Surfactant assisted exfoliation of near infrared fluorescent silicate nanosheets.

Fluorophores that emit light in the near infrared (NIR) are advantageous in photonics and imaging due to minimal light scattering, absorption, phototoxicity and autofluorescence in this spectral region. The layered silicate Egyptian blue (CaCuSi4O10) emits as a bulk material bright and stable fluorescence in the NIR and is a promising NIR fluorescent material for (bio)photonics. Here, we demonstrate a surfactant-based (mild) exfoliation procedure to produce nanosheets (EB-NS) of high monodispersity, heights down to 1 nm and diameters <20 nm in large quantities. The approach combines planetary ball milling, surfactant assisted bath sonication and centrifugation steps. It avoids the impurities that are typical for the harsh conditions of tip-sonication. Several solvents and surfactants were tested and we found the highest yield for sodium dodecyl benzyl sulfate (SDBS) and water. The NIR fluorescence emission (λem ≈ 930-940 nm) is not affected by this procedure, is extremely stable and is not affected by quenchers. This enables the use of EB-NS for macroscopic patterning/barcoding of materials in the NIR. In summary, we present a simple and mild route to NIR fluorescent nanosheets that promise high potential as NIR fluorophores for optical applications.

Covalently Functionalized Egyptian Blue Nanosheets for Near-Infrared Bioimaging.

Fluorophores emitting in the near-infrared (NIR) wavelength region present optimal characteristics for photonics and especially bioimaging. Unfortunately, only few NIR fluorescent materials are known, and even fewer are biocompatible. For this reason, the scientific interest in designing NIR fluorophores is very high. Egyptian Blue (CaCuSi4O10, EB) is an NIR fluorescent layered silicate that can be exfoliated into fluorescent nanosheets (EB-NS). So far, its surface chemistry has not been tailored, but this is crucial for colloidal stability and biological targeting. Here, we demonstrate covalent surface functionalization of EB nanosheets (EBfunc) via Si-H activation using hydrosilanes with variable functionalities. In the first part of this work, EB-NS are grafted with the visible fluorescent pyrene (Pyr) moieties to demonstrate conjugation by colocalization of the Vis/NIR fluorescence on the (single) EB-NS level. Next, the same grafting procedure was repeated and validated with carboxyl group (COOH)-containing hydrosilanes. These groups serve as a generic handle for further (bio)functionalization of the EB-NS surface. In this way, folic acid (FA) could be conjugated to EB-NS, allowing the targeting of folic acid receptor-expressing cancer cells. These results highlight the potential of this surface chemistry approach to modify EB-NS, enabling targeted NIR imaging for biomedical applications.

Preparation, properties and applications of near-infrared fluorescent silicate nanosheets.

The layered silicates Egyptian Blue (CaCuSi4O10, EB), Han Blue (BaCuSi4O10, HB) and Han Purple (BaCuSi2O6, HP) are known as historic pigments, but they also possess novel optoelectronic properties with great potential for fundamental research and technology. They fluoresce in the near-infrared (NIR) range and can be exfoliated into two-dimensional (2D) nanomaterials (i.e. nanosheets, NS) which retain the photophysical properties of the bulk materials. These and other characteristics fuel the growing excitement of the scientific community about these materials. EB-, HB- and HP-NS have been used in various applications ranging from smart inks, energy storage, bioimaging, to phototherapy and more. In this review article, we report the fundamental properties of these low-dimensional silicate nanomaterials, discuss applications and outline perspectives for the future.

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.

Photophysical properties and fluorescence lifetime imaging of exfoliated near-infrared fluorescent silicate nanosheets.

The layered silicates Egyptian Blue (CaCuSi4O10, EB), Han Blue (BaCuSi4O10, HB) and Han Purple (BaCuSi2O6, HP) emit as bulk materials bright and stable fluorescence in the near-infrared (NIR), which is of high interest for (bio)photonics due to minimal scattering, absorption and phototoxicity in this spectral range. So far the optical properties of nanosheets (NS) of these silicates are poorly understood. Here, we exfoliate them into monodisperse nanosheets, report their physicochemical properties and use them for (bio)photonics. The approach uses ball milling followed by tip sonication and centrifugation steps to exfoliate the silicates into NS with lateral size and thickness down to ≈ 16-27 nm and 1-4 nm, respectively. They emit at ≈ 927 nm (EB-NS), 953 nm (HB-NS) and 924 nm (HP-NS), and single NS can be imaged in the NIR. The fluorescence lifetimes decrease from ≈ 30-100 µs (bulk) to 17 µs (EB-NS), 8 µs (HB-NS) and 7 µs (HP-NS), thus enabling lifetime-encoded multicolor imaging both on the microscopic and the macroscopic scale. Finally, remote imaging through tissue phantoms reveals the potential for bioimaging. In summary, we report a procedure to gain monodisperse NIR fluorescent silicate nanosheets, determine their size-dependent photophysical properties and showcase the potential for NIR photonics.

Remote near infrared identification of pathogens with multiplexed nanosensors.

Infectious diseases are worldwide a major cause of morbidity and mortality. Fast and specific detection of pathogens such as bacteria is needed to combat these diseases. Optimal methods would be non-invasive and without extensive sample-taking/processing. Here, we developed a set of near infrared (NIR) fluorescent nanosensors and used them for remote fingerprinting of clinically important bacteria. The nanosensors are based on single-walled carbon nanotubes (SWCNTs) that fluoresce in the NIR optical tissue transparency window, which offers ultra-low background and high tissue penetration. They are chemically tailored to detect released metabolites as well as specific virulence factors (lipopolysaccharides, siderophores, DNases, proteases) and integrated into functional hydrogel arrays with 9 different sensors. These hydrogels are exposed to clinical isolates of 6 important bacteria (Staphylococcus aureus, Escherichia coli,…) and remote (≥25 cm) NIR imaging allows to identify and distinguish bacteria. Sensors are also spectrally encoded (900 nm, 1000 nm, 1250 nm) to differentiate the two major pathogens P. aeruginosa as well as S. aureus and penetrate tissue (>5 mm). This type of multiplexing with NIR fluorescent nanosensors enables remote detection and differentiation of important pathogens and the potential for smart surfaces.

Exfoliated near infrared fluorescent silicate nanosheets for (bio)photonics.

Imaging of complex (biological) samples in the near-infrared (NIR) is beneficial due to reduced light scattering, absorption, phototoxicity, and autofluorescence. However, there are few NIR fluorescent materials known and suitable for biomedical applications. Here we exfoliate the layered pigment CaCuSi4O10 (Egyptian Blue, EB) via ball milling and facile tip sonication into NIR fluorescent nanosheets (EB-NS). The size of EB-NS can be tailored to diameters <20 nm and heights down to 1 nm. EB-NS fluoresce at 910 nm and the fluorescence intensity correlates with the number of Cu2+ ions. Furthermore, EB-NS display no bleaching and high brightness compared with other NIR fluorophores. The versatility of EB-NS is demonstrated by in-vivo single-particle tracking and microrheology measurements in Drosophila melanogaster embryos. EB-NS can be uptaken by plants and remotely detected in a low-cost stand-off detection setup. In summary, EB-NS have the potential for a wide range of bioimaging applications.

Multispectral near infrared absorption imaging for histology of skin cancer.

Multispectral imaging combines the spectral resolution of spectroscopy with the spatial resolution of imaging and is therefore very useful for biomedical applications. Currently, histological diagnostics use mainly stainings with standard dyes (eg, hematoxylin + eosin) to identify tumors. This method is not applicable in vivo and provides low amounts of chemical information. Biomolecules absorb near infrared light (NIR, 800-1700 nm) at different wavelengths, which could be used to fingerprint tissue. Here, we built a NIR multispectral absorption imaging setup to study skin tissue samples. NIR light (900-1500 nm) was used for homogenous wide-field transmission illumination and detected by a cooled InGaAs camera. In this setup, images I(x, y, λ) from dermatological samples (melanoma, nodular basal-cell carcinoma, squamous-cell carcinoma) were acquired to distinguish healthy from diseased tissue regions. In summary, we show the potential of multispectral NIR imaging for cancer diagnostics.

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.

Chirality enriched carbon nanotubes with tunable wrapping via corona phase exchange purification (CPEP).

Single-walled carbon nanotubes (SWCNTs) have unique photophysical properties and serve as building blocks for biosensors, functional materials and devices. For many applications it is crucial to use chirality-pure SWCNTs, which requires sophisticated processes. Purification procedures such as wrapping by certain polymers, phase separation, density gradient centrifugation or gel chromatography have been developed and yield distinct SWCNT species wrapped by a specific polymer or surfactant. However, many applications require a different organic functionalization (corona) around the SWCNTs instead of the one used for the purification process. Here, we present a novel efficient and straightforward process to gain chirality pure SWCNTs with tunable functionalization. Our approach uses polyfluorene (PFO) polymers to enrich certain chiralities but the polymer is removed again and finally exchanged to any desired organic phase. We demonstrate this concept by dispersing SWCNTs in poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(6,6'-{2,2'-bipyridine})] (PFO-BPy), which is known to preferentially solubilize (6,5)-SWCNTs. Then PFO-BPy is removed and recycled, while letting the SWCNTs adsorb/agglomerate on sodium chloride (NaCl) crystals, which act as a toluene-stable but water-soluble filler material. In the last step these purified SWCNTs are redispersed in different polymers, surfactants and ssDNA. This corona phase exchange purification (CPEP) approach was also extended to other PFO variants to enrich and functionalize (7,5)-SWCNTs. CPEP purified and functionalized SWCNTs display monodisperse nIR spectra, which are important for fundamental studies and applications that rely on spectral changes. We show this advantage for SWCNT-based nIR fluorescent sensors for the neurotransmitter dopamine and red-shifted sp3 defect peaks . In summary, CPEP makes use of PFO polymers for chirality enrichment but provides access to chirality enriched SWCNTs functionalized in any desired polymer, surfactant or biopolymer.