Fluorescence-based Method Research Articles

Highly sensitive and simple fluorescence-based method for trace level residue determination of pinoxaden in agricultural and environmental samples: Experimental and DFT study

Keeping in view the toxicity of pesticides, development of analytical methods for their determination in environmental samples is the need of the day. Herein, a simple, fast and sensitive method for the determination of pinoxaden has been described. The kinetically stable, Fe(III) based complex of pinoxaden was formed at room temperature showing maximum fluorescence intensity at 337 nm with 0.2 µg mL−1 of Fe(III) at pH 9. The complex shows a linear response in the concentration range of 1 × 10−4-1 × 10−3 µg mL−1 with a coefficient of determination R2 = 0.9917, limit of detection and limit of quantification of 8.1 × 10−5 and 2.4 × 10−4 µg mL−1, respectively, and relative standard deviation and maximum matrix effect of 0.021% and 2.13%. Density functional theory simulations were used to understand the nature of Fe(III) based complex of pinoxaden. The larger negative interaction energy of −1.11 eV evince that the Fe(III) strongly coordinated with the N-atom shared by oxadiazopine ring and the pyrazole ring and the O atom of –CO group of side chain, which are consistent with the experimental results. Interference study shows that among the metal ions studied, only Cu(II), Co(II) and Pb(II) show reasonable interference effect. The proposed approach was applied to quantify pinoxaden in the formulation and real agricultural and environmental samples. The results indicate excellent recovery in the range of 89.44 ± 1.38–97.51 ± 2.62 % for wheat, barley, water and soil samples. In soil samples no residue was quantified while in the wheat and barley samples the residue found was below the maximum residue limit. Under optimized experimental conditions, the proposed method was found to be simple, fast, reasonably interference free and has trace level sensitivity. The method was found to be comparable and even superior to some literature methods for determination of pinoxaden in complex matrices.

Fluorescent protein lifetimes report densities and phases of nuclear condensates during embryonic stem-cell differentiation

Fluorescent proteins (FP) are frequently used for studying proteins inside cells. In advanced fluorescence microscopy, FPs can report on additional intracellular variables. One variable is the local density near FPs, which can be useful in studying densities within cellular bio-condensates. Here, we show that a reduction in fluorescence lifetimes of common monomeric FPs reports increased levels of local densities. We demonstrate the use of this fluorescence-based variable to report the distribution of local densities within heterochromatin protein 1α (HP1α) in mouse embryonic stem cells (ESCs), before and after early differentiation. We find that local densities within HP1α condensates in pluripotent ESCs are heterogeneous and cannot be explained by a single liquid phase. Early differentiation, however, induces a change towards a more homogeneous distribution of local densities, which can be explained as a liquid-like phase. In conclusion, we provide a fluorescence-based method to report increased local densities and apply it to distinguish between homogeneous and heterogeneous local densities within bio-condensates.

Spectrofluorimetric and circular dichroism approaches for sulfasalazine determination using serum proteins: JMP optimization and quantitative studies

Circular dichroism (CD) based spectroscopic methods (zero and second-order derivative), as well as the fluorescence-based method, were developed for the assay of sulfasalazine (SZ) in pharmaceutical formulation and in biological sample using the proteins bovine serum albumin (BSA) and human serum albumin (HSA). The proposed methods are simple and selective for SZ determination. Fluorescence spectra of proteins (method A) show a peak at 340 nm, and a zero order CD scan (normal CD) shows a negative peak at 208 nm (method B). The calibration curve (method A) shows linearity in between 2.91 and 7.41 μg mL−1 for the BSA-SZ system and between 2.44 and 7.83 μg mL−1 for the HSA-SZ system. For method B, the linearity is between 3.38 and 6.10 μg mL−1 for BSA-SZ system, while it is between 2.91 and 6.10 μg mL−1 for the HSA-SZ system. The derivative CD spectrum i.e., second order CD spectrum (D2) exhibits two bands, positive band at 208 nm (method C) and another negative band at 195 nm (method D). Linear calibration curves for CD based methods were in the concentration range of 3.38–6.10 μg mL−1 for BSA-SZ and 2.91–6.10 μg mL−1 for HSA-SZ systems. ICH guidelines were used for the validation of the proposed methods. To demonstrate their suitability for sulfasalazine quality control in its dosage forms, the proposed methods were compared to the reference method.

Synthesis and Anticancer Activity of Novel Dual Inhibitors of Human Protein Kinases CK2 and PIM-1.

CK2 and PIM-1 are serine/threonine kinases involved in the regulation of many essential processes, such as proliferation, differentiation, and apoptosis. Inhibition of CK2 and PIM-1 kinase activity has been shown to significantly reduce the viability of cancer cells by inducing apoptosis. A series of novel amino alcohol derivatives of parental DMAT were designed and synthesized as potent dual CK2/PIM-1 inhibitors. Concomitantly with the inhibition studies toward recombinant CK2 and PIM-1, the influence of the obtained compounds on the viability of three human carcinoma cell lines, i.e., acute lymphoblastic leukemia (CCRF-CEM), human chronic myelogenous leukemia (K-562), and breast cancer (MCF-7), as well as non-cancerous cells (Vero), was evaluated using an MTT assay. Induction of apoptosis and cell cycle progression after treatment with the most active compound and a lead compound were studied by flow-cytometry-based assay. Additionally, autophagy induction in K-562 cells and intracellular inhibition of CK2 and PIM-1 in all the tested cell lines were evaluated by qualitative/quantitative fluorescence-based assay and Western blot method, respectively. Among the newly developed inhibitors, 1,1,1-trifluoro-3-[(4,5,6,7-tetrabromo-1H-benzimidazol-2-yl)amino]propan-2-ol demonstrates the highest selectivity and the most prominent proapoptotic properties towards the studied cancer cells, especially towards acute lymphoblastic leukemia, in addition to inducing autophagy in K-562 cells.

Illuminating Bacterial Contamination in Water Sources: The Power of Fluorescence-Based Methods.

Bacterial contamination of water sources is a significant public health concern, and therefore, it is important to have accurate and efficient methods for monitoring bacterial concentration in water samples. Fluorescence-based methods, such as SYTO 9 and PI staining, have emerged as a promising approach for real-time bacterial quantification. In this review, we discuss the advantages of fluorescence-based methods over other bacterial quantification methods, including the plate count method and the most probable number (MPN) method. We also examine the utility of fluorescence arrays and linear regression models in improving the accuracy and reliability of fluorescence-based methods. Overall, fluorescence-based methods offer a faster, more sensitive, and more specific option for real-time bacterial quantification in water samples.

The Impact of Fruit Ripeness on the Lower Oxygen Limit, Chlorophyll Fluorescence and Fermentation Behavior in Apples

Dynamic Controlled Atmosphere-Chlorophyll Fluorescence storage (DCA-CF) uses a fluorescence-based measurement method to detect fermentation in apples (Malus × domestica BORKH.) caused by low-oxygen levels at an early stage. In recent years, it has been observed that individual apples of the same variety and origin can exhibit different fermentation behavior when stored under completely identical conditions. The causes of the different fermentation behavior must be found in order to be able to use DCA storage optimally. This study aimed to find the causes of the different fermentation behaviors of individual apples. Our results show that fruit ripeness can affect the lower oxygen limit (LOL), especially immediately after harvest, when the starch degradation in the fruit is not yet complete. A significant increase in the LOL was observed in ‘Elstar’ (2020: 0.3 kPa, 0.6 kPa, 0.9 kPa; 2021: 0.3 kPa, 0.4 kPa, 0.6 kPa). ‘Braeburn’ also exhibited this behavior regarding the LOL at a lower level. The LOL could not be identified for some of the fruit (varying from 12.5% to 41.7% of the examined apples) previously stored in Ultra Low Oxygen (ULO) storage for 4 months. Also, the chlorophyll content in the apple skin influences the fluorescence measurement method. Within 2 weeks, the chlorophyll content in the apple skin was halved. If the chlorophyll content drops, the reliability of the fluorescence measurement also decreases. It turned out that apples with an Fv/Fm < 0.7 were unsuitable for valid LOL identification.

A cellulose-based material as a fluorescent sensor for Cr(VI) detection and investigation of antimicrobial properties of its encapsulated form in two different MOFs

It is crucial to detect toxic chromium ions quickly, reliably, sensitively and at low concentrations. In recent years, fluorescence-based methods have been developed for the rapid detection and determination of toxic ions such as chromium. In present work, we focused on the development of a cellulose-based fluorescent probe (Cel-Nap) for the determination of Cr(VI). The fluorescent probe bearing the 1,8-naphthalimide group displayed a low LOD of 1.07 μM for Cr(VI) in the working range of 0.33 × 10−5–3.22 × 10−5 M. The fluorescence and antibacterial properties of UiO-66-Cel-Nap and ZIF-8-Cel-Nap materials prepared by encapsulating Cel-Nap with 2 different MOF types (UiO-66 and ZIF-8) were investigated. While it was found that ZIF-8-based materials had better antimicrobial properties compared to those of UiO-66, it was determined that materials containing Ag+ were more effective against microbial than those containing AgNPs. It was found that the most effective material was ZIF-8-Cel-Nap-Ag+ and it had a significant antibacterial effect against E. coli at a MIC value of 0.0024 mg/mL.

Optical Nanoscopy Enabled by Nanophotonics

Optical nanoscopy employs the principles of nanophotonics to improve the spatiotemporal resolution by manipulating light at the nanoscale. By integrating nanophotonic structures, such as nanoscale waveguides and metal/dielectric nanostructures, with fluorescence-based imaging methods, optical nanoscopy achieves sub-diffraction-limit resolution. This rapidly developing research field holds exciting potential for advancing our understanding of the nanoscopic world and revealing insights beyond conventional optical microscopy.

Protein-RNA interactions: from mass spectrometry to drug discovery.

Proteins and RNAs are fundamental parts of biological systems, and their interactions affect many essential cellular processes. Therefore, it is crucial to understand at a molecular and at a systems level how proteins and RNAs form complexes and mutually affect their functions. In the present mini-review, we will first provide an overview of different mass spectrometry (MS)-based methods to study the RNA-binding proteome (RBPome), most of which are based on photochemical cross-linking. As we will show, some of these methods are also able to provide higher-resolution information about binding sites, which are important for the structural characterisation of protein-RNA interactions. In addition, classical structural biology techniques such as nuclear magnetic resonance (NMR) spectroscopy and biophysical methods such as electron paramagnetic resonance (EPR) spectroscopy and fluorescence-based methods contribute to a detailed understanding of the interactions between these two classes of biomolecules. We will discuss the relevance of such interactions in the context of the formation of membrane-less organelles (MLOs) by liquid-liquid phase separation (LLPS) processes and their emerging importance as targets for drug discovery.

Correlative light-electron microscopy using small gold nanoparticles as single probes

Correlative light-electron microscopy (CLEM) requires the availability of robust probes which are visible both in light and electron microscopy. Here we demonstrate a CLEM approach using small gold nanoparticles as a single probe. Individual gold nanoparticles bound to the epidermal growth factor protein were located with nanometric precision background-free in human cancer cells by light microscopy using resonant four-wave mixing (FWM), and were correlatively mapped with high accuracy to the corresponding transmission electron microscopy images. We used nanoparticles of 10 nm and 5 nm radius, and show a correlation accuracy below 60 nm over an area larger than 10 µm size, without the need for additional fiducial markers. Correlation accuracy was improved to below 40 nm by reducing systematic errors, while the localisation precision is below 10 nm. Polarisation-resolved FWM correlates with nanoparticle shapes, promising for multiplexing by shape recognition in future applications. Owing to the photostability of gold nanoparticles and the applicability of FWM microscopy to living cells, FWM-CLEM opens up a powerful alternative to fluorescence-based methods.

Label-Free Analysis of Organelle Interactions Using Organelle-Specific Phase Contrast Microscopy (OS-PCM)

Organelles are highly dynamic and fulfill their function by constant motion and cooperation with each other. Current methods rely on fluorescence, leading to short observation time (via photobleaching) and experimental complexity (via multiple labeling). While label-free microscopes promise a paradigm change in this regard, the spatiotemporal resolutions and specificity are still insufficient to study organelle interactions. Using mitochondria and lysosome as examples, we demonstrate that our organelle-specific phase contrast microscopy (OS-PCM) can achieve automatic analysis of dynamic metrics of multiple organelles as well as their interactions from unlabeled cells for the first time. Compared to fluorescence-based methods, our method is gentle and holds great promise for label-free visualization and analysis of pan-organelle dynamics and interactions, with minimum perturbation to the cell.

Substantial dimerized G-quadruplex signal units engineered by cutting-mediated exponential rolling circle amplification for ultrasensitive and label-free detection of exosomes

Sensitive and accurate determination of tumor-derived exosomes from complicated biofluids is an important prerequisite for early tumor diagnosis through exosome-based liquid biopsy. Herein, a label-free fluorescence immunoassay protocol for ultrasensitive detection of exosomes was developed by engineering substantial dimerized guanine-quadruplex (Dimer-G4) signal units via in situ cutting-mediated exponential rolling circle amplification (CM-ERCA). First, exosomes were captured and enriched via immunomagnetic separation. Then, molecular recognition was built by the formation of antibody−aptamer sandwich immunocomplex through the specific binding of the designed aptamer-primers with the targeted exosomes. The accuracy of exosome detection was significantly improved by the specific recognition of two typical exosomal protein markers simultaneously. Eventually, in situ CM-ERCA was triggered by a perfect match between the multifunctional circular DNA template and the aptamer-primer on exosomal surface. Amplicons of CM-ERCA loaded with Dimer-G4 were exponentially accumulated during continuous cyclic amplification, dramatically lighting up the thioflavin T (ThT) and generating substantial Dimer-G4 signal units. As a result, ultrasensitive detection of exosomes with the detection limit down to 2.4 × 102 particles/mL was achieved due to the fluorescence enhancement of substantial Dimer-G4 signal units, which is ahead of most of available fluorescence-based methods reported currently. In addition, the intense fluorescence emission and favorable anti-interference of the proposed immunoassay supports identification of exosomes direct in human serums, overcoming the limitations of conventional G4/ThT in serum analysis and revealing its potential for exosome-based liquid biopsy.

Point‐of‐care devices based on fluorescence imaging and spectroscopy for tumor margin detection during breast cancer surgery: Towards breast conservation treatment

Fluorescence-based methods are highly specific and sensitive and have potential in breast cancer detection. Simultaneous fluorescence imaging and spectroscopy during intraoperative procedures of breast cancer have great advantages in detection of tumor margin as well as in classification of tumor to healthy tissues. Intra-operative real-time confirmation of breast cancer tumor margin is the aim of surgeons, and therefore, there is an urgent need for such techniques and devices which fulfill the surgeon's priorities. In this article, we propose the development of fluorescence-based smartphone imaging and spectroscopic point-of-care multi-modal devices for detection of invasive ductal carcinoma in tumor margin during removal of tumor. These multimodal devices are portable, cost-effective, noninvasive, and user-friendly. Molecular level sensitivity of fluorescence process shows different behavior in normal, cancerous and marginal tissues. We observed significant spectral changes, such as, red-shift, full-width half maximum (FWHM), and increased intensity as we go towards tumor center from normal tissue. High contrast in fluorescence images and spectra are also recorded for cancer tissues compared to healthy tissues. Preliminary results for the initial trial of the devices are reported in this article. A total 44 spectra from 11 patients of invasive ductal carcinoma (11 spectra for invasive ductal carcinoma and rest are normal and negative margins) are used. Principle component analysis is used for the classification of invasive ductal carcinoma with an accuracy of 93%, specificity of 75% and sensitivity of 92.8%. We obtained an average 6.17 ± 1.66 nm red shift for IDC with respect to normal tissue. The red shift and maximum fluorescence intensity indicates p < 0.01. These results described here are supported by histopathological examination of the same sample. In the present manuscript, simultaneous fluorescence-based imaging and spectroscopy is accomplished for the classification of IDC tissues and breast cancer margin detection.

Serial nanomaterial synthesis from Ilex paraguariensis biomass for efficient Cr6+ adsorption and detection

This study presents the use of Ilex paraguariensis, an agro-industrial waste, to obtain multiple nanomaterials through serial processes. The materials synthesized included organic-coated magnetite (Fe3O4@YM), Carbon dots (CD), SiO2 materials, and a magnetite-CD composite (Fe3O4@CD). The characterization of these materials showed that they have nanosphere-like geometries with mean diameters ranging from 4.5 nm to 19.7 nm. The versatility of these materials was demonstrated by using them to develop both Cr6+ adsorbents and sensors. CDs were used as Cr6+ sensors, while all Fe or Si-containing materials were used as Cr6+ adsorbents. CD showed a fluorescence-based method for Cr6+ quantification in the range 1.00–243.00 μM with a detection limit of 0.58 μM. Of all the adsorbents, Fe3O4@YM and Fe3O4@CD showed the highest number of adsorption sites at 161.29 and 312.50 mg Cr6+ g−1, which was attributed to the higher surface area and concentration of carboxylic and phenolic groups on the latter material. The economic feasibility of the materials was evaluated through the intensive enhancement price for the adsorption process (φ). It was found that the materials with the highest adsorption capacity are not necessarily the ones with the lowest required economic investment. Fe3O4@YM was shown to have higher economic feasibility considering both synthesis and adsorption processes with values of 8.4 and 5.8 mg Cr6+ USD−1. This approach demonstrates the potential of using agro-industrial residues for the green synthesis of multiple nanomaterials with different applications, useful for fully developing a remediation process.

Identification of West Nile virus RNA-dependent RNA polymerase non-nucleoside inhibitors by real-time high throughput fluorescence screening

West Nile virus (WNV) is a re-emergent mosquito-borne RNA virus that causes major outbreaks of encephalitis around the world. However, there is no therapeutic treatment to struggle against WNV, and the current treatment relies on alleviating symptoms. Therefore, due to the threat virus poses to animal and human health, there is an urgent need to come up with fast strategies to identify and assess effective antiviral compounds. A relevant target when developing drugs against RNA viruses is the viral RNA-dependent RNA polymerase (RdRp), responsible for the replication of the viral genome within a host cell. RdRps are key therapeutic targets based on their specificity for RNA and their essential role in the propagation of the infection. We have developed a fluorescence-based method to measure WNV RdRp activity in a fast and reliable real-time way. Interestingly, rilpivirine has shown in our assay inhibition of the WNV RdRp activity with an IC50 value of 3.3 μM and its antiviral activity was confirmed in cell cultures. Furthermore, this method has been extended to build up a high-throughput screening platform to identify WNV polymerase inhibitors. By screening a small chemical library, novel RdRp inhibitors 1–4 have been identified. When their antiviral activity was tested against WNV in cell culture, 4 exhibited an EC50 value of 2.5 μM and a selective index of 12.3. Thus, rilpivirine shows up as an interesting candidate for repurposing against flavivirus. Moreover, the here reported method allows the rapid identification of new WNV RdRp inhibitors.

Specific fluorescent signatures for body fluid identification using fluorescence spectroscopy

Non-invasive, rapid, on-site detection and identification of body fluids is highly desired in forensic investigations. The use of fluorescence-based methods for body fluid identification, have so far remain relatively unexplored. As such, the fluorescent properties of semen, serum, urine, saliva and fingermarks over time were investigated, by means of fluorescence spectroscopy, to identify specific fluorescent signatures for body fluid identification. The samples were excited at 81 different excitation wavelengths ranging from 200 to 600 nm and for each excitation wavelength the emission was recorded between 220 and 700 nm. Subsequently, the total emitted fluorescence intensities of specific fluorescent signatures in the UV–visible range were summed and principal component analysis was performed to cluster the body fluids. Three combinations of four principal components allowed specific clustering of the body fluids, except for fingermarks. Blind testing showed that 71.4% of the unknown samples could be correctly identified. This pilot study shows that the fluorescent behavior of ageing body fluids can be used as a new non-invasive tool for body fluid identification, which can improve the current guidelines for the detection of body fluids in forensic practice and provide the robustness of methods that rely on fluorescence.

Two-dimensional measurements of receptor-ligand interactions.

Gaining insight into the two-dimensional receptor-ligand interactions, which play a significant role in various pivotal biological processes such as immune response and cancer metastasis, will deepen our understanding of numerous physiological and pathological mechanisms and contribute to biomedical applications and drug design. A central issue involved is how to measure the in situ receptor-ligand binding kinetics. Here, we review several representative mechanical-based and fluorescence-based methods, and briefly discuss the strengths and weaknesses for each method. In addition, we emphasize the great importance of the combination of experimental and computational methods in studying the receptor-ligand interactions, and further studies should focus on the synergistic development of experimental and computational methods.

Multi-marker profiling of extracellular vesicles using streaming current and sequential electrostatic labeling

High heterogeneity in the membrane protein expression of small extracellular vesicles (sEVs) means that bulk methods relying on antibody-based capture for expression analysis have a drawback that each type of antibody may capture a different sub-population. An improved approach is to capture a representative sEV population, without any bias, and then perform a multiplexed protein expression analysis on this population. However, such a possibility has been largely limited to fluorescence-based methods. Here, we present a novel electrostatic labelling strategy and a microchip-based all-electric method for membrane protein analysis of sEVs. The method allows us to profile multiple surface proteins on the captured sEVs using alternating charge labels. It also permits the comparison of expression levels in different sEV-subtypes. The proof of concept was tested by capturing sEVs both non-specifically (unbiased) as well as via anti-CD9 capture probes (biased), and then profiling the expression levels of various surface proteins using the charge labelled antibodies. The method is the first of its kind, demonstrating an all-electrical and microchip based method that allows for unbiased analysis of sEV membrane protein expression, comparison of expression levels in different sEV subsets, and fractional estimation of different sEV sub-populations. These results were also validated in parallel using a single-sEV fluorescence technique.

Lighting Up the Heritage Sciences: The Past and Future of Laser-Induced Fluorescence Spectroscopy in the Field of Cultural Goods

With the rapid scientific and technological changes that occur every day, a new kind of necessity, real-time, rapid, and accurate detection methods, preferably also non- or minimally invasive and non-destructive, has emerged. One such method is laser-induced fluorescence spectroscopy (LIF), applied in various fields of activity in recent decades, ranging from industry and biochemistry to medicine and even heritage sciences. Fluorescence-based spectroscopic methods have all of the above-mentioned characteristics, and their functionality has been proven in many studies. Yet, they have not known great success as other molecular techniques. This paper is a short synthesis of the role of the laser-induced fluorescence spectroscopy technique in heritage sciences, the main applications in this field, along with its advantages and limitations. The article focuses on the most common types of lasers used, the merging of two or more methods into hybrid techniques, the enhancement of the analytical capabilities of LIF and post-processing methods, and also explores some future development possibilities of LIF.

Single-molecule fluorescence methods for protein biomarker analysis.

Proteins have been considered key building blocks of life. In particular, the protein content of an organism and a cell offers significant information for the in-depth understanding of the disease and biological processes. Single-molecule protein detection/sequencing tools will revolutionize clinical (proteomics) research, offering ultrasensitivity for low-abundance biomarker (protein) detection, which is important for the realization of early-stage disease diagnosis and single-cell proteomics. This improved detection/measurement capability delivers new sets of techniques to explore new frontiers and address important challenges in various interdisciplinary areas including nanostructured materials, molecular medicine, molecular biology, and chemistry. Importantly, fluorescence-based methods have emerged as indispensable tools for single protein detection/sequencing studies, providing a higher signal-to-noise ratio (SNR). Improvements in fluorescent dyes/probes and detector capabilities coupled with advanced (image) analysis strategies have fueled current developments for single protein biomarker detections. For example, in comparison to conventional ELISA (i.e., based on ensembled measurements), single-molecule fluorescence detection is more sensitive, faster, and more accurate with reduced background, high-throughput, and so on. In comparison to MS sequencing, fluorescence-based single-molecule protein sequencing can achieve the sequencing of peptides themselves with higher sensitivity. This review summarizes various typical single-molecule detection technologies including their methodology (modes of operation), detection limits, advantages and drawbacks, and current challenges with recent examples. We describe the fluorescence-based single-molecule protein sequencing/detection based on five kinds of technologies such as fluorosequencing, N-terminal amino acid binder, nanopore light sensing, and DNA nanotechnology. Finally, we present our perspective for developing high-performance fluorescence-based sequencing/detection techniques.