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.

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.

PLA2G15 is a Lysosomal BMP Hydrolase with Ester Position Specificity and its Targeting Ameliorates Lysosomal Disease.

Lysosomes catabolize lipids and other biological molecules, a function essential for cellular and organismal homeostasis. Key to lipid catabolism in the lysosome is bis(monoacylglycero)phosphate (BMP), a major lipid constituent of intralysosomal vesicles (ILVs) and a stimulator of lipid-degrading enzymes. BMP levels are altered in a broad spectrum of human conditions, including neurodegenerative diseases. Although BMP synthase was recently discovered, it has long been thought that BMP's unique stereochemistry confers resistance to acid phospholipases, a requirement for its role in the lysosome. Here, we demonstrate that PLA2G15, a major lysosomal phospholipase, efficiently hydrolyzes BMP with primary esters regardless of stereochemistry. Interestingly, we discover that BMP's unique esterification position is what confers resistance to hydrolysis. Purified PLA2G15 catabolizes most BMP species derived from cell and tissue lysosomes under acidic conditions. Furthermore, PLA2G15 catalytic activity against synthesized BMP stereoisomers with primary esters was comparable to its canonical substrates. Conversely, BMP with secondary esters is intrinsically stable in vitro and requires acyl migration for hydrolysis in lysosomes. Consistent with our biochemical data, PLA2G15-deficient tissues and cells accumulate multiple BMP species, a phenotype reversible by supplementing wildtype PLA2G15 but not its catalytically dead mutant. Increasing BMP levels by targeting PLA2G15 reverses the cholesterol accumulation phenotype in Niemann Pick Disease Type C (NPC1) patient fibroblasts and significantly ameliorate disease pathologies in NPC1-deficient mice leading to extended lifespan. Our findings establish the rules that govern the stability of BMP in the lysosome and identify PLA2G15 as a lysosomal BMP hydrolase and as a potential target for modulating BMP levels for therapeutic intervention.

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.

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.

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.

Duplex Labeling and Manipulation of Neuronal Proteins Using Sequential CRISPR/Cas9 Gene Editing.

CRISPR/Cas9-mediated knock-in methods enable the labeling of individual endogenous proteins to faithfully determine their spatiotemporal distribution in cells. However, reliable multiplexing of knock-in events in neurons remains challenging because of cross talk between editing events. To overcome this, we developed conditional activation of knock-in expression (CAKE), allowing efficient, flexible, and accurate multiplex genome editing in rat neurons. To diminish cross talk, CAKE is based on sequential, recombinase-driven guide RNA (gRNA) expression to control the timing of genomic integration of each donor sequence. We show that CAKE is broadly applicable to co-label various endogenous proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels and neurotransmitter receptor subunits. To take full advantage of CAKE, we resolved the nanoscale co-distribution of endogenous synaptic proteins using super-resolution microscopy, demonstrating that their co-organization depends on synapse size. Finally, we introduced inducible dimerization modules, providing acute control over synaptic receptor dynamics in living neurons. These experiments highlight the potential of CAKE to reveal new biological insight. Altogether, CAKE is a versatile method for multiplex protein labeling that enables the detection, localization, and manipulation of endogenous proteins in neurons.Significance StatementAccurate localization and manipulation of endogenous proteins is essential to unravel neuronal function. While labeling of individual proteins is achievable with existing gene editing techniques, methods to label multiple proteins in neurons are limiting. We introduce a new CRISPR/Cas9 strategy, CAKE, achieving faithful duplex protein labeling using sequential editing of genes. We use CAKE to visualize the co-localization of essential neuronal proteins, including cytoskeleton components, ion channels and synaptic scaffolds. Using super-resolution microscopy, we demonstrate that the co-organization of synaptic scaffolds and neurotransmitter receptors scales with synapse size. Finally, we acutely modulate the dynamics of synaptic receptors using labeling with inducible dimerization domains. Thus, CAKE mediates accurate duplex endogenous protein labeling and manipulation to address biological questions in neurons.

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.

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons.

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.

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.

SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse.

Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders ("synaptopathies"). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org).

Functional Analysis of Genetic Variation in the SECIS Element of Thyroid Hormone Activating Type 2 Deiodinase.

CONTEXT: Thyroid hormone is important for normal brain development. The type 2 deiodinase (D2) controls thyroid hormone action in the brain by activating T4 to T3. The enzymatic activity of D2 depends on the incorporation of selenocysteine for which the selenocysteine-insertion sequence (SECIS) element located in the 3' untranslated region is indispensable. We hypothesized that mutations in the SECIS element could affect D2 function, resulting in a neurocognitive phenotype. OBJECTIVE: To identify mutations in the SECIS element of DIO2 in patients with intellectual disability and to test their functional consequences. DESIGN, SETTING, AND PATIENTS: The SECIS element of DIO2 was sequenced in 387 patients with unexplained intellectual disability using a predefined pattern of thyroid function tests. SECIS element read-through in wild-type or mutant D2 was quantified by a luciferase reporter system in transfected cells. Functional consequences were assessed by quantifying D2 activity in cell lysate or intact cell metabolism studies. RESULTS: Sequence analysis revealed 2 heterozygous mutations: c.5703C>T and c.5730A>T, which were also present in the unaffected family members. The functional evaluation showed that both mutations did not affect D2 enzyme activity in cell lysates or intact cells, although the 5730A>T mutation decreased SECIS element read-through by 75%. In the patient harboring the c.5730A>T variant, whole genome sequencing revealed a pathogenic deletion of the STXBP1 gene. CONCLUSIONS: We report on two families with mutations in the SECIS element of D2. Although functional analysis showed that nucleotide 5730 is important for normal SECIS element read-through, the two variants did not segregate with a distinct phenotype.

RIM C2B Domains Target Presynaptic Active Zone Functions to PIP2-Containing Membranes.

Rapid and efficient synaptic vesicle fusion requires a pool of primed vesicles, the nearby tethering of Ca2+ channels, and the presence of the phospholipid PIP2 in the target membrane. Although the presynaptic active zone mediates the first two requirements, it is unclear how fusion is targeted to membranes with high PIP2 content. Here we find that the C2B domain of the active zone scaffold RIM is critical for action potential-triggered fusion. Remarkably, the known RIM functions in vesicle priming and Ca2+ influx do not require RIM C2B domains. Instead, biophysical experiments reveal that RIM C2 domains, which lack Ca2+ binding, specifically bind to PIP2. Mutational analyses establish that PIP2 binding to RIM C2B and its tethering to the other RIM domains are crucial for efficient exocytosis. We propose that RIM C2B domains are constitutive PIP2-binding modules that couple mechanisms for vesicle priming and Ca2+ channel tethering to PIP2-containing target membranes.