Nucleotide-Bearing Benzylidene-Tetrahydroxanthylium Near-IR Fluorophore for Sensing DNA Replication,Secondary Structures and Interactions

Thymidine triphosphate bearing benzylidene-tetrahydroxanthylium near-IR fluorophore linked to the 5-methyl group via triazole was synthesized through the CuAAC reaction and was used for polymerase synthesis of labelled DNA probes. The fluorophore lights up upon incorporation to DNA (up to 348-times) presumably due to interactions in major groove and the fluorescence further increases in the single-stranded oligonucleotide. The labelled dsDNA senses binding of small molecules and proteins by a strong decrease of fluorescence. The nucleotide was used as a light-up building block in real-time PCR for detection of SARS-CoV-2 virus.     

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

We report the design, synthesis, and characterization of binary oligonucleotide probes for mRNA detection. The probes were designed to avoid common problems found in standard binary probes such as direct excitation of the acceptor fluorophore and overlap between the donor and acceptor emission spectra. Two different probes were constructed that contained an array of either two or three dyes and were characterized using steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and fluorescence depolarization measurements. The three-dye binary probe (BP-3d) consists of a Fam fluorophore which acts as a donor, collecting light and transferring it as energy to Tamra, which subsequently transfers energy to Cy5 when the two probes are hybridized to mRNA. This design allows the use of 488 nm excitation, which avoids the direct excitation of Cy5 and at the same time provides a good fluorescence resonance energy transfer (FRET) efficiency. The two-dye binary probe system (BP-2d) was constructed with Alexa488 and Cy5 fluorophores. Although the overlap between the fluorescence of Alexa488 and the absorption of Cy5 is relatively low, FRET still occurs due to their close physical proximity when the probes are hybridized to mRNA. This framework also decreases the direct excitation of Cy5 and reduces the fluorescence overlap between the donor and the acceptor. Picosecond time-resolved spectroscopy showed a reduction in the fluorescence lifetime of donor fluorophores after the formation of the hybrid between the probes and target mRNA. Interestingly, BP-2d in the presence of mRNA shows a slow rise in the fluorescence decay of Cy5 due to a relatively slow FRET rate, which together with the reduction in the Alexa488 lifetime provides a way to improve the signal to background ratio using time-resolved fluorescence spectra (TRES). In addition, fluorescence depolarization measurements showed complete depolarization of the acceptor dyes (Cy5) for both BP-3d (due to sequential FRET steps) and BP-2d (due to the relatively low FRET rate) in the presence of the mRNA target.     

Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes

Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through F?rster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.5-beta-actin-3'BHQ1 and 5'FAM-beta-actin-3'BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.     

Intramolecular Long‐Distance Nucleophilic Reactions as a Rapid Fluorogenic Switch Applicable to the Detection of Enzymatic Activity

Long‐distance intramolecular nucleophilic reactions are promising strategies for the design of fluorogenic probes to detect enzymatic activity involved in lysine modifications. However, such reactions have been challenging and hence have not been established. In this study, we have prepared fluorogenic peptides that induce intramolecular reactions between lysine nucleophiles and electrophiles in distal positions. These peptides contain a lysine and fluorescence‐quenched fluorophore with a carbonate ester, which triggers nucleophilic transesterification resulting in fluorogenic response. Transesterification occurred under mild aqueous conditions despite the presence of a long nine‐amino‐acid spacer between the lysine and fluorophore. In addition, one of the peptides showed the fastest reaction kinetics with a half‐life time of 3.7 min. Furthermore, the incorporation of this fluorogenic switch into the probes allowed rapid fluorogenic detection of histone deacetylase (HDAC) activity. These results indicate that the transesterification reaction has great potential for use as a general fluorogenic switch to monitor the activity of lysine‐targeting enzymes.     

A Far-Red Molecular Rotor Fluorogenic Trehalose Probe for Live Mycobacteria Detection and Drug-Susceptibility Testing

Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green-emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose-based fluorogenic probe featuring a molecular rotor turn-on fluorophore with bright far-red emission (RMR-Tre). RMR-Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no-wash, low-background fluorescence detection of live mycobacteria. Aided by the red-shifted molecular rotor fluorophore, RMR-Tre exhibited up to a 100-fold enhancement in M. tuberculosis labeling compared to existing fluorogenic trehalose probes. We show that RMR-Tre reports on M. tuberculosis drug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.     

Metal-enhanced fluorescent probes based on silver nanoparticles and its application in IgE detection

In this paper, a novel metal plasmon coupled with an aptamer–nucleotide hybridized probe was fabricated and applied for protein detection. The specific aptamer and single-strand oligonucleotide were chemically bound to silver nanoparticles (AgNPs), and Cy5-labeled, complementary single-strand oligonucleotides were hybridized with the particle-bound oligonucleotides. The hybridized DNA duplexes were regarded as rigid rods that separated the fluorophore Cy5 and the surface of AgNPs to reduce the competitive quenching. Using a model system comprising human immunoglobulin E (IgE) as the analyte and goat antihuman IgE as immobilized capture antibody on glass slides, we demonstrate that the detection performance of the synthetic probe was superior to the aptamer-based fluorescent probes. The results showed a good linear correlation for human IgE in the range from 10 ng/ml to 6.25 μg/ml. The detection limit obtained was 1 ng/ml, which was 50 times lower than that using Cy5 oligonucleotide/aptamer hybrid duplex (Probe2) due to the metal-enhanced fluorescence effect. This new strategy opens the possibility for the preparation of high-sensitivity detection probes based on metal nanoparticles.     

A new method for the detection of ATP using a quantum-dot-tagged aptamer

Fluorescence resonance energy transfer (FRET) between a quantum dot as donor and an organic fluorophore as acceptor has been widely used for detection of nucleic acids and proteins. In this paper, we developed a new method, characterized by 605-nm quantum dot (605QD) fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease, to detect adenosine triphosphate (ATP). The new method involved the use of three different oligonucleotides: 3′-biotin-modified DNA that binds to streptavidin-conjugated 605QD; 3′-Cy5-labelled DNA; and a capture DNA consisting of an ATP aptamer and a sequence which could hybridize with both 3′-biotin-modified DNA and 3′-Cy5-labelled DNA. In the absence of the target ATP, the capture DNA binds to 3′-biotin-modified DNA and 3′-Cy5-labelled DNA, bringing quantum dot and Cy5 into close proximity for greater FRET efficiency. When ATP is introduced, the release of the 3′-Cy5-labelled DNA from the hybridization complex took place, triggering 605QD fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease. Taken together, the virtue of FRET pair 605QD/Cy5 and the property of aptamer-specific conformation change caused by aptamer–ATP interaction, combined with the fluorescence intensity change of both 605QD and Cy5, provide prerequisites for simple and convenient ATP detection. Zhang Chen and Guang Li contributed equally to this work.     

Optical modulation and selective recovery of Cy5 fluorescence

Fluorescence modulation offers the opportunity to detect low-concentration fluorophore signals within high background. Applicable from the single-molecule to bulk levels, we demonstrate long-wavelength optical depopulation of dark states that otherwise limit Cy5 fluorescence intensity. By modulated excitation of a long-wavelength Cy5 transient absorption, we dynamically modulate Cy5 emission. The frequency dependence enables specification of the dark-state timescales enabling optical-demodulation-based signal recovery from high background. These dual-laser illumination schemes for high-sensitivity fluorescence-signal recovery easily improve signal-to-noise ratios by well over an order of magnitude, largely by discrimination against background. Previously limited to very specialized dyes, our utilization of long-lived dark states in Cy5 enables selective detection of this very common single-molecule and bulk fluorophore. Although, in principle, the "dark state" can arise from any photoinduced process, we demonstrate that cis-trans photoisomerization, with its unique transient absorption and lifetime enables this sensitivity boosting, long-wavelength modulation to occur in Cy5. Such studies underscore the need for transient absorption studies on common fluorophores to extend the impact of fluorescence modulation for high-sensitivity fluorescence imaging in a much wider array of applications.     

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N‐substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si‐containing analogue of Q‐rhodamine. This probe is the first example of a “caged” Si‐rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red‐shifted to allow multicolor imaging. The dye is a useful label for super‐resolution imaging and constitutes a new scaffold for far‐red fluorogenic molecules.     

In Situ Localization of Enzyme Activity in Live Cells by a Molecular Probe Releasing a Precipitating Fluorochrome

Current enzyme‐responsive, fluorogenic probes fail to provide in situ information because the released fluorophores tend to diffuse away from the reaction sites. The problem of diffusive signal dilution can be addressed by designing a probe that upon enzyme conversion releases a fluorophore that precipitates. An excited‐state intramolecular proton transfer (ESIPT)‐based solid‐state fluorophore HTPQ was developed that is strictly insoluble in water and emits intense fluorescence in the solid state, with λ _ex/em=410/550 nm, thus making it far better suited to use with a commercial confocal microscope. HTPQ was further utilized in the design of an enzyme‐responsive, fluorogenic probe (HTPQA), targeting alkaline phosphatase (ALP) as a model enzyme. HTPQA makes possible diffusion‐resistant in situ detection of endogenous ALP in live cells. It was also employed in the visualizing of different levels of ALP in osteosarcoma cells and tissue, thus demonstrating its interest for the diagnosis of this type of cancer.     

A sialic acid-targeted near-infrared theranostic for signal activation based intraoperative tumor ablation

Agents enabling tumor staging are valuable for cancer surgery. Herein, a targetable sialic acid-armed near-infrared profluorophore (SA-pNIR) is reported for fluorescence guided tumor detection. SA-pNIR consists of a sialic acid entity effective for in vivo tumor targeting and a profluorophore which undergoes lysosomal acidity-triggered fluorogenic isomerization. SA-pNIR displays a number of advantageous biomedical properties in mice, e.g. high tumor-to-normal tissue signal contrast, long-term retention in tumors and low systemic toxicity. In addition, SA-pNIR effectively converts NIR light into cytotoxic heat in cells, suggesting tumor-activatable photothermal therapy. With high performance tumor illumination and lysosome-activatable photothermal properties, SA-pNIR is a promising agent for detection and photothermal ablation of surgically exposed tumors.     

High-contrast and real-time visualization of membrane proteins in live cells with malachite green-based fluorogenic probes

Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green(MG), an organic dye widely used in pigment industry, as a switchable fluorophore to monitor membrane enzymes or noncatalytic proteins in live cells. Conformationally flexible MG is non-fluorescent in aqueous solution, yet covalent binding with endogenous proteins of cells significantly enhances its fluorescence at 670...     

Detection of Helicobacter pylori with a nanobiosensor based on fluorescence resonance energy transfer using CdTe quantum dots

We report on a method for the sensitive determination of Helicobacter that is based on fluorescence resonance energy transfer using two oligonucleotide probes labeled with CdTe quantum dots (QDs) and 5-carboxytetramethylrhodamine (Tamra) respectively. QDs labeled with an amino-modified first oligonucleotide, and a Tamra-labeled second oligonucleotide were added to the DNA targets upon which hybridization occurred. The resulting assembly brings the Tamra fluorophore (the acceptor) and the QDs (the donor) into close proximity and causes fluorescence resonance energy transfer (FRET) to occur upon photoexcitation of the donor. In the absence of target DNA, on the other hand, the probes are not ligated, and no emission by the Tamra fluorophore is produced due to the lack of FRET. The feasibility of the method was demonstrated by the detection of a synthetic 210-mer nucleotide derived from Helicobacter on a nanomolar level. This homogeneous DNA detection scheme is simple, rapid and efficient, does not require excessive washing and separation steps, and is likely to be useful for the construction of a nanobiosensor for Helicobacter species.

A Dendron-Based Fluorescence Turn-On Probe for Tumor Detection

Specifically amplifying the emission signals of optical probes in tumors is an effective way to improve the tumor-imaging sensitivity and contrast. In this paper, the first case of dendron-based fluorescence turn-on probes mediated by a Förster resonance energy transfer (FRET) mechanism is reported. Dendrons up to the fourth generation with a hydrophilic oligo(ethylene glycol) scaffold are synthesized by a solid-phase synthesis strategy, and show precise and defect-free chemical structures. To construct the fluorescence turn-on probe, one Cy5.5 molecule is conjugated to the focal of a G3 dendron through a robust linkage and eight Black Hole Quencher 3 (BHQ-3) molecules are conjugated to its periphery through a PEG chain bearing a reductively cleavable disulfide linkage. By in vitro and in vivo experiments, it is demonstrated that the fluorescence of the dendron-based probe can be activated effectively and rapidly in the reductive environments of tumor cells and tissues, and the probe thus exhibits amplified tumor signals and weak normal tissue signals. Compared with the reported nanoscale turn-on probes, the dendron-based probe has several significant advantages, such as well-defined chemical structure, precisely controllable fluorophore/quencher conjugation sites and ratio, desirable chemical stability, and reproducible pharmacokinetic and pharmacological profiles, and is very promising in tumor detection.     

Surface modified microarray chip and laser induced fluorescence microscopy to detect DNA cleavage

This work describes a quantitative method to detect DNA damage in the presence of Pb and Cd ions using a surface modified microarray chip and a laser induced fluorescence microscopy (LIFM). The detection was carried out by the immobilization of a single-stranded DNA oligomer, tagged with a Cy5 fluorophore on a polydimethylsiloxane (PDMS) microarray chip followed by LIFM. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) was attached as a cross-linker via the formation of covalent amide bonds. Then, the single-stranded DNA oligomer containing Cy5 as a fluorophore and thiol functional groups at both terminals, was bonded to the linker by reaction with sulfhydryl group. As the DNA oligomers were reacted with metal ions of Pb and Cd, the un-cleaved DNA oligomers were quantitatively identified by monitoring Cy5 fluorescence. Cadmium showed a quenching constant of 0.84 in the Stern–Volmer plot, whereas lead gave 0.22, indicating that cadmium ions suppress fluorescence more than lead ions. When optimized, fluorescence reductions of 23% (± 2.1) for Pb and 25% (± 1.4) for Cd were observed in air and decreased to almost < 5.0% in a radical scavenger of 5 mM. The cleaved DNA was also confirmed by MALDI-TOF-MS. In result, this experimental method using a microarray chip with surface modification provided quantitative determination of DNA oligomer damage with reproducible results, significantly reduced sample volumes and analysis times.     

A Strategy for Specific Fluorescence Imaging of Monoamine Oxidase A in Living Cells

Monoamine oxidase (MAO) has two isoforms, MAO‐A and MAO‐B, which show different functions, and thus selective fluorescence imaging is important for biological studies. Currently, however, specific detection of MAO‐A remains a great challenge. Herein, we report a new strategy for specific imaging of MAO‐A through the design of fluorogenic probes combining the characteristic structure of an inhibitor of the target enzyme along with propylamine as a recognition moiety. The high specificity of our representative probe is demonstrated by imaging MAO‐A in different live cells such as SH‐SY5Y (high levels of MAO‐A) and HepG2 (high levels of MAO‐B), and further validated by western blot analyses. The superior specificity of the probe may enable the accurate detection of MAO‐A in complex biosystems. Importantly, the use of the characteristic structure of an inhibitor, as demonstrated in this work, may serve as a general strategy to design specific recognition moieties for fluorogenic probes for enzymes.     

SNAP-tag fluorogenic probes for wash free protein labeling

In this review, we described the design strategies of SNAP-tag fl uorogenic probes with turn-on fl uorescence responses, which minimized the fl uorescence background and allowed for direct imaging in living cells without wash-out steps. These probes can apply in real-time analysis of protein localization, dynamics, and protein– protein interactions in living cells. Furthermore, the excellent fl uorescent properties made it possible to apply some of the probes in super-resolution fl uorescence imaging.     

Chemical speciation of MeHg+ and Hg2+ in aqueous solution and HEK cells nuclei by means of DNA interacting fluorogenic probes

Selected new fluorogenic probes that interact in different ways with Hg²⁺ and MeHg⁺ have been prepared and used for the chemical speciation of both cations in aqueous solution as well as in HEK293 cells. The best selective speciation of Hg²⁺ and MeHg⁺ has been achieved by in vitro approaches based on fluorogenic probes supported in cultured cells, due to the particular sensitivity of the HEK293 cells to permeation by Hg²⁺, MeHg⁺ and the fluorogenic probes. In particular, MeHg⁺ was selectively detected in cell nuclei by probe JG45.     

Ultrafluorogenic Coumarin–Tetrazine Probes for Real‐Time Biological Imaging

We have developed a series of new ultrafluorogenic probes in the blue‐green region of the visible‐light spectrum that display fluorescence enhancement exceeding 11 000‐fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal trans‐cyclooctene(TCO)–tetrazine chemistry platform. By exploiting highly efficient through‐bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported for any bioorthogonal fluorogenic dyes. No‐wash, fluorogenic imaging of diverse targets including cell‐surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds, with minimal background signal and no appreciable nonspecific binding, opening the possibility for in vivo sensing.     

Turn‐On Fluorogenic Probes for the Selective and Quantitative Detection of the Cyanide Anion from Natural Sources

We report new indene derivatives that are good fluorogenic probes for the cyanide anion, one of which is a highly selective and sensitive fluorogenic probe for the fluorescent detection—as well as reliable quantification—of the cyanide anion in water or buffer, with a 10³‐fold increase of fluorescence and low detection limit. It is therefore useful for the quantification of natural cyanide from aqueous extracts of green almond seeds, thus proving that the system is suitable for fast detection and quantification of cyanide from natural sources.