Significant Fluorescence Enhancement Research Articles

Plasmonic Approach to Fluorescence Enhancement of Mesoporous Silica-Coated Gold Nanorods for Highly Sensitive Influenza A Virus Detection Using Lateral Flow Immunosensor.

Rapid diagnostic tests based on the lateral flow immunoassay (LFI) enable early identification of viral infection, owing to simple interpretation, short turnaround time, and timely isolation of patients to minimize viral transmission among communities. However, the LFI system requires improvement in the detection sensitivity to match the accuracy of nucleic acid amplification tests. Fluorescence-based LFIs are more sensitive and specific than absorption-based LFIs, but their performance is significantly affected by fundamental issues related to the quantum yield and photobleaching of fluorophores. Metal-enhanced fluorescence (MEF), which is a plasmonic effect in the vicinity of metallic nanoparticles, can be an effective strategy to improve the detection sensitivity of fluorescence-based LFIs. The key factors for obtaining a strong plasmonic effect include the distance and spectral overlap of the metal and fluorophore in the MEF system. In this study, MEF probes were designed based on core-shell nanostructures employing a gold nanorod core, mesoporous silica shell, and cyanine 5 fluorophore. To optimize the efficiency of MEF probes incorporated on the LFI platform (MEF-LFI), we experimentally and theoretically investigated the distance dependence of plasmonic coupling between cyanine 5 and gold nanorods by adjusting the shell thickness, resulting in significant fluorescence enhancement. The proposed MEF-LFI enabled highly sensitive detection of influenza A virus (IAV) nucleocapsid protein with a detection limit of 0.52 pg mL-1 within 20 min and showed high specificity and accuracy for determining IAV clinical samples. Overall, our findings demonstrate the potential of this method as an effective tool for molecular diagnosis under emergency conditions.

A cucurbit[8]uril-based fluorescent probe for the selective detection of pymetrozine

Herein, we report a simple fluorescence-enhanced system for the selective recognition and determination of the insecticide pymetrozine. 1H NMR spectroscopic data indicate that 1,2-bis(4-pyridyl)ethylene (BPE) is partially encapsulated in the cavity of the cucurbit[8]uril (Q[8]) in aqueous solution, forming a stable 1:2 host-guest inclusion complex. Good evidence is also provided by other characterization techniques including single crystal X-ray diffraction, UV–Vis and fluorescence spectroscopies. This host-guest inclusion complex shows weak fluorescence in aqueous solution. Interestingly, the addition of pymetrozine greatly enhanced the fluorescence of the host-guest inclusion complex. In contrast, no significant fluorescence enhancement was observed on addition of 10 other pesticides. The concentration of pymetrozine in aqueous solution was easily detected based on the linear relationship between fluorescence intensity and pymetrozine concentration. Therefore, this paper reports a new method to identify and determine pymetrozine by fluorescence enhancement.

Phenothiazine/xanthene-based fluorescent probes for trace and real-time detection of HClO in cells and complex environmental water samples

Hypochlorous acid (HClO) is a vital bioactive molecule. Abnormal levels of HClO can affect the body's immune system and pathological processes, leading to many diseases such as kidney disease and neurodegenerative diseases. Therefore, it is very important and necessary to develop a rapid and sensitive HClO detection method. In this work, we propose a novel phenothiazine-xanthene fluorophore, which has flexible adjustability and outstanding responsiveness to HClO. Using phenothiazine-xanthene fluorophore, a “turn-on” probe 7-ethyl-1,2,3,7-tetrahydrochromeno[2,3-b]phenothiazine-4-carbaldehyde (PTZ-CHO) was designed and synthesized for the trace analysis of HClO. In the presence of HClO, the sulfur atom on phenothiazine is rapidly oxidized to the sulfoxide, and a significant fluorescence enhancement (about 100-fold) was observed. Moreover, the advantages including fast response (<10 s), ultra-high sensitivity (limit of detection = 6 nM), strong specificity, and splendid light stability can all be achieved by PTZ-CHO. Benefitting from these advantages, this unique HClO-responding probe has been successfully applied to the detection of HClO in Hela cells, RAW 264.7 cells, and environmental water samples.

Graphene oxide coatings enhance fluorescence signals in a lateral flow immunoassay for the detection of UCH-L1, a marker for trauma brain injury

We present a lateral flow immunoassay (LFA) for the quantitative, fluorescence-based detection of the trauma brain injury (TBI) biomarker ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) that features a layer of graphene oxide (GO) particles over the test zone of nitrocellulose (NC) strips. The introduction of 80 ng of GO at test lines increased fluorescence signals 2–3-fold. This was attributed to an increase in the immobilization of capture antibodies in the top layer of NC, which increased the density of recognition complexes at the surface and hence the signals reaching the reader used. A linear concentration–response relationship was observed in the 0–200 pg/mL UCH-L1 range with an inter assay CoV of 2.65 %. The LOD and LOQ values of 11.1 pg/mL and 33.5 pg/mL were compatible with threshold levels of UCH-L1. Finally, mock plasma samples with UCH-L1 levels characteristic of TBI patients with negative (60 pg/mL) and positive (130 pg/mL) CT scans were successfully analyzed. Storage stability testing further showed that GO did not affect the biological activity of the capture antibody. In summary, we demonstrate that the use of GO particles in the top layers of the test zone of NC strips improves signal intensity, which could potentially enhance the accuracy and efficiency of LFA-based diagnosis.

Rapid particle concentration for immunofluorescence enhancement by a surface acoustic wave microchip

At the initial stages of pathogenesis, biomarkers are generated in an insignificant concentration. Hence, the rapid screening for these low-abundance indicators is immensely critical for early disease diagnosis. Enhancement of the immunofluorescence signal emitted from little concentrated biomarkers can aid their detection among other molecules in complex body fluids. Here, we developed an advanced bead-based immunosensing technique combining surface acoustic wave (SAW) for a rapid and sensitive on-chip detection of the diabetic retinopathy biomarker lipocalin 1. As a result, the implementation of the SAW microchip-enabled particle mixing, separation, and concentration in the droplet delivered a 3.6-fold enhanced immunofluorescence signal within 5 s. The integration of fluorescent signal enhancement by SAW, along with the high sensitivity of the immunoassay, featured a limit of detection of 40 pg/mL in 3 min with a sample volume as low as 4 µL. A simple addition of cover glass on the SAW microchip allowed the immuno-sensing process to be completed without any direct sample contact with the interdigital transducer, minimizing the risk of cross-contamination and damage to the chip due to frequent washing. This study demonstrated the potential of the developed SAW device and insights into its utility as an early diagnostic tool.

A simple quinolimide-based fluorescent sensor for formaldehyde and its applications in test strips and living cells

A simple fluorescent sensor H2 containing quinolimide as a fluorophore and hydrazine as a reaction site was designed and synthesized for the detection of formaldehyde (FA). H2 had the high selectivity and sensitivity to FA through the pseudo-first-order reaction (k = 0.048 min−1), exhibiting the significantly enhanced fluorescence signal. The quantitative determination range of H2 for FA was 0–180 μM, and the LOD of FA is 1.7 μM. Moreover, H2-loaded test strips were demonstrated in the visualization of sensing FA. And H2 was successfully applied for imaging FA in living cells.

An acridone-derived fluorescent off-on probe for detection and in vivo imaging of nitroreductase

Understanding the functions of enzymes in various physiological processes is important, but the design of signaling probes for fast analysis of enzymatic activity is particularly challenging. Herein, a fluorescence-enhanced probe, 10-methyl-2-nitro-acridone (MNA), was synthesized and applied to analyze nitroreductase (NTR) activity in vitro and in vivo. The detection mechanism is based on the nitro group in MNA reacting toward NTR with high reactivity and generating 10-methyl-2-amino- acridone (MAA) accompanied by an obvious fluorescence signal enhancement at 525 nm emission. The probe shows low cytotoxicity, fast response, and high selectivity and sensitivity with a limit of detection as low as 150 ng·mL−1. The probe was also employed for two-photon fluorescence imaging of NTR in zebrafish in vivo revealing the distribution of NTR. Versus existing NTR probes, the proposed probe shows favorable analytical performance including near-infrared light excitation with no other byproducts produced after the reaction. The superior properties of this signaling probe allow it to become a fluorescence imaging candidate in other biosystems.

Silver nanoparticles supported on magnetic iron oxide as fluorescence substrates for efficient detection of dyes

Currently, fluorescence detection has become a hot spot in the field of life science research. However, the fluorescence detection system faces some challenges arising from increased complex degree of detected substances, low sensitivity and difficult reusability of fluorescence substrates, as well easily being affected by external environment interference, causing that there is an urgent demand for developing new and sensitive fluorescence substrates. In this work, Fe3O4 nanoparticles were first prepared via a facile hydrothermal method and then silver-deposited Fe3O4 nanocomposites were fabricated. As a result, the significant fluorescent enhancement was realized for as-obtained Fe3O4/Ag as substrates, in which Ag nanoparticles played a vital role in surface enhanced fluorescence (SEF) effect mainly depending on their surface plasmon resonance to produce local electromagnetic enhancement. In addition, the liquid fluorescent substrate showed the stronger fluorescent signals in comparison with the solid fluorescent substrate that was subject to aggregation-caused quenching effect, which could be evaluated by the difference of enhancement factors for detecting Rhodamine B dyes which reached 42.9-fold and 12.5-fold, respectively, as well as by the difference of fluorescence stability for 10.0% and 35.9% intensity decay rate, respectively. Therefore, such composite materials will provide bright ideas and guides for particular applications in fluorescence detection.

A portable and intelligent logic detector for simultaneous and in-situ detection of Al3+ and fluoride in groundwater

To develop a convenient and intelligent detector for simultaneous and in-situ detection of Al3+ and F- in groundwater, a novel organic probe called RBP has been prepared. With the increase of Al3+, RBP showed a significant fluorescence enhancement at 588 nm, and the detection limit was 0.130 mg/L. After combining with fluorescent internal standard CDs, the fluorescence of RBP-Al-CDs at 588 nm was quenched due to the replace of F- for Al3+, while the CDs at 460 nm remained unchanged, and the detection limit was 0.0186 mg/L. For convenient and intelligent detection, an RBP-based logic detector has been developed for simultaneous detection of Al3+ and F-. Within the ultra-trace, low concentration, and high concentration range of Al3+ and F-, the logic detector can achieve rapid feedback on their concentration levels (“U”, “L” and “H”) through different output modes of the signal lamps. The development of logical detector is of great significance for studying the in-situ chemical behavior of Al3+ and F- and for daily household detection.

Host-guest interaction of tryptophane with acid-functionalized calix[4]pyrrole: a fluorescence-based study

Functionalized calix[4]pyrroles are at forefront of host-guest aided molecular sensors. They offer unique platform for flexible functionalization to develop receptors suitable for different applications. In this context, calix[4]pyrrole derivative (TACP) was functionalized with an acidic group to investigate its binding behavior with different amino acids. The acid functionalization facilitated host-guest interactions through hydrogen bonding and increase the solubility of ligand in 90% aqueous media. The results indicated that TACP exhibited significant fluorescence enhancement in the presence of tryptophan while no considerable changes were observed with other amino acids. The other complexation properties such as LOD and LOQ were determined to be 25 µM and 22 µM respectively with 1:1 stoichiometry. In addition, the proposed binding phenomena were further confirmed through computational docking studies and NMR complexation study. Overall, this work highlights the potential of acid functionalization in developing molecular sensors for amino acid detection using calix[4]pyrrole derivatives. Communicated by Ramaswamy H. Sarma

Aptamer-based regulation of DNA polymerase activity for the detection of protein-small molecule interactions

Herein, we developed a novel method for identifying protein-small molecule interactions (PSMIs) based on the switching of DNA polymerase activity. This strategy employs the small molecule-linked DNA probe (SMP) that could bind to and disrupt the DNA polymerase aptamer probe (DAP) capable of inhibiting DNA polymerase activity. Without the binding of the target protein, the SMP is readily degraded by the applied exonuclease I (exo I), preventing its binding to the DAP. The DAP then freely inhibits the polymerase activity through its specific binding to the polymerase. In the presence of the target protein, however, the binding between the target protein and the small molecule protects the SMP against the exo I-catalyzed degradation. The intact SMP now binds to the DAP and disrupts its active aptameric structure, preventing the DAP from inhibiting the polymerase activity. The uninhibited DNA polymerase catalyzes the primer extension reaction in conjugation with the TaqMan probe (TP), leading to a significant fluorescence enhancement. Taking advantage of this elaborate design principle, we successfully identified the two model PSMIs, streptavidin/biotin (SA/BTN) and anti-digoxigenin/digoxigenin (ADIG/DIG), as low as 4.01 nM and 6.72 nM, respectively, with excellent specificity. We further validated its practical applicability by reliably identifying the SA/BTN interaction in heterogeneous human serum specimens.

Synthesis of spirobifluorene derivatives via metal catalyzed C[sbnd]N and C[sbnd]C cross couplings: useful substrate for cadmium ion sensing

We report, a simple synthetic strategy involving C—N and C–C cross-coupling reactions starting with spirobifluorene (SBF) catalyzed by transition metal (Cu and Pd) catalysts. The promising applications of SBF derivatives inspired us to carry out this investigation. Various heterocyclic coupling partners having electron-rich and electron-deficient functionalities (e.g., pyrrole, pyrazole, imidazole, triazole, and phenylboronic acids) are used in this study. The extended conjugation present in the cross-coupling products makes them suitable candidates for fluorescent study. Among the synthesized substrates, spirobifluorene- terphenyl amine (SBF-TA) emission spectra displayed a significant fluorescence enhancement against the Cd(II) ion which has a detection limit of 20 nM.

Cascade-Activated AIEgen-Peptide Probe for Noninvasively Monitoring Chymotrypsin-like Activity of Proteasomes in Cancer Cells.

Noninvasive monitoring of chymotrypsin-like (ChT-L) activity of proteasomes is of great significance for the diagnosis and prognosis of various cancers. However, commercially available proteasome probes usually lack adequate cancer-cell selectivity. To noninvasively monitor ChT-L activity of proteasomes in living cells, we rationally designed a cascade-activated AIEgen-peptide probe (abbreviated as TPE-1p), which self-assembled in aqueous solution to exhibit bright fluorescence in response to sequential treatment of alkaline phosphatase (ALP) and ChT-L. Transmission electron microscopy, enzymatic kinetics, and in vitro fluorescence experiments validated that TPE-1p was efficiently dephosphorylated by ALP to generate TPE-1, which was recognized by ChT-L in the proteasome, and transformed to form nanofibers with strong fluorescence signals. Cell imaging experiments revealed that bright blue fluorescence was observed in TPE-1p-treated HeLa cells, whereas NIH3T3 and HepG2 cells showed less fluorescence at the same condition. The enhanced fluorescence signals in HeLa cells were attributed to the high activities of endogenous ALP and ChT-L. Moreover, TPE-1p was utilized to noninvasively assess the inhibition efficiency of a ChT-L inhibitor (bortezomib, abbreviated as Btz) in HeLa cells. Significant correlation was found between the fluorescence signals of TPE and the viabilities of Btz-treated cells in concentration ranges from 0 to 1 μM, indicating that TPE-1p could be employed to predict the activity of ChT-L inhibitors. The design of the cascade-activated AIEgen-peptide probe provides a viable approach for noninvasively monitoring the ChT-L activity of proteasomes in living cells, which facilitates high-throughput screening of ChT-L inhibitors in cancer therapy.

A “turn-on” Schiff base fluorescent probe for recognizing Zn2+ and its application in indictor and imaging of zebrafish

A novel fluorescent probe was designed and synthesized by using benzothiazole ring as fluorophore to specifically recognize Zn2+ in aqueous solution. In methanol and aqueous mixed solution, the probe had a significant fluorescence enhancement response to zinc ions and indicated high selectivity and sensitivity. Through fluorescence titration, 1H NMR titration, Job’s plot, HRMS data and DFT calculation, the binding ratio of the probe to Zn2+ was 2: 1, and the detection limit was 2.85 × 10-8 M. The probe was further applied to actual water sample to detect Zn2+, and the medium-speed filter paper was combined with probe solution to make an indicator to detect Zn2+ more conveniently and quickly. More importantly, the probe could detect Zn2+ in zebrafish from the results of fluorescence imaging, which proved that the probe had a broader prospect in biological field.

Study on the protective effect of RNA-binding motif protein 3 in mild hypothermia oxygen-glucose deprivation/reoxygenation cell model

Mild hypothermia is proven neuroprotective in clinical practice. While hypothermia leads to the decrease of global protein synthesis rate, it upregulates a small subset of protein including RNA-binding motif protein 3 (RBM3). In this study, we treated mouse neuroblastoma cells (N2a) with mild hypothermia before oxygen-glucose deprivation/reoxygenation (OGD/R) and discovered the decrease of apoptosis rate, down-regulation of apoptosis-associated protein and enhancement of cell viability. Overexpression of RBM3 via plasmid exerted similar effect while silencing RBM3 by siRNAs partially reversed the protective effect exerted by mild hypothermia pretreatment. The protein level of Reticulon 3(RTN3), a downstream gene of RBM3, also increased after mild hypothermia pretreatment. Silencing RTN3 weakened the protective effect of mild hypothermia pretreatment or RBM3 overexpression. Also, the protein level of autophagy gene LC3B increased after OGD/R or RBM3 overexpression while silencing RTN3 decreased this trend. Furthermore, immunofluorescence observed enhanced fluorescence signal of LC3B and RTN3 as well as a large number of overlaps after RBM3 overexpressing. In conclusion, RBM3 plays a cellular protective role by regulating apoptosis and viability via its downstream gene RTN3 in the hypothermia OGD/R cell model and autophagy may participate in it.

Compute-and-Release Logic-Gated DNA Cascade Circuit for Accurate Cancer Cell Imaging.

Accurate identification of cancer cells is an essential prerequisite for cancer diagnosis and subsequent effective curative interventions. The logic-gate-assisted cancer imaging system that allows a comparison of expression levels between biomarkers, rather than just reading biomarkers as inputs, returns a more comprehensive logical output, improving its accuracy for cell identification. To fulfill this key criterion, we develop a compute-and-release logic-gated double-amplified DNA cascade circuit. This novel system, CAR-CHA-HCR, consists of a compute-and-release (CAR) logic gate, a double-amplified DNA cascade circuit (termed CHA-HCR), and a MnO2 nanocarrier. CAR-CHA-HCR, a novel adaptive logic system, is designed to logically output the fluorescence signals after computing the expression levels of intracellular miR-21 and miR-892b. Only when miR-21 is present and its expression level is above the threshold CmiR-21 > CmiR-892b, the CAR-CHA-HCR circuit performs a compute-and-release operation on free miR-21, thereby outputting enhanced fluorescence signals to accurately image positive cells. It is capable of comparing the relative concentrations of two biomarkers while sensing them, thus allowing accurate identification of positive cancer cells, even in mixed cell populations. Such an intelligent system provides an avenue for highly accurate cancer imaging and is potentially envisioned to perform more complex tasks in biomedical studies.

Fluorescent macrocycle-dye-anchor conjugates for sensing phospholipids in biomembranes

Negatively charged phospholipids can serve as biomarkers for a number of biological processes allowing detection of apoptosis in animal cells, drug-induced phospholipidosis as well as detection of bacteria. Selective detection of phospholipids thus represents an important but challenging goal. The development of fluorescent probes capable of selective recognition of specific phospholipids can be a promising solution to the problem. Along this line, we have developed the macrocycle-dye-anchor conjugates, in which the macrocycle recognizes the phosphate residue, hydrocarbon anchors ensure hydrophobic interaction with the membrane, and environment-sensitive fluorescent dye functions as a reporter unit. To the best of our knowledge, this is the first report of synthetic fluorescent probes for specific phospholipids using the macrocycle-dye-anchor concept. We have studied the effect of the structure of the anchor and the macrocycle on the resulting fluorescent response in the presence of two types of lipid vesicles: phosphatidylcholine as a model of healthy cells and phosphatidylcholine and phosphatidylserine (1:1), mimicking apoptotic cells. The properties of the obtained compounds were analyzed in various model media and in HeLa cells. The response of fluorescent probes has been found to be strongly dependent on the macrocycle structure, length of the anchor, nature of the buffer, and pH. The probes showed significant fluorescence enhancement in apoptotic cells, thus providing the proof of concept for the macrocycle-dye-anchor design.

Optical Fiber Axicon Enabled Plasmofluidic Trapping: Sensing With Enhanced Figure of Merit

In this letter, trapping of colloidal gold nanoparticles (AuNPs) using an optical fiber axicon, and its use in plasmon-enhanced sensing have been reported. The AuNPs of 40 nm size were trapped in a Bessel beam from an optical fiber negative axicon coupled with a laser of 532 nm wavelength. The fluorescence signal from a mixture of AuNPs and Rhodamine 6G (Rh6G) of varying concentrations was studied on a custom-made optical setup. Control experiments on an optical fiber with Gaussian beam illumination, and on NPs of varying sizes were performed to achieve optimal performance. An overall plasmonic enhancement factor of 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sup> per molecule in the fluorescence signal was estimated. A performance enhancement of about 480 times was achieved through trapping, as compared to normal optical fiber illumination. A limit of detection (LoD) of 10 ng/ml of Rh6G was achieved. This study is useful in the development of application-specific metasurfaces, and biosensors using structured beam illumination of plasmonic-fluids. This study also shows that structured illumination can lead to manifold increase in plasmon enhanced fluorescence signal.

A DNAzyme-enhanced nonlinear hybridization chain reaction for sensitive detection of microRNA

As a typical biomarker, the expression of microRNA is closely related to the occurrence of cancer. However, in recent years, the detection methods have had some limitations in the research and application of microRNAs. In this paper, an autocatalytic platform was constructed through the combination of a nonlinear hybridization chain reaction and DNAzyme to achieve efficient detection of microRNA-21. Fluorescently labeled fuel probes can form branched nanostructures and new DNAzyme under the action of the target, and the newly formed DNAzyme can trigger a new round of reactions, resulting in enhanced fluorescence signals. This platform is a simple, efficient, fast, low-cost, and selective method for the detection of microRNA-21, which can detect microRNA-21 at concentrations as low as 0.004nM and can distinguish sequence differences by single-base differences. In tissue samples from patients with liver cancer, the platform shows the same detection accuracy as real-time PCR but with better reproducibility. In addition, through the flexible design of the trigger chain, our method could be adapted to detect other nucleic acid biomarkers.

A fluorescent receptor for alkylammonium ions based on an anthryl-linked triazole-modified hexahomotrioxacalix[3]arene

A novel C3-symmetrical anthracene-functionalized hexahomotrioxacalix[3]arene L was synthesized via Click chemistry and its chemosensing properties with alkylammonium ions were investigated. The significant fluorescence enhancements were observed with addition of alkylammonium ions (including primary and tertiary ones), indicating a high selectivity for alkylammonium ions. Our studies demonstrated that the detection limit on fluorescence response of the sensor to is in the 10–6 M range. The mechanism of the interaction between the L and alkylammonium ions has been investigated in detail by 1H NMR spectroscopic titration experiments. Density functional theory (DFT) quantum chemical calculations were conducted to estimate the electronic interaction energies (ΔIE) between the chemosensor L and alkylammonium ions.