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)-grafted-poly(ethylene glycol) (PLL-g-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. The biosensing functionality of the glycan-remodeled antibodies was demonstrated in a sandwich immunosensor for procalcitonin. The results show that glycan-remodeled antibodies enable oriented immobilization and biosensing functionality with low nonspecific binding on antifouling polymer substrates.

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. Experiments were performed wherein only the particles or only the surfaces were aged in order to clarify how each individual component changes over time. Furthermore, distributions of particle motion patterns and switching activity were studied to reveal how particle populations change over timespans of several days. For a cortisol sensor with anticortisol antibodies on the particles and cortisol analogues on the sensing surface, the leading hypotheses for the long-term changes are (i) that the particles lose antibodies and develop nonspecific interactions and (ii) that analogue molecules dissociate from the sensing surface. The developed methodologies and the acquired insights pave a way for realizing sensors that can operate over long timespans.

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. The technique is based on the optical tracking of particle motion and uses an antibody sandwich configuration. Experimental results show how the analyte concentration influences the particle diffusivity and characteristic response time of the sensor, and how the sensitivity range depends on the antibody functionalization density. Furthermore, the data clarifies how antibodies supplemented in solution can shorten the characteristic response time. Finally, we demonstrate association rate-based sensing as a first step towards continuous monitoring of picomolar TNF-α concentrations, over a period of 2 h with delay times under 15 min. The insights from this research will enable the development of continuous monitoring sensors using high-affinity binders, providing the sensitivity and speed needed in applications like cytokine monitoring.

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. By comparing characteristic response times at different incubation configurations, the data clarifies how two distinct reaction pathways play a role in the sandwich immunosensor, namely, analyte binding first to particles and thereafter to the substrate, and analyte binding first to the substrate and thereafter to a particle. For a concrete biosensor design, we found that the biosensor is dominated by the reaction pathway where analyte molecules bind first to the substrate and thereafter to a particle. Within this pathway, the binding of a particle to the substrate-bound analyte dominates the sensor response time. Thus, the probability of a particle interacting with the substrate was identified as the main direction to improve the speed of the biosensor while maintaining good sensitivity. We expect that the developed immunosensor and research methodology can be generally applied to understand the reaction mechanisms and optimize the kinetic properties of sandwich immunosensors with particle labels.

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. Phage-display antibody fragments were developed for the measurement of small molecules, as exemplified with the measurement of glycoalkaloids (GAs) in potato fruit juice. The recombinant antibodies were selected for use in a competition-based biosensor with single-molecule resolution, called biosensing by particle motion, using assay architectures with free particles as well as tethered particles. The resulting sensor measures GAs in the micromolar range, is reversible, has a measurement response time below 5 min, and enables continuous monitoring of GAs in protein-rich solutions for more than 20 h with concentration measurement errors below 15%. The demonstrated biosensor gives the perspective to enable a variety of monitoring and control strategies based on continuous measurement of small molecules in industrial food processes.

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. The method records digital events due to single-molecule interactions and enables biomarker monitoring at picomolar to micromolar concentrations without consuming any reagents. We demonstrate the affinity-based sensing methodology for DNA-based sandwich and competition assays, and for an antibody-based cortisol assay. Additionally, the sensor can be dried, facilitating storage over weeks while maintaining its sensitivity. We foresee that this will enable the development of continuous monitoring sensors for applications in fundamental research, for studies on organs on a chip, for the monitoring of patients in critical care, and for the monitoring of industrial processes and bioreactors as well as ecological systems.

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. The sensor is based on measuring single-molecule binding and unbinding events of tethered particles. The particles are functionalized with antibodies and the substrate with cortisol-analogues, causing binding and unbinding events to occur between particles and substrate. The frequency of binding events is reduced when cortisol is present in the solution as it blocks the binding sites of the antibodies. The sensor responds to cortisol in the high nanomolar to low micromolar range and can monitor cortisol concentrations over multiple hours. Results are shown for cortisol monitoring in filtered and in microdialysis-sampled human blood plasma.

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. Simulations are performed for sensors with diffusion-based sampling (e.g., a sensor patch on the skin) and advection-based sampling (e.g., a sensor connected to a catheter). The simulations clarify how transport processes and molecular binding kinetics result in concentration gradients and time delays in the sensor system. Using these simulations, measured and true concentration-time profiles of insulin were compared as a function of sensor design parameters. The results lead to guidelines on how biomolecular monitoring sensors can be designed for optimal bioanalytical performance in terms of concentration and time properties.

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. We demonstrate that low-concentration biomolecules can be monitored with small time delays, short time intervals, and in principle over an endless time span.

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. The biofunctionalization involves covalent coupling by click chemistry to PLL-g-PEG bottlebrush polymers. The polymer is grafted to a surface by multivalent electrostatic interactions, while the poly(ethylene glycol) suppresses nonspecific binding of biomolecules. With this biofunctionalization strategy, we demonstrate the continuous monitoring of single-stranded DNA and a medically relevant small-molecule analyte (creatinine), in sandwich and competitive assays, in buffer and in filtered blood plasma, with picomolar, nanomolar, and micromolar analyte concentrations, and with continuous sensor operation over 10 h.

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. From atomic force microscopy-based single-molecule force spectroscopy, we find that individual silica-binding peptides have high unbinding rates. Nevertheless, from quartz crystal microbalance measurements, we find that the self-assembled polypeptide brushes cannot easily be rinsed off. From atomic force microscopy imaging and bulk dynamic light scattering, we find that the binding to silica induces fibrillar self-assembly of the peptides. Hence, we conclude that the unexpected stability of these self-assembled polypeptide brushes is at least in part due to peptide-peptide interactions of the silica-binding blocks at the silica surface.

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. Single-molecule techniques are used to quantify stochastic, interparticle, and intraparticle variabilities, in order to show how these variabilities collectively contribute to reactivity variability per particle, and how the influence of each contributor changes as a function of the system parameters such as particle interaction area, the particle size, the targeting moiety density, and the number of particles. The results give insights into the consequences of superpositional heterogeneities for the reactivity variability in biomedical applications and give guidelines on how the precision can be optimized in the presence of multiple independent sources of variability.

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. This reversible switching yields binary transitions between two highly reproducible states, enabling reliable quantification of the single-molecule kinetics. The multiplexing functionality is encoded per particle via the dissociation characteristics of the nanoswitch, while the target concentration is revealed by the association characteristics. We demonstrate by experiments and simulations the multiplexed, continuous monitoring of oligonucleotide targets, at picomolar concentrations in buffer and in filtered human blood plasma.

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. At a pulling speed of 1 µm/s, the MpAFP extender domain fails at an unfolding force Fu = 348 ± 37 pN and MhLap at Fu = 306 ± 51 pN in buffer with 10 mM Ca2+. These forces place both extender domains well above the mechanical stability of many other ß-sandwich domains in mechanostable proteins. We propose that the increased stability of MpAFP and MhLap is due to a combination of both hydrogen bonding between parallel terminal strands and intra-molecular coordination of calcium ions.

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. The data reveal how the association rate depends on the particle velocity, particle density, and particle assembly characteristics. Interestingly, single particles appear to exhibit target depletion zones near their surface, caused by the high density of capture molecules. The depletion effects are even more limiting in cases with high particle densities. The depletion effects are overcome and protein capture rates are enhanced by applying dynamic particle actuation, resulting in an increase in the association rate constants by up to 2 orders of magnitude.

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. We have designed an experiment in which repeated motion trajectories are measured on pairs of particles. The experiment gives an independent quantification of the interfacial drag coefficient (10-7-10-4 Ns/m) and of the interparticle capillary forces (0.1-10 pN). We observed that the attractive capillary force depends on the amount of polymer in the oil phase and on the particle pair. However, the attraction appears to be independent of the surface rheology, with changes over a wide range of apparent viscosity values due to aging. Given the direction (attraction), the range (∼µm), and the distance dependence (∼1/S5) of the observed interparticle force, we interpret the force as being caused by quadrupolar deformations of the fluid-fluid interface induced by particle surface roughness. The results suggest that capillary forces are equilibrated in the early stages of interface aging and thereafter do not change anymore, even though strong changes in surface rheology still occur. The described experimental approach is powerful for studying dissipative as well as conservative forces of micro- and nanoparticles at fluid-fluid interfaces for systems out of equilibrium.

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. The use of local dipole-dipole forces makes the system insensitive to fluid flow and suited for simultaneously recording many particles in parallel over a long period of time. We apply IPM to study the time-dependent adsorption of an oil-soluble amino-modified silicone polymer at a water-oil interface using carboxylated magnetic particles. At low polymer concentration the carboxylated particles remain on the water side of the water-oil interface, while at high polymer concentrations the particles transit into the oil phase. Both conditions show a drag coefficient that does not depend on time. However, at intermediate polymer concentrations data show an increase of the interfacial drag coefficient as a function of time, with an increase over more than three orders of magnitude (10(-7) to 10(-4) N s m(-1)), pointing to a strong polymer-polymer interaction at the interface. The time-dependence of the interfacial drag appears to be highly sensitive to the polymer concentration and to the ionic strength of the aqueous phase. We foresee that IPM will be a very convenient technique to study fluid-fluid interfaces for a broad range of materials systems.

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. We further developed a thumb pressure-actuated applicator-assisted npMNA insertion method, which enables anchoring of MNs in the skin by an adhesive in one handling step. Using a set of simple artificial skin models, we found a puncture efficiency of this insertion method a factor three times higher than by applying thumb pressure on the npMNA base plate directly. In addition, this new method facilitated zero MN-breakage due to a well-defined force distribution exerted onto the MNs and the closely surrounding area prior to bringing the adhesive into contact with the skin. Owing to the fact that such parameter space exists, we can conclude that npMNAs by soft lithography are a platform technology for MN assembly into a patch.

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. The induced fluid flow is directly related to the stirring rate and the amount of magnetic particle chains. The observed behavior is caused by the magnetic and hydrodynamic interactions between the magnetic microparticles and the fluid. We utilized the phenomenon of swarming particles to enhance biochemical assays with magnetic capture particles (4000 µL(-1)) and IgG targets (500 pM). When compared to a reference system of sedimented magnetic capture particles, magnetic actuation leads to both a ~9 times increase in the initial assay kinetics as well as a ~7 times increase of target capture signal after 30 minutes.

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. We discuss the use of magnetic particles to mix fluids, to capture specific analytes, to concentrate analytes, to transfer analytes from one solution to another, to label analytes, to perform stringency and washing steps, and to probe biophysical properties of the analytes, distinguishing methodologies with fluid flow and without fluid flow (stationary microfluidics). Our review focuses on efforts to combine and integrate different magnetically actuated assay steps, with the vision that it will become possible in the future to realize integrated lab-on-chip biosensing assays in which all assay process steps are controlled and optimized by magnetic forces.