Fluorescent Polystyrene Microspheres Research Articles

Detection of Tetracycline with a CRISPR/Cas12a Aptasensor Using a Highly Efficient Fluorescent Polystyrene Microsphere Reporter System.

CRISPR-based diagnostics use the CRISPR-Cas system trans-cleavage activity to identify specific target sequences. When activated, this activity cleaves surrounding reporter molecules, producing a detectable signal. This technique has great specificity, sensitivity, and rapid detection, making it an important molecular diagnostic tool for medical and infectious disease applications. Despite its potential, the present CRISPR/Cas system has challenges with its single-stranded DNA reporters, characterized by low stability and limited sensitivity, restricting effective application in complex biological settings. In this work, we investigate the trans-cleavage activity of CRISPR/Cas12a on substrates utilizing fluorescent polystyrene microspheres to detect tetracycline. This innovative discovery led to the development of microsphere probes addressing the stability and sensitivity issues associated with CRISPR/Cas biosensing. By attaching the ssDNA reporter to polystyrene microspheres, we discovered that the Cas12a system exhibits robust and sensitive trans-cleavage activity. Further work revealed that the trans-cleavage activity of Cas12a on the microsphere surface is significantly dependent on the concentration of the ssDNA reporters. Building on these intriguing discoveries, we developed microsphere-based fluorescent probes for CRISPR/Cas aptasensors, which showed stability and sensitivity in tetracycline biosensing. We demonstrated a highly sensitive detection of tetracycline with a detection limit of 0.1 μM. Finally, the practical use of a microsphere-based CRISPR/Cas aptasensor in spiked food samples was proven successful. These findings highlighted the remarkable potential of microsphere-based CRISPR/Cas aptasensors for biological research and medical diagnosis.

A Study on the Growth and Physiological Toxicity Effects of the Combined Exposure of Microplastics and Cadmium on the Vicia faba L. Seedlings.

To investigate the toxicological effects of polystyrene microplastics (PS-MPs), cadmium (Cd), and their combined contamination on the growth and physiological responses of V. faba seedlings, this experiment employed a hydroponic method. The Hoagland nutrient solution served as the control, changes in root growth, physiological and biochemical indicators of V. faba seedlings under different concentrations of PS-MPs (10, 100mg/L) alone and combined with 0.5mg/L Cd. The results demonstrated that the root biomass, root vitality, generation rate of superoxide radicals (O2·-), malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity increased with increasing concentration under the influence of PS-MPs alone, while the soluble sugar content and peroxidase (POD) activity decreased. In the combined treatment with Cd, the trends of these indicators are generally similar to the PS-MPs alone treatment group. However, root vitality and SOD activity showed an inverse relationship with the concentration of PS-MPs. Furthermore, laser confocal and electron microscopy scanning revealed that the green fluorescent polystyrene microspheres entered the root tips of the V. faba and underwent agglomeration in the treatment group with a low concentration of PS-MPs alone and a high concentration of composite PS-MPs with Cd.

Abstract 2018: Tumor treating fields (TTFields) disrupt cancer cell invasion by impacting cell-ECM traction forces

Abstract Introduction: Tumor Treating Fields (TTFields) are an anti-cancer treatment that use low intensity, alternating electric fields. Anti-mitotic treatment effects of TTFields have been, to date, considered the primary anti-cancer effect. Recent studies have shown additional impacts on cancer cell invasion and migration, among other effects, resulting in a complex anti-tumor response derived from a combination of factors. By deploying computational imaging tools to assess experimental conditions that apply TTFields treatment to 3D cancer multicellular spheroid cultures, the goal of our study is to elucidate important mechanisms and effect sizes on anti-tumor effects relating to the modulation of cancer cell-extracellular matrix (ECM) biophysical interactions. Methods: 3D multicellular tumor spheroids (MTS) are cultured using GFP-tagged U-87 MG glioma cells. A single 3D MTS is placed in a cell culture insert and embedded in collagen I ECM at 2.25 mg/mL. Fluorescent polystyrene microspheres are dispersed in the ECM to facilitate subsequent tracking of displacement fields induced by the MTS-ECM construct. Fluorescence microscopy imaging is acquired for all spheroids prior to treatment initiation. Then, with 3 replicates in each treatment condition, TTFields are applied for a duration of 48 hours at 200 kHz frequency with a no treatment condition as a control. At 48 hours, we acquire fluorescence microscopy images to track spheroid growth and bead-field displacement. To determine deformation fields induced by the cancer spheroid, we compare each image at 48 hours to their respective baseline image. Deformation fields are analyzed using a custom-developed analysis code that performs an initial alignment of the baseline and specific time point images using a global rigid registration. Next, we perform a B-spline based nonrigid image registration to generate an estimate of the deformation field vector at each pixel location within the image. Deformation intensity is measured within a circular region of interest (r = 900 µm) centered at the spheroid core. Results: The average magnitude of displacement for treatment and control groups is 0.52±0.2 µm and 1.07±0.1 µm, respectively. Analysis of deformation fields reveals a significant difference (p < 0.1) in the magnitude of deformation present between treatment and control groups. Conclusions: Implementation of our computational imaging analysis methods using rigid and nonrigid registration, we observe a 69.6% decrease in the magnitude of deformation in TTFields treated spheroids compared to control spheroids. Due to the decrease in deformation induced by cancer spheroids treated with TTFields, we conclude that TTFields impact cancer cell traction forces applied to the ECM to disrupt cancer cell invasion. Citation Format: Samantha M. Short, Haley J. Bowers, Jared A. Weis. Tumor treating fields (TTFields) disrupt cancer cell invasion by impacting cell-ECM traction forces [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 2018.

Response of garlic (Allium sativum L.) to the combined toxicity of microplastics and arsenic

The extensive utilization of mulch films in agricultural settings, coupled with the persistence of microplastic remnants in soil following the natural degradation of plastics, has given rise to detrimental microplastic impacts on crops. Arsenic (As) contamination in the environment is known to accumulate in crops through aquatic pathways or soil. Garlic (Allium sativum L.), a globally popular crop and seasoning, contains alliin, a precursor of its flavor compounds with medicinal properties. While alliin exhibits antimicrobial and antioxidant effects in garlic, its response to microplastics and arsenic has not been thoroughly investigated, specifically in terms of microplastic or As uptake. This study aimed to explore the impact of varied stress concentrations of microplastics on the toxicity, migration, and accumulation of As compounds. Results demonstrated that polystyrene (PS) fluorescent microspheres, with an 80 nm diameter, could permeate garlic bulbs through the root system, accumulating within vascular tissues and intercellular layers. Low concentrations of PS (10 and 20 mg L−1) and As (2 mg L−1) mitigated the production and accumulation of reactive oxygen species (ROS) and antioxidant enzymes in garlic. Conversely, garlic exhibited reduced root vigor, substance uptake, and translocation when treated with elevated As concentrations (4 mg L−1) in conjunction with PS concentrations of 40 and 80 mg L−1. An escalation in PS concentration facilitated As transport into bulbs but led to diminished As accumulation and biomass in the root system. Notably, heightened stress levels weakened garlic's antioxidant defense system, encompassing sulfur allicin and phytochelatin metabolism, crucial for combating the phytotoxicity of PS and As. In summary, PS exerted a detrimental influence on garlic, exacerbating As toxicity. The findings from this study offer insights for subsequent investigations involving Liliaceae plants.

Light sheet fluorescence microscopy with active optical manipulation.

We present a light sheet fluorescence microscopy (LSFM) with active optical manipulation by using linear optical tweezers (LOTs). In this method, two coaxially transmitting laser beams of different wavelengths are shaped using cylindrical lenses to form a linear optical trapping perpendicular to the optical axis and an excitation light sheet (LS) parallel to the optical axis, respectively. Multiple large-sized polystyrene fluorescent microspheres are stably captured by LOTs, and their rotation angles around specific rotation axes are precisely controlled. During a sample rotation, the stationary excitation LS scans the sample to obtain fluorescence sectioning images of the sample at different angles.

Dynamic speckle illumination wide-field fluorescence microscopy with actively optical manipulation of rotational angles.

We present a dynamic speckle illumination wide-field fluorescence microscopy (DSIWFM) combined with a line optical tweezers (LOTs) for rotational fluorescence sectioning imaging. In this method, large polystyrene fluorescent microspheres are stably trapped with LOTs, and precisely manipulated to rotate around a specific rotation axis. During the rotation process, multiple raw fluorescence images of trapped microspheres are obtained with dynamic speckle illumination. The root-mean-square (RMS) algorithm is used to extract the drastically changing fluorescent signals in the focal plane to obtain the fluorescence sectioning images of the samples at various angles. The influence of speckle granularity on the image quality of fluorescence sectioning images is experimentally analyzed. The rotational fluorescence sectioning images obtained by DSIWFM with LOTs could provide an alternative technique for applications of biomedical imaging.

Upflow Direct Filtration and Downflow Direct Filtration: A Comparison with Emphasis on the Removal of Aerobic Spore-Forming Bacteria and Fluorescent Microspheres as Surrogates for Cryptosporidium Oocyst Removal

Monitoring the removal of Cryptosporidium oocysts in water treatment plants is a challenge, particularly in developing countries, and the use of surrogates has proven necessary. Two surrogates already investigated and recognized by the scientific community are aerobic spore-forming bacteria (ASFB) and fluorescent polystyrene microspheres (ME). In this context, this study evaluates and compares the performance of upflow direct filtration and downflow direct filtration, with emphasis on the removal of ASFB and ME as a surrogate of Cryptosporidium oocyst removal. Pilot filtration experiments were carried out with low turbidity Paranoá Lake water (average—3.7 NTU) with ASFB varying from 7 × 102 to 5.5 × 103 CFU/100 mL. In some experiments, water was spiked with microspheres, aiming for 105 ME/L. Two operational conditions were evaluated: (1) filters operating with the same filtration rate (5 m/h); (2) filters operating with the same flow rate of 59.41 L/h. In general, the downflow filter presented a slightly higher removal efficiency than the upflow filter but the differences were not considered statistically significant in most cases. The removal efficiency of microspheres (3 to 5 log) was higher than that of ASFB (1.2 to 4 log) in both filters, corroborating that the removal of ASFB is a conservative surrogate of the removal of Cryptosporidium.

Quantitative determination of four mycotoxins in cereal by fluorescent microsphere based immunochromatographic assay.

Mycotoxins are secondary metabolites produced by fungi, which have serious effects on humans and animals. In this study, we selected the monodispersed polystyrene fluorescent microspheres with good luminescence performance and strong stability as markers to conjugate with four mycotoxins antibodies for preparing fluorescent probes. We have developed a fluorescent microsphere based immunochromatographic assay (FMICA) to detect sensitively and quickly zearalenone (ZEN), aflatoxin B1 (AFB1 ), fumonisin B1 (FB1 ), and ochratoxin A (OTA) in cereal. Under optimal experimental conditions, the procedure of this method can be completed within 10min. The limit of detection (LOD) of FMICA for ZEN, AFB1 , FB1 , and OTA is 0.072, 0.093, 0.32, and 0.19 μg L-1 , respectively. And FMICA has good specificity and no cross-reactivity with other mycotoxins. Four mycotoxins in naturally contaminated cereal samples (corn, rice, and oat) are detected by this method, and the results are highly consistent with that of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The developed FMICA has good accuracy, high sensitivity, simplicity, convenience, rapidity, and low cost, and it could be employed for sensitive and quantitative detecting of mycotoxins in cereal on-site. © 2022 Society of Chemical Industry.

Europium(III)-Based Fluorescent Microspheres with Styrene Copolymerization toward an Enhanced Photoluminescence Performance

Rare earth-based polystyrene (PS) fluorescent microspheres, which vigorously combine the merits of rare earth elements and polystyrene microspheres, hold great potential for applications in the state-of-the-art technology and tremendous efforts have been devoted to the photoluminescence field. Herein, a 5-acrylamido-1,10-phenanthroline (Aphen) and Eu(III)-based polystyrene fluorescent microsphere was developed via a facial copolymerization strategy. The optimal complexes of Eu(β-NTA)3(Aphen) and Eu(BFA)3(TPPO)2 were fabricated as the luminescent materials. Specifically, Eu(β-NTA)3(Aphen)-PS was modified for the first time to enhance the photoluminescence property, which effectively integrated the Aphen with styrene monomer. Meanwhile, Eu(BFA)3(TPPO)2 was encapsulated into PS microspheres for the realization of Eu(BFA)3(TPPO)2-PS via an ubiquitous embedding process. The fluorescent intensity of Eu(β-NTA)3(Aphen)-PS was notably reinforced along with an increasing concentration of the rare earth complexes. However, the fluorescence intensity of Eu(BFA)3(TPPO)2-PS was decreased when the concentration was 50 mg due to the saturated solubility of the complexes. The obtained copolymerization fluorescent microspheres could prevent the premature leakage of the fluorescent complexes in a natural environment and showed an outstanding photoluminescence efficiency.

Deploying holey rGO-based membranes for MPs removal

The serious environmental issue caused by the rising levels of microplastics (MPs) pollution has aroused much more attention globally, therefore, an effective strategy needs to be established to eliminate MPs from the water. Herein, we first report the preparation of membranes based on holey reduced graphene oxide (h-rGO) nanosheets for efficient removal of MPs in wastewater. Compared with those untreated rGO, the h-rGO nanosheets show a porous structure with an average size of 27.3 nm, which is a benefit for construction membrane for separation of MPs with better water flux (37.19 L m−2 h−1 kPa−1). Employing the fluorescent polystyrene (FP) microspheres and PS beads as model MPs, the as-resulted h-rGO membranes exhibit extremely high MPs removal efficiency of 99.9%. In the case of treatment of the industrial MPs containing wastewater, almost all MPs could be captured by the h-rGO membranes which were confirmed by stereomicroscope. Taking advantage of their physicochemical stability and exceptional MPs removal efficiency, the h-rGO membranes may have great potentials for the removal of MPs in wastewater.

Porous organic polymers (POPs) membrane via thiol-yne click chemistry for efficient particulate matter capture and microplastics separation

In viewing the serious environmental issues caused by microplastics and air pollution, it is of great significance to capture microplastics in water and harmful particulate matter (PM) in atmosphere for maintaining human health. In this study, we report the synthesis of porous composite membrane, which was fabricated using 1,4-phenyldimercaptans and 1,3,5-triacetylbenzene as building blocks via a click chemistry, for microplastics filtration and PM capture. By using polystyrene fluorescent microspheres with a diameter of 0.1 μm as simulated microplastics, the filtration efficiency of the as-resulted porous composite membrane (PCM) was measure to be 90.92% by fluorescence analysis. The results obtained from infrared microspectrography also show that the PCM has superior removal performance for microplastics in terms to urban domestic wastewater. In the case of PM capture, the removal efficiencies of PCM for PM 2.5 and PM 10 were measured as high as 99.90% ± 0.42% and 99.93% ± 0.26%, respectively. Moreover, its removal efficiency for PM can remain nearly unchanged within 30 h. Owing to the flexible kapok fibers as supporting layer of PCM, the PCM possesses good mechanical properties and could be prepared without limitation in size and shape on a large scale under mild conditions, thus showing great potential for practical applications of microplastic removal and PM capture. • A porous composite membrane based on porous organic polymers (POPs) was prepared. • The POPs was synthesized via a thiol-yne click chemistry. • The porous composite membrane has excellent flexibility and mechanical strength. • The porous composite membrane possesses high efficiency for PM capture. • The porous composite membrane shows high efficiency for microplastics separation.

Lattice light sheets generated with a firmly arranged dielectric regular hexagonal pyramid array.

We present a firmly arranged dielectric regular hexagonal pyramid array to generate lattice light sheets with high conversion efficiency and low stray light. Both the size and working distance of the lattice light sheets can be modulated by changing the structural parameters. We experimentally recorded the lattice light sheets illumination, which is consistent with the corresponding simulation. To evaluate the imaging quality, we compared the light field generated with and without structure by using polystyrene fluorescent microspheres. This study provides a potential method for the building of light sheet fluorescence microscopy with high resolution and low phototoxicity.

Effects of BNO 1016 on ciliary transport velocity and cell culture surface liquid height of sinonasal epithelial cultures

BackgroundBNO 1016 is an ethanolic extract of a mixture of five herbs that has been sold in different formulations for decades in the European market and more recently, in the United States market as an over-the-counter treatment for rhinosinusitis. Previous studies indicated activation of chloride secretion and increase in ciliary beat frequency by BNO 1016 but the functional consequences on mucociliary transport velocity and airway surface liquid homeostasis are unknown. This study intends to examine the effects of BNO 1016 on these properties in vitro.ResultsHuman sinonasal epithelial cells were grown at an air-liquid interface, with addition of BNO 1016 basolaterally in each experiment. Polystyrene fluorescent microspheres were added to the apical surface of the culture, and distance traveled across the surface of the culture over a fixed time period was measured using live imaging. BNO 1016 concentrations of 50 μg/ml and 500 μg/ml were tested. Basolateral application of compound resulted in a non-dose-dependent increase in culture surface liquid height compared to controls at 30 min, and this effect persisted through the one-hour duration of the experiment (p < 0.01). Basolateral application of BNO 1016 also resulted in a non-dose-dependent increase in microsphere transport velocity at 45 and 60 min following compound application (p < 0.01).ConclusionsBasolateral application of BNO 1016 at a concentration mimicking post-ingestion serum levels appears to elicit increases in cell culture surface liquid height and mucociliary clearance, as assessed by microsphere transport velocity. These properties can potentially be leveraged for therapeutic efficacy in diseases affecting mucus production and mucociliary transport.

Recovery of Hypoxic Regions in a Rat Model of Microembolism

ObjectivesEndovascular treatment (EVT) has become the standard of care for acute ischemic stroke. Despite successful recanalization, a limited subset of patients benefits from the new treatment. Human MRI studies have shown that during removal of the thrombus, a shower of microclots is released from the initial thrombus, possibly causing new ischemic lesions. The aim of the current study is to quantify tissue damage following microembolism. Materials and methodsIn a rat model, microembolism was generated by injection of a mixture of polystyrene fluorescent microspheres (15, 25 and 50 µm in diameter). The animals were killed at three time-points: day 1, 3 or 7. AMIRA and IMARIS software was used for 3D reconstruction of brain structure and damage, respectively. ResultsMicroscopic analysis of 500 µm thick brain volumes revealed multiple ischemic, hypoxic and infarcted regions. Both the number and total volume of hypoxic and ischemic areas declined over time, whereas the infarcts remained. ConclusionsMicroembolism induces ischemia, hypoxia and infarction. Infarcted areas persist, but hypoxic regions recover over time suggesting that repair processes in the brain rescue the regions at risk.

Synthesis and experimental study of liquid dispersions of magnetic fluorescent polystyrene microspheres

Multiplex microsphere-based immunofluorescence assay is a reliable, accurate, and highly sensitive method for the detection of various biomolecules. However, for the moment, the wide application of the method in clinical practice is prevented by the high cost of reagents for analysis - magnetic spectrally encoded microspheres. Therefore, an urgent task is the development of new methods for the synthesis of microspheres with the required properties. The aim of this study was the creation of new magnetic fluorescent microspheres suitable for use in multiplex immunoassay.Samples of magnetic fluorescent polystyrene microspheres were synthesized by dispersion polymerization and two-stage swelling methods. Experimental studies of geometric parameters, fluorescence, magnetic properties of the synthesized microspheres have been carried out.The results of the studies have shown that microspheres synthesized by dispersion polymerization are promising for the use in immunofluorescence analysis. The obtained results can be used for the development of new diagnostic multiplex test systems based on spectrally encoded microspheres.

Thinner and Longer Working Distance Light Sheet Illumination and Microscopic Imaging

Light sheet fluorescence microscopy has become a basic tool in biology and medical research with its fast imaging speed, low phototoxicity and high spatiotemporal resolution. Here, we report a novel method to generate multiplane parallel light sheets with a micro structure named isosceles triangular array. The thickness of light sheet in each plane can approach 3.12 μm along with the working distance of 152.20 μm directly generated with the isosceles triangular array. We experimentally recorded the multiplane parallel light sheets illumination, which is consistent with the corresponding simulation by using ray-tracing method. To evaluate the imaging quality, we compared the light field generated with and without projected phase mask onto the spatial light modulator by using polystyrene fluorescent microspheres nanoparticles and transgenic zebrafish. This study provides a potential method for the building of volumetric imaging light sheet fluorescence microscopy with high resolution and low phototoxicity.

Detection of Circulating Tumor Cells by Fluorescence Microspheres-Mediated Amplification.

Here we describe a fluorescent microspheres-based separation and analysis that enables the isolation of circulating tumor cells (CTCs) from whole blood of patients with metastatic cancer and the identification of isolated CTCs in situ without immunostaining. This approach uses antibody-functionalized fluorescent polystyrene (PS) microspheres that can selectively bind to CTCs. The binding of CTCs and fluorescent PS microspheres leads to the formation of complexes of CTCs and fluorescent PS microspheres, thereby the CTCs are size-amplified and labeled simultaneously. A pyramidal microcavity array (PMCA) is fabricated using microfabrication technology to create a precise microfilter structure with a high aspect ratio. The PMCA filter device can effectively isolate microspheres-labeled CTCs, while allow hematologic cells to deform and pass through. Using this approach, CTCs are isolated and identified in 15 of 18 patients with metastatic colorectal cancer. This approach will open new possibilities for CTCs isolation and identification and can serve a versatile platform to facilitate CTCs analysis in diverse biomedical applications.

The impact of contact lens wear on particulate clearance from the ocular surface

Purpose Contact lens-related infection and inflammation is thought to be related to the aggregation of bacteria between the contact lens and the corneal surface during wear. In this clinical study, fluorescent polystyrene microspheres were utilised to compare the particulate clearance rate with and without contact lens wear.

Inherently fluorescent polystyrene microspheres as a fluorescent probe for highly sensitive determination of chromium (VI) and mercury (II) ions

Nowadays developing fluorescent probes that are cheaper, nontoxic, robust and easy to use for determination of heavy metal ions with high selectivity and sensitivity is still an ongoing challenge. In this paper, inherently fluorescent aminated polystyrene microspheres (PS-NH2) were fabricated from cross-linked polystyrene (PS) microspheres through acetylation, oximation and reduction, which can serve as a selective and sensitive fluorescence (FL) probe for the determination of chromium (VI) (Cr(VI)) and mercury (II) (Hg2+) ions. The photostability, selectivity and sensitivity of the fluorescent PS-NH2 microspheres were studied in detail. The feasibility of PS-NH2 microspheres as a fluorescent probe was conducted by determining Cr(VI) or Hg2+ in real water samples. Results revealed that no photoleaching or leaking issues were observed on PS-NH2 microspheres. The fluorescent probe could analyze Cr(VI) and Hg2+ separately in water samples with high sensitivity and good accuracy in the range of 0.01–1 μM. The detection limits for Cr(VI) and Hg2+ were 9.1 nM and 9.9 nM, respectively. Additionally, two possible FL quenching mechanisms were proposed for Cr(VI) and Hg2+. This work provides a new platform for accurate determination of Cr(VI) and Hg2+ in environmental fields.

A novel guidewire-integrated embolic protection filter device with a handy-folding system: In vitro and in vivo performance assessment.

We developed a novel guidewire-integrated filter device with a handy-folding system (IFD: Inoue filter device). In vitro and in vivo studies were conducted to evaluate the feasibilityof the IFD. Although distal atheromatous and thrombotic embolizations remain unresolved critical issues during catheter interventions, distal protection devices are infrequently used partly because of reduced lower maneuverability. In the in vitro experiment, we created an experimental circulation model composed of silicone latex tubes, a reservoir, and a roller pump. After the filter device was deployed in the tube, polystyrene fluorescent microspheres were injected and the capture rate was calculated. Ten trials were performed using the IFD and Spider FX. In the in vivo study, five independent operators deployed, and they retrieved the IFD in swine common iliac and internal carotid arteries. The procedural success rate as well as the delivery and retrieval time was evaluated. In the in vitro study, the mean capture rate was 94% and 35% in the IFD and Spider groups, respectively. In the in vivo study, all procedures were successful, with no complications. The mean delivery time was 281 ± 87 s and 194 ± 67 s and the mean retrieval time was 24 ± 9 and 13 ±1 s in the left internal carotid and the left common iliac arteries, respectively. Although further studies and improvements are required, the study results indicate that the IFD is feasible.