How Highly Heterogeneous Sensors with Single-Molecule Resolution can Result in Robust Continuous Monitoring Over Long Time Spans.

Biomolecular sensors with single-molecule resolution are composed of multitudes of transducers that measure state changes related to single-molecular binding and unbinding events. Conventionally, signals are aggregated from many individual transducers in order to achieve sufficient statistics. However, by aggregating signals, transducer-to-transducer differences are lost and heterogeneities cannot be studied.

Revealing Spatial Molecular Heterogeneity of High-Density Biofunctionalized Surfaces Using DNA-PAINT.

The quantification and control of molecular densities and distributions on biofunctionalized surfaces are key for enabling reproducible functions in biosciences. Here, we describe an analysis methodology for quantifying the density and spatial distribution of high-density biofunctionalized surfaces, with densities in the order of 10-10 biomolecules per μm area, in a short measurement time. The methodology is based on single-molecule DNA-PAINT imaging combined with simulation models that compensate for lifetime and spatial undersampling effects, resulting in three distinct molecule counting methods and a statistical test for spatial distribution.

Molecular Origins of Long-Term Changes in a Competitive Continuous Biosensor with Single-Molecule Resolution.

Biosensing by particle motion is a biosensing technology that relies on single-molecule interactions and enables the continuous monitoring of analytes from picomolar to micromolar concentration levels. However, during sensor operation, the signals are observed to change gradually. Here, we present a comprehensive methodology to elucidate the molecular origins of long-term changes in a particle motion sensor, focusing on a competitive sensor design under conditions without flow.

Oriented Antibody Coupling to an Antifouling Polymer Using Glycan Remodeling for Biosensing by Particle Motion.

Biosensors based on immobilized antibodies require molecular strategies that (i) couple the antibodies in a stable fashion while maintaining the conformation and functionality, (ii) give outward orientation of the paratope regions of the antibodies for good accessibility to analyte molecules in the biofluid, and (iii) surround the antibodies by antibiofouling molecules. Here, we demonstrate a method to achieve oriented coupling of antibodies to an antifouling poly(l-lysine)--poly(ethylene glycol) (PLL--PEG) substrate, using glycan remodeling to create antibody-DNA conjugates. The coupling, orientation, and functionality of the antibodies were studied using two analysis methods with single-molecule resolution, namely single-molecule localization microscopy and continuous biosensing by particle motion.

Towards continuous monitoring of TNF-α at picomolar concentrations using biosensing by particle motion.

The ability to continuously monitor cytokines is desirable for fundamental research studies and healthcare applications. Cytokine release is characterized by picomolar circulating concentrations, short half-lives, and rapid peak times. Here, we describe the characteristics and feasibility of a particle-based biosensing technique for continuous monitoring of TNF-α at picomolar concentrations.

Sandwich Immunosensor Based on Particle Motion: How Do Reactant Concentrations and Reaction Pathways Determine the Time-Dependent Response of the Sensor?

To control and optimize the speed of a molecular biosensor, it is crucial to quantify and understand the mechanisms that underlie the time-dependent response of the sensor. Here, we study how the kinetic properties of a particle-based sandwich immunosensor depend on underlying parameters, such as reactant concentrations and the size of the reaction chamber. The data of the measured sensor responses could be fitted with single-exponential curves, with characteristic response times that depend on the analyte concentration and the binder concentrations on the particle and substrate.

Real-Time Immunosensor for Small-Molecule Monitoring in Industrial Food Processes.

Industrial food processes are monitored to ensure that food is being produced with good quality, yield, and productivity. For developing innovative real-time monitoring and control strategies, real-time sensors are needed that can continuously report chemical and biochemical data of the manufacturing process. Here, we describe a generalizable methodology to develop affinity-based biosensors for the continuous monitoring of small molecules in industrial food processes.

Reversible Immunosensor for the Continuous Monitoring of Cortisol in Blood Plasma Sampled with Microdialysis.

Cortisol is a steroid hormone involved in a wide range of medical conditions. The level of the hormone fluctuates over time, but with traditional laboratory-based assays, such dynamics cannot be monitored in real time. Here, a reversible cortisol sensor is reported that allows continuous monitoring of cortisol in blood plasma using sampling by microdialysis.

Continuous biomarker monitoring with single molecule resolution by measuring free particle motion.

There is a need for sensing technologies that can continuously monitor concentration levels of critical biomolecules in applications such as patient care, fundamental biological research, biotechnology and food industry, as well as the environment. However, it is fundamentally difficult to develop measurement technologies that are not only sensitive and specific, but also allow monitoring over a broad concentration range and over long timespans. Here we describe a continuous biomolecular sensing methodology based on the free diffusion of biofunctionalized particles hovering over a sensor surface.

Real-Time Monitoring of Biomolecules: Dynamic Response Limits of Affinity-Based Sensors.

Sensors for monitoring biomolecular dynamics in biological systems and biotechnological processes in real time, need to accurately and precisely reconstruct concentration-time profiles. This requirement becomes challenging when transport processes and biochemical kinetics are important, as is typically the case for biomarkers at low concentrations. Here, we present a comprehensive methodology to study the concentration-time profiles generated by affinity-based sensors that continuously interact with a biological system of interest.

Sensing Methodology for the Rapid Monitoring of Biomolecules at Low Concentrations over Long Time Spans.

Studies on the dynamics of biological systems and biotechnological processes require measurement techniques that can reveal time dependencies of concentrations of specific biomolecules, preferably with small time delays, short time intervals between subsequent measurements, and the possibility to record over long time spans. For low-concentration biomolecules, these requirements are very challenging since low-concentration assays are typically slow and require new reagents in every assay. Here, we present a sensing methodology that enables rapid monitoring of picomolar and sub-picomolar concentrations in a reversible affinity-based assay, studied using simulations.

Click-Coupling to Electrostatically Grafted Polymers Greatly Improves the Stability of a Continuous Monitoring Sensor with Single-Molecule Resolution.

Sensing technologies for the real-time monitoring of biomolecules will allow studies of dynamic changes in biological systems and the development of control strategies based on measured responses. Here, we describe a molecular architecture and coupling process that allow continuous measurements of low-concentration biomolecules over long durations in a sensing technology with single-molecule resolution. The sensor is based on measuring temporal changes of the motion of particles upon binding and unbinding of analyte molecules.

Self-Assembly of Elastin-like Polypeptide Brushes on Silica Surfaces and Nanoparticles.

Control over the placement and activity of biomolecules on solid surfaces is a key challenge in bionanotechnology. While covalent approaches excel in performance, physical attachment approaches excel in ease of processing, which is equally important in many applications. We show how the precision of recombinant protein engineering can be harnessed to design and produce protein-based diblock polymers with a silica-binding and highly hydrophilic elastin-like domain that self-assembles on silica surfaces and nanoparticles to form stable polypeptide brushes that can be used as a scaffold for later biofunctionalization.

How Reactivity Variability of Biofunctionalized Particles Is Determined by Superpositional Heterogeneities.

The biofunctionalization of particles with specific targeting moieties forms the foundation for molecular recognition in biomedical applications such as targeted nanomedicine and particle-based biosensing. To achieve a high precision of targeting for nanomedicine and high precision of sensing for biosensing, it is important to understand the consequences of heterogeneities of particle properties. Here, we present a comprehensive methodology to study with experiments and simulations the collective consequences of particle heterogeneities on multiple length scales, called superpositional heterogeneities, in generating reactivity variability per particle.

Multiplexed Continuous Biosensing by Single-Molecule Encoded Nanoswitches.

Single-molecule techniques have become impactful in bioanalytical sciences, though the advantages for continuous biosensing are yet to be discovered. Here we present a multiplexed, continuous biosensing method, enabled by an analyte-sensitive, single-molecular nanoswitch with a particle as a reporter. The nanoswitch opens and closes under the influence of single target molecules.

Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps.

To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two bacterial adhesins MpAFP and MhLap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends.

How Actuated Particles Effectively Capture Biomolecular Targets.

Because of their high surface-to-volume ratio and adaptable surface functionalization, particles are widely used in bioanalytical methods to capture molecular targets. In this article, a comprehensive study is reported of the effectiveness of protein capture by actuated magnetic particles. Association rate constants are quantified in experiments as well as in Brownian dynamics simulations for different particle actuation configurations.

Interparticle Capillary Forces at a Fluid-Fluid Interface with Strong Polymer-Induced Aging.

We report on a measurement of forces between particles adsorbed at a water-oil interface in the presence of an oil-soluble polymer. The cationic polymer interacts electrostatically with the negatively charged particles, thereby modulating the particle contact angle and the magnitude of capillary attraction between the particles. However, polymer adsorption to the interface also generates an increase in the apparent interfacial viscosity over several orders of magnitude in a time span of a few hours.

Interfacial rheometry of polymer at a water-oil interface by intra-pair magnetophoresis.

We describe an interfacial rheometry technique based on pairs of micrometer-sized magnetic particles at a fluid-fluid interface. The particles are repeatedly attracted and repelled by well-controlled magnetic dipole-dipole forces, so-called interfacial rheometry by intra-pair magnetophoresis (IPM). From the forces (∼pN), displacements (∼μm) and velocities (∼μm s(-1)) of the particles we are able to quantify the interfacial drag coefficient of particles within a few seconds and over very long timescales.

Insertion Process of Ceramic Nanoporous Microneedles by Means of a Novel Mechanical Applicator Design.

Arrays of microneedles (MNAs) are integrated in an out-of-plane fashion with a base plate and can serve as patches for the release of drugs and vaccines. We used soft-lithography and micromolding to manufacture ceramic nanoporous (np)MNAs. Failure modes of ceramic npMNAs are as yet poorly understood and the question remained: is our npMNA platform technology ready for microneedle (MN) assembly into patches? We investigated npMNAs by microindentation, yielding average crack fracture forces above the required insertion force for a single MN to penetrate human skin.

Strong vortical flows generated by the collective motion of magnetic particle chains rotating in a fluid cell.

Magnetic microparticles, assembled into chains that are actuated with rotating magnetic fields, can be used as microstirrers to promote fluid transport and biochemical reactions in microfluidic systems. We show that, within a certain range of magnetic field rotation frequency, the microstirrers exhibit a coherent collective motion: the rotating magnetic particle chains move throughout the volume of a flat fluid cell and generate very strong (~1 mm s(-1)) and global (9 mm) vortical fluid flows, with many eddy-type substructures that fluctuate continuously in time, resembling turbulent flow. The collective motion makes the microstirrers not only defy gravity, but also move against magnetic field gradients.

Integrated lab-on-chip biosensing systems based on magnetic particle actuation--a comprehensive review.

The demand for easy to use and cost effective medical technologies inspires scientists to develop innovative lab-on-chip technologies for point-of-care in vitro diagnostic testing. To fulfill medical needs, the tests should be rapid, sensitive, quantitative, and miniaturizable, and need to integrate all steps from sample-in to result-out. Here, we review the use of magnetic particles actuated by magnetic fields to perform the different process steps that are required for integrated lab-on-chip diagnostic assays.

A method for time-resolved measurements of the mechanics of phagocytic cups.

The internalization of matter by phagocytosis is of key importance in the defence against bacterial pathogens and in the control of cancerous tumour growth. Despite the fact that phagocytosis is an inherently mechanical process, little is known about the forces and energies that a cell requires for internalization. Here, we use functionalized magnetic particles as phagocytic targets and track their motion while actuating them in an oscillating magnetic field, in order to measure the translational and rotational stiffnesses of the phagocytic cup as a function of time.

Disaggregation of microparticle clusters by induced magnetic dipole-dipole repulsion near a surface.

Ensembles of magnetic particles are known to align and aggregate into multi-particle clusters in an applied magnetic field, and the physical laws governing these processes are well described in literature. However, it has been elusive how to achieve the opposite process, i.e.