Increase In Fluorescence Signal Research Articles

Development of an assay to quantify tranexamic acid levels in plasma

Dysregulations during blood clot breakdown (fibrinolysis) during vascular trauma can lead to excessive blood loss. Tranexamic acid (TXA) is an inhibitor of fibrinolysis that works by blocking the interaction between plasminogen and fibrin degradation products (FDPs) – a key step in fibrinolysis. Despite the widespread usage, there are no tests available in a clinical setting to monitor TXA levels. We developed a fluorescence resonance energy transfer (FRET)-based assay to quantify TXA concentrations in plasma by using 1) fluorescently labeled plasminogen, and 2) FDPs labelled with a fluorescence quencher. Once plasminogen binds the FDPs, the fluorescent signal is quenched. TXA causes plasminogen to dissociate from the FDPs, thus increasing fluorescence signal in a dose-dependent manner. The dose response was sensitive between 1 and 100 μM (0.16 and 15.7 mg/L). The intraassay and interassay variabilities were determined to be 5.7% and 3.0%, respectively. Limit of detection was estimated to be 0.28 μM (0.044 mg/L). When tested for measuring known levels of TXA added to plasma samples, the ratio between measured and expected TXA concentration was 1.0151. Our study demonstrates a novel assay that can rapidly quantify TXA concentrations in plasma samples, thus demonstrating its potential as an in-hospital tool.

Hypoxia-Induced Mitochondrial ROS and Function in Pulmonary Arterial Endothelial Cells.

Pulmonary artery endothelial cells (PAECs) are a major contributor to hypoxic pulmonary hypertension (PH) due to the possible roles of reactive oxygen species (ROS). However, the molecular mechanisms and functional roles of ROS in PAECs are not well established. In this study, we first used Amplex UltraRed reagent to assess hydrogen peroxide (H2O2) generation. The result indicated that hypoxic exposure resulted in a significant increase in Amplex UltraRed-derived fluorescence (i.e., H2O2 production) in human PAECs. To complement this result, we employed lucigenin as a probe to detect superoxide (O2-) production. Our assays showed that hypoxia largely increased O2- production. Hypoxia also enhanced H2O2 production in the mitochondria from PAECs. Using the genetically encoded H2O2 sensor HyPer, we further revealed the hypoxic ROS production in PAECs, which was fully blocked by the mitochondrial inhibitor rotenone or myxothiazol. Interestingly, hypoxia caused an increase in the migration of PAECs, determined by scratch wound assay. In contrast, nicotine, a major cigarette or e-cigarette component, had no effect. Moreover, hypoxia and nicotine co-exposure further increased migration. Transfection of lentiviral shRNAs specific for the mitochondrial Rieske iron-sulfur protein (RISP), which knocked down its expression and associated ROS generation, inhibited the hypoxic migration of PAECs. Hypoxia largely increased the proliferation of PAECs, determined using Ki67 staining and direct cell number accounting. Similarly, nicotine caused a large increase in proliferation. Moreover, hypoxia/nicotine co-exposure elicited a further increase in cell proliferation. RISP knockdown inhibited the proliferation of PAECs following hypoxia, nicotine exposure, and hypoxia/nicotine co-exposure. Taken together, our data demonstrate that hypoxia increases RISP-mediated mitochondrial ROS production, migration, and proliferation in human PAECs; nicotine has no effect on migration, increases proliferation, and promotes hypoxic proliferation; the effects of nicotine are largely mediated by RISP-dependent mitochondrial ROS signaling. Conceivably, PAECs may contribute to PH via the RISP-mediated mitochondrial ROS.

Fluorescence-Based Monitoring of Early-Stage Aggregation of Amyloid-β, Amylin Peptide, Tau, and α-Synuclein Proteins.

Early-stage aggregates of amyloid-forming proteins, specifically soluble oligomers, are implicated in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Protein aggregation is typically monitored by fluorescence using the amyloid-binding fluorophore thioflavin T (ThT). Thioflavin T interacts, however, preferentially with fibrillar amyloid structures rather than with soluble, early-stage aggregates. In contrast, the two fluorophores, aminonaphthalene 2-cyanoacrylate-spiropyran (AN-SP) and triazole-containing boron-dipyrromethene (taBODIPY), were reported to bind preferentially to early-stage aggregates of amyloidogenic proteins. The present study compares ThT with AN-SP and taBODIPY with regard to their ability to monitor early stages of aggregation of four different amyloid-forming proteins, including amyloid-β (Aβ), tau protein, amylin, and α-synuclein. The results show that the three fluorophores vary in their suitability to monitor the early aggregation of different amyloid-forming proteins. For instance, in the presence of Aβ and amylin, the fluorescence intensity of AN-SP increased at an earlier stage of aggregation than the fluorescence of ThT, albeit with only a small fluorescence increase in the case of AN-SP. In contrast, in the presence of tau and amylin, the fluorescence intensity of taBODIPY increased at an earlier stage of aggregation than the fluorescence of ThT. Finally, α-synuclein aggregation could only be monitored by ThT fluorescence; neither AN-SP nor taBODIPY showed a significant increase in fluorescence over the course of aggregation of α-synuclein. These results demonstrate the ability of AN-SP and taBODIPY to monitor the formation of early-stage aggregates from specific amyloid-forming proteins at an early stage of aggregation, although moderate increases in fluorescence intensity, relatively large uncertainties in fluorescence values, and limited solubility of both fluorophores limit their usefulness for some amyloid proteins. The capability to monitor early aggregation of some amyloid proteins, such as amylin, might accelerate the discovery of aggregation inhibitors to minimize the formation of toxic oligomeric species for potential therapeutic use.

Colorimetric and fluorescent dual-mode detection of Escherichia coli using Fe3O4 nanoparticle coated with fluorescein-labeled aptamer

Ensuring the reliable and accurate detection of Escherichia coli (E. coli) is essential for maintaining food safety and safeguarding human health. In this work, a dual-mode sensor that integrates colorimetric and fluorescence detection was developed for the precise identification of E. coli. Fe3O4 nanoparticles (NPs) were coated with a fluorescein-labeled aptamer (FAM-Apt) to enhance their peroxidase-like activity under neutral pH conditions. Simultaneously, this coating also effectively suppressed the fluorescence emitted by FAM. Upon exposure to E. coli, specific binding of the bacteria causes the detachment of FAM-Apt from the Fe3O4 NPs surface, resulting in the suppression of Fe3O4 NPs catalytic activity under neutral pH and the alleviation of the quenching effect on FAM fluorescence. This leads to a simultaneous decrease in the colorimetric signal and an increase in fluorescence signal, facilitating the development of a dual-mode detection method for E. coli with an impressive limit of detection (LOD) of 10 CFU/mL in the colorimetric mode and 6 CFU/mL in the fluorescence mode. This approach highlights the untapped potential of Fe3O4 NPs as a peroxidase mimic in dual-mode sensors, offering a promising strategy for E. coli detection with significant implications for enhancing food safety and public health practices.

(Invited) C60-Based Switchable Fluorescent Probes

Photodynamic therapy (PDT), an emerging non-invasive tumor treatment, requires suitable photosensitizer (PS) molecules that generate reactive oxygen species (ROSs) efficiently under long wavelength of light with deep tissue penetration, ideally activated in the diseased tissue. C60 is known as an excellent acceptor molecule in both electron- and energy-transfer reactions and exhibits biocompatibility and efficient ROS generation, rendering it a promising candidate as a PS for PDT.In this study, we developed a C60-based dual-functional molecular probe containing a water-soluble C60-peptide and a donor fluorophore connected with a ligand peptide specific for matrix metalloproteinases (MMP) (Figure 1). We expect that such a molecular probe can be used in both diagnostic imaging and concurrent treatment. The probe revealed increased fluorescence signal and 1O2 generation in the presence of MMP2/9 enzymes that are known to be overexpressed in tumor cells. Furthermore, in vitro cellular tests demonstrated the probe's abilities in imaging MMP activity and photocytotoxicity on MMP-expressing tumor cells. Figure 1

On the Feasibility of Using an Acedane-Based Fluorescent Probe to Monitor Hydrogen Sulfide in Primary Neuronal Cultures

Hydrogen sulfide (H₂S), which, under physiological conditions, exists in cells mainly in the form of anion HS–, is considered as a gaseous transmitter of inter- and intracellular signals along with nitrogen oxide and carbon monoxide. Analysis of the dynamics of H₂S content in living cells is impossible without creating sensitive and specific probes. Several acedan-based compounds have been synthesized in the group of K.H. Ahn (Singha et al., 2015. Anal. Chem. 87 (2), 1188–1195). In the presence of H₂S these probes attach to the sulfhydrilic group and form fluorescent carbocyclic compounds. The carbocyclic derivative of P3, compound csP3, was found to be optimal for fluorescence-microscopic studies in terms of spectral characteristics and response time to H₂S. In this work, we tested the suitability of csP3 to record H₂S changes in buffers mimicking the salt composition of the intracellular environment and in primary neuronal culture cells from rat cerebral cortex. It was found that reducing the polarity of the solution by adding dimethyl sulfoxide (30% by volume) caused a blue shift of the emission by ~10 nm and a twofold increase in fluorescence intensity. The csP3 fluorescence depends on the salt composition and increases in the presence of bicarbonate (NaHCO₃, 10 mM). Addition of P3 or csP3 to the neuronal culture caused a rapid increase in fluorescence, which was followed by a slow increase in fluorescence signal after 3–5 min. Glutamate (10 μM, in the presence of 10 μM glycine, 0 Mg2+) increased probe fluorescence, but only in those neurons in which delayed deregulation of calcium homeostasis did not occur. We conclude that the product of the reaction of P3 with H₂S is sensitive to a change in the salt composition of the intracellular medium and can be redistributed in cells between water and more hydrophobic environment. This means that an increase in P3 fluorescence in cells, especially after the addition of glutamate to neurons, does not necessarily indicate an increase in H₂S concentration. To confirm the feasibility of using P3 and structurally related probes as quantitative indicators of H₂S presence, additional studies of the properties of these compounds are needed.

Self-Internal Standard Fluorescence for Ultrasensitive Detecting of mtDNA to Evaluate Matrilineal Genetic Defect Levels.

Mitochondrial DNA (mtDNA) is a unique genetic material characterized by maternal inheritance. It possesses a circular structure devoid of histone protection and exhibits low cellular abundance, which poses great challenges for its sensitive and selective detection at the living cell level. Herein, we have designed three bis-naphthylimide probes with varying linker lengths (NANn-OH, n = 0, 2, 6), facilitating the formation of distinct twisted or folded molecular conformations in the free state. These probes emit the red fluorescence around 627 nm with different fluorescence quantum yields (ΦNAN0-OH = 0.0016, ΦNAN2-OH = 0.0136, and ΦNAN6-OH = 0.0125). When encountering mtDNA (0.4-3.4 μg/mL), these probes undergo conformational changes depending on the length of the attached C-strand and exhibit a gradually increasing fluorescence signal around 453 nm. The fluorescence intensity increased to 13.5-fold, 1.9-fold, and 8.2-fold, respectively. Notably, the red fluorescence intensities around 627 nm remain constant throughout this process, thus serving as an inherent correction mechanism for proportional fluorescence signal enhancement to improve selectivity and sensitivity. NAN0-OH, NAN2-OH, and NAN6-OH showed good linearity for mtDNA in the range of 0.4-3.4 μg/mL with detection limits of LODNAN0-OH = 1.04 μg/mL, LODNAN2-OH = 1.10 μg/mL, and LODNAN6-OH = 1.15 μg/mL. Cellular experiments reveal that NAN6-OH effectively monitors curcumin-induced mtDNA damage in HepG-2 cells while enabling monitoring of genetic mtDNA damage. We anticipate that this tool holds significant potential for the precise evaluation of maternal genetic defects, thereby enhancing hypersensitive assessment in clinical medicine.

Endogenous mRNA-Driven "One-To-More" Signal Amplification of DNA Probe for Intracellular miR155 Sensing.

The detection of specific intracellular microRNAs could be potentially helpful in understanding the underlying mechanisms of cancer metastasis and invasion. MiRNAs are usually present in lower expression levels, especially in early stage of cancer. Here, we proposed a "one-to-more" amplification strategy for miRNA imaging, by virtue of DNA strand displacements with dual-amplification. This approach involves leveraging high-abundance endogenous mRNA as fuel strand to drive cascade reactions between DNA strands for amplification, enabling the monitoring of low-abundance intracellular microRNA155. Notably, in comparison to the traditional "one-to-one" signal triggering mode, our "one-to-more" amplification strategy led to a remarkable 11.8-fold increase in fluorescence signal. Our approach not only demonstrates a high sensitivity and specificity in detecting miR155, but also allows for discrimination of miR155 expression levels in different cell lines. With the advantages of intracellular signal amplification and reduced background signal, this approach holds substantial potential in the early diagnosis of cancer.

A high-throughput screen in mESCs to identify the cross-talk between signaling, endocytosis, and pluripotency.

Embryonic stem cell fate is regulated by various cellular processes. Recently, the process of endocytosis has been implicated in playing a role in the maintenance of self-renewal and pluripotency of mouse embryonic stem cells. A previous siRNA-based screen interrogated the function of core components of the endocytic machinery in maintaining the pluripotency of embryonic stem cells, revealing a crucial role for clathrin mediated endocytosis. A number of other proteins involved in key signaling pathways have also been shown to both regulate and be regulated by endocytosis. We collated a list of 1141 genes in connection to the term "endocytosis" from Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), excluding those previously interrogated, and examined the effect of their knockdown on the pluripotency of mouse embryonic stem cells (mESCs) using levels of green fluorescent protein driven by the Oct4 promoter. We used high-throughput screening followed by an automatedMATrix LABoratory(MATLAB)-based analysis pipeline and assessed changes in GFP fluorescence as a readout for ESC pluripotency. Through this screen we identified a number of genes, many hitherto not associated with stem cell pluripotency, which upon knockdown either resulted in a significant increase or decrease of GFP fluorescence. We further present validation for some of these hits. We present a workflow aimed to identify genes involved in signaling pathways which can be regulated by endocytosis, and that affect the pluripotency of ESCs.

Rational design of a near-infrared fluorescent probe for monitoring butyrylcholinesterase activity and its application in development of inhibitors.

Butyrylcholinesterase (BChE) is widely expressed in multiple tissues and has a vital role in several key human disorders, such as Alzheimer's disease and tumorigenesis. However, the role of BChE in human disorders has not been investigated. Thus, to quantitatively detect and visualize dynamical variations in BChE activity is essential for exploring the biological roles of BChE in the progression of a number of human disorders. Herein, based on the substrate characteristics of BChE, we customized and synthesized three near-infrared (NIR) fluorescent probe substrates with cyanine-skeleton, and finally selected a NIR fluorescence probe substrate named CYBA. The CYBA demonstrated a significant increase in fluorescence when interacting with BChE, but mainly avoided AChE. Upon the addition of BChE, CYBA could be specifically hydrolyzed to TBO, resulting in a significant NIR fluorescence signal enhancement at 710nm. Systematic evaluation revealed that CYBA exhibited exceptional chemical stability in complex biosamples and possessed remarkable selectivity and sensitivity towards BChE. Moreover, CYBA was successfully applied for real-time imaging of endogenous BChE activity in two types of nerve-related living cells. Additionally, CYBA demonstrated exceptional stability in the detection of complex biological samples in plasma recovery studies (97.51%-104.01%). Furthermore, CYBA was used to construct a high-throughput screening (HTS) method for BChE inhibitors using human plasma as the enzyme source. We evaluated inhibitory effects of a series of natural products and four flavonoids were identified as potent inhibitors of BChE. Collectively, CYBA can serve as a practical tool to track the changes of BChE activity in complicated biological environments due to its excellent capabilities.

A new Eosin Y-based ‘turn[sbnd]on’ fluorescent sensor for ratiometric sensing of toxic mercury ion (Hg2+) offering unaided eye detection and its antibacterial activity

A unique fluorescent molecule (ND-S) was obtained from Eosin Y in two simple yet high yielding steps (1). ND-S has special metal ion sensing ability, such that it can selectively detect toxic Hg2+ present in very low concentration in aqueous solutions in the presence of other competing metal ions. The host-guest complexation is ratiometric and is associated with significant increase in fluorescence during the process. Isothermal titration calorimetry (ITC) experiments provided thermodynamic parameters related to interaction between ND-S and Hg2+. Using inductively coupled plasma mass spectrometry (ICP-MS), the Hg2+(aq) removal efficiency of ND-S was estimated to be 99.88%. Appreciable limit of detection (LOD = 7.4 nM) was observed. Other competing ions did not interfere with the sensing of Hg2+ by ND-S. The effects of external stimuli (temperature and pH) were studied. Besides, the complex (ND-M), formed by 1:1 coordination of ND-S and Hg2+ was found to be effective against the survival of Gram-positive bacteria (S. aureus and B. subtilis) with a high selectivity index. Moreover, bacterial cell death mechanism was studied systematically. Overall, we have shown the transformation of a toxic species (Hg2+), extracted from polluted water by a biocompatible sensor (ND-S), into an effective and potent antibacterial agent (ND-M).

Protein biomarker detection via differential dynamic microscopy

We demonstrate a pioneering application of differential dynamic microscopy (DDM) for disease diagnosis by monitoring the dynamic signal of probe beads. When the target antibody binds to our sensitized probe beads, it allows these originally non-fluorescent beads to be indirectly decorated with fluorescently labeled secondary antibodies. Using standard epi-fluorescence microscopy without the need for purification, DDM reliably detects the resulting increase in fluorescence signal from the probe beads. It effectively discerns this signal from the background, which includes residual free fluorescent secondary antibodies. As a proof-of-concept, polystyrene beads (500 nm in diameter) were conjugated with the receptor binding domain of SARS-CoV-2’s spike protein to quantify immunoglobulin G1-specific antibodies against it in three different media of increasing complexity (phosphate buffer, model saliva and fetal bovine serum). Our results show that as the medium becomes more complex, more dilution and/or longer incubation time may be needed to overcome the competition effect between the target and interfering proteins. By optimizing imaging parameters and sample preparation we could surpass clinically relevant limits of detection in both saliva and serum samples. In contrast to conventional diagnostic techniques including ELISA and PCR, DDM brings many advantages including simplicity of the preparation protocol, high speed, and versatility without requiring very expensive or complicated equipment. As such, DDM-based disease diagnostics has the potential to be utilized in many laboratories, especially in remote regions with limited resources and point-of-use applications may also be feasible by miniaturizing the imaging setup.

Investigation of biofluorescence produced by the red king crab Paralithodes camtschaticus

AbstractBiofluorescence is widely documented in marine organisms, yet few studies exist for decapods. After observing the king crab Paralithodes camtschaticus biofluoresces, we carried out studies on two separately maintained groups of male crabs under controlled conditions. Hyperspectral imaging on Group 1 (n = 18) examined the exoskeleton, whereas fluorospectrometry examined the hemolymph of Group 2 animals (n = 19). Both groups were investigated for fluorescence signals before and after exposure to a live shipping transportation simulation. The spines, chelae, eyestalks and cervical grooves of the cardiac region of P. camtschaticus fluoresce in the green spectra (∼500 nm), while the arthrodial membranes of the joints fluoresce in the red spectrum (∼680 nm). After the shipping simulation, we observed a significant decrease in fluorescence in the eyestalks (p = 0.009), while the cervical grooves showed a less significant change (p = 0.01). The hemolymph examined with 21 excitation wavelengths (250–350 nm) emitted fluorescence in ∼400–550 nm spectrum. We found a significant increase (p < 0.05) in fluorescence for 16 excitation wavelengths after transport simulation. The results presented in this study indicate that king crab fluorescence changes due to external stimuli. Fluorospectroscopy or hyperspectral imaging technology may serve as an effective early indicator of preclinical stress in these commercially important decapods.

Abstract 1021: Defining total protease activity profiles in pancreatic cancer using a FRET substrate array

Abstract Proteases are an important component of many biological processes in the human body, and as such makes them a well-known subject to explore when investigating a variety of disease states from cancer to cardiovascular disease. In all stages of cancer, including early development, proteases play critical roles in almost every aspect for processes such as invasion, promotion of tumor growth, and metastasis. This makes screening of circulating proteases a potentially powerful tool for early detection and prognosis for a variety of cancers. The human proteome, however, is very complex, with around 600 proteases in total, and at least 234 of them found in circulation. Additionally, proteases often act on more than one substrate, cross reactively cleaving several substrates at different rates, further adding to the complexity of cleavage patterns of the human proteome. Previous liquid biopsies for cancer detection based on protease cleavage patterns focused mainly on detecting the activities of specific proteases, using single substrates to examine the effects of well-studied cancer-related proteases such as MMPs and cathepsins. In this study, we developed a method for screening cleavage patterns of proteases active in plasma samples that deviated from this norm. Due to the complexity of the human proteome, rather than developing a specific substrate for a specific protease, we utilized a large array of substrates to screen for cleavage patterns in plasma samples of cancer patients aimed at not one specific protease, but a wide range that may be present. In this proof of concept study, we used a commercially available internally quenched set of 360 substrates composed of 8 amino acids and a FRET pair. Initially, we optimized this assay to measure protease activity in the complex plasma environment. Next, we screened several plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC) or pancreatitis, and screen negative samples using the 360 substrate set assay and identified 5 top substrates that were proficient in identifying PDAC from other conditions. Finally, we performed a validation study with a larger cohort (45 screen negative, 26 PDAC, 19 pancreatitis) using the selected substrates to determine the diagnostic performance of the assay. All five of the selected substrates showed 1.3-1.8-fold increase in fluorescence signal in PDAC compared to screen negative and pancreatitis. Amongst the cancer samples, the substrates showed no significant difference between stage, supporting possible applications for early detection. Additionally, area under the curve analysis of the five substrates gave a score of 0.8. In summary, in this study, we reported a novel approach for the detection of cancer exploiting the protease cleavage patterns in plasma samples. We believe that this approach holds great potential for detecting substrates and proteases for early detection of PDAC and other cancers. Citation Format: Morgan Stewart, Arnaud Quentel, Jose Montoya Mira, Elise Manalo-Hall, Srivathsan Ranganathan, Bruce Branchaud, Fehmi Civitci, Eugene Tu, Jared Fischer, Yu-Jui Chiu, Adem Yildirim. Defining total protease activity profiles in pancreatic cancer using a FRET substrate array [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 1021.

Evaluation of the Remineralization Capacity of Water-based Silver Fluoride

Silver diamine fluoride, which can arrest dental caries, is alkaline and may cause mild soft tissue irritation. Water-based silver fluoride has a neutral pH, which is closer to the physiological range, and is biocompatible for use in the oral environment. This study aimed to evaluate the effect of water-based silver fluoride on remineralizing early enamel lesions by comparing it with other fluoride agents through microhardness and quantitative light-induced fluorescence measurements. An in vitro study with intact bovine incisors was performed. Artificial enamel lesions were induced and subjected to microhardness and quantitative light-induced fluorescence testing. Specimens were randomly divided into 4 groups for treatment. The specimens in group I were treated with water-based silver fluoride and potassium iodide, group II with silver diamine fluoride and potassium iodide, group III with sodium fluoride varnish, and group IV with distilled water. After 8 days of pH cycling, the specimens were subjected to microhardness and quantitative light-induced fluorescence testing. Water-based silver fluoride and silver diamine fluoride showed the greatest increases in microhardness and quantitative light-induced fluorescence, with no significant differences between the two. Sodium fluoride varnish also exhibited a significant increase in microhardness and quantitative light-induced fluorescence, but the differences were smaller than those for water-based silver fluoride and silver diamine fluoride. Water-based silver fluoride is considered useful in a clinical setting for remineralizing enamel lesions, with the advantages of no risk of tissue burn and improved taste and smell.

Determination of Lead in Bee Products by Solid Surface Fluorescence Using Complexation and Coacervation at Room Temperature Processes. An Environmental Friendly Methodology.

An accurate, economic and green methodology for Pb(II) monitoring in bee products is proposed. Complexed metal traces were preconcentrated on Nylon membranes using the coacervation phenomenon based on room temperature reaction between the cationic surfactant hexadecyltrimethylammonium bromide and the bile salt sodium cholate. The increase in solid surface fluorescence signal of dyes 8-hydroxyquinoleine and o-phenanthroline due to Pb(II) presence was used for the metal quantification. Experimental variables that influence on preconcentration step and fluorimetric sensitivity were optimized using uni-varied assays. Pb(II) concentration was determined on membranes by solid surface fluorescence at λem = 470nm (λexc = 445nm), using a solid sample holder. The calibration at optimal experimental conditions showed a LOD of 4.2 × 10-4mg Kg-1 with a linear range of 1.28 × 10-3mg Kg-1 to 8.73mg Kg-1 and was successfully applied to Pb(II) quantification in different bee products produced in central west region of Argentina. The proposed methodology was applied to all samples after appropriate dilution. Accuracy methodology was evaluated by comparison of the obtained results with those found by ICP-MS, with percentage relative error under 8%. The precision was better than 0.0344 CV for Pb(II) determination.

Strain-Modulated and Nanorod-Waveguided Fluorescence in Single Zinc Oxide Nanorod-Based Immunodetection.

Mechanical strain has been shown to be a versatile and tunable means to control various properties of nanomaterials. In this work, we investigate how strain applied to individual ZnO nanorods (NRs) can affect the fluorescence signals originated from external sources of bioanalytes, which are subsequently coupled and guided onto the NRs. Specifically, we determine how factors such as the NR length and protein concentration can influence the strain-induced changes in the waveguided fluorescence intensity along the NRs. We employ a protein of tumor necrosis factor-α (TNF-α) and a fluorophore-labeled antibody in a model immunoassay reaction, after which Alexa488-TNF-α immunocomplex is formed on ZnO NRs. We elucidate the relationships between the types as well as amounts of strain on the NRs and the fluorescence intensity originated from the Alexa488-TNF-α immunocomplexes. We show that tensile (compressive) strain applied to the NR leads to an increase (decrease) in the waveguided fluorescence signals. By assessing important optical phenomena such as fluorescence intensification on nanorod ends (FINE) and degree of FINE (DoF), we confirm their linear dependence with both the types and amounts of strain. Furthermore, the strain-induced changes in both FINE and DoF are found to be independent of protein concentration. We determine that NR length plays a critical role in obtaining high strain-dependence of the measured fluorescence signals. Particularly, we ascertain that longer NRs yield larger changes in both FINE and DoF in response to the applied strain, relative to shorter ones. In addition, longer NRs permit higher linear correlation between the protein concentration and the waveguided fluorescence intensity. These outcomes provide valuable insight into exploiting strain to enhance the detection of optical signals from bioanalytes, thus enabling their quantifications even at ultra-trace levels. Coupled with the use of individual ZnO NRs demonstrated in our measurements, this work may contribute to the development of a miniaturized, highly sensitive biosensor whose signal transduction is best optimized by the application of strain.

Development of optical probes with excellent intracellular retention

Small-molecule based activatable fluorescence probes for detecting specific enzyme activity with high sensitivity can visualize the expression site of marker genes and cancers where the enzyme is highly expressed. However, the enzyme-catalyzed fluorescent hydrolysis product easily leaks out and diffuses from the reaction site, making it difficult to perform long-term tracking and immunohistochemical analysis which needs washing/fixation procedure. Our group have focused on quinone methide chemistry and developed series of activatable fluorescence probes with excellent intracellular retention that are converted to quinone-methide or aza-quinone-methide intermediates upon reaction with enzymes, which are then react with intracellular nucleophiles such as proteins and glutathione to be retained in cells and to exhibit significant increase in fluorescence. Based on this molecular design, we have developed fluorescence probes targeting β-galactosidase and γ-glutamyltranspeptidase with different colors. We also developed photo-functional probes such as activatable photosensitizers and caged fluorophores. These probes can visualize or kill target enzyme-expressing cells with high selectivity by suppressing the leakage of hydrolysis products from target cells, and fluorescence imaging in combination with immunostaining was possible due to the high tolerance of the obtained fluorescence signal even after washing and fixation.

Photophysical analysis of dual fluorescence and phosphorescence emissions observed for semi-aliphatic polyimides at lower temperatures.

The photoluminescence (PL) properties of four types of blue fluorescent semi-aliphatic polyimides (PIs) derived from aromatic dianhydrides (ODPA, BPDA, HQDEA, and BPADA) and an alicyclic diamine (DCHM) were investigated at temperatures ranging from room temperature (RT, 298 K) to 30 K to analyse the origins of their non-radiative relaxation (NR) processes. These PIs exhibited significant increases in fluorescence (FL) intensity and lifetimes when lowering the temperature, stabilising below 100 K. The PIs containing ether (-O-) linkages showed a shoulder peak at around 500 nm below 150 K, which is attributable to phosphorescence (PH). These results show that the NR deactivation at RT includes three processes: intersystem crossing (ISC) from the excited singlet (S1) to the triplet (T1) state, temperature-dependent NR from the S1 state, which becomes suppressed below around 100 K, and temperature-independent NR. Based on the analyses of the temperature dependences, polymer structures, and quantum chemical analysis of molecular orbitals, we contemplate that the temperature-dependent NR is attributable to the excitation quenching by defect states mediated by excitation migration, and the temperature-independent NR may be caused by the deactivation of the excited state induced by molecular vibrations.

Fluorescence and colorimetric analysis of β-estradiol based on aptamer assembled spherical nucleic acids.

Detecting β-estradiol (E2) in environmental monitoring is a complex task due to its status as a significant environmental contaminant. The detection methods require precision, sensitivity, and the ability to be conducted on-site without expensive instrumentation. Herein, we developed a novel approach using E2 aptamer assembled spherical nucleic acids (SNAs), which combines the sensitivity of fluorescence and the simplicity of colorimetry. Initially, a fluorescein (FAM)-labeled DNA aptamer is attached to the surface of gold nanoparticles (AuNPs) through hybridization with thiol-labeled DNA, resulting in fluorescence quenching. However, when E2 is present, the aptamer specifically binds to it, displacing from the thiol-DNA and releasing from the AuNP's surface. This leads to the recovery of fluorescence, allowing for quantitative detection of E2 by measuring the increase in fluorescence signal. Additionally, E2 detection can also be achieved visually using ultraviolet light. For colorimetric analysis, we introduce another set of AuNPs modified with thiol-DNA complementary to the DNA remaining on the surface of the previous AuNPs. When E2 triggers the release of the aptamer, the DNA on both AuNPs hybridized to each other, causing the aggregation of AuNPs and resulting in a distinct color change from red to purple. The detection limits for fluorescence and colorimetric analyses are 1 nM and 5 nM, respectively. We successfully applied this biosensing strategy to determine E2 concentrations in tap water and serum samples. Furthermore, our assay exhibits high selectivity towards E2 over other estrogens. Overall, this innovative approach provides an effective and versatile method for convenient on-site monitoring of E2.