Higher Fluorescence Intensity Research Articles

Development of a TaqMan polymerase chain reaction detection method for the precise identification and quantification of an attenuated Eimeria maxima vaccine strain in poultry

Avian coccidiosis, a parasitic disease prevalent in poultry, is caused by Eimeria species and leads to significant economic losses. The use of attenuated live oocyst vaccines has been adopted as an alternative to the use of anticoccidial drugs. However, the accurate detection and differentiation of vaccine strains from virulent ones remain challenging. Therefore, this study presents a novel TaqMan polymerase chain reaction (PCR) detection method that offers enhanced sensitivity, specificity, and reproducibility compared with traditional PCR techniques. Through whole-genome resequencing and bioinformatics analysis, we identified a molecular marker gene, Em_marker6, with a unique 21-base pair deletion specific to the Eimeria maxima attenuated vaccine strain. Optimized primers and probes targeting this marker enabled rapid quantification cycle value achievement and high fluorescence intensity. The standard curve’s slope of −3.540 and correlation coefficient of 0.9971 confirmed precise quantification capabilities. The TaqMan PCR method detected as few as 30 plasmid DNA copies and 50 oocysts per reaction, outperforming traditional PCR techniques by an order of magnitude. No cross-reactivity was observed with other E. maxima wide-type strains or common intestinal pathogens, ensuring the exclusive detection of the E. maxima EMPY vaccine strain. Weekly testing over 3 weeks demonstrated minimal variability, indicating robust consistency in the method’s application. Testing on 61 clinical samples revealed a 57.38% positivity rate for E. maxima species and 13.11% for the vaccine strain. The Em_marker6 gene exhibited genetic stability across multiple generations, confirming the detection method’s robust stability for the attenuated E. maxima vaccine strain. This study significantly advances the field of avian coccidiosis research and control by providing a valuable tool for monitoring vaccine purity and preventing inadvertent infections in vaccinated flocks, aligning with global efforts to curb antibiotic use in animal feed.

Development of a FRET aptasensor based on MoS2-doped Zn-MOF as luminophore for selective detection of cadmium in aqueous solutions.

A robust "on-off" fluorescent aptasensor was developed using nanohybrids of molybdenum sulfide (MoS2) quantum dot (QD)-doped zinc metal-organic frameworks (Zn-MOF) for selective and sensitive detection of cadmium ions (Cd2+) in water. This nanohybrid (MoS2@Zn-MOF), synthesized via "bottle around the ship" methodology, exhibited a high-intensity fluorescence emission centered at 430nm (λEm) (blue) on excitation at 320nm (λEx). Further, the conjugation of this fluorophore to phosphate-modified cadmium aptamer (Cd-2-2) was achieved through carbodiimide reaction. The hybridization of prepared sensing probe (MoS2@Zn-MOF/Cd-2-2 aptamer) was done with dabcyl-conjugated complementary DNA (cDNA), acting as energy donor-acceptor pair in the fluorescence resonance energy transfer (FRET) system. This hybridizationcauses the fluorescence quenching of the nanohybrid. In the presence of Cd2+, the aptamer from the fabricated nano-biosensing probe binds to these ions, resulting in release of dabcyl-cDNA oligomer. This release of dabcyl-cDNA oligomer from the sensing probes restores the fluorescence of the nanohybrid. Under optimized conditions (sensing probe/dabcyl-cDNA ratio 1/7, pH 7.4, and temp 28°C), the sensing probe showed a fast response time of 1min. The fluorescence intensity of the nanohybrid can be utilizedto determine the concentration of Cd2+. The proposed aptasensor achieved highly sensitive detection of Cd2+ with a limit of detection (LOD) of 0.24ppb over the range of 1 × 10-9 to 1 × 10-4M along with minimal effects of interferences (e.g., Hg2+, Pb2+, and Zn2+) and good reproducibility. The designed aptasensor based on MoS2@Zn-MOF nanofluorophore offers a highly sensitive and selective approach for rapid screening of metal ions in aqueous environments.

A low-molecular-weight, long-wavelength emissive Coumarin: Green synthesis, spectral properties, fluorescence enhancement and solvatochromism application

In this study, a novel low-molecular-weight coumarin fluorescent dye, 3,4-dicyano-7-diethylaminocoumarin, was designed. This dye features a donor-π-bridge-acceptor (D-π-A) conjugated structure, which confers superior fluorescence properties.The dye was synthesized using a simple, efficient, and environmentally friendly two-step process, facilitating the direct conversion of the key intermediate into the final product via simultaneous addition and elimination reactions. A comprehensive structural characterization of the dye was undertaken, and its UV absorption and fluorescence emission properties were extensively analyzed using spectroscopic techniques, with structural explanations of the fluorescence activities provided by the Density Functional Theory (DFT) method.Experimental results revealed that this coumarin dye emits strong fluorescence, characterized by a long wavelength, a large Stokes shift, and high fluorescence intensity. It exhibits excellent fluorescent performance in various solvents. Despite experiencing fluorescence quenching in its solid state, the formation of an inclusion complex with β-cyclodextrin restores strong red fluorescence, presenting new possibilities for biofluorescence imaging. Notably, this coumarin dye also demonstrates a significant solvatochromic effect. This effect enables rapid visual discrimination among multiple sets of structurally and chemically similar organic solvents. Consequently, the dye holds promise as a versatile solvatochromic fluorescent probe for a broad spectrum of applications.

Synthesis and characterizations of MoS2 nanoflowers and spectroscopic study of their interaction with bovine serum albumin.

Molybdenum disulfide nanoflowers (MoS2 NFs) were prepared by hydrothermal method. The prepared MoS2 NFs was characterized by SEM, TEM, XRD, specific surface areas, Raman and XPS. The characterization results show that the flower-like spherical MoS2 is composed of many ultra-thin nanosheets with an average diameter of about 300-400 nm. MoS2 NFs also exhibits excellent absorption and high fluorescence intensity. In order to explore the biological behavior of MoS2 NFs, the interaction between MoS2 NFs and bovine serum albumin (BSA) was studied by UV-Vis absorption, fluorescence, synchronous fluorescence spectra, and cyclic voltammetry. The results of absorption and fluorescence show that MoS2 NFs and BSA interact strongly through the formation of complexes. The Stern-Volmer constant and the quenching constant was calculated about 3.79×107 L mol-1 and 3.79×1015 L mol-1 s-1, respectively. The synchronous fluorescence implied that MoS2 in the complex may mainly bind to tryptophan residues of BSA. The cyclic voltammograms indicated that the addition of BSA makes electron reduction of MoS2 NFs more difficult than the corresponding free state. These experimental results clarified the effective transportation and elimination of MoS2 NFs in the body by binding to BSA, and can provide useful guideline for estimating MoS2 NFs as a drug carrier.

Synthesis, characterization and biological activities of nitrophenol-substituted phosphazene dendrimers

This study introduces the synthesis and characterization of two novel nitrophenol-substituted phosphazene dendrimers, [N3P3(OC6H5NO2)6] (III) and [N3P3(OC6H5(NO2)2)5O]ˉ.C6H16N+.H2O (IV), derived from hexachlorocyclotriphosphazene (HCCP). The structures of the compounds were determined using elemental analysis, infrared spectroscopy, and X-ray crystallography. The optical properties of the crystals were investigated by photophysical methods. The thermal properties of the compounds were measured by thermogravimetry-differential thermal analysis (TG-DTA). The antibacterial and anticancer properties of the compounds were tested based on the antibacterial tests against Gram-negative (Escherichia coli ATCC 8739 G(-)) and Gram-positive (Staphylococcus aureus ATCC 6538 G(+), Bacillus subtilis 2a ATCC 23,857 G(+)) bacteria, as well as the MTT assay test on K562 leukemia cells (ATCC: CCL-243) and MCF-7 breast cancer cells (ATCC: HTB-22). The presence of six homogeneous 2-nitrophenol substitutions in compound III reduces the fluorescence of the phosphazene ring. The results showed that compound IV has a higher fluorescence intensity and similar toxicity to MCF-7 and HDF cells. The structural differences and the number of nitro groups in the nitrophenol ligands account for these variations. Our results suggest that these cyclophosphazene derivatives have promising properties for biomedical applications and further studies are warranted to explore their mechanisms of action and therapeutic potential.

Solvent Effects on the Absorption and Emission Spectra of the [5-amino-1-bromoindolizin-3-yl](4-bromophenyl)methanone Molecule.

The effects of solvent on the absorption and emission spectra and dipole moments of the 5ABBM have been extensively studied in a series of solvents. The dipole moments in the excited state are observed to be greater than those in the ground-state in all the solvents studied for the chosen molecule. The dipole moment increase in the excited singlet state ranges from 2.42 to 24.14 D. The experimentally calculated ground state and excited state dipole moments were determined using the solvatochromatic shifts in the absorption and emission spectra as a function of dielectric constant (ɛ) and refractive index (n). These data are used to estimate the excited-state dipole moment using the experimentally determined ground-state dipole moment. A series of fifteen different organic solvents (toluene, methanol, n-butyl alcohol, ethyl acetate, DMS, acetonitrile, benzene, isopropyl alcohol, water, DMF, DCM, DIO, THF, ethanol, and octanol) were investigated at constant dye concentrations. Small changes in the fluorescence spectrum were observed for the different solvents; the highest fluorescence intensity was observed for DMS, and the lowest was observed for water. The Stokes shift in different solvents was studied for the 5ABBM molecule. This results in molecule being more polar in the excited state than in the ground state for the solvents used. The ground state dipole moments, HOMO-LUMO, and molecular electrostatic potential maps were also computed via ab initio calculations and evaluated via Gaussian 09W software.

Mechano-responsive and Acid Stimuli-responsive Luminescence of a Hydrogen-Bonded Organic Frameworks

Stimuli-responsive materials are capable of changing their chemical or physical properties when exposed to external stimuli, showing a wide range of potential applications such as drug delivery, sensing, smart coatings, and artificial muscles. Here, a micron-sized hydrogen-bonded organic framework PFC-1 was constructed and its mechanical and acid stimuli-responsive luminescence were investigated. It was found that the fluorescence of PFC-1 aqueous suspension is affected HCl concentrations. The pristine PFC-1 aqueous suspension exhibits an emission peak with high fluorescence intensity at 440 nm and two weak emission shoulders at 475 nm and 535 nm, respectively. Upon the addition of hydrochloric acid, there is a gradual reduction in the peak intensities at 440 nm and 475 nm, while the peak intensity at 535 nm remains relatively unchanged, resulting in a progressive shift in fluorescence color from blue to green. Mechanical grinding induces approximate 30 nm blue-shift in the fluorescence emission peak, a change that is reversible upon immersing the powder in ethanol for 60 minutes. This work contributes to the development of stimuli-responsive hydrogen-bonded organic framework materials with potential applications in acid and pressure fluorescence sensing.

Fluorescence-guided surgery using cetuximab-800CW in patients with penile carcinoma.

To investigate the feasibility of fluorescence molecular imaging (FMI), using cetuximab-800CW, as an intraoperative tool to determine surgical margins in penile squamous cell carcinoma (PSCC). A total of 11 patients with PSCC received 75 mg cetuximab followed by 15 mg cetuximab-800CW 2 days before surgery. FMI of the whole excision specimen and tissue slices was performed. Fluorescence visualisation was correlated to histopathology. Based on tumour and healthy tissue regions of interest, mean fluorescence intensity was calculated for each individual patient. Significant differences between tumour and healthy mean fluorescence intensity were found with tumour-to-background ratios of a median (IQR) of 1.51 (0.99) and a mean (SD) of 1.51 (0.32) in the excision specimen and tissue slices, respectively. One patient showed a high relative fluorescence intensity with a signal-to-background ratio of 1.79, corresponding to a tumour-positive margin on fresh frozen sectioning. In this Phase I study we showed that cetuximab-800CW seems suitable to discriminate PSCC from background tissue. The tracer was well tolerated, and no false positive spots were seen.

Effects of a Larger N‐Heterocyclic Ring in Rofecoxib Analogues on AIE Activity and Multifunctional Applications

AbstractTwo analogues of rofecoxib, 2‐Pyr‐ROF and 2‐Pip‐ROF, were designed according to the strategy of heterocyclic ring enlargement and facilely synthesized in a facile manner by Knoevenagel reactions. The first compound, 2‐Pyr‐ROF, had low quantum yields (QY) of 3.0 % in the solid states. After replacing the pyrrolidinyl with piperidinyl, the resulting 2‐Pip‐ROF had a significantly increased QY, with a value of 28 %. The root‐mean‐square deviation (RMSD) values between S0 and S1 of 2‐Pyr‐ROF and 2‐Pip‐ROF in tetrahydrofuran were 0.51, 0.35 Å, respectively. As compared with 2‐Pyr‐ROF, compound 2‐Pip‐ROF exhibited a smaller RMSD, which indicated a lower non‐radiative transition rate. A single‐crystal X‐ray diffraction study and Hirshfeld surfaces analysis revealed that 2‐Pip‐ROF had a more nonplanar molecule structure and a weaker π‐π interaction, which might result in a higher fluorescence intensity and QY. Owing to its better aggregation‐induced emission (AIE) behavior, compound 2‐Pip‐ROF showed more multi‐functional applications, including mechanochromism, acidochromism, and lipid droplets imaging ability. These results imply that the strategy of heterocyclic ring enlargement provides a new paradigm for the design of new luminogens with AIE properties.

Hemicyanine-Phenothiazine Based Highly Selective Ratiometric Fluorescent Probes for Detecting Hypochlorite Ion in Fruits, Vegetables and Beverages.

Hypochloric acid (HClO) is a reactive oxygen species (ROS) that functions as a bacteriostatic and disinfectant in food production. Excessive levels of ClO-, however, have been linked to various health issues, including cardiovascular diseases (Halliwell and Gutteridge in Oxford University press, USA, 2015), arthritis, and neurodegenerative diseases (Heinzelmann and Bauer in Biol Chem. 391(6):675-693, 2010). Therefore, synthesizing highly selective and sensitive probes for rapidly detecting endogenous ClO- in daily foods is currently a popular research topic (Kalyanaraman et al. in Redox Biol. 15:347-362, 2018;Winterbourn in Nat Chem Biol. 4(5):278-286, 2008; Turrens in J Physiol. 552(2):335-344, 2003). Thus, we have developed two highly selective ratiometric fluorescent probes (Probe1 and Probe2) based on indole-phenothiazine to detect ClO- in common vegetables, fruits and beverages qualitatively and quantitatively. Moreover, Both Probe1 and Probe2 have shown good specificity and stability, with high fluorescence intensity and long duration (Feng et al. in Adv Sci. 5:1800397, 2018; Wei et al. in Angew Chem. 131(14):4595-4599, 2019; Baruah et al. in J Mater Chem B, 2022).

Development of polymeric nanoparticles associated with SPION and curcumin: a versatile magnetic and fluorescent platform for Life Science applications

Magnetic nanoparticles are prominent in biomedical applications due to their adjustable properties through surface functionalization and inherent magnetism. In the present manuscript, multifunctional nanoparticles were synthesized by combining polymer with a fluorophore and magnetic nanoparticles, resulting in a magnetic and fluorescent nanomaterial. A miniemulsion polymerization technique was employed, using the comonomers methyl methacrylate (MMA) and acrylic acid (AA) in the presence of superparamagnetic iron oxide nanoparticles (SPIONs) and natural turmeric extract (curcumin). Physicochemical characterizations revealed efficient encapsulation of SPIONs within the polymer matrices while maintaining superparamagnetic behavior. The nanosystems exhibited ILP values consistent with those commonly used for magnetic hyperthermia applications, ranging from 0.13 – 0.32 nHm2 kg−1. Additionally, a high fluorescence intensity in the blue/green region was observed, alongside no cell toxicity and substantial internalization in human osteosarcoma cells (OSAS2). Given their intrinsic magnetic and fluorescent properties exhibited, the nanosystems can be considered potential candidates for cell tracking and as a platform in nanotheranostics (MRI, magnetic hyperthermia, and photodynamic therapy).

Salt Tolerance Assessment in Triticum Aestivum and Triticum Durum.

Salt stress is a multicomponent phenomenon; it includes many processes that directly or indirectly affect the plant. Attempts have been made to comprehensively consider the processes of salt stress in plants Triticum aestivum (variety Orenburgskaya 22) and Triticum durum (variety Zolotaya). The study used methods of light and fluorescence microscopy, methods of immunofluorodetection, expression of DNA methyltransferase genes, genes of the ion transporter and superoxide dismutase families, as well as biochemical determination of total antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. According to morphometric indicators, the Orenburgskaya 22 variety showed greater tolerance to the action of 150 mM NaCl than the Zolotaya variety. The level of expression of genes of the HKT ion transporter family in the Orenburgskaya 22 variety is higher than in the Zolotaya variety. It was found that the expression of the DNA methyltransferase gene DRM2.1, which post-translationally methylates cytosine residues, is 22.3 times higher in Zolotaya compared to Orenburg 22 when exposed to salt. The accumulation of toxic ions is accompanied by an increase in reactive oxygen species (ROS) and increased damage to root tissue, especially in the Zolotaya variety. Using fluorescence microscopy using the Carboxy-H2DFF marker in the Orenburgskaya 22 variety at high NaCl concentrations, the highest fluorescence intensity was determined in the cap zone; in the Zolotaya variety-in the zones of the cap and root meristem. Excess ROS is more successfully removed in the Orenburgskaya 22 variety, which has a higher level of antioxidant activity (AOA), as well as the level of expression of the Cu/ZnSOD and MnSOD superoxide dismutase genes. Using programmed cell death (PCD) markers based on the release of cytochrome c from mitochondria into the cytoplasm, DNA breakage and the release of phosphatidylserine from mitochondria, the degree of damage to root cells was assessed in both wheat varieties. It has been proven that wheat cell death occurs through the mitochondrial pathway. It was noted that the salt-sensitive variety Zolotaya had a significant number of necrotic cells. Based on the data obtained, it was concluded that the Orenburgskaya 22 variety exhibits greater resistance to salinity than the Zolotaya variety. These data may be of practical importance for enhancing protective mechanisms under abiotic stress.

Fluorescent BF2 Complexes Inspired by Ageladine A

Abstract. The pH‐dependent UV/Vis absorption and fluorescence of several analogs of the marine natural product ageladine A were investigated. Besides ageladine A itself, its BF2 complex, and the corresponding debrominated analogs were included, as were the chloro analog of the debromoageladine‐BF2 complex, an imidazolone analog, and a triazole derivative, which lack the free amino group. Importantly, the pH‐dependence of the fluorescence of the chloro analog closely resembles that of the natural product ageladine A, but with about 25‐fold higher fluorescence intensity and a maximum between pH 3 and 6. The imidazolone derivative behaves in a complementary manner and shows fluorescence only above pH 6, but with less intensity than ageladine A. The calculated Stokes shifts agrees well with the experiment, which underlines the accuracy of the DFT calculation method (ωB97XD/TApr‐cc‐pVDZ, water, PCM). The combination of BF2 units with arylated imidazo[4,5‐c]pyridine structural motifs encourages further research on more strongly fluorescent derivatives of comparable molecular size.

A highly sensitive ratiometric fluorescent probe for detecting HSO3−/SO32− and viscosity change based on FRET/TICT mechanism

BackgroundSulfur dioxide (SO2) is a significant gas signaling molecule in organisms, and viscosity is a crucial parameter of the cellular microenvironment. They are both involved in regulating many physiological processes in the human body. However, abnormalities in SO2 and viscosity levels are associated with various diseases, such as cardiovascular disease, lung cancer, respiratory diseases, neurological disorders, diabetes and Alzheimer's disease. Hence, it is essential to explore novel and efficient fluorescent probes for simultaneously monitoring SO2 and viscosity in organisms. ResultsWe selected quinolinium salt with good stability, high fluorescence intensity, good solubility and low cytotoxicity as the fluorophore and developed a highly sensitive ratiometric probe QQD to identify SO2 and viscosity changes based on Förster resonance energy transfer/twisted intramolecular charge transfer (FRET/TICT) mechanism. Excitingly, compared with other probes for SO2 detection, QQD not only identified HSO3−/SO32− with a large Stokes shift (218 nm), low detection limit (1.87 μM), good selectivity, high energy transfer efficiency (92 %) and wide recognition range (1.87–200 μM), but also identified viscosity with a 26-fold fluorescence enhancement and good linearity. Crucially, QQD was applied to detect HSO3−/SO32− and viscosity in actual water and food samples. In addition, QQD had low toxicity and good photostability for imaging HSO3−/SO32− and viscosity in cells. These results confirmed the feasibility and reliability of QQD for HSO3−/SO32− and viscosity imaging and environmental detection. SignificanceWe reported a unique ratiometric probe QQD for detecting HSO3−/SO32− and viscosity based on the quinolinium skeleton. In addition to detecting HSO3−/SO32− and viscosity change in actual water and food samples, QQD could also monitor the variations of HSO3−/SO32− and viscosity in cells, which provided an experimental basis for further exploration of the role of SO2 derivatives and viscosity in biological systems.

Integration of a biocompatible metal-phenolic network and fluorescence microspheres as labels for sensitive and stable detection of carbendazim with a lateral flow immunoassay

High fluorescence intensity microspheres such as aggregation-induced emission fluorescence microspheres (AIEFM) have improved the sensitivity of lateral flow immunoassay (LFIA). The preparation of immune probes in LFIA usually adopts the chemical coupling strategy with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for antibody coupling, which has the problems of low coupling efficiency, tedious coupling process, and poor repeatability. A biocompatible metal-phenolic network (MPN), which contains large amounts of phenols and galloyl groups, could easily, quickly, and stably couple with antibodies. Herein, we proposed a strategy based on MPN modification on ultrabright AIEFM surface as a novel label for the rapid detection of carbendazim. The limit of detection of AIEFM@MPN-LFIA was 0.019 ng/mL, which was 4.9 times lower than that of AIEFM-LFIA. In spiked samples, the average recoveries of AIEFM@MPN-LFIA ranged from 80% to 118% (coefficient of variation <13.45%). Therefore, AIEFM@MPN was a promising signal label that could improve the detection performance of LFIA.

Abstract 3102: A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University

Abstract Background: Drug development is important for cancer therapy to improve patient outcomes. In most cases, drug development starts in discovering candidate compounds from a chemical library in high-throughput screening (HTS). Tohoku University Graduate School of Pharmaceutical Science owns the original chemical library consisting of thousands of original chemical compounds. P-glycoprotein (P-gp), a major drug efflux transporter mediates multi-drug resistance (MDR) in cancer cells. Therefore, P-gp modulators could revere MDR and increase chemosensitivity. The aim of this study is to reveal an adequate method of HTS for discovering potent P-gp modulators from the chemical library in Tohoku University. Methods: The chemical library in Tohoku University had 5861 compounds in the present study, and the collection contains over 7 thousand compounds in 2023. P-gp overexpressing KB-V1, human epidermal carcinoma cell lines were obtained, and calcein AM, P-gp substrate was employed for fluorescent substrate efflux assays. A plate reader-based calcein AM efflux assay for HTS was established to select candidate compounds as P-gp modulators. After the primary HTS, structures of the candidate compounds were analyzed, then several analogous compounds were extracted from the chemical library. For the validation of the hit compounds in HTS and the analogs, a flow cytometry-based calcein AM efflux assay was conducted. In addition, reversal effects of P-gp modulators on the cytotoxicity of paclitaxel were measured in a cell proliferation assay. Results: Of 5861 compounds in the chemical library, 53 compounds showed 2-fold or higher fluorescence intensity, and then 13 compounds showed 3-fold or higher fluorescence intensity in a plate reader-based assay for the HTS. Especially 2 candidate compounds showed over 5-fold fluorescence intensity. In the analysis for structures of the candidate compounds, 20 analogous compounds were extracted from the chemical library. To verify the result of the plate reader-based assay, a flow cytometry-based assay was conducted for 53 hits and 20 analogs. As a result, the 2 candidate compounds that selected in the plate reader-based assay showed 60-fold, the highest fluorescence intensity in a flow cytometry-based assay. The other compounds showed less fluorescence intensity than these 2 candidates. In addition, both candidates enhanced the paclitaxel-induced cytotoxicity in a cell proliferation assay. Conclusions: A plate reader-based calcein AM efflux assay in HTS was an adequate method for discovering potent P-gp modulators from the chemical library in Tohoku University. Citation Format: Norihiko Sugisawa, Shinobu Ohnuma, Takayuki Doi, Michiaki Unno. A high-throughput screening method for discovering potent P-glycoprotein modulators from the chemical library in Tohoku University [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3102.

Polymer Microspheres Copolymerized with Deep Red Fluorescent Molecules as a Label for Lateral Flow Immunochromatography.

The development of fluorescently labeled microspheres is a critical aspect of advancing the technology of lateral flow immunochromatography (LFIA) for biological detection. Nevertheless, potential interference posed by the background fluorescence originating from the nitrocellulose (NC) membrane would significantly impact the sensitivity and accuracy of microsphere-based detection in LFIA. In this work, an attempt was made to extend the π-conjugated system and asymmetric structure of rhodamine fluorophore, resulting in the synthesis of dye molecules (RB2) incorporating double bonds, which can reach an absolute photoluminescence quantum yield (PLQY) of 30.01% in EtOH. Subsequently, carboxyl group functionalized fluorescent microspheres were prepared in a two-step copolymerization via soap-free emulsion polymerization. The obtained microspheres were characterized by scanning electron microscopy, transmission electron microscopy, DLS, Fourier transform infrared spectroscopy, ultraviolet spectrophotometry, and fluorescence spectrophotometry. The results showed that RB2 was successfully copolymerized into the microspheres, and the resulting microspheres had good dispersion and stability with high red fluorescence intensity (λabs ∼ 610 nm, λem ∼ 660 nm). Utilizing these microspheres, the resulting lateral flow immunoassay was successfully found to detect SARS-CoV-2 N protein with a detection limit of 2.5 pg/mL and the linear concentration spanning from 2.5 pg/mL to 10 ng/mL. The results confirm the effectiveness of the synthetic fluorescent microspheres as the label for LFIA.

Tumor-Targeted Squaraine Dye for Near-Infrared Fluorescence-Guided Photodynamic Therapy.

Many efforts have been made to develop near-infrared (NIR) fluorescent dyes with high efficiency for the NIR laser-induced phototherapy of cancer. However, the low tumor targetability and high nonspecific tissue uptake of NIR dyes in vivo limit their applications in preclinical cancer imaging and therapy. Among the various NIR dyes, squaraine (SQ) dyes are widely used due to their high molar extinction coefficient, intense fluorescence, and excellent photostability. Previously, benzoindole-derived SQ (BSQ) was prepared by incorporating carboxypentyl benzoindolium end groups into a classical SQ backbone, followed by conjugating with cyclic RGD peptides for tumor-targeted imaging. In this study, we demonstrate that the structure-inherent tumor-targeting BSQ not only shows a high fluorescence quantum yield in serum but also exhibits superior reactive oxygen species (ROS) generation capability under the 671 nm laser irradiation for effective photodynamic therapy (PDT) in vitro and in vivo. Without targeting ligands, the BSQ was preferentially accumulated in tumor tissue 24 h post-injection, which was the optimal timing of the laser irradiation to induce increments of ROS production. Therefore, this work provides a promising strategy for the development of photodynamic therapeutic SQ dyes for targeted cancer therapy.

Fluorescence intensity of composite resin for direct veneer removal using a fluorescence-aided identification technique.

This laboratory study evaluated the influence of the fluorescence intensity of composite resins on additional tooth wear and the presence of restorative material in different dental thirds during the retreatment of direct veneers. The crown dimensions of 60 bovine incisors were reduced to 10 × 8 mm. The teeth were classified according to the fluorescence intensity of the composites: low (LOW) (TPH Spectra), medium (MED) (Opallis), and high (HIGH) (Essentia) groups. The teeth were divided according to the removal methods: conventional (CON) and fluorescence-aided identification technique (FIT). The specimens were scanned (T0), received veneer preparation, and scanned again (T1). After restorations, the composites were removed and the teeth were scanned (T2). Measurement assessments between T1 and T2 were performed to determine additional wear, presence of residual areas, and the average between additional wear and the presence of residual areas. Kruskal Wallis, Mann-Whitney, Friedman, 2-way ANOVA, and post-Tukey tests were performed (α < 0.05). The comparison of composite resins indicated a smaller area of additional wear and greater residue presence in the HIGH group than the LOW group for both techniques in the cervical third. Regarding removal methods, the FIT produced greater additional wear than the CON method for the LOW and MED groups in the middle and cervical thirds. The incisal third exhibited greater additional wear than the other thirds. Composite resins with high fluorescence intensity removed using FIT had less tooth wear. The incisal third was the most affected area for direct veneer removal procedures. A FIT has been proposed for composite resin removal; however, the different fluorescence intensities of composite resins can influence tooth wear caused during this procedure.

Impact of stepwisely reducing settling time on the formation and performance of aerobic granular sludge

The impact of gradually reducing the settling time strategy on the formation, performance, and microbial succession of aerobic granular sludge (AGS) was investigated in a sequencing batch reactor (SBR). The results showed that the short settling time accelerated the granulation of AGS and the granulation was completed within 35 d. When the settling time was 10 min, the granular sludge had good settling performance and significant biological activity, and the average particle size and integrity coefficient increased to 1.017 mm and 93.3 %. The shortening of settling time stimulated bacteria to secrete more extracellular polymeric substances (EPS). Three-dimensional fluorescence spectrum analysis showed that aromatic proteins had high fluorescence intensity. According to the analysis of high-throughput sequencing, Rhodanobacter and Klebsiella were the dominant bacteria, and carbohydrate metabolism and amino acid metabolism were significantly enhanced, which may play an important role in maintaining the stability of AGS.