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.

Continuous Biosensing to Monitor Acute Systemic Inflammation, a Diagnostic Need for Therapeutic Guidance.

Continuous monitoring of acute inflammation can become a very important next step for guiding therapeutic interventions in severely ill patients. This Perspective discusses the current medical need for patients with acute inflammatory diseases and the potential of continuous biosensing technologies. First, we discuss biomarkers that could help to monitor the state of a patient with acute systemic inflammation based on theoretical studies and empirical data.

Mathematical model of the inflammatory response to acute and prolonged lipopolysaccharide exposure in humans.

One in five deaths worldwide is associated with sepsis, which is defined as organ dysfunction caused by a dysregulated host response to infection. An increased understanding of the pathophysiology of sepsis could provide improved approaches for early detection and treatment. Here we describe the development and validation of a mechanistic mathematical model of the inflammatory response, making use of a combination of in vitro and human in vivo data obtained from experiments where bacterial lipopolysaccharide (LPS) was used to induce an inflammatory response.

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.

Continuous Biosensor Based on Particle Motion: How Does the Concentration Measurement Precision Depend on Time Scale?

Continuous biosensors measure concentration-time profiles of biomolecular substances in order to allow for comparisons of measurement data over long periods of time. To make meaningful comparisons of time-dependent data, it is essential to understand how measurement imprecision depends on the time interval between two evaluation points, as the applicable imprecision determines the significance of measured concentration differences. Here, we define a set of measurement imprecisions that relate to different sources of variation and different time scales, ranging from minutes to weeks, and study these using statistical analyses of measurement data.

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 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.

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.

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.

Accurate staging of non-metastatic colon cancer with CT: the importance of training and practice for experienced radiologists and analysis of incorrectly staged cases.

Purpose: To investigate whether locoregional staging of colon cancer by experienced radiologists can be improved by training and feedback to minimize the risk of over-staging into the context of patient selection for neoadjuvant therapy and to identify potential pitfalls of CT staging by characterizing pathologic traits of tumors that remain challenging for radiologists.

Methods: Forty-five cases of stage I-III colon cancer were included in this retrospective study. Five experienced radiologists evaluated the CTs; 5 baseline scans followed by 4 sequential batches of 10 scans.

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.

Real-Time Detection of State Transitions in Stochastic Signals from Biological Systems.

Robust analysis of signals from stochastic biomolecular processes is critical for understanding the dynamics of biological systems. Measured signals typically show multiple states with heterogeneities and a wide range of state lifetimes. Here, we present an algorithm for robust detection of state transitions in experimental time traces where the properties of the underlying states are unknown.

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.

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.

Multivalent weak interactions enhance selectivity of interparticle binding.

Targeted drug delivery critically depends on the binding selectivity of cargo-transporting colloidal particles. Extensive theoretical work has shown that two factors are necessary to achieve high selectivity for a threshold receptor density: multivalency and weak interactions. Here, we study a model system of DNA-coated particles with multivalent and weak interactions that mimics ligand-receptor interactions between particles and cells.

Inter-particle biomolecular reactivity tuned by surface crowders.

The rate at which colloidal particles can form biomolecular bonds controls the kinetics of applications such as particle-based biosensing, targeted drug delivery and directed colloidal assembly. Here we study how the reactivity of the particle surface depends on its molecular composition, quantified by the inter-particle rate of aggregation in an optomagnetic cluster experiment. Particles were functionalized with DNA or with proteins for specific binding, and with polyethylene glycol as a passive surface crowder.

[18F]FDG PET/CT in the staging of inflammatory breast cancer: A systematic review.

Up to 78 % of patients with inflammatory breast cancer (IBC) present with axillary lymph node involvement and up to 40 % with distant metastases. Previous studies indicate that 2-deoxy-2-(F)fluoro-d-glucose ([F]FDG) positron emission tomography/computed tomography (PET/CT) might be used for initial staging in patients with inflammatory breast cancer (IBC). In other cancer types, [F]FDG PET/CT has been demonstrated to be a sensitive technique, providing complementary information on locoregional and distant disease to conventional imaging modalities.

Continuous Small-Molecule Monitoring with a Digital Single-Particle Switch.

The ability to continuously measure concentrations of small molecules is important for biomedical, environmental, and industrial monitoring. However, because of their low molecular mass, it is difficult to quantify concentrations of such molecules, particularly at low concentrations. Here, we describe a small-molecule sensor that is generalizable, sensitive, specific, reversible, and suited for continuous monitoring over long durations.