A fluorescent nanosensor paint detects dopamine release at axonal varicosities with high spatiotemporal resolution.

The neurotransmitter dopamine (DA) controls multiple behaviors and is perturbed in several major brain diseases. DA is released from large populations of specialized structures called axon varicosities. Determining the DA release mechanisms at such varicosities is essential for a detailed understanding of DA biology and pathobiology but has been limited by the low spatial resolution of DA detection methods. We used a near-infrared fluorescent DA nanosensor paint, adsorbed nanosensors detecting release of dopamine (AndromeDA), to detect DA secretion from cultured murine dopaminergic neurons with high spatial and temporal resolution. We found that AndromeDA detects discrete DA release events and extracellular DA diffusion and observed that DA release varies across varicosities. To systematically detect DA release hotspots, we developed a machine learning­based analysis tool. AndromeDA permitted the simultaneous visualization of DA release for up to 100 dopaminergic varicosities, showing that DA release hotspots are heterogeneous and occur at only ∼17% of all varicosities, indicating that many varicosities are functionally silent. Using AndromeDA, we determined that DA release requires Munc13-type vesicle priming proteins, validating the utility of AndromeDA as a tool to study the molecular and cellular mechanism of DA secretion.

Ratiometric Imaging of Catecholamine Neurotransmitters with Nanosensors.

Neurotransmitters are important signaling molecules in the brain and are relevant in many diseases. Measuring them with high spatial and temporal resolutions in biological systems is challenging. Here, we develop a ratiometric fluorescent sensor/probe for catecholamine neurotransmitters on the basis of near-infrared (NIR) semiconducting single wall carbon nanotubes (SWCNTs). Phenylboronic acid (PBA)-based quantum defects are incorporated into them to interact selectively with catechol moieties. These PBA-SWCNTs are further modified with poly(ethylene glycol) phospholipids (PEG-PL) for biocompatibility. Catecholamines, including dopamine, do not affect the intrinsic E11 fluorescence (990 nm) of these (PEG-PL-PBA-SWCNT) sensors. In contrast, the defect-related E11* emission (1130 nm) decreases by up to 35%. Furthermore, this dual functionalization allows tuning selectivity by changing the charge of the PEG polymer. These sensors are not taken up by cells, which is beneficial for extracellular imaging, and they are functional in brain slices. In summary, we use dual functionalization of SWCNTs to create a ratiometric biosensor for dopamine.

Signal Amplification and Near-Infrared Translation of Enzymatic Reactions by Nanosensors.

Enzymatic reactions are used to detect analytes in a range of biochemical methods. To measure the presence of an analyte, the enzyme is conjugated to a recognition unit and converts a substrate into a (colored) product that is detectable by visible (VIS) light. Thus, the lowest enzymatic turnover that can be detected sets a limit on sensitivity. Here, we report that substrates and products of horseradish peroxidase (HRP) and ß-galactosidase change the near-infrared (NIR) fluorescence of (bio)polymer modified single-walled carbon nanotubes (SWCNTs). They translate a VIS signal into a beneficial NIR signal. Moreover, the affinity of the nanosensors leads to a higher effective local concentration of the reactants. This causes a non-linear sensor-based signal amplification and translation (SENSAT). We find signal enhancement up to ≈120x for the HRP substrate p-phenylenediamine (PPD), which means that reactions below the limit of detection in the VIS can be followed in the NIR (≈1000 nm). The approach is also applicable to other substrates such as 3,3'-5,5'-tetramethylbenzidine (TMB). An adsorption-based theoretical model fits the observed signals and corroborates the sensor-based enhancement mechanism. This approach can be used to amplify signals, translate them into the NIR and increase sensitivity of biochemical assays.

Near-Infrared Fluorescent Biosensors Based on Covalent DNA Anchors.

Semiconducting single-walled carbon nanotubes (SWCNTs) are versatile near-infrared (NIR) fluorophores. They are noncovalently modified to create sensors that change their fluorescence when interacting with biomolecules. However, noncovalent chemistry has several limitations and prevents a consistent way to molecular recognition and reliable signal transduction. Here, we introduce a widely applicable covalent approach to create molecular sensors without impairing the fluorescence in the NIR (>1000 nm). For this purpose, we attach single-stranded DNA (ssDNA) via guanine quantum defects as anchors to the SWCNT surface. A connected sequence without guanines acts as flexible capture probe allowing hybridization with complementary nucleic acids. Hybridization modulates the SWCNT fluorescence and the magnitude increases with the length of the capture sequence (20 > 10 ≫ 6 bases). The incorporation of additional recognition units via this sequence enables a generic route to NIR fluorescent biosensors with improved stability. To demonstrate the potential, we design sensors for bacterial siderophores and the SARS CoV-2 spike protein. In summary, we introduce covalent guanine quantum defect chemistry as rational design concept for biosensors.

High Sensitivity Near-Infrared Imaging of Fluorescent Nanosensors.

Biochemical processes are fast and occur on small-length scales, which makes them difficult to measure. Optical nanosensors based on single-wall carbon nanotubes (SWCNTs) are able to capture such dynamics. They fluoresce in the near-infrared (NIR, 850-1700 nm) tissue transparency window and the emission wavelength depends on their chirality. However, NIR imaging requires specialized indium gallium arsenide (InGaAs) cameras with a typically low resolution because the quantum yield of normal Si-based cameras rapidly decreases in the NIR. Here, an efficient one-step phase separation approach to isolate monochiral (6,4)-SWCNTs (880 nm emission) from mixed SWCNT samples is developed. It enables imaging them in the NIR with high-resolution standard Si-based cameras (>50× more pixels). (6,4)-SWCNTs modified with (GT)10 -ssDNA become highly sensitive to the important neurotransmitter dopamine. These sensors are 1.7× brighter and 7.5× more sensitive and allow fast imaging (<50 ms). They enable high-resolution imaging of dopamine release from cells. Thus, the assembly of biosensors from (6,4)-SWCNTs combines the advantages of nanosensors working in the NIR with the sensitivity of (Si-based) cameras and enables broad usage of these nanomaterials.

Near-Infrared Fluorescence Lifetime Imaging of Biomolecules with Carbon Nanotubes.

Single-walled carbon nanotubes (SWCNTs) are versatile near infrared (NIR) fluorescent building blocks for biosensors. Their surface is chemically tailored to respond to analytes by a change in fluorescence. However, intensity-based signals are easily affected by external factors such as sample movements. Here, we demonstrate fluorescence lifetime imaging microscopy (FLIM) of SWCNT-based sensors in the NIR. We tailor a confocal laser scanning microscope (CLSM) for NIR signals (>800 nm) and employ time correlated single photon counting of (GT)10 -DNA functionalized SWCNTs. They act as sensors for the important neurotransmitter dopamine. Their fluorescence lifetime (>900 nm) decays biexponentially and the longer lifetime component (370 ps) increases by up to 25 % with dopamine concentration. These sensors serve as paint to cover cells and report extracellular dopamine in 3D via FLIM. Therefore, we demonstrate the potential of fluorescence lifetime as a readout of SWCNT-based NIR sensors.

The power from within - understanding the driving forces of neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) are one of the most intriguing discoveries in immunological research of the past few years. After their first description in 2004, the number of research articles on how NETs affect immunodefense, and also how they contribute to an ever-growing number of diseases, has skyrocketed. However, tempting as it may seem to plunge into pharmaceutical approaches to tamper with NET formation, our understanding of this complex process is still incomplete. Important concepts such as the context-dependent dual functions of NETs, in that they are both inflammatory and anti-inflammatory, or the major intra- and extracellular forces driving NET formation, are only emerging. In this Review, we summarize key aspects of our current understanding of NET formation (also termed NETosis), emphasize biophysical aspects and focus on three key principles - rearrangement and destabilization of the plasma membrane and the cytoskeleton, alterations and disassembly of the nuclear envelope, and chromatin decondensation as a driving force of intracellular reorganization.

Prospects of Fluorescent Single-Chirality Carbon Nanotube-Based Biosensors.

Semiconducting single-wall carbon nanotubes (SWCNTs) fluoresce in the near-infrared (NIR), and the emission wavelength depends on their structure (chirality). Interactions with other molecules affect their fluorescence, which has successfully been used for SWCNT-based molecular sensors. So far, most such sensors are assembled from crude mixtures of different SWCNT chiralities, which causes polydisperse sensor responses as well as spectral congestion and limits their performance. The advent of chirality-pure SWCNTs is about to overcome this limitation and paves the way for the next generation of biosensors. Here, we discuss the first examples of chirality-pure SWCNT-based fluorescent biosensors. We introduce routes to such sensors via aqueous two-phase extraction-assisted purification of SWCNTs and highlight the critical interplay between purification and surface modification procedures. Applications include the NIR detection and imaging of neurotransmitters, reactive oxygen species, lipids, bacterial motives, and plant metabolites. Most importantly, we outline a path toward how such monodisperse (chirality-pure) sensors will enable advanced multiplexed sensing with enhanced bioanalytical performance.

NIR-emitting benzene-fused oligo-BODIPYs for bioimaging.

Near-infrared (NIR) fluorophores are emerging tools for biophotonics because of their reduced scattering, increased tissue penetration and low phototoxicity. However, the library of NIR fluorophores is still limited. Here, we report the NIR fluorescence of two benzene-fused oligo-BODIPYs in their hexameric (H) and octameric (O) forms. These dyes emit bright NIR fluorescence (H: maxima 943/1075 nm, O: maxima 976/1115 nm) that can be excited in the NIR (H = 921 nm, O = 956 nm) or non-resonantly over a broad range in the visible region. The emission bands of H show a bathochromic shift and peak sharpening with increasing dye concentration. Furthermore, the emission maxima of both H and O shift up to 20 nm in solvents of different polarity. These dyes can be used as NIR ink and imaged remotely on the macroscopic level with a stand-off distance of 20 cm. We furthermore demonstrate their versatility for biophotonics by coating microscale beads and performing microrheology via NIR video particle tracking (NIR-VPT) in biopolymer (F-actin) networks. No photodamaging of the actin filaments takes place, which is typically observed for visible fluorophores and highlights the advantages of these NIR dyes.

Biosensing with Fluorescent Carbon Nanotubes.

Biosensors are powerful tools for modern basic research and biomedical diagnostics. Their development requires substantial input from the chemical sciences. Sensors or probes with an optical readout, such as fluorescence, offer rapid, minimally invasive sensing of analytes with high spatial and temporal resolution. The near-infrared (NIR) region is beneficial because of the reduced background and scattering of biological samples (tissue transparency window) in this range. In this context, single-walled carbon nanotubes (SWCNTs) have emerged as versatile NIR fluorescent building blocks for biosensors. Here, we provide an overview of advances in SWCNT-based NIR fluorescent molecular sensors. We focus on chemical design strategies for diverse analytes and summarize insights into the photophysics and molecular recognition. Furthermore, different application areas are discussed-from chemical imaging of cellular systems and diagnostics to in vivo applications and perspectives for the future.

Detection and Imaging of the Plant Pathogen Response by Near-Infrared Fluorescent Polyphenol Sensors.

Plants use secondary metabolites such as polyphenols for chemical defense against pathogens and herbivores. Despite their importance in plant pathogen interactions and tolerance to diseases, it remains challenging to detect polyphenols in complex plant tissues. Here, we create molecular sensors for plant polyphenol imaging that are based on near-infrared (NIR) fluorescent single-wall carbon nanotubes (SWCNTs). We identified polyethylene glycol-phospholipids that render (6,5)-SWCNTs sensitive (Kd =90 nM) to plant polyphenols (tannins, flavonoids, …), which red-shift (up to 20 nm) and quench their emission (ca. 1000 nm). These sensors report changes in total polyphenol level after herbivore or pathogen challenge in crop plant systems (Soybean Glycine max) and leaf tissue extracts (Tococa spp.). We furthermore demonstrate remote chemical imaging of pathogen-induced polyphenol release from roots of soybean seedlings over the time course of 24 h. This approach allows in situ visualization and understanding of the chemical plant defense in real time and paves the way for plant phenotyping for optimized polyphenol secretion.

Sensing with Chirality-Pure Near-Infrared Fluorescent Carbon Nanotubes.

Semiconducting single-wall carbon nanotubes (SWCNTs) fluoresce in the near-infrared (NIR) region, and the emission wavelength depends on their chirality (n,m). Interactions with the environment affect the fluorescence and can be tailored by functionalizing SWCNTs with biopolymers such as DNA, which is the basis for fluorescent biosensors. So far, such biosensors have been mainly assembled from mixtures of SWCNT chiralities with large spectral overlap, which affects sensitivity as well as selectivity and prevents multiplexed sensing. The main challenge to gain chirality-pure sensors has been to combine approaches to isolate specific SWCNTs and generic (bio)functionalization approaches. Here, we created chirality-pure SWCNT-based NIR biosensors for important analytes such as neurotransmitters and investigated the effect of SWCNT chirality/handedness as well as long-term stability and sensitivity. For this purpose, we used aqueous two-phase extraction (ATPE) to gain chirality-pure (6,5)-, (7,5)-, (9,4)-, and (7,6)-SWCNTs (emission at ∼990, 1040, 1115, and 1130 nm, respectively). An exchange of the surfactant sodium deoxycholate (DOC) to specific single-stranded (ss)DNA sequences yielded monochiral sensors for small analytes (dopamine, riboflavin, ascorbic acid, pH). DOC residues impaired sensitivity, and therefore substantial removal was necessary. The assembled monochiral (6,5)-SWCNTs were up to 10 times brighter than their nonpurified counterparts, and the ssDNA sequence determined the absolute fluorescence intensity as well as colloidal (long-term) stability and selectivity for the analytes. (GT)40-(6,5)-SWCNTs displayed the maximum fluorescence response to the neurotransmitter dopamine (+140%, Kd = 1.9 × 10-7 M) and a long-term stability of >14 days. The specific ssDNA sequences imparted selectivity to the analytes mostly independent of SWCNT chirality and handedness of (±) (6,5)-SWCNTs, which allowed a predictable design. Finally, multiple monochiral/single-color SWCNTs were combined to achieve ratiometric/multiplexed sensing of the important analytes dopamine, riboflavin, H2O2, and pH. In summary, we demonstrated the assembly, characteristics, and potential of monochiral (single-color) SWCNTs for NIR fluorescence sensing applications.

Monitoring Plant Health with Near-Infrared Fluorescent H2O2 Nanosensors.

Near-infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) were designed and interfaced with leaves of Arabidopsis thaliana plants to report hydrogen peroxide (H2O2), a key signaling molecule associated with the onset of plant stress. The sensor nIR fluorescence response (>900 nm) is quenched by H2O2 with selectivity against other stress-associated signaling molecules and within the plant physiological range (10-100 H2O2 µM). In vivo remote nIR imaging of H2O2 sensors enabled optical monitoring of plant health in response to stresses including UV-B light (-11%), high light (-6%), and a pathogen-related peptide (flg22) (-10%), but not mechanical leaf wounding (<3%). The sensor's high biocompatibility was reflected on similar leaf cell death (<5%) and photosynthetic rates to controls without SWCNT. These optical nanosensors report early signs of stress and will improve our understanding of plant stress communication, provide novel tools for precision agriculture, and optimize the use of agrochemicals in the environment.

High-resolution imaging of cellular dopamine efflux using a fluorescent nanosensor array.

Intercellular communication via chemical signaling proceeds with both spatial and temporal components, but analytical tools, such as microfabricated electrodes, have been limited to just a few probes per cell. In this work, we use a nonphotobleaching fluorescent nanosensor array based on single-walled carbon nanotubes (SWCNTs) rendered selective to dopamine to study its release from PC12 neuroprogenitor cells at a resolution exceeding 20,000 sensors per cell. This allows the spatial and temporal dynamics of dopamine release, following K+ stimulation, to be measured at exceedingly high resolution. We observe localized, unlabeled release sites of dopamine spanning 100 ms to seconds that correlate with protrusions but not predominately the positive curvature associated with the tips of cellular protrusions as intuitively expected. The results illustrate how directionality of chemical signaling is shaped by membrane morphology, and highlight the advantages of nanosensor arrays that can provide high spatial and temporal resolution of chemical signaling.

Near-Infrared Imaging of Serotonin Release from Cells with Fluorescent Nanosensors.

Serotonin is an important neurotransmitter involved in various functions of the nervous, blood, and immune system. In general, detection of small biomolecules such as serotonin in real time with high spatial and temporal resolution remains challenging with conventional sensors and methods. In this work, we designed a near-infrared (nIR) fluorescent nanosensor (NIRSer) based on fluorescent single-walled carbon nanotubes (SWCNTs) to image the release of serotonin from human blood platelets in real time. The nanosensor consists of a nonbleaching SWCNT backbone, which is fluorescent in the beneficial nIR tissue transparency window (800-1700 nm) and a serotonin binding DNA aptamer. The fluorescence of the NIRSer sensor (995 nm emission wavelength for (6,5)-SWCNTs) increases in response to serotonin by a factor up to 1.8. It detects serotonin reversibly with a dissociation constant of 301 nM ± 138 nM and a dynamic linear range in the physiologically relevant region from 100 nM to 1 µM. As a proof of principle, we detected serotonin release patterns from activated platelets on the single-cell level. Imaging of the nanosensors around and under the platelets enabled us to locate hot spots of serotonin release and quantify the time delay (≈ 21-30 s) between stimulation and release in a population of platelets, highlighting the spatiotemporal resolution of this nanosensor approach. In summary, we report a nIR fluorescent nanosensor for the neurotransmitter serotonin and show its potential for imaging of chemical communication between cells.

Quantum Defects as a Toolbox for the Covalent Functionalization of Carbon Nanotubes with Peptides and Proteins.

Single-walled carbon nanotubes (SWCNTs) are a 1D nanomaterial that shows fluorescence in the near-infrared (NIR, >800 nm). In the past, covalent chemistry was less explored to functionalize SWCNTs as it impairs NIR emission. However, certain sp3 defects (quantum defects) in the carbon lattice have emerged that preserve NIR fluorescence and even introduce a new, red-shifted emission peak. Here, we report on quantum defects, introduced using light-driven diazonium chemistry, that serve as anchor points for peptides and proteins. We show that maleimide anchors allow conjugation of cysteine-containing proteins such as a GFP-binding nanobody. In addition, an Fmoc-protected phenylalanine defect serves as a starting point for conjugation of visible fluorophores to create multicolor SWCNTs and in situ peptide synthesis directly on the nanotube. Therefore, these quantum defects are a versatile platform to tailor both the nanotube's photophysical properties as well as their surface chemistry.

Nanobody-Conjugated Nanotubes for Targeted Near-Infrared In Vivo Imaging and Sensing.

Fluorescent nanomaterials such as single-walled carbon nanotubes (SWCNTs) have many advantages in terms of their photophysics, but it is difficult to target them to specific locations in living systems. In contrast, the green fluorescent protein (GFP) has been genetically fused to proteins in many cells and organisms. Therefore, GFP can be seen not only as a fluorophore but as a universal target/handle. Here, we report the conjugation of GFP-binding nanobodies to DNA-wrapped SWCNTs. This approach combines the targeting capabilities of GFP-binding nanobodies and the nonbleaching near-infrared fluorescence (850-1700 nm) of SWCNTs. These conjugates allow us to track single Kinesin-5-GFP motor proteins in developing embryos of Drosophila melanogaster. Additionally, they are sensitive to the neurotransmitter dopamine and can be used for targeted sensing of dopamine in the nm regime.

Carbon Nanotubes Encapsulated in Coiled-Coil Peptide Barrels.

Specific functionalization of 1D nanomaterials such as near infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) is essential for colloidal stability and tailoring of their interactions with the environment. Here, we show that de novo designed alpha-helical coiled-coil peptide barrels (αHBs) with appropriate pores encapsulate and solubilize SWCNTs. In contrast, barrels without or with narrow pores showed a much smaller efficiency. Absorption/fluorescence spectroscopy and atomic force microscopy indicate that the SWCNTs are incorporated into the αHB's pore. The resulting hybrid SWCNT@αHBs display periodic surface coverage with a 40 nm pitch and remain fluorescent in the nIR. This approach presents a novel concept to encapsulate, discriminate and functionalize SWCNTs non-covalently with peptides and holds great promise for future applications in bioimaging or drug delivery.

Tuning Selectivity of Fluorescent Carbon Nanotube-Based Neurotransmitter Sensors.

Detection of neurotransmitters is an analytical challenge and essential to understand neuronal networks in the brain and associated diseases. However, most methods do not provide sufficient spatial, temporal, or chemical resolution. Near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) have been used as building blocks for sensors/probes that detect catecholamine neurotransmitters, including dopamine. This approach provides a high spatial and temporal resolution, but it is not understood if these sensors are able to distinguish dopamine from similar catecholamine neurotransmitters, such as epinephrine or norepinephrine. In this work, the organic phase (DNA sequence) around SWCNTs was varied to create sensors with different selectivity and sensitivity for catecholamine neurotransmitters. Most DNA-functionalized SWCNTs responded to catecholamine neurotransmitters, but both dissociation constants (Kd) and limits of detection were highly dependent on functionalization (sequence). Kd values span a range of 2.3 nM (SWCNT-(GC)15 + norepinephrine) to 9.4 µM (SWCNT-(AT)15 + dopamine) and limits of detection are mostly in the single-digit nM regime. Additionally, sensors of different SWCNT chirality show different fluorescence increases. Moreover, certain sensors (e.g., SWCNT-(GT)10) distinguish between different catecholamines, such as dopamine and norepinephrine at low concentrations (50 nM). These results show that SWCNTs functionalized with certain DNA sequences are able to discriminate between catecholamine neurotransmitters or to detect them in the presence of interfering substances of similar structure. Such sensors will be useful to measure and study neurotransmitter signaling in complex biological settings.

Nanosensors for neurotransmitters.

Neurotransmitters are an important class of messenger molecules. They govern chemical communication between cells for example in the brain. The spatiotemporal propagation of these chemical signals is a crucial part of communication between cells. Thus, the spatial aspect of neurotransmitter release is equally important as the mere time-resolved measurement of these substances. In conclusion, without tools that provide the necessary spatiotemporal resolution, chemical signaling via neurotransmitters cannot be studied in greater detail. In this review article we provide a critical overview about sensors/probes that are able to monitor neurotransmitters. Our focus are sensing concepts that provide or could in the future provide the spatiotemporal resolution that is necessary to 'image' dynamic changes of neurotransmitter concentrations around cells. These requirements set the bar for the type of sensors we discuss. The sensor must be small enough (if possible on the nanoscale) to provide the envisioned spatial resolution and it should allow parallel (spatial) detection. In this article we discuss both optical and electrochemical concepts that meet these criteria. We cover techniques that are based on fluorescent building blocks such as nanomaterials, proteins and organic dyes. Additionally, we review electrochemical array techniques and assess limitations and possible future directions.