolecule sensing

Nanoscale electrical biosensors are promising tools for diagnostics and high-throughput screening systems. The electrical signal allows label-free assays with a high signal-to-noise ratio and fast real-time measurements. The challenge in developing such biosensors lies in functionally connecting a molecule detector to an electrical switch. Advances in this field have relied on synthetic ion-conducting pores and modified ion channels that are not yet suitable for biomolecule screening. Here we report the design and characterization of a novel bioelectric-sensing platform engineered by coupling an ion channel, which serves as the electrical probe, to G-protein-coupled receptors (GPCRs), a family of receptors that detect molecules outside the cell. These ion-channel-coupled receptors may potentially detect a wide range of ligands recognized by natural or altered GPCRs, which are known to be major pharmaceutical targets. This could form a unique platform for label-free drug screening.     

Improvement of biomolecule quantification precision and use of a single-element aspheric objective lens in fluorescence correlation spectroscopy

We found a way to increase the precision with which biomolecules present at concentrations below 10(-10) M can be quantified by fluorescence correlation spectroscopy (FCS). The effectiveness of the way was demonstrated experimentally by using a single-element aspheric objective lens, which was newly developed to reduce the cost of FCS instruments. In the first part of this paper, the relative standard deviation (RSD) of FCS-based concentration measurements is estimated theoretically by an analytical approximation assuming the detection volume profiles in FCS setups to be Gaussian and by molecular simulations in which more realistic profiles are calculated from physical parameters of the measurement setups. In a limit of infinitely bright molecules and zero background emission, the analytical approximation predicts that the RSD at a concentration is minimized when the mean number of molecules in a detection volume is approximately 0.5. A detection volume of the order of 10(-13) L thus gives smaller RSD values for concentrations from 10(-11) to 10(-10) M than does one of the order of 10(-15) L, which is widely used in FCS. This prediction is supported by the molecular simulations, taking into account the finite molecule brightness and background emission. In the second part of the paper, the RSD is evaluated experimentally with an FCS setup with a detection volume of 1.1 x 10(-13) L. The newly developed objective lens, serving as the bottom of the sample cell in this setup, has a large numerical aperture (0.9) without using immersion liquid. When a calibration line was made by 30-s FCS measurements of Cy3-labeled, 112-mer single-stranded DNA solutions, the RSD roughly agreed with the simulation result and was less than 0.1 for DNA concentrations from 2 x 10(-11) to 10(-10) M.     

Helix-coiled gold nanowires for molecular sensing

Helix-coiled gold nanowires were fabricated by a templating route using unique composite templates consisting of anodic aluminum oxide (AAO) nanotubular membrane and confined mesoporous silica therein. A different degree of confinement energy induces a different degree of helix curvature of confined porous silica nanochannels in an AAO, which works as a hard template for the electrochemical deposition of gold, thereby rationally enabling a different degree of helix curvature of gold nano-replicas. From surface-enhanced Raman scattering experiments, we first found that helix-coiled gold nanowires show more distinctly enhanced molecule sensing efficiency than those from simple smooth gold nanowires, and gold nanowires with the narrower lateral width show more enhanced molecule sensing efficiency than those of thicker width helix nanowires.     

Fluorescence near gold nanoparticles for DNA sensing

We investigated fluorescence quenching and enhancement near gold nanoparticles (GNP) of various sizes using fluorescently labeled hairpin DNA probes of different lengths. A closed hairpin caused intimate contact between the fluorophore and the gold, resulting in an efficient energy transfer (quenching). Upon hybridization with complementary DNA, the DNA probes were stretched yielding a strong increase in fluorescence signal. By carefully quantifying the amount of bound fluorescent probes and the GNP concentrations, we were able to determine the quenching and enhancement efficiencies. We also studied the size and distance dependence theoretically, using both FDTD simulations and the Gersten-Nitzan model and obtained a good agreement between experiments and theory. On the basis of experimental and theoretical studies, we report over 96.8% quenching efficiency for all particle sizes tested and a maximal signal increase of 1.23 after DNA hybridization. The described results also demonstrate the potential of gold nanoparticles for label free DNA sensing.     

Time-resolved fluorescence spectroscopy for chemical sensors

A family of sensors is presented with fluorescence decay-time measurements used as the sensing technique. The concept is to take a single fluorophore with a suitably long fluorescence decay time as the basic building block for numerous different sensors. Analyte recognition can be performed by different functional groups that are necessary for selective interaction with the analyte. To achieve this, the principle of excited-state electron transfer is applied with pyrene as the fluorophore. Therefore the same instrumentation based on a small, ambient air-nitrogen laser and solid-state electronics can be used to measure different analytes, for example, oxygen, pH, carbon dioxide, potassium, ammonium, lead, cadmium, zinc, and phosphate.     

Binding kinetics of biomolecule interaction at ultralow concentrations based on gold nanoparticle enhancement

Measuring the kinetic constants of protein-protein interactions at ultralow concentrations becomes critical in characterizing biospecific affinity, and exploring the feasibility of clinical diagnosis with respect to detection sensitivity, efficiency and accuracy. In this study, we propose a method that can calculate the binding constants of protein-protein interactions in sandwich assays at ultralow concentrations at the pg/mL level, using a localized surface plasmon coupled fluorescence fiber-optic biosensor (LSPCF-FOB). We discuss a two-compartment model to achieve reaction-limited kinetics under the stagnant conditions of the reaction chamber. The association rate constant, dissociation rate constant, and the equilibrium dissociation constant, that is, k(a), k(d), K(D), respectively, of the kinetics of binding between total prostate-specific antigen (t-PSA) and anti-t-PSA at concentrations from 0.1 pg/mL to 1 ng/mL, were measured either in PBS or in human serum. This is the first time that k(a), k(d), and K(D) have been measured at such a low concentration range in a complex sample such as human serum.     

Dithizone modified gold nanoparticles films for potentiometric sensing

For the first time, application of a membrane composed of gold nanoparticles decorated with complexing ligand for potentiometric sensing is shown. Gold nanoparticles drop cast from a solution form a porous structure on a substrate electrode surface. Sample cations can penetrate the gold nanoparticles layer and interact with ligand acting as a charged ionophore, resulting in Nernstian potentiometric responses. Anchoring of complexing ligand on the gold surface abolishes the necessity of ionophore application. Moreover, it opens the possibility of preparation of potentiometric sensors using chelators of significantly different selectivity patterns further enhanced by the absence of polymeric membrane matrix. This was clearly seen, for example, for gold nanoparticles stabilizing the applied ligand-dithizone-thiol conformation leading to a high potentiometric selectivity toward copper ions, much higher than that of ionophores typically used to induce selectivity for polymeric ion-selective membranes.     

High-throughput single-cell fluorescence spectroscopy

A high-throughput method for measuring single-cell fluorescence spectra is presented. Upon excitation with a 488 nm argon-ion laser many bacterial cells were imaged by a 20x microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grating, and an intensified charge-coupled device (ICCD) camera simultaneously recorded the zero and the first orders of the fluorescence from each cell. Single-cell fluorescence spectra were reconstructed from the distance between zero-order and first-order maxima as well as the length and the pixel intensity distribution of the first-order images. By using this approach, the emission spectrum of E. coli cells expressing green fluorescent protein (GFP) was reconstructed. Also, fluorescence spectra of E. coli cells expressing non-fluorescent apo-subunits of R-phycoerythrin (R-PE) were recorded after incubation of the cells with phycoerythrobilin (PEB) chromophore. The fluorescence spectra are in good agreement with results obtained on the same cells using a fluorescence spectrometer or a fluorescence microscope. When spectra are to be acquired, this approach has a higher throughput, better sensitivity, and better spectral resolution compared to flow cytometry.     

Spatial fluorescence cross-correlation spectroscopy

We present an alternative method for diffusion measurements of fluorescent species in solution by use of confocal microscopy and fluorescence correlation spectroscopy techniques. It consists of making a time and spatial dual correlation in which one detects the fluorescence signals from two nearby separate confocal volumes and cross correlates them. To improve the spatial discrimination between the two confocal volumes we propose filtering of fluorescence photocounts by rejecting the fluorescence background, which corresponds to particles located far from the center of the detection volumes.     

Impedance spectroscopy on xerogel layer for chemical sensing

This paper deals with a new approach for toluene detection in water based on conductivity measurement on a xerogel layer deposited on Si/SiO₂ electrode. The conductivity changes is evaluated by means of resistance and phase variation of absolute impedance of the TEOS xerogel layer in contact with small amounts of toluene in water. The electrical properties of such xerogel layer can be measured with impedance spectroscopy technique and modelised with electrical model. The molecule structure of such layer has been obtained with Fourier transformed infrared spectroscopy. For toluene detection, impedance spectroscopy shows a decrease of the membrane resistance over time and an increase of the phase. This decrease can be attributed to the conductivity and dielectric constant variation. A detection limit of 100 ppm and a dynamic range 100 ppm–0.7% are obtained in our experimental conditions.     

Reversible fluorescence quenching in carbon nanotubes for biomolecular sensing

Biosensing applications of single-walled carbon nanotubes have been demonstrated in solid-state device structures. Bioanalyte sensing schemes based on coupling of reversible nanotube fluorescence quenching to redox reactions paired to enzymatic peroxide generation have also been pursued. Here we show a new approach to highly sensitive nanotube-based optical sensing. Single-walled carbon nanotubes interacting with dye-ligand conjugates--a redox-active dye molecule that is covalently bound to a biological receptor ligand (such as biotin in this case)--showed fluorescence quenching. Further interaction between the receptor ligand on the conjugates and target analytes (avidin in this case) induced the recovery of the quenched fluorescence, forming the basis of the sensing scheme. Nanomolar sensitivity was attained with high specificity for the target analyte. This is a versatile approach because a wide range of conjugation possibilities exists between the potential receptors and redox quenchers.     

Multiple-fiber probe design for fluorescence spectroscopy in tissue

The fiber-optic probe is an essential component of many quantitative fluorescence spectroscopy systems, enabling delivery of excitation light and collection of remitted fluorescence in a wide variety of clinical and laboratory situations. However, there is little information available on the role of illumination--collection geometry to guide the design of these components. Therefore we used a Monte Carlo model to investigate the effect of multifiber probe design parameters--numerical aperture, fiber diameter, source--collection fiber separation distance, and fiber-tissue spacer thickness--on light propagation and the origin of detected fluorescence. An excitation wavelength of 400 nm and an emission wavelength of 630 nm were simulated. Noteworthy effects included an increase in axial selectivity with decreasing fiber size and a transition with increasing fiber-tissue spacer size from a subsurface peak in fluorophore sensitivity to a nearly monotonic decrease typical of single-fiber probes. We provide theoretical evidence that probe design strongly affects tissue interrogation. Therefore application-specific customization of probe design may lead to improvements in the efficacy of fluorescence-based diagnostic devices.     

Electrochemical sensing using quantum-sized gold nanoparticles

This paper describes the electrocatalytic activity of quantum-sized thiolate protected Au(25) nanoparticles and their use in electrochemical sensing. The Au(25) film modified electrode exhibited excellent mediated electrocatalytic activity that was utilized for amperometric sensing of biologically relevant analytes, namely, ascorbic acid and uric acid. The electron transfer dynamics in the Au(25) film was examined as a function of Au(25) concentration, which manifested the dual role of Au(25) as an electronic conductor as well as a redox mediator. The electron transfer study has further revealed the correlation between the electronic conductivity of the Au(25) film and the sensing sensitivity.     

Horseradish peroxidase functionalized fluorescent gold nanoclusters for hydrogen peroxide sensing

The fluorescence of metal nanoclusters provides an amusing optic feature to be applied in various fields. However, rational design of dual functional fluorescent metal nanoclusters directed by active enzyme with targeted application remains little explored. In this work, we report a new strategy to construct enzyme functionalized fluorescent gold nanoclusters via a biomineralization process for the detection of hydrogen peroxide. Horseradish peroxidase (HRP) was used as a model functional template to direct the synthesis of fluorescent gold nanoclusters (Au NCs) at physiological conditions to form HRP-Au NCs bioconjugates. We found that the fluorescence of HRP-Au NCs can be quenched quantitatively by adding H(2)O(2), indicating that HRP enzyme remains active and enables catalytic reaction of HRP-Au NCs and H(2)O(2). Upon the addition of H(2)O(2) under optimal conditions, the fluorescence intensity quenched linearly over the range of 100 nM to 100 μM with high sensitivity (LOD = 30 nM, S/N = 3). This study would be potentially extended to other functional proteins to generate dual functional nanoclusters and applied to real time monitoring of biologically important targets in living cells.     

Infrared spectroscopy for chemically specific sensing in silicon-based microreactors

Fourier transform infrared (FT-IR) spectroscopy in a multiple internal reflection (MIR) geometry is integrated with silicon-based microreactors to allow detection of a wide range of chemical species while taking advantage of inexpensive batch fabrication techniques applicable to silicon substrates. The microreactors are fabricated in silicon and glass using standard microfabrication and selective etching techniques. The small ( approximately 1 cm side) reactor size provides access to nearly the full mid-IR frequency region with MIR-FT-IR, allowing us to probe both solution-phase and surface-bound chemical transformations. The wide applicability of this approach is demonstrated with two representative test cases: kinetics of acid-catalyzed ethyl acetate hydrolysis and amidization of surface-tethered amine groups.     

Fluorescence lifetime spectroscopy for pH sensing in scattering media

Fluorescence lifetime spectroscopy in the presence of tissuelike scattering is demonstrated from measurements of phase and modulation ratio as a function of modulation frequency using a pH-sensitive dye, Carboxy Seminaphthofluorescein-1 (C-SNAFL-1). From the optical diffusion equation describing the propagation and generation of fluorescence within solutions of 0.5 microM C-SNAFL-1 containing 2.0% (by volume) of Intralipid as a scatterer, the values of the average lifetime of C-SNAFL-1 were determined as the solution pH varied between 5 and 9. Average lifetime values were found to match those measured using traditional phase-modulation measurement in nonscattering media. Furthermore, the robustness of the spectroscopic technique was demonstrated by conducting lifetime measurements at varying scatterer concentrations (1.5-3.0 vol % Intralipid). These results confirm the approach for analytical sensing in scattering media via fluorescence lifetime kinetics in order to track changes in analyte concentrations.     

Glucose sensing using near-infrared surface-enhanced Raman spectroscopy: gold surfaces, 10-day stability, and improved accuracy

This research presents the achievement of significant milestones toward the development of a minimally invasive, continuously monitoring, glucose-sensing platform based on the optical quantitation of glucose in interstitial fluid. We expand our initial successes in the measurement of glucose by surface-enhanced Raman scattering (SERS), demonstrating substantial improvements not only in the quality and optical properties of the substrate system itself but also in the robustness of the measurement methodology and the amenability of the technique to compact, diode laser-based instrumentation. Herein, we compare the long-term stability of gold to silver film over nanosphere (AuFON, AgFON) substrates functionalized with a partitioning self-assembled monolayer (SAM) using both electrochemical and SERS measurements. AuFONs were found to be stable for a period of at least 11 days. The switch to AuFONs not only provides a more stable surface for SAM formation but also yields better chemometric results, with improved calibration and validation over a range of 0.5-44 mM (10-800 mg/dL). Measured values for glucose concentrations in phosphate-buffered saline (pH approximately 7.4) based on 160 independent SERS measurements on AuFONs have a root-mean-square error of prediction of 2.7 mM (49.5 mg/dL), with 91% of the values falling within an extended A-B range on an expanded Clarke error grid. Furthermore, AuFONs exhibit surface plasmon resonances at longer wavelengths than similar AgFONs, which make them more efficient for SERS at near-infrared wavelengths, enabling the use of low-power diode lasers in future devices.     

Characterization of time-resolved fluorescence response measurements for distributed optical-fiber sensing

A distributed optical-fiber sensing system based on pulsed excitation and time-gated photon counting has been used to locate a fluorescent region along the fiber. The complex Alq3 and the infrared dye IR-125 were examined with 405 and 780 nm excitation, respectively. A model to characterize the response of the distributed fluorescence sensor to a Gaussian input pulse was developed and tested. Analysis of the Alq3 fluorescent response confirmed the validity of the model and enabled the fluorescence lifetime to be determined. The intrinsic lifetime obtained (18.2±0.9 ns) is in good agreement with published data. The decay rate was found to be proportional to concentration, which is indicative of collisional deactivation. The model allows the spatial resolution of a distributed sensing system to be improved for fluorophores with lifetimes that are longer than the resolution of the sensing system.     

Fluorescence coincidence spectroscopy for single-molecule fluorescence resonance energy-transfer measurements

Single-molecule fluorescence resonance energy transfer (FRET) is commonly used to probe different conformations and conformational dynamics of single biomolecules. However, the analysis of raw burst traces is not always straightforward. The presence of a "zero peak" and the skewness of peaks at high and low FRET efficiencies in proximity ratio histograms make the accurate evaluation of the histogram a challenging task. This is further compounded by the difficulty associated with siting two fluorophores in optimal range of each other. Here we present an alternative method of analysis, based on handling coincident FRET photon bursts, that addresses these problems. In addition, we demonstrate methods to enhance coincidence levels and thus the accuracy of FRET determination: the use of dual-color excitation, including direct excitation of the acceptor fluorophore; the addition of a remote dye to the biomolecule, not involved in the FRET process; or a combination of the two. We show the advantages of dual excitation by studying several labeled double-stranded DNA samples as FRET models. This method extends the application of single-molecule FRET to more complicated biological systems where only a small fraction of complexes are fully assembled.