Use of high frequency electrorotation to identify cytoplasmic changes in cells non-disruptively.

In this paper we demonstrate how the use of frequencies ranging from 50 kHz to 5 GHz in the analysis of cells by electrorotation can open the path to the identification of differences not detectable by conventional set-ups. Earlier works usually reported electrorotation devices operating below 20 MHz, limiting the response obtained to properties associated with the cell membrane. Those devices are thus unable to resolve the physiological properties in the cytoplasm. We used microwave-based technology to extend the frequency operation to 5 GHz. At high frequencies (from tens of MHz to GHz), the electromagnetic signal passes through the membrane and allows probing the cytoplasm. This enables several applications, such as cell classification, and viability analysis. Additionally, the use of conventional microfabrication techniques reduces the cost and complexity of analysis, compared to other non-invasive methods. We demonstrated the potential of this set-up by identifying two different populations of T-lymphocytes not distinguishable through visual assessment. We also assessed the effect of calcein on cell cytoplasmic properties and used it as a controlled experiment to demonstrate the possibility of this method to detect changes happening predominantly in the cytoplasm.

Amplification Efficiency and Template Accessibility as Distinct Causes of Rain in Digital PCR: Monte Carlo Modeling and Experimental Validation.

Partitions in digital PCR (dPCR) assays do not reach the detection threshold at the same time. This heterogeneity in amplification results in intermediate endpoint fluorescence values (i.e., rain) and misclassification of partitions, which has a major impact on the accuracy of nucleic acid quantification. Rain most often results from a reduced amplification efficiency or template inaccessibility; however, exactly how these contribute to rain has not been described. We developed and experimentally validated an analytical model that mechanistically explains the relationship between amplification efficiency, template accessibility, and rain. Using Monte Carlo simulations, we show that a reduced amplification efficiency leads to broader threshold cycle (Ct) distributions that can be fitted using a log-normal probability distribution. From the fit parameters, the amplification efficiency can be calculated. Template inaccessibility, on the other hand, leads to a different rain pattern, in which a distinct exponential tail in the Ct distribution can be observed. Using our model, it is possible to determine if the amplification efficiency, template accessibility, or another source is the main contributor of rain in dPCR assays. We envision that this model will facilitate and speed up dPCR assay optimization and provide an indication for the accuracy of the assay.

Measuring mass of nanoparticles and viruses in liquids with nanometer-scale pores.

Nanopores have recently been developed for the detection and physical characterization of nanoparticles, viruses, proteins, nucleic acids, and other macromolecules in liquids. The method provides the ability to rapidly estimate the size and electrical charge of analytes over a wide range of concentration, potentially with small sample volumes and low cost. Here, we use the technique to measure the mass of nanoparticles and viruses and their sedimentation. The analyte sedimentation-time measurement provides an estimate for the nanoparticle mass-density. We also show that the method can be used with samples at low concentration and in small volumes.

Toward point-of-care diagnostics: Running enzymatic assays on a photonic waveguide-based sensor chip with a portable, benchtop measurement system.

We demonstrate a portable, compact system to perform absorption-based enzymatic assays at a visible wavelength of 639 nm on a photonic waveguide-based sensor chip, suitable for lab-on-a-chip applications. The photonic design and fabrication of the sensor are described, and a detailed overview of the portable measurement system is presented. In this publication, we use an integrated photonic waveguide-based absorbance sensor to run a full enzymatic assay. An assay to detect creatinine in plasma is simultaneously performed on both the photonic sensor on the portable setup and on a commercial microplate reader for a clinically relevant creatinine concentration range. We observed a high correlation between the measured waveguide propagation loss and the optical density measurement from the plate reader and measured a limit-of-detection of 4.5 µM creatinine in the sensor well, covering the relevant clinical range for creatinine detection.

Boosting the figure-of-merit of LSPR-based refractive index sensing by phase-sensitive measurements.

Localized surface plasmon resonances possess very interesting properties for a wide variety of sensing applications. In many of the existing applications, only the intensity of the reflected or transmitted signals is taken into account, while the phase information is ignored. At the center frequency of a (localized) surface plasmon resonance, the electron cloud makes the transition between in- and out-of-phase oscillation with respect to the incident wave. Here we show that this information can experimentally be extracted by performing phase-sensitive measurements, which result in linewidths that are almost 1 order of magnitude smaller than those for intensity based measurements. As this phase change is an intrinsic property of a plasmon resonance, this opens up many possibilities for boosting the figure-of-merit (FOM) of refractive index sensing by taking into account the phase of the plasmon resonance. We experimentally investigated this for two model systems: randomly distributed gold nanodisks and gold nanorings on top of a continuous gold layer and a dielectric spacer and observed FOM values up to 8.3 and 16.5 for the respective nanoparticles.

Ultralocal modification of surface plasmons properties in silver nanocubes.

The plasmonic properties of individual subwavelength-sized silver nanocubes are mapped with nanometric spatial resolution by means of electron energy-loss spectroscopy in a scanning transmission electron microscope. Three main features with different energies and spatial behavior (two peaked at the corners, one on the edges) are identified and related to previous measurements on ensemble or individual nanoparticles. The highly subwavelength mapping of the energy position and intensity of the excitations shows that the surface plasmon modes, localized at specific areas of the particles, for example, the corners or the edges, are modified by their size, the presence of a substrate, and the very local environment. Helped by discrete dipole approximation numerical simulations, we discuss how local modifications of the environment affect the global modes of the particles. In particular, we show both experimentally and theoretically that absorption resonances at different corners of the same nanocube are largely independent of each other in energy and intensity. Our findings provide a better understanding of the spatial coherence of the surface plasmons in nanoparticles but also give useful insights about their roles in the nanoparticle sensing properties.

Unraveling the impact of nano-scaling on silicon field-effect transistors for the detection of single-molecules.

Electrolyte-gated silicon field-effect transistors (FETs) capable of detecting single molecules could enable high-throughput molecular sensing chips to advance, for example, genomics or proteomics. For solid-gated silicon FETs it is well-known that nano-scaled devices become sensitive to single elementary charges near the silicon-oxide interface. However, in electrolyte-gated FETs, electrolyte screening strongly reduces sensitivity to charges near the gate oxide. The question arises whether nano-scaling electrolyte-gated FETs can entail a sufficiently large signal-to-noise ratio (SNR) for the detection of single molecules. We enhanced a technology computer-aided design tool with electrolyte screening models to calculate the impact of the FET geometry on the single-molecule signal and FET noise. Our continuum FET model shows that a sufficiently large single-molecule SNR is only obtained when nano-scaling all FET channel dimensions. Moreover, we show that the expected scaling trend of the single-molecule SNR breaks down and no longer results in improvements for geometries approaching the decananometer size. This is the characteristic size of the FET channel region modulated by a typical molecule. For gate lengths below 50 nm, the overlap of the modulated region with the highly conductive junctions leads to saturation of the SNR. For cross-sections below 10-30 nm, SNR degrades due to the overlap of the modulated region with the convex FET corners where a larger local gate capacitance reduces charge sensitivity. In our study, assuming a commercial solid-state FET noise amplitude, we find that a suspended nanowire FET architecture with 35 nm length and 5 × 10 nm2 cross-section results in the highest SNR of about 10 for a 15-base DNA oligo in a 15 mM electrolyte. In contrast with typical silicon nanowire FET sensors which possess micron-scale gate lengths, we find it to be key that all channel dimensions are scaled down to the decananometer range.

Electric field gradient focusing with electro-osmotic flow to reduce analyte dispersion: Concept and numerical investigation.

In proteomics, the need to precisely examine the protein compounds of small samples, requires sensitive analytical methods which can separate and enrich compounds with high precision. Current techniques require a minimal analysis time to obtain satisfactory compound separation where longer analysis time means better separation of compounds. But, molecular diffusion will create broadening of the separated compound bands over time, increasing the peak width, and thus reducing the resolution and the enrichment. Electric field gradient focusing (EFGF) is a separation technique, in which proteins are simultaneously separated and enriched by balancing a gradient electrostatic force with a constant hydrodynamic drag force. Because of this balance, analytes are continuously pushed back to their focusing point, limiting the time-dependent peak broadening due to molecular diffusion. Current EFGF techniques are however still suffering from peak broadening because of flow-profile inhomogeneities. In this paper, we propose to use AC electro-osmotic flow (AC EOF) to create a homogeneous flow in EFGF. The interference between the electric field gradient and the AC EOF was thoroughly analysed and the concept was validated using numerical simulations. The results show that a plug flow is obtained on top of a small, distorted boundary layer. While applying different DC electric fields in the electrolyte, a constant flow velocity can be obtained by including a DC offset to the electrodes generating the AC EOF. The plug flow is then maintained over the whole separation channel length, while an electric field gradient is applied. This way, the flow-induced contribution to peak broadening can be minimized in EFGF devices. By modelling the separation of green fluorescent protein (GFP) and R-Phycoerythrin (R-PE), it was shown that the peak width of separated compounds can be reduced and that the separation resolution can be improved, compared to current EFGF methods.

Measuring the electric charge and zeta potential of nanometer-sized objects using pyramidal-shaped nanopores.

Nanometer-scale pores are capable of detecting the size and concentration of nanometer-sized analytes at low concentrations upon analyzing their translocation through the pore, in small volumes and over a short time without labeling. Here, we present a simple, widely applicable, robust, and precise method to measure the zeta-potential of different nano-objects using nanopores. Zeta-potential i.e., a quantity that represents electrical charge in nanocolloids, is an important property in manufacturing of pharmaceuticals, inks, foams, cosmetics, and food. Its use is also imperative in understanding basic properties of complex dispersions including blood, living organisms, and their interaction with the environment. The characterization methods for zeta-potential are limited. Using the nanopore technique, the zeta-potential and the charge of nanoparticles can be measured independently of other parameters, such as particle size. This simple method is based on measuring the duration of the translocation of analytes through a nanopore as a function of applied voltage. A simple analytical model has been developed to extract the zeta-potential. This method is able to detect and differentiate nanometer-sized objects of similar size; it also enables the direct and precise quantitative measurement of their zeta-potential. We have applied this method to a wide range of different nanometer-sized particles and compared the results with values measured by commercially available tools. Furthermore, potential capability of this method in detection and characterization of virions is shown by measuring the low zeta-potential of HIV and EBV viruses.

The Significance of Nonlinear Screening and the pH Interference Mechanism in Field-Effect Transistor Molecular Sensors.

Electrolyte screening is well known for its detrimental impact on the sensitivity of liquid-gated field-effect transistor (FET) molecular sensors and is mostly described by the linearized Debye-Hückel model. However, charged and pH-sensitive FET sensing surfaces can limit the FET molecular sensitivity beyond the Debye-Hückel screening formalism. Pre-existing surface charges can lead to the breakdown of Debye-Hückel screening and induce enhanced nonlinear Poisson-Boltzmann screening. Moreover, the charging of the pH-sensitive surface groups interferes with biomolecule sensing resulting in a pH interference mechanism. With analytical equations and TCAD simulations, we highlight that the Debye-Hückel approximation can underestimate screening and overestimate FET molecular sensitivity by more than an order of magnitude. Screening strengthens significantly beyond Debye-Hückel in the proximity of even moderately charged surfaces and biomolecule charge densities (≥1 × 1012 q/cm2). We experimentally show the strong impact of both nonlinear screening and the pH interference effect on charge-based biomolecular sensing using a model system based on the covalent binding of single-stranded DNA on silicon FET sensors. The DNA signal increases from 24 mV at pH 7 to 96 mV at pH 3 in 1.5 mM PBS for a DNA density of 7 × 1012 DNA/cm2. Our model quantitatively explains the signal's pH dependence with roughly equal nonlinear screening and pH interference contributions. This work shows the importance of reducing the net charge and the pH sensitivity of the sensing surface to improve molecular sensing. Therefore, tailoring the gate dielectric and functional layer of FET sensors is a promising route to strong silicon FET molecular sensitivity boosts.

Rapid Quantification of Plasma Creatinine Using a Novel Kinetic Enzymatic Assay.

BACKGROUND: Enzymatic assays are among the most common diagnostic tests performed in the clinical laboratory. Enzymatic substrate analysis is most commonly measured using endpoint methods; however, modulating the reaction kinetics allows fine control of the reaction rate, which can be adjusted based on specific monitoring technologies. METHODS: We developed and optimized an enzymatic method for measurement of creatinine in plasma, using commonly paired enzymes of creatininase (Crtnnase), creatinase (Crtase), sarcosine oxidase (SOX), ascorbate oxidase (AOX), and horseradish peroxidase (HRP). The novel aspect of the assay is that it is fast and uses SOX as the limiting enzyme. The assay performance was assessed with respect to precision, accuracy, and interferences. RESULTS: The intrarun %CV (n = 12) was approximately 5% for each concentration tested, with biases ranging from -3 to -9%. The interrun %CV (n = 39) ranged from 5 to 8%, with biases ranging from -2 to -6%. During the accuracy assessment (n = 127), only 4 samples did not meet the minimum acceptability criteria. Minimal interference was observed, except at low creatinine concentrations with elevated creatine. CONCLUSION: Our novel and versatile enzymatic assay to measure plasma creatinine using kinetic analysis with SOX as the limiting enzyme is rapid (<2 mins), sensitive, and specific and demonstrates excellent concordance with the laboratory standard. We anticipate this rapid kinetic assay to be compatible with emerging technologies in the field of portable diagnostic devices, such as the usage of silicon photonics to monitor biochemical reactions.

Ion current rectification, limiting and overlimiting conductances in nanopores.

Previous reports on Poisson-Nernst-Planck (PNP) simulations of solid-state nanopores have focused on steady state behaviour under simplified boundary conditions. These are Neumann boundary conditions for the voltage at the pore walls, and in some cases also Donnan equilibrium boundary conditions for concentrations and voltages at both entrances of the nanopore. In this paper, we report time-dependent and steady state PNP simulations under less restrictive boundary conditions, including Neumann boundary conditions applied throughout the membrane relatively far away from the nanopore. We simulated ion currents through cylindrical and conical nanopores with several surface charge configurations, studying the spatial and temporal dependence of the currents contributed by each ion species. This revealed that, due to slow co-diffusion of oppositely charged ions, steady state is generally not reached in simulations or in practice. Furthermore, it is shown that ion concentration polarization is responsible for the observed limiting conductances and ion current rectification in nanopores with asymmetric surface charges or shapes. Hence, after more than a decade of collective research attempting to understand the nature of ion current rectification in solid-state nanopores, a relatively intuitive model is retrieved. Moreover, we measured and simulated current-voltage characteristics of rectifying silicon nitride nanopores presenting overlimiting conductances. The similarity between measurement and simulation shows that overlimiting conductances can result from the increased conductance of the electric double-layer at the membrane surface at the depletion side due to voltage-induced polarization charges. The MATLAB source code of the simulation software is available via the website http://micr.vub.ac.be.

Sensitive in vivo cell detection using size-optimized superparamagnetic nanoparticles.

Magnetic nanoparticle (MNP) enabled cell visualization with magnetic resonance imaging (MRI) is currently an intensively studied area of research. In the present study, we have synthesized polyethylene glycolated (PEG) MNPs and validated their suitability as MR cell labeling agents in in vitro and in vivo experiments. The labeling of therapeutic potent mesenchymal stem cells (MSCs) with small core and large core MNPs was evaluated. Both MNPs were, in combination with a transfection agent, stably internalized into the MSCs and didn't show an effect on cell metabolism. The labeled cells showed high contrast in MRI phantom studies. For quantification purposes, the MRI contrast generating properties of cells labeled with small core MNPs were compared with large core MNPs and with the commercial contrast agent Endorem. MSCs labeled with the large core MNPs showed the highest contrast generating properties in in vitro phantom studies and in in vivo intracranial stereotactic injection experiments, confirming the size-relaxivity relationship in biological systems. Finally, the distribution of MSCs pre-labeled with large core PEGylated MNPs was visualized non-invasively with MRI in a glioma model.

Synthetic antiferromagnetic nanoparticles as potential contrast agents in MRI.

We present the top-down synthesis of a novel type of MRI T2 contrast agent with great control over size and shape using a colloidal lithography technique. The resulting synthetic antiferromagnetic nanoparticles (SAF-NPs) yield improved relaxivities compared to superparamagnetic iron oxide alternatives (SPIONs). For T2 weighted imaging, the outer sphere relaxation theory has shown that the sensitivity of a T2 contrast agent is dependent on the particle size with an optimal size that exceeds the superparamagnetic limit of SPIONs. With the use of the interlayer exchange coupling effect, the SAF-NPs presented here do not suffer from this limit. Adjusting the outer sphere relaxation theory for spherical particles to SAF-NPs, we show both theoretically and experimentally that the SAF-NP size can be optimized to reach the r2 maximum. With measured r2 values up to 355 s(-1) mM(-1), our SAF-NPs show better performance than commercial alternatives and are competitive with the state-of-the-art. This performance is confirmed in an in vitro MRI study on SKOV3 cells.

Plasmonic modes of metallic semishells in a polymer film.

The symmetry-broken geometry and variation of metal composition of semishells induce new plasmonic properties. A system of separated metallic semishells embedded in a poly(dimethylsiloxane) polymer film provides an ideal platform to investigate the localized surface plasmon resonance modes of semishells. We demonstrate experimentally that silver, gold, copper, and aluminum semishells can offer distinct plasmonic responses due to the wide range of their material parameters. Numerical calculations combined with the plasmon hybridization theory render us a clear understanding and assignment of the plasmonic modes of the semishells.

Fabrication, characterization, and optical properties of gold nanobowl submonolayer structures.

We report on a versatile method to fabricate hollow gold nanobowls and complex gold nanobowls (with a core) based on an ion milling and a vapor HF etching technique. Two different sized hollow gold nanobowls are fabricated by milling and etching submonolayers of gold nanoshells deposited on a substrate, and their sizes and morphologies are characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Optical properties of hollow gold nanobowls with different sizes are investigated experimentally and theoretically, showing highly tunable plasmon resonance ranging from the visible to the near-infrared region. Additionally, finite difference time domain (FDTD) calculations show an enhanced localized electromagnetic field around hollow gold nanobowl structures, which indicates a potential application in surface-enhanced Raman scattering (SERS) spectroscopy for biomolecular detection. Finally, we demonstrate the fabrication of complex gold nanobowls with a gold nanoparticle core which offers the capability to create plasmon hybridized nanostructures.