Fluorescent Sensors for Tetracycline Detection in Aqueous Medium: A Mini-Review.

Tetracycline (TC) is a commonly used antibiotic in human therapy and animal husbandry. Public concerns about TC residues inflated due to their negative impact on the environment, food, and human health concerns. To ensure human health and safety, there is a need for fluorogenic chemosensors that can easily detect TC antibiotics with high selectivity and sensitivity in the aqueous medium. This mini-review discusses the progress and achievements in several fluorometric antibiotic tetracycline detection methods. Fluorogenic chemosensors for tetracycline antibiotics with easy-to-use, high selectivity, and sensitivity have been essentially required to regulate food safety and secure human health and safety. Moreover, we gave more attention to the practical applicability of chemosensors for tetracycline antibiotics in food and water quality assessment. This article starts with a section that constitutes an overview of the problems of antibiotics and the typical features of traditional techniques of antibiotic detection. It then goes on to describe up-to-date optical methods for the selective detection and efficient removal of TC. These methods involve a variety of platforms, like tetraphenylethylene polymers, metal complexes, self-assembled CuNCs, and hydrogel. The article also discusses the practical applicability of chemosensors for tetracycline antibiotics in food and water quality.

Development of a fluorometric and colorimetric dual-mode sensing platform for acid phosphatase assay based on Fe3+ functionalized CuInS2/ZnS quantum dots.

BACKGROUND: The spectral dual-mode response towards analyte has been attracted much attention, benefiting from the higher detection accuracy of such strategy in comparison to single signal readout. However, the currently reported dual-mode sensors for acid phosphatase (ACP) activity are still limited, and most of them more or less exist some deficiencies, such as complicated construction procedure, high-cost, poor biocompatibility, aggregation-caused quenching and limited emission capacity. RESULTS: Herein, we employed Fe3+ functionalized CuInS2/ZnS quantum dots (CIS/ZnS QDs) as nanosensor to develop a novel fluorometric and colorimetric dual-mode assay for ACP activity, combing with ACP-triggered hydrolysis of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA). The Fe3+ binding to CIS/ZnS QDs can be reduced into Fe2+ during the determination, resulting in the dramatically weakened photoinduced electron transfer (PET) effect and the disappearance of competition absorption. Thus, a highly sensitive ACP assay in the range of 0.22-12.5 U L-1 through fluorescence "turn-on" mode has been achieved with a detection of limit (LOD) of 0.064 U L-1. Meanwhile, the ACP activity can also be quantified by spectrophotometry based on the chromogenic reaction of the formed Fe2+ with 1,10-phenanthroline (Phen). Moreover, the designed nanosensor with good biocompatibility was successfully applied to image and monitor the ACP levels in living cells. SIGNIFICANCE: We believe that the proposed method has remarkable advantages and potential application for ACP assay in terms of the high accuracy, simplicity, low cost, as well as its adequate sensitivity.

Dual-functionalization of fluorescent carbon dots via cyclodextrin and aminosilane for visual detection of ß-glucuronidase and bioimaging.

A sensitive method for the detection of ß-glucuronidase was established using functionalized carbon dots (ß-CD-SiCDs) as fluorescent probes. The ß-CD-SiCDs were found to be obtained through in situ autopolymerization by mixing the solutions of methyldopa, mono-6-ethylenediamine-ß-cyclodextrin and N-(ß-aminoethyl)-γ-aminopropyltrimethoxysilane at room temperature. The method has the characteristics of low energy consumption, simple and rapid. ß-CD-SiCDs exhibited green fluorescence at 515 nm emission with a quantum yield of 7.9 %. 4-nitrophenyl-ß-D-glucuronide was introduced as a substrate for ß-glucuronidase to generate p-nitrophenol. Subsequently, p-nitrophenol self-assembled with ß-CD-SiCDs through host-guest recognition to form a stable inclusion complex, resulting in the fluorescence quenching of ß-CD-SiCDs. The linear range of ß-CD-SiCDs for detecting ß-glucuronidase activity was 0.5-60 U L-1 with a detection limit of 0.14 U L-1. For on-site detection, gel reagents were prepared by a simple method and the images were visualized and quantified by taking advantage of smartphones, avoiding the use of large instrumentation. The constructed fluorescence sensing platform has the benefits of easy operation and time saving, and has been successfully used for the detection of ß-glucuronidase activity in serum and cell imaging.

A spatially-controlled DNA triangular prism nanomachine for AND-gated intracellular imaging of ATP in acidic microenvironment.

A DNA triangular prism nanomachine (TPN)-based logic device for intracellular AND-gated imaging of adenosine triphosphate (ATP) has been constructed. By using i-motif sequences and ATP-binding aptamers as logic control units, the TPN logic device is qualified to respond to the acidic environment and ATP in cancer cell lysosomes. Once internalized into the lysosome, the specific acidic microenvironment in lysosome causes the i-motif sequence to fold into a tetramer, resulting in compression of DNA tri-prism. Subsequently, the split ATP aptamer located at the tip of the collapsed triangular prism binds stably to ATP, which results in the fluorescent dyes (Cy3 and Cy5) modified at the ends of the split aptamer being in close proximity to each other, allowing Förster Resonance Energy Transfer (FRET) to occur. The FRET signals are excited at a wavelength of 543 nm and can be collected within the emission range of 646-730 nm. This enables the precise imaging of ATP within a cell. We also dynamically operate AND logic gates in living cells by modulating intracellular pH and ATP levels with the help of external drugs. Owing to the AND logic unit on TPN it can simultaneously recognize two targets and give corresponding intelligent logic judgment via imaging signal output. The accuracy of molecular diagnosis of cancer can be improved thus eliminating the false positive signal of single target-based detection. Hence, this space-controlled TPN-based logical sensing platform greatly avoids sensitivity to extracellular targets during the cell entry process, providing a useful tool for high-precision imaging of the cancer cell's endogenous target ATP.

Photophysical Study of Heteroatom-Doped Carbon Dots-MnO2-Based Nanosensor: Selective Detection of Glutathione in the Nanomolar Level.

Heteroatom doping on carbon dots (Cdots) has been developed as an efficient approach to modify its optical and electronic properties. The four different types of heteroatom-doped Cdots (undoped Cdots (u-Cdots, nitrogen-doped Cdots (N-Cdots), sulfur-doped Cdots (Cdots), nitrogen, sulfur codoped Cdots (N, S-Cdots)) have been synthesized through a simple heat treatment of 5 min. Among four different heteroatoms doped nanosensors, N, S-Cdots with MnO2 nanospheres (Mn NS) showed one of the best fluorescents "on-off-on" nanosensors for selective sensing of glutathione (GSH) and cell imaging. N, S-Cdots showed a high fluorescence quantum yield, good photostability, ionic strength, and pH stability. N, S-Cdots with Mn NS demonstrated extremely high fluorescence quenching efficiency and the maximum fluorescence recovery rate after adding GSH to the produced solution. The photophysical study of N, S-Cdots-Mn NS used as a sensor confirms the inner filter effect (IFE) quenching mechanism between them. The developed sensor has an 80 nM limit of detection (LOD) for GSH. The heteroatom-doped framework of Cdots plays a significant role in the sensitive detection of GSH. N, S-Cdots-Mn NS have good permeability, biocompatibility, and low toxicity, due to which it was used in the intracellular imaging of GSH in living cells. The prepared sensor is rapid, economical, less toxic, and highly applicable in diagnosing diseases.

Recent Progress of Spectroscopic Probes for Peroxynitrite and Their Potential Medical Diagnostic Applications.

Peroxynitrite (ONOO-) is a crucial reactive oxygen species that plays a vital role in cellular signal transduction and homeostatic regulation. Determining and visualizing peroxynitrite accurately in biological systems is important for understanding its roles in physiological and pathological activity. Among the various detection methods, fluorescent probe-based spectroscopic detection offers real-time and minimally invasive detection, high sensitivity and selectivity, and easy structural and property modification. This review categorizes fluorescent probes by their fluorophore structures, highlighting their chemical structures, recognition mechanisms, and response behaviors in detail. We hope that this review could help trigger novel ideas for potential medical diagnostic applications of peroxynitrite-related molecular diseases.

A selective fluorescent probe for hydrogen sulfide from a series of flavone derivatives and intracellular imaging.

In this work, through the orthogonal design of two fluorophores and two recognition groups, a series of fluorescent probes were developed from the flavone derivatives for hydrogen sulfide (H2S). The probe FlaN-DN stood out from the primarily screening on the selectivity and response intensities. It could respond to H2S with both the chromogenic and fluorescent signals. Among the recent reported probes for the H2S detection, FlaN-DN indicated the most highlighted advantages including the rapid response (within 200 s) and the high response multiplication (over 100 folds). FlaN-DN was sensitive to the pH condition, thus could be applied to distinguish the cancer micro-environment. Moreover, FlaN-DN suggested practical capabilities including a wide linear range (0-400 µM), a relatively high sensitivity (limit of detection 0.13 µM), and high selectivity towards H2S. As a low cytotoxic probe, FlaN-DN achieved the imaging in living HeLa cells. FlaN-DN could detect the endogenous generation H2S and visualize the dose-dependent responses to the exogenous H2S level. This work provided a typical case of natural-sourced derivatives as functional implements, which might inspire the future investigations.

Carbon dots doped with nitrogen as an ultrasensitive fluorescent probe for thrombin activity monitoring and inhibitor screening.

A simple and sensitive fluorometric assay based on nitrogen-doped carbon dots (N-CDs) was developed for the determination of thrombin (TB) activity in human serum samples and living cells. The novel N-CDs were prepared by a facile one-pot hydrothermal method using 1,2-ethylenediamine and levodopa as precursors. Such N-CDs exhibited green fluorescence with excitation/emission peaks at 390/520 nm and a high fluorescence quantum yield of approximately 39.2%. H-D-Phenylalanyl-L-pipecolyl-Larginine-p-nitroaniline-dihydrochloride (S-2238) was hydrolyzed by TB to produce p-nitroaniline which was capable of quenching the fluorescence of N-CDs due to an inner filter effect. This assay was used to detect TB activity with a low detection limit of 11.3 fM. The proposed sensing method was then expanded to the TB inhibitor screening and exhibited excellent applicability. As a typical TB inhibitor, argatroban was determined in a concentration as low as 1.43 nM. The method has also been successfully employed for the determination of TB activity in living HeLa cells. This work showed significant potential for TB activity assay in clinical and biomedicine applications.

Ultra-sensitive biosensor based on CRISPR-Cas12a and Endo IV coupled DNA hybridization reaction for uracil DNA glycosylase detection and intracellular imaging.

As an essential biomarker associated with various diseases, Uracil-DNA Glycosylase (UDG) detection is vital for disease diagnosis, treatment selection, and prognosis assessment. In recent years, the signal amplification effect of the CRISPR-Cas12a trans-cleaved single-stranded DNA probe has provided an available strategy for constructing highly sensitive biosensors. However, its superior trans-cleavage activity has become a "double-edged sword" for building biosensors that can amplify the target signal while also amplifying the leakage signal, causing out of control. Therefore, the construction of structurally simple, extremely low-background, highly sensitive CRISPR-Cas12a-based biosensors is an urgent bottleneck problem in the field. Here, we applied CRISPR-Cas12a with a DNA hybridization reaction to develop a simple, rapid, low background, and highly sensitive method for UDG activity detection. It has no PAM restriction and the detection limit is as low as 2.5 × 10-6 U/mL. As far as we know, this method is one of the most sensitive methods for UDG detection. We also used this system to analyze UDG activity in tumor cells (LOD: 1 cell/uL) and to evaluate the ability to screen for UDG inhibitors. Furthermore, we verified the possibility of intracellular UDG activity imaging by transfecting the biosensors to the cells. We believe this novel sensor has good clinical application prospects and will effectively broaden the application space of CRISPR-Cas12a.

How sticky? How tight? How hot? Imaging probes for fluid viscosity, membrane tension and temperature measurements at the cellular level.

We review the progress made in imaging probes for three important physical parameters: viscosity, membrane tension, and temperature, all of which play important roles in many cellular processes. Recent evidences showed that cell migration speed can be modulated by extracellular fluid viscosity; membrane tension contributes to the regulation of cell motility, exo-/endo-cytosis, and cell spread area; and temperature affects neural activity and adipocyte differentiation. We discuss the techniques implementing imaging-based probes to measure viscosity, membrane tension, and temperature at subcellular resolution dynamically. The merits and shortcomings of each technique are examined, and the future applications of the recently developed techniques are also explored.

Challenges of intracellular visualization using virtual and augmented reality.

Microscopy image observation is commonly performed on 2D screens, which limits human capacities to grasp volumetric, complex, and discrete biological dynamics. With the massive production of multidimensional images (3D + time, multi-channels) and derived images (e.g., restored images, segmentation maps, and object tracks), scientists need appropriate visualization and navigation methods to better apprehend the amount of information in their content. New modes of visualization have emerged, including virtual reality (VR)/augmented reality (AR) approaches which should allow more accurate analysis and exploration of large time series of volumetric images, such as those produced by the latest 3D + time fluorescence microscopy. They include integrated algorithms that allow researchers to interactively explore complex spatiotemporal objects at the scale of single cells or multicellular systems, almost in a real time manner. In practice, however, immersion of the user within 3D + time microscopy data represents both a paradigm shift in human-image interaction and an acculturation challenge, for the concerned community. To promote a broader adoption of these approaches by biologists, further dialogue is needed between the bioimaging community and the VR&AR developers.

Molecular to Supramolecular Self-Assembled Luminogens for Tracking the Intracellular Organelle Dynamics.

Deciphering the dynamics of intracellular organelles has gained immense attention due to their subtle control over diverse, complex biological processes such as cellular metabolism, energy homeostasis, and autophagy. In this context, molecular materials, including small-organic fluorescent probes and their supramolecular self-assembled nano-/microarchitectures, have been employed to explore the diverse intracellular biological events. However, only a handful of fluorescent probes and self-assembled emissive structures have been successfully used to track different organelle's movements, circumventing the issues related to water solubility and long-term photostability. Thus, the water-soluble molecular fluorescent probes and the water-dispersible supramolecular self-assemblies have emerged as promising candidates to explore the trafficking of the organelles under diverse physiological conditions. In this review, we have delineated the recent progress of fluorescent probes and their supramolecular self-assemblies for the elucidation of the dynamics of diverse cellular organelles with a special emphasis on lysosomes, lipid droplets, and mitochondria. Recent advancement in fluorescence lifetime and super-resolution microscopy imaging has also been discussed to investigate the dynamics of organelles. In addition, the fabrication of the next-generation molecular to supramolecular self-assembled luminogens for probing the variation of microenvironments during the trafficking process has been outlined.

Fluorescence imaging of FEN1 activity in living cells based on controlled-release of fluorescence probe from mesoporous silica nanoparticles.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease, which catalyzes the removal of 5' overhanging DNA flap from a specific DNA structure. FEN1 has been considered as an important biomarker for cancer diagnosis since it is over-expressed in various types of human tumor cells and closely related to cancer development. Nanoprobes gradually become basic tools for analyzing biomarkers variations in vivo. Here, we utilized aminoated mesoporous silica nanoparticles (NH2-MSNs) with a rich porous structure as the fluorescence nanoprobes to entrap the rhodamine 6G (Rh6G) molecules. Then gold nanoparticles linked specific single-stranded DNA (AuNPs-ssDNA) as a molecular gate was used to coat the NH2-MSNs surface. The fluorescence signal was weak when the fluorescence molecules were blocked by the AuNPs-ssDNA. In the presence of FEN1, it recognized and cleaved the specific ssDNA to release the Rh6G from NH2-MSNs, which resulted in recovered fluorescence signals. Thus, the sensitive detection of FEN1 activity was realized by controlled-release of Rh6G. The fluorescence signal showed a good linear relationship with the logarithm of FEN1 activity ranging from 0.05 to 1.75 U with a detection limit of 0.03 U. Moreover, confocal imaging demonstrated that the proposed biosensor could distinguish tumor cells from normal cells. Therefore, this technique contributes to clinical diagnostic and therapeutic monitoring.

A fluorescent probe for monitoring sulfite in living cells with large Stokes shift and rapid response.

Sulfite (SO32-) is considered as a monitor of a wide range of physiological processes. However, cells and tissues are adversely affected when the body ingests high level of sulfite. Here, we designed and synthesized a "turn on" fluorescent probe ImiSft-1 with 2-cyano-N-methylacetamide as the specific recognition site of SO32-. This probe predominantly achieved high response intensity to SO32- and desirable properties such as large Stokes shift (∼180 nm), fast response time (within 15 s), and high sensitivity (LOD = 0.12 µM). Importantly, the probe was highly selective for sulfite from other bio-species including biological thiols. Other functional properties included broad pH adaptability (5.0-10.0) and low cytotoxicity. Given these advantages and the fluorescence imaging in living MCF-7 cells, it was demonstrated that probe ImiSft-1 could monitor the changes of sulfite concentration in living cells.

Rational design of a fluorescent probe for specific sensing of hydrogen peroxide/glucose and intracellular imaging applications.

A new type of dye with advantages of high selectivity and sensitivity is formed by using the strategy of hybridization between the luminescent unit and recognition unit. Based on this strategy, we exploit a novel dye bonding the benzopyrylium salt as a luminescent unit and phenylboronate group as a response site, which is served as a fluorescent probe 1 for specific recognition of hydrogen peroxide in biological application. Probe 1 employs a unique recognition switch, phenylboronate unit, to"turn-on"a highly specific and rapid fluorescence response toward hydrogen peroxide combined with the 1,6-rearrangement elimination reaction strategy. Meanwhile, probe 1 has the ability to glucose assay by taking advantage of glucose oxidase/glucose enzymatic reaction. What's more, the probe 1 is capable of tracking endogenous hydrogen peroxide in living cells and intracellular imaging. Therefore, the newly developed bioprobe 1 is expected to be used to monitor hydrogen peroxide and glucose levels in complex organisms.

Closing-upon-repair DNA tetrahedron nanoswitch for FRET imaging the repair activity of 8-oxoguanine DNA glycosylase in living cells.

In situ imaging the repair activity of 8-oxoguanine (8-OG) DNA glycosylase in living cells is important as it is associated with genetic mutation. However, the existing imaging methods confront the interference of intracellular nuclease and resulting in false positive signal. Here, a closing-upon-repair DNA tetrahedron nanoswitch (CRTN) was designed for FRET imaging the repair activity of 8-OG DNA glycosylase in living cells with high specificity and accuracy. CRTN comprised a DNA tetrahedron, a recognition strand modified with 8-OG bases, and a reporting strand designed as hairpin structure and labeled with Cy3/Cy5 dual fluorophores. Initially, the DNA tetrahedron was linked with the reporting strand hybridized to the recognition strand, separating the Cy3 donor and Cy5 acceptor into FRET-invalid distance. Upon repair the 8-OG bases by 8-OG DNA glycosylase, CRTN could undergo a structure change from the open to closed state. Specifically, the reporting strand was dissociated from the recognition strand under the action of 8-OG DNA glycosylase and folded into hairpin structure, bringing the Cy3 donor and Cy5 acceptor into FRET-valid proximity with the generation of FRET signal, which could prevent false positive signal arising from nuclease degradation. CRTN exhibited the feasibility for detecting 8-OG DNA glycosylase activity in vitro with good sensitivity and selectivity. More importantly, CRTN could enter cells without any transfection for FRET imaging the repair activity of intracellular 8-OG DNA glycosylase with high specificity and accuracy. This approach provided a promising tool for deeper understanding 8-OG DNA glycosylase function and further studying genetic mutation-related diseases.

Fluorescein isothiocyanate-doped conjugated polymer nanoparticles for two-photon ratiometric fluorescent imaging of intracellular pH fluctuations.

Herein, we report a two-photon ratiometric fluorescent pH nanosensor based on conjugated polymer poly(9,9-di-n-octylfluorenyl-2,7-diyl) (PFO) nanoparticles loaded with pH-sensitive fluorescein isothiocyanate (FITC) for intracellular pH monitoring. The obtained nanosensor (FITC-PFO NPs) possesses high sensitivity, excellent stability, good reversibility, favorable two-photon excitability and low cytotoxicity. The ratiometric fluorescence of FITC and PFO (F517/F417) in FITC-PFO NPs solution shows an efficient pH-sensitive response over the pH range from 3 to 10 (pKa = 6.43) under two-photon excitation. Additionally, the FITC-PFO NPs is successfully applied for ratiometric imaging of intracellular pH and its fluctuation in both one-photon and two-photon excitation modes. Overall, the two-photon pH nanosensor based on FITC-PFO NPs exhibits great potential in crucial physiological and biological processes related to intracellular pH fluctuations.

Coordination-driven reversible supramolecular assembly formation at biological pH: Trace-level detection of Hg2+ and I- ions in real life samples.

Pyridine coupled bisbenzimidazole probe has been developed for colorimetric sensing of heavy metal pollutants in the aqueous medium. Mechanistic investigation indicates that Hg2+ ions (detection limit: 7.5 ppb) bind to the pyridyl nitrogen ends and form linear supramolecular assembly. Red-shifted absorption and fluorescence maxima upon addition of Hg2+ ions were observed, presumably caused by charge transfer interaction and coordination-driven planarization of the biphenyl backbone. Additionally, the in-situ formed mercury complex was utilized for selective recognition of iodide ion (detection limit: 20.2 ppb). Considering its high sensitivity, the present system was utilized in analysing Hg2+ in natural water and in presence of albumin protein. The high recovery values ranging from 95 to 98% with substantially low relative standard deviation (<4%) confirm the suitability of the present method in estimating trace-level of Hg2+ even in real-life samples. Imaging of intracellular Hg2+ ion was also achieved in cervical cancer cells. Low-cost paper strips are designed for rapid, on-site detection of Hg2+ without engaging any sophisticated analytical tools or trained personnel.

A dual-response fluorescent probe for discriminative sensing of hydrazine and bisulfite as well as intracellular imaging with different emission.

Bisulfite and hydrazine are harmful to the environment safety and human health. Therefore, it is of great value to develop a smart fluorescent probe with high selectivity for detection of bisulfite and hydrazine. In our report, a dual-response fluorescent probe EDBI with high selectivity, rapid response, and low detection limit for discriminative determination HSO3- and N2H4 was exploited. The probe EDBI is capable of distinctive sensing HSO3- and N2H4 based on nucleophilic addition reactions by taking advantage of ratiometric fluorescence and fluorescence "on-off" mode, respectively. The dual-responses behaviors of probe EDBI toward HSO3- and N2H4 were attribute to different reaction sites, which it has been confirmed by HRMS. More importantly, cytotoxicity experiment authenticated that probe possesses low toxicity and good penetration. The probe EDBI with excellent performance, it was successfully employed to distinguishable sense HSO3- and N2H4 in living cells by diverse channel patterns. Therefore, this simple dual-response fluorescence probe is expected to be used for real-time monitoring bisulfite and hydrazine in biological samples.

Self-Protected DNAzyme Walker with a Circular Bulging DNA Shield for Amplified Imaging of miRNAs in Living Cells and Mice.

Abnormal expression of miRNAs is often detected in various human cancers. DNAzyme machines combined with gold nanoparticles (AuNPs) hold promise for detecting specific miRNAs in living cells but show short circulation time due to the fragility of catalytic core. Using miRNA-21 as the model target, by introducing a circular bulging DNA shield into the middle of the catalytic core, we report herein a self-protected DNAzyme (E) walker capable of fully stepping on the substrate (S)-modified AuNP for imaging intracellular miRNAs. The DNAzyme walker exhibits 5-fold enhanced serum resistance and more than 8-fold enhanced catalytic activity, contributing to the capability to image miRNAs much higher than commercial transfection reagent and well-known FISH technique. Diseased cells can accurately be distinguished from healthy cells. Due to its universality, DNAzyme walker can be extended for imaging other miRNAs only by changing target binding domain, indicating a promising tool for cancer diagnosis and prognosis.