Delivery and reveal of localization of upconversion luminescent microparticles and quantum dots in the skin in vivo by fractional laser microablation, multimodal imaging, and optical clearing

Delivery and spatial localization of upconversion luminescent microparticles [Y 2 O 3 ;Yb, Er] (mean size ~1.6 μm) and quantum dots (QDs) (CuInS 2 ZnS nanoparticles coated with polyethylene glycol-based amphiphilic polymer, mean size ~20 nm) inside rat skin was studied in vivo using a multimodal optical imaging approach. The particles were embedded into the skin dermis to the depth from 300 to 500 μm through microchannels performed by fractional laser microablation. Low-frequency ultrasound was applied to enhance penetration of the particles into the skin. Visualization of the particles was revealed using a combination of luminescent spectroscopy, optical coherence tomography, confocal microscopy, and histochemical analysis. Optical clearing was used to enhance the image contrast of the luminescent signal from the particles. It was demonstrated that the penetration depth of particles depends on their size, resulting in a different detection time interval (days) of the luminescent signal from microparticles and QDs inside the rat skin in vivo. We show that luminescent signal from the upconversion microparticles and QDs was detected after the particle delivery into the rat skin in vivo during eighth and fourth days, respectively. We hypothesize that the upconversion microparticles have created a long-time depot localized in the laser-created channels, as the QDs spread over the surrounding tissues

Assessment of ITS1, ITS2, 5′-ETS, and trnL-F DNA Barcodes for Metabarcoding of Poaceae Pollen

Grass pollen is one of the major causes of allergy. Aerobiological monitoring is a necessary element of the complex of anti-allergic measures, but the similar pollen morphology of Poaceae species makes it challenging to discriminate species in airborne pollen mixes, which impairs the quality of aerobiological monitoring. One of the solutions to this problem is the metabarcoding approach employing DNA barcodes for taxonomical identification of species in a mix by high-throughput sequencing of the pollen DNA. A diverse set of 14 grass species of different genera were selected to create a local reference database of nuclear ITS1, ITS2, 5′-ETS, and plastome trnL-F DNA barcodes. Sequences for the database were Sanger sequenced from live field and herbarium specimens and collected from GenBank. New Poaceae-specific primers for 5′-ETS were designed and tested to obtain a 5′-ETS region less than 600 bp long, suitable for high-throughput sequencing. The DNA extraction method for single-species pollen samples and mixes was optimized to increase the yield for amplification and sequencing of pollen DNA. Barcode sequences were analyzed and compared by the barcoding gap and intra- and interspecific distances. Their capability to correctly identify grass pollen was tested on artificial pollen mixes of various complexity. Metabarcoding analysis of the artificial pollen mixes showed that nuclear DNA barcodes ITS1, ITS2, and 5′-ETS proved to be more efficient than the plastome barcode in both amplification from pollen DNA and identification of grass species. Although the metabarcoding results were qualitatively congruent with the actual composition of the pollen mixes in most cases, the quantitative results based on read-counts did not match the actual ratio of pollen grains in the mixes

Assessment of ITS1, ITS2, 5′-ETS, and <i>trnL-F</i> DNA Barcodes for Metabarcoding of Poaceae Pollen

Grass pollen is one of the major causes of allergy. Aerobiological monitoring is a necessary element of the complex of anti-allergic measures, but the similar pollen morphology of Poaceae species makes it challenging to discriminate species in airborne pollen mixes, which impairs the quality of aerobiological monitoring. One of the solutions to this problem is the metabarcoding approach employing DNA barcodes for taxonomical identification of species in a mix by high-throughput sequencing of the pollen DNA. A diverse set of 14 grass species of different genera were selected to create a local reference database of nuclear ITS1, ITS2, 5′-ETS, and plastome trnL-F DNA barcodes. Sequences for the database were Sanger sequenced from live field and herbarium specimens and collected from GenBank. New Poaceae-specific primers for 5′-ETS were designed and tested to obtain a 5′-ETS region less than 600 bp long, suitable for high-throughput sequencing. The DNA extraction method for single-species pollen samples and mixes was optimized to increase the yield for amplification and sequencing of pollen DNA. Barcode sequences were analyzed and compared by the barcoding gap and intra- and interspecific distances. Their capability to correctly identify grass pollen was tested on artificial pollen mixes of various complexity. Metabarcoding analysis of the artificial pollen mixes showed that nuclear DNA barcodes ITS1, ITS2, and 5′-ETS proved to be more efficient than the plastome barcode in both amplification from pollen DNA and identification of grass species. Although the metabarcoding results were qualitatively congruent with the actual composition of the pollen mixes in most cases, the quantitative results based on read-counts did not match the actual ratio of pollen grains in the mixes

Synthesis of Hydrophilic CuInS₂/ZnS Quantum Dots with Different Polymeric Shells and Study of Their Cytotoxicity and Hemocompatibility

In this work, there is a detailed description of the whole process of biocompatible CIS/ZnS QDs production. Special attention was paid to the stability of QDs against photooxidation. It was shown that Cu/In ratio greatly affected not only nanocrystals PLQYs but photostability as well. CIS/ZnS QDs with Cu/In = 1:4 ratio showed high photostability under UV illumination both in toluene and aqueous solutions. Meanwhile, photoluminescence of CIS/ZnS QDs with Cu/In = 1:1 ratio was completely quenched after several hours under UV illumination, though their initial QY was as high as 40% with peak maximum at 740 nm. QDs were transferred to water by polymer encapsulation and were subsequently modified with polyethers Jeffamines, cheap analogues of PEG-derivatives. Three types of hydrophilic QDs differing in size, PEG content, and surface charge were obtained for further investigation and comparison of their cytotoxicity and hemocompatibility. It was shown that both leucocytes size distribution and coagulation activation change after introduction of polyethers into QDs polymeric shell, while red blood cells and platelets size distribution as well as hemolysis rate did not show any different results among different QDs and the polymer itself. All three types of QDs showed only slight cytotoxicity. Confocal microscopy proves penetration of hydrophilic CIS/ZnS QDs inside cells, so the low QDs cytotoxocity cannot be explained by low cellular uptake of the QDs and indicated low QDs toxicity in general

Delivery and reveal of localization of upconversion luminescent microparticles and quantum dots in the skin in vivo by fractional laser microablation, multimodal imaging, and optical clearing

Delivery and spatial localization of upconversion luminescent microparticles [Y2O3:Yb, Er] (mean size ∼1.6  μm) and quantum dots (QDs) (CuInS2/ZnS nanoparticles coated with polyethylene glycol-based amphiphilic polymer, mean size ∼20  nm) inside rat skin was studied in vivo using a multimodal optical imaging approach. The particles were embedded into the skin dermis to the depth from 300 to 500  μm through microchannels performed by fractional laser microablation. Low-frequency ultrasound was applied to enhance penetration of the particles into the skin. Visualization of the particles was revealed using a combination of luminescent spectroscopy, optical coherence tomography, confocal microscopy, and histochemical analysis. Optical clearing was used to enhance the image contrast of the luminescent signal from the particles. It was demonstrated that the penetration depth of particles depends on their size, resulting in a different detection time interval (days) of the luminescent signal from microparticles and QDs inside the rat skin in vivo. We show that luminescent signal from the upconversion microparticles and QDs was detected after the particle delivery into the rat skin in vivo during eighth and fourth days, respectively. We hypothesize that the upconversion microparticles have created a long-time depot localized in the laser-created channels, as the QDs spread over the surrounding tissues

Hydrophilic, Bright CuInS₂ Quantum Dots as Cd-Free Fluorescent Labels in Quantitative Immunoassay

We report on the synthesis of core–shell CuInS₂/ZnS quantum dots (QDs) in organic solution, their encapsulation with a PEG-containing amphiphilic polymer, and the application of the resulting water-soluble QDs as fluorescent label in quantitative immunoassay. By optimizing the methods for core synthesis and shell growth, CuInS₂/ZnS QDs were obtained with a quantum yield of 50% on average after hydrophilization. After conjugation with an aflatoxin B1-protein derivative, the obtained QDs were used as fluorescent labels in microplate immunoassay for the quantitative determination of the mycotoxin aflatoxin B1. QDs-based immunoassay showed higher sensitivity compared to enzyme-based immunoassay