Real-time continuous monitoring of dynamic concentration profiles studied with biosensing by particle motion.

Real-time monitoring-and-control of biological systems requires lab-on-a-chip sensors that are able to accurately measure concentration-time profiles with a well-defined time delay and accuracy using only small amounts of sampled fluid. Here, we study real-time continuous monitoring of dynamic concentration profiles in a microfluidic measurement chamber. Step functions and sinusoidal oscillations of concentrations were generated using two pumps and a herringbone mixer. Concentrations in the bulk of the measurement chamber were quantified using a solution with a dye and light absorbance measurements. Concentrations near the surface were measured using a reversible cortisol sensor based on particle motion. The experiments show how the total time delay of the real-time sensor has contributions from advection, diffusion, reaction kinetics at the surface and signal processing. The total time delay of the studied real-time cortisol sensor was ∼90 seconds for measuring 63% of the concentration change. Monitoring of sinusoidal cortisol concentration-time profiles showed that the sensor has a low-pass frequency response with a cutoff frequency of ∼4 mHz and a lag time of ∼60 seconds. The described experimental methodology paves the way for the development of monitoring-and-control in lab-on-a-chip systems and for further engineering of the analytical characteristics of real-time continuous biosensors.

High-Throughput Single-Molecule Sensors: How Can the Signals Be Analyzed in Real Time for Achieving Real-Time Continuous Biosensing?

Single-molecule sensors collect statistics of single-molecule interactions, and the resulting data can be used to determine concentrations of analyte molecules. The assays are generally end-point assays and are not designed for continuous biosensing. For continuous biosensing, a single-molecule sensor needs to be reversible, and the signals should be analyzed in real time in order to continuously report output signals, with a well-controlled time delay and measurement precision. Here, we describe a signal processing architecture for real-time continuous biosensing based on high-throughput single-molecule sensors. The key aspect of the architecture is the parallel computation of multiple measurement blocks that enables continuous measurements over an endless time span. Continuous biosensing is demonstrated for a single-molecule sensor with 10,000 individual particles that are tracked as a function of time. The continuous analysis includes particle identification, particle tracking, drift correction, and detection of the discrete timepoints where individual particles switch between bound and unbound states, yielding state transition statistics that relate to the analyte concentration in solution. The continuous real-time sensing and computation were studied for a reversible cortisol competitive immunosensor, showing how the precision and time delay of cortisol monitoring are controlled by the number of analyzed particles and the size of the measurement blocks. Finally, we discuss how the presented signal processing architecture can be applied to various single-molecule measurement methods, allowing these to be developed into continuous biosensors.

Conformation switching of single native proteins revealed by nanomechanical probing without a pulling force.

Protein conformational changes are essential to biological function, and the heterogeneous nature of the corresponding protein states provokes an interest to measure conformational changes at the single molecule level. Here we demonstrate that conformational changes in single native proteins can be revealed by non-covalent antibody-targeting of specific domains within the protein, using nanomechanical probing without an applied pulling force. The protein of interest was captured between a particle and a substrate and three properties were quantified: the twist amplitude related to an applied torque, torsional compliance related to rotational Brownian motion, and translational Brownian displacement. Calcium-dependent conformation switching was studied in native human cardiac troponin, a heterotrimer protein complex that regulates the contraction and relaxation of heart muscle cells and is also a key biomarker for diagnosing myocardial infarction. The data reveal a change in mechanical properties upon conformation switching from the non-saturated to the calcium-saturated state, which in cardiomyocytes gives myosin motor proteins access to actin filaments. A clear increase was observed in the molecular stiffness for the calcium-saturated protein conformation. Using libraries of monoclonal antibodies, the nanomechanical probing of conformation by antibody targeting opens avenues for characterizing single native protein complexes for research as well as for diagnostic applications.

Continuous biomarker monitoring by particle mobility sensing with single molecule resolution.

Healthcare is in demand of technologies for real-time sensing in order to continuously guard the state of patients. Here we present biomarker-monitoring based on the sensing of particle mobility, a concept wherein particles are coupled to a substrate via a flexible molecular tether, with both the particles and substrate provided with affinity molecules for effectuating specific and reversible interactions. Single-molecular binding and unbinding events modulate the Brownian particle motion and the state changes are recorded using optical scattering microscopy. The technology is demonstrated with DNA and protein as model biomarkers, in buffer and in blood plasma, showing sensitivity to picomolar and nanomolar concentrations. The sensing principle is direct and self-contained, without consuming or producing any reactants. With its basis in reversible interactions and single-molecule resolution, we envisage that the presented technology will enable biosensors for continuous biomarker monitoring with high sensitivity, specificity, and accuracy.

Nanoscale Interparticle Distance within Dimers in Solution Measured by Light Scattering.

We demonstrate a novel approach to quantify the interparticle distance in colloidal dimers using Mie scattering. The interparticle distance is varied in a controlled way by changing the ionic strength of the solution and the magnetic attraction between the particles. The measured scaling behavior is interpreted using an energy-distance model that includes the repulsive electrostatic and attractive magnetic interactions. The center-to-center distances of particles with a 525 nm radius can be determined with a root-mean-square accuracy of 12 nm. The data show that the center-to-center distance is larger by 83 nm compared to perfect spheres. The underlying distance offset can be attributed to repulsion by charged protrusions caused by particle surface roughness. The measurement method accurately quantifies interparticle distances that can be used to study cluster formation and colloid aggregation in complex systems, e.g., in biosensing applications.

Particle Motion Analysis Reveals Nanoscale Bond Characteristics and Enhances Dynamic Range for Biosensing.

Biofunctionalized colloidal particles are widely used as labels in bioanalytical assays, lab-on-chip devices, biophysical research, and in studies on live biological systems. With detection resolution going down to the level of single particles and single molecules, understanding the nature of the interaction of the particles with surfaces and substrates becomes of paramount importance. Here, we present a comprehensive study of motion patterns of colloidal particles maintained in close proximity to a substrate by short molecular tethers (40 nm). The motion of the particles (500-1000 nm) was optically tracked with a very high localization accuracy (below 3 nm). A surprisingly large variation in motion patterns was observed, which can be attributed to properties of the particle-molecule-substrate system, namely the bond number, the nature of the bond, particle protrusions, and substrate nonuniformities. Experimentally observed motion patterns were compared to numerical Monte Carlo simulations, revealing a close correspondence between the observed motion patterns and properties of the molecular system. Particles bound via single tethers show distinct disc-, ring-, and bell-shaped motion patterns, where the ring- and bell-shaped patterns are caused by protrusions on the particle in the direct vicinity of the molecular attachment point. Double and triple tethered particles exhibit stripe-shaped and triangular-shaped motion patterns, respectively. The developed motion pattern analysis allows for discrimination between particles bound by different bond types, which opens the possibility to improve the limit of detection and the dynamic range of bioanalytical assays, with a projected increase of dynamic range by nearly 2 orders of magnitude.

Distance within colloidal dimers probed by rotation-induced oscillations of scattered light.

Aggregation processes of colloidal particles are of broad scientific and technological relevance. The earliest stage of aggregation, when dimers appear in an ensemble of single particles, is very important to characterize because it opens routes for further aggregation processes. Furthermore, it represents the most sensitive phase of diagnostic aggregation assays. Here, we characterize dimers by rotating them in a magnetic field and by recording the angle dependence of light scattering. At small scattering angles, the scattering cross section can be approximated by the total cross-sectional area of the dimer. In contrast, at scattering angles around 90 degrees, we reveal that the dependence of the scattering cross section on the dimer angle shows a series of peaks per single 2π rotation of the dimers. These characteristics originate from optical interactions between the two particles, as we have verified with two-particle Mie scattering simulations. We have studied in detail the angular positions of the peaks. It appears from simulations that the influence of particle size polydispersity, Brownian rotation and refractive index on the angular positions of the peaks is relatively small. However, the angular positions of the peaks strongly depend on the distance between the particles. We find a good correspondence between measured data and calculations for a gap of 180 nm between particles having a diameter of 1 micrometer. The experiment and simulations pave the way for extracting distance-specific data from ensembles of dimerizing colloidal particles, with application for sensitive diagnostic aggregation assays.

Probing the force-induced dissociation of aptamer-protein complexes.

Aptamers are emerging as powerful synthetic bioreceptors for fundamental research, diagnostics, and therapeutics. For further advances, it is important to gain a better understanding of how aptamers interact with their targets. In this work, we have used magnetic force-induced dissociation experiments to study the dissociation process of two different aptamer-protein complexes, namely for hIgE and Ara h 1. The measurements show that both complexes exhibit dissociation with two distinct regimes: the dissociation rate depends weakly on the applied force at high forces but depends stronger on force at low forces. We attribute these observations to the existence of at least one intermediate state and at least two energy barriers in the aptamer-protein interaction. The measured spontaneous dissociation rate constants were validated with SPR using both Biacore and fiber optic technology. This work demonstrates the potential of the magnetic force-induced dissociation approach for an in-depth study of the dissociation kinetics of aptamer-protein bonds, which is not possible with SPR technologies. The results will help in the development and expansion of aptamers as bioaffinity probes.

High-efficiency dielectrophoretic ratchet.

Brownian ratchets enable the use of thermal motion in performing useful work. They typically employ spatial asymmetry to rectify nondirected external forces that drive the system out of equilibrium (cf. running marbles on a shaking washboard). The major application foreseen for Brownian ratchets is high-selectivity fractionation of particle or molecule distributions. Here, we investigate the functioning of an important model system, the on/off ratchet for water-suspended particles, in which interdigitated finger electrodes can be switched on and off to create a time-dependent, spatially periodic but asymmetric potential. Surprisingly, we find that mainly dielectrophoretic rather than electrophoretic forces are responsible for the ratchet effect. This has major implications for the (a)symmetry of the ratchet potential and the settings needed for optimal performance. We demonstrate that by applying a potential offset the ratchet can be optimized such that its particle displacement efficiency reaches the theoretical upper limit corresponding to the electrode geometry and particle size. Efficient fractionation based on size selectivity is therefore not only possible for charged species, but also for uncharged ones, which greatly expands the applicability range of this type of Brownian ratchet.

Quantification of protein-ligand dissociation kinetics in heterogeneous affinity assays.

Biochemical affinity assays inherently involve interactions of heterogeneous nature. We report a methodology to discriminate between and accurately characterize specific and nonspecific interactions in force-induced dissociation assays. Ligand-coupled superparamagnetic particles are incubated on surfaces coated with a mixture of specific receptors and nonspecifically interacting proteins. Consequently, a mixed population of surface bound particles is formed with different binding natures. Magnetic field gradients are used to apply translational forces on the bound particles. Using a multicomponent dissociation analysis, we are able to make a distinction between weak nonspecific interactions, strong nonspecific interactions, and specific interactions. We validate the model by comprehensive experiments in which the biochemical components and applied forces are varied. The low-force data yield reliable values for the spontaneous dissociation rates of single-molecule specific bonds, and at high forces, the bond barriers are modified by the applied force. The results generate a new perspective for applications of magnetic force affinity assays in studies of heterogeneous molecular biorecognition.

One-step homogeneous magnetic nanoparticle immunoassay for biomarker detection directly in blood plasma.

Assay technologies capable of detecting low biomarker concentrations in complex biological samples are fundamental for biological research and for applications in medical diagnostics. In this paper we address the challenge to perform protein biomarker detection homogeneously in one single step, applying a minute amount of reagent directly into whole human blood plasma, avoiding any sample dilution, separation, amplification, or fluid manipulation steps. We describe a one-step homogeneous assay technology based on antibody-coated magnetic nanoparticles that are spiked in very small amount directly into blood plasma. Pulsed magnetic fields and a double-linker molecular architecture are used to generate high biomarker-induced binding and low nonspecific binding between the nanoparticles. We demonstrate dose-response curves for prostate specific antigen (PSA) measured in undiluted human blood plasma with a detection limit of 400-500 femtomol/L, in a total assay time of 14 min and an optically probed volume of only 1 nL. We explain the dose-response curves with a model based on discrete binding of biomarker molecules onto the nanoparticles, which allows us to extract reaction parameters for the binding of biomarker molecules onto the nanoparticles and for the biomarker-induced binding between nanoparticles. The demonstrated analytical performance and understanding of the nanoparticle assay technology render it of interest for a wide range of applications in quantitative biology and medical diagnostics.

Roughness and ordering at the interface of oxidized polystyrene and water.

For the first time, atomistically detailed molecular dynamics calculations revealed molecular ordering of the water-oxidized atactic polystyrene (aPS) interface. Both ordering of the water molecules and the phenyl rings occur. In addition, the natural roughness of the surface has been simulated and compared to experimental values. The composition of the simulated aPS films is based on spin-coated aPS films that have been oxidized and characterized experimentally. The aPS surfaces are oxidized with ultraviolet-ozone radiation and have been characterized by XPS, AFM, and water contact angle measurements. XPS measurements show that the oxygen content in the sample increases rapidly with exposure and reaches saturation near 24 at. % of oxygen. The molecular dynamics simulations show smoothening of an hydrophobic aPS surface upon transition from vacuum to water. The smoothening decreases with increasing hydrophilicity. The calculations reveal ordering of oxidized phenyl rings for aPS surfaces in water. The order increases with increasing hydrophilicity. Additionally, we investigated the water structure near the aPS-water interface as a function of the surface hydrophilicity. With increasing hydrophilicity, the density of water at the aPS-water interface increases. The water density profile is steeper in the presence of hydrophobic aPS. The water shows an ordered layer near both the hydrophobic and hydrophilic surfaces; the position of this layer shifts toward the interface with increasing hydrophilicity.

Frequency-selective rotation of two-particle nanoactuators for rapid and sensitive detection of biomolecules.

We describe an optomagnetic bionanotechnology for rapid and sensitive solution-based affinity assays. Nanoactuators made from bioactive magnetic nanoparticles undergo rotational motion in the volume of a fluid under frequency-controlled magnetic actuation. The nanoactuators show a time-dependent scattering cross-section to an incoming light beam. We demonstrate that the temporal behavior of the scattered light intensity relates to the number, the magnetic properties and the size distribution of the nanoactuators, independently revealing the average value and variation in the magnetic properties of the nanoparticles as well as the concentration of nanoactuators. The method is applied to detect biomolecules in fluid by interparticle binding. In a total assay time of less than 3 min, we demonstrate a limit of detection lower than 400 fM in buffer and 5 pM in human plasma.

Sensitivities of InGaAsP photonic crystal membrane nanocavities to hole refractive index.

The sensitivities of resonant wavelengths of photonic crystal (PhC) membrane nanocavities with embedded InAs quantum dots to the ambient refractive index are reported for use in (bio) chemical sensing. The resonances for the different modes of several point-defect type cavities are obtained by photoluminescence measurements. Systematic trends of the variation of sensitivity with increase of the overlap of the modes with the PhC holes are observed for varying cavity type as well as for a given mode within a cavity type. A maximum sensitivity of approximately 300 nm/RIU (refractive index unit) is observed, corresponding to approximately 25% mode overlap with the holes and complete infiltration with the aqueous solution.

Magnetically controlled rotation and torque of uniaxial microactuators for lab-on-a-chip applications.

We demonstrate the controlled rotation and torque generated by uniaxial magnetic microactuators formed by two bound superparamagnetic particles in a fluid. The torque and rotation are precisely controlled by rotating magnetic fields, generated by an external electromagnet or by on-chip current wires. We present the magnetic energy equations and the equations of motion for two-particle microactuators, with contributions from the permanent and induced magnetic moments of the particles. A comparison of theory and experiments allows an estimation of the different moments with accuracy better than 10% across a wide frequency range. At low frequencies and low magnitudes of the applied magnetic field, both the permanent and induced moments of the particles have contributions to the torque. At either high fields or high frequencies, the torque is dominated by the induced moment. The predictability of the torque is highest in the regime of low frequencies and high field, where the torque has a large magnitude and is determined by the magnetic shape anisotropy of the microactuator. A comparison of rotation in bulk fluid and on a chip surface shows an increase of friction by a factor 9 originating from the surface proximity. The detailed understanding of the torque and rotation of two-particle uniaxial magnetic microactuators opens a range of possibilities in lab-on-a-chip applications, such as the actuation of single molecules, fluid mixing in microfluidic chambers, and novel cluster-based assays.

Micro-fluidic actuation using magnetic artificial cilia.

We demonstrate advanced fluid manipulations using magnetic polymeric artificial cilia on the walls of a microfluidic channel. In nature, cilia are little hairs covering the surface of micro-organisms which enable them to manipulate a fluid on the micro-scale. The asymmetric movement of natural cilia is crucial to obtain a net fluid flow. We have developed a ferromagnetic polymer made from iron nanoparticles and polydimethylsiloxane, and describe a process that can structure the material into high aspect ratio lying artificial cilia with a length of 300 microm. These artificial cilia were actuated with a homogeneous rotating magnetic field (micro(0)H < 50 mT) generated with a compact external electromagnet. An asymmetric movement involving torsion could be created when the cilia were provided with a remanent magnetisation perpendicular to the plane of rotation of the magnetic field vector. The artificial cilia could be actuated in fluid up to a frequency of approximately 50 Hz. In an aqueous solution in a microfluidic chamber we were able to generate rotational as well as translational fluid movements with fluid velocities up to approximately 0.5 mm s(-1).

The mutual diffusion coefficient for (meth)acrylate monomers as determined with a nuclear microprobe.

The value of the mutual diffusion coefficient DV of two acrylic monomers is determined with nuclear microprobe measurements on a set of polymer films. These films have been prepared by allowing the monomers to diffuse into each other for a certain time and subsequently applying fast ultraviolet photo-polymerization, which freezes the concentration profile. The monomer diffusion profiles are studied with a scanning 2.1 MeV proton microprobe. Each monomer contains a marker element, e.g., Cl and Si, which are easily detected with proton induced x-ray emission. From the diffusion profiles, it is possible to determine the mutual diffusion coefficient. The mutual diffusion coefficient is dependent of concentration, which is concluded from the asymmetry in the Cl- and Si-profiles. A linear dependence of the mutual diffusion coefficient on the composition is used as a first order approximation. The best fits are obtained for a value of b=(0.38+/-0.15), which is the ratio of the diffusion coefficient of 1,3-bis(3-methacryloxypropyl)-1, 1,3,3-tetramethyldisiloxane in pure 2-chloroethyl acrylate and the diffusion coefficient of 2-chloroethyl acrylate in pure 1,3-bis(3-methacryloxypropyl)-1,1,3,3-tetramethyldisiloxane. Under the assumption of a linear dependence of the mutual diffusion coefficient DV on monomer composition, it follows that DV = (2.9+/-0.6)10(-10) m(2)/s at a 1:1 monomer ratio. With Flory-Huggins expressions for the monomer chemical potentials, one can derive approximate values for the individual monomer diffusion coefficients.