A 1,8-naphthalimide-derived fluorogenic probe was reported to label SNAP-tag fusion proteins in living cells. The probe can rapidly label a SNAP-tag and exhibit a fluorescence increase of 36-fold due to the additive effects of environment sensitivity of fluorophores and inhibition of photo-induced electron transfer from O6-benzylguanine to the fluorophore. The labeling of intracellular proteins has been successfully achieved without a wash-out procedure.

Near-infrared (NIR) fluorescence imaging technology calls for highly bright and photostable emissive materials for long-term and real-time bioimaging and medical diagnosis. Herein, we report that four aminocyanine dyes were covalently encapsulated inside silica nanoparticles by reverse microemulsion using different linkage methods. The fluorescence brightness and photostability of the obtained fluorescent silica nanoparticles (FSNPs) were found to have direct correlation with the used covalent encapsulation methods, especially the number of anchoring sites of the encapsulated dyes. The aminocyanine dye 4-Si contains three anchoring sites for embedding into the silica nanoparticles, and provides FSNP-4 – the FSNP with the best brightness and photostability.

•The probe displayed a fluorescence turn-on response to label SNAP-tag.•The probe exhibited the fast record labeling rate among known fluorogenic probes.•The probe enables no-wash protein imaging in live cells.SNAP-tag is one of most popular genetically encoded protein tags that can be labeled with fluorescent molecules for visualizing a protein of interest in live cells. Fluorogenic probes keep dark until they label protein tags, significantly improving the signal-to-noise ratio to image proteins without wash-out step. However, most of reported fluorogenic probes for SNAP-tag suffered from the low or mild labeling rate comparing with non-fluorogenic ones. In this paper, we reported a 4-amino-naphthliamide derived fluorogenic probe for SNAP-tag, which exhibited the fast record labeling rate among fluorogenic probes. Finally, we applied this probe to image proteins in mitochondria and nucleus in live cells without wash-out steps.The fluorogenic probe for SNAP-tag displayed a fast labeling rate.Download high-res image (175KB)Download full-size image

•A substrate-inspired fluorescent probe for human albumin (HA) has been developed on the basis of its enzymatic activity.•BNPB exhibits excellent selectivity, high sensitivity and good reactivity under physiological conditions.•BNPB has been successfully applied to measure native HA in complex biological systems.•BNPB can be used for two-photon imaging of HA reabsorption in living cells.Human albumin (HA) displays crucial roles in maintaining health and fighting diseases. Accurate determination of native HA in plasma or non-plasma samples are of immense significance in both basic research and clinical practice. Herein, a novel ratiometric two-photon fluorescent probe (N-butyl-4-(4-phenyl-benzoyloxy) 1,8-naphthalimide, BPBN) has been designed and developed for highly selective and sensitive sensing the enzymatic activities of HA, on the basis of its unique pseudo-esterase feature. BPBN exhibits excellent selectivity, high sensitivity and good reactivity under physiological conditions. As an enzymatic activity-based probe, BPBN can distinguish between native HA and denatured HA, while the currently used dye-binding method cannot. The probe has been successfully applied to measure native HA in plasma samples and the secreted HA in the hepatocyte culture supernatant. BPBN has also been used for two-photon imaging of HA reabsorption in living renal cells, and the results demonstrate that this probe exhibits good cell permeability, low cytotoxicity and high imaging resolution. All these findings suggest that BPBN can be reliably used for the highly selective and sensitive detection of native HA in complex biological samples, as well as for exploring HA-associated biological processes and the physiological functions of native HA in living cells.Download high-res image (195KB)Download full-size image

A rapid-response fluorescent probe ACDM was developed for the selective and sensitive detection of human albumin (HA) via binding onto a non-drug binding site. ACDM was successfully used to detect trace HA in various biological samples including diluted plasma and cell culture supernatants.

•A near-infrared fluorescent probe for carboxylesterase 2 (CE2) has been designed.•This probe exhibits good specificity, ultrahigh sensitivity, and fast reaction kinetics.•This newly probe can be used for sensing CE2 in living cells and animals.A near-infrared fluorescent probe (DDAB) for highly selective and sensitive detection of carboxylesterase 2 (CE2) has been designed, synthesized, and systematically studied both in vitro and in vivo. Upon addition of CE2, the ester bond of DDAB could be rapidly cleaved and then release a near-infrared (NIR) fluorophore DDAO, which brings a remarkable yellow-to-blue color change and strong NIR fluorescence emission in physiological solutions. The newly developed probe exhibits excellent properties including good specificity, ultrahigh sensitivity and high imaging resolution. Moreover, DDAB has been applied to measure the real activities of CE2 in complex biological samples, as well as to screen CE2 inhibitors by using tissue preparations as the enzymes sources. The probe has also been successfully used to detect endogenous CE2 in living cells and in vivo for the first time, and the results demonstrate that such detection is highly reliable. All these prominent features of DDAB make it holds great promise for further investigation on CE2-associated biological process and for exploring the physiological functions of CE2 in living systems.A colorimetric near-infrared fluorescent probe (DDAB) for highly selective and sensitive detection of carboxylesterase 2 (CE2) has been designed and well-characterized. This newly developed probe can be used for sensing CE2 in living cells and animals, and holds great promise for exploring the biological functions of CE2 in complex biological systems.

Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure–selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems.

In this study, a two-photon ratiometric fluorescent probe NCEN has been designed and developed for highly selective and sensitive sensing of human carboxylesterase 2 (hCE2) based on the catalytic properties and substrate preference of hCE2. Upon addition of hCE2, the probe could be readily hydrolyzed to release 4-amino-1,8-naphthalimide (NAH), which brings remarkable red-shift in fluorescence (90 nm) spectrum. The newly developed probe exhibits good specificity, ultrahigh sensitivity, and has been successfully applied to determine the real activities of hCE2 in complex biological samples such as cell and tissue preparations. NCEN has also been used for two-photon imaging of intracellular hCE2 in living cells as well as in deep-tissues for the first time, and the results showed that the probe exhibited high ratiometric imaging resolution and deep-tissue imaging depth. All these findings suggested that this probe holds great promise for applications in bioimaging of endogenous hCE2 in living cells and in exploring the biological functions of hCE2 in complex biological systems.Keywords: bioimaging; carboxylesterase 2; ratiometric fluorescent probe; specific sensing; two-photon

The emission intensities of coumarin 545 solution exhibit a low temperature dependence, with a record-low temperature coefficient of only ∼0.025% per °C. This monomer-aggregate coupled fluorescence system can be used for ratiometric temperature measurements with high spatial and temporal resolutions; three different working modes have been demonstrated.

•A ratiometric fluorescent probe has been developed for UGT1A1 for the first time.•The probe triggers remarkable changes in the color and fluorescence spectrum.•The probe can monitor the enzyme activity of UGT1A1 in complex biological systems.•The probe can also be used to image endogenous UGT1A1 in living cells.This study aimed to develop a practical ratiometric fluorescent probe for highly selective and sensitive detection of human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most important phase II enzymes. 4-Hydroxy-1,8-naphthalimide (HN) was selected as the fluorophore for this study because it possesses intramolecular charge transfer (ICT) feature and displays outstanding optical properties. A series of N-substituted derivatives with various hydrophobic, acidic and basic groups were designed and synthesized to evaluate the selectivity of HN derivatives toward UGT1A1. Our results demonstrated that the introduction of an acidic group to HN could significantly improve the selectivity of UGT1A1. Among the synthesized fluorescent probes, NCHN (N-3-carboxy propyl-4-hydroxy-1,8-naphthalimide) displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following UGT1A1-catalyzed glucuronidation. UGT1A1-catalyzed NCHN-4-O-glucuronidation generated a single fluorescent product with a high quantum yield (Φ=0.688) and brought remarkable changes in both color and fluorescence in comparison with the parental substrate. The newly developed probe has been successfully applied for sensitive measurements of UGT1A1 activities in human liver preparations, as well as for rapid screening of UGT1A1 modulators, using variable enzyme sources. Furthermore, its potential applications for live imaging of endogenous UGT1A1in cells have also been demonstrated.

•A ratiometric fluorescent ESIPT probe for selective detection of human CE2 was developed for the first time.•The probe can be selectively hydrolyzed by CE2 to release 3-hydroxylflavone which brings the remarkable changes in fluorescence spectrum.•The probe can be used to efficiently detect the real activity of CE2 in biological samples.•The probe also can monitor the real function of endogenous CE2 in living cells.A new ratiometric florescence probe derived from 3-hydroxyflavone (3-HF) has been developed for selective and sensitive detection of human carboxylesterase 2 (CE2). The probe is designed by modulating the excited state intramolecular proton transfer (ESIPT) emission of 3-HF via introducing of 4-ethylbenzoyloxy group. Under physiological conditions, probe 1 displays satisfying stability with very low background signal, but it can be selectively hydrolyzed by CE2 to release free 3-HF which brings remarkable changes in fluorescence spectrum. Both reaction phenotyping and chemical inhibition assays demonstrate that probe 1 is highly selective for CE2 over other human hydrolases including carboxylesterase 1, cholinesterases and paraoxonases. Probe 1 has been applied successfully to measure the real activities of CE2 in human biological samples, as well as to screen CE2 inhibitors by using tissue preparations as the enzymes sources. Additionally, probe 1 is cell membrane permeable and can be used for cellular imaging of endogenous CE2 in living cells. All of these features make it possible to serve as a promising tool for exploring the individual differences in biological function of CE2, as well as for rapid screening of selective and potent inhibitors of CE2 for further clinical use.

A highly selective long-wavelength fluorescent probe TCFB has been developed for the detection of hCE2. The probe can be used for real-time monitoring of hCE2 activity in complex biological systems.

Three imidazolium derivatives 3–5 were designed and synthesized, in which naphthaimidazolium group acted as both fluorophore and anion receptor. Compound 3 exhibited high selectivity for F− in CH3CN solution over all the other anions and acted as a ratiometric fluorescent probe for F− with an enhanced blue-shift in emission. However, the fluorescence of compound 4 and 5 displayed a quenched blue-shift in emission with fluoride ion and could be quenched by some other tested anions, where the degree of quenching depended on the characteristic of the anions. More importantly, only compound 3 could detect F− in DMSO–water (95:5, v/v) aqueous solution ratiometrically. Based on the analysis of the results of 1H-NMR and 19F-NMR, it was deduced that compound 3 bound with F− mainly by the force of hydrogen bonding, while compound 4 and 5 coordinated with F− through electrostatic interaction.

Hydrogen sulfide (H2S) is an endothelial gasotransmitter which has been extensively studied recently in various physiological processes. H2S can induce lysosomal membrane destabilization leading to an autophagic event of precipitation apoptosis coupled with calpain activation, thus ensuring cellular demise. In this study, we developed a lysosome-targetable fluorescent probe for the recognition of H2S with considerable fluorescence enhancement. Through introducing a lysosome-targetable group 4-(2-aminoethyl)-morpholine into the H2S probe N-imide termus of 4-azide-1,8-naphthalimide, the new compound Lyso-AFP can recognize H2S in lysosomes. This probe emerges as a more biocompatible analysis tool with low poison by-product than reported H2S fluorescent probes.

•A highly selective ratiometric fluorescent probe of hCE1 was synthesized and well-characterized for the first time.•Isoform specific hydrolysis mediated by hCE1 leads to a large emission shift with a desirable red-shifted.•The fluorescence intensity ratio (488 nm/368 nm) exhibited a good linearity increasing with both protein concentration and time.•The probe was successfully applied in measuring hCE1 activity and screening the inhibitors of hCE1 in a heterogeneous mixture containing multi-enzymes.•The probe was cell membrane permeable and can be used to image the hCE1 activity in living cells.A new ratiometric fluorescent probe derived from 2-(2-hydroxy-3-methoxyphenyl) benzothiazole (HMBT) has been developed for selective monitoring of human carboxylesterase 1 (hCE1). The probe is designed by introducing benzoyl moiety to HMBT. The prepared latent spectroscopic probe 1 displays satisfying stability under physiological pH conditions with very low background signal. Both the reaction phynotyping and chemical inhibition assays demonstrated that hCE1 mediated the specific cleavage of the carboxylic ester bond of probe 1 in human biological samples. The release of HMBT leads to a remarkable red-shifted emission in fluorescence spectrum (120 nm large emission shift). Furthermore, human cell-based assays show that probe 1 is cell membrane permeable, and it can be used for bioassay and cellular imaging of hCE1 activity in HepG2 cells. These findings lead to the development of a simple and sensitive fluorescent method for measurement of hCE1 activity in vitro or in living cells, in the presence of additional enzymes or endogenous compounds.

In this work, a 1,8-naphthalimide-derived fluorescent probe for H2S based on the thiolysis of dinitrophenyl ether is reported. This probe exhibits turn-on fluorescence detection of H2S in bovine serum and lysosome-targetable fluorescent imaging of H2S with excellent selectivity.

A fluorescent chemosensor N-n-butyl-3,4-diamino-1,8-naphthalimide (DAN) for detecting NO was developed on the basis of 1,8-naphthalimide with a similar structure of o-phenylenediamine as a NO reaction site. Due to the interaction between the photoinduced electron transfer (PET) and the intramolecular charge transfer (ICT), the chemosensor exhibits a remarkable enhancement in the emission intensity that is ca. 160-fold increase and a blue shift in the emission wavelength after the addition of NO. Meanwhile, it displays a colorimetric response accompanied with a color change from yellow to colorless. The chemosensor DAN shows a high selectivity for NO in the presence of various reactive nitrogen species (RNS) and reactive oxygen species (ROS). Furthermore, DAN can be used for bioimaging of NO in living cells.Graphical abstractHighlights► A fluorescent chemosensor for detecting nitric oxide (NO) was synthesized. ► The chemosensor was extremely sensitive to NO with about 160-fold fluorescence enhancement. ► It can rapidly and selectively detect NO in a colorometric way. ► The molecule can penetrate cell membrane easily and make fast fluorescence bioimaging.

•A two-photon fluorescent probe for hydrogen sulfide was synthesized.•The probe is extremely sensitive to H2S with 37-fold emission enhancement.•The probe has an excellent selectivity for H2S.•The probe can detect H2S in bovine serum.•The probe can image H2S in living cells.Fluorescent probes for hydrogen sulfide have received considerable attention because of the biological significance of H2S recognized recently. Two-photo microscopy offers advantages of increased penetration depth, localized excitation, and prolonged observation time. However, two-photon fluorescent probes for H2S are still rare. In this work, we introduced a dinitrophenyl ether group into the 4-position of 1,8-naphthalimide, which acts as the H2S reactive site, to efficiently yield compound NI–NHS as a two-photo fluorescent probe for H2S. The probe NI–NHS has a high selectivity for H2S over competitive anions and sulfide-containing analytes. This probe exhibits turn-on fluorescence detection of H2S in bovine serum and two-photon fluorescent imaging of H2S in living cells.

Hydrogen sulfide has emerged as an important biological messenger and much attention has been paid to the design of fluorescent probes for H2S to meet the requirement of accurate measurement of H2S. In this work, a new red emission fluorescent probe for H2S was developed based on the reduction reaction of azide with H2S to amine with the fluorophore of dicyanomethylenedihydrofuran because of its long excitation and emission wavelength. The probe has a high selectivity for H2S over competitive anions and sulfide-containing analytes. Finally, the probe was applied to sense H2S in living cells.

A simply synthesized 4-aminonaphthalimide derivative 1 expresses both polarity and viscosity sensitive fluorescence spectra, indicating its potential usage as an environmentally sensitive fluorescence probe. By comparing the fluorescence behavior of 1 with that of a known 4-aminonaphthalimide derivative 2, it was found that the substitution of the 4-amino group has profound influence on the environmentally sensitive fluorescence properties of 4-aminonaphthalimide.A simply synthesized, small sized 4-aminonaphthalimide derivative exhibits diverse environmentally sensitive fluorescence spectra.

To establish a new strategy for the development of fluorescent viscosity sensors by the principle of photoinduced electron transfer, three 4-aminonaphthalimide based D-(CH2)2-A molecules were designed and synthesized. The effects of solvent polarity and viscosity on the fluorescence spectra of these compounds were investigated. These compounds exhibit viscosity-sensitive fluorescence spectra in ethylene glycol–glycerol mixtures. A mechanism about exciplex formation was proposed to explain their viscosity sensitivity.

A series of novel acenaphthopyrazine derivatives was synthesized from acenaphthylene-1,2-dione via three steps, including bromination, cyclization, and SNArH reaction. These new compounds exhibited potential antiproliferative activity against MCF-7 cells in vitro, and 3-[2-(dimethylamino)ethylamino]acenaphtho[1,2-b]pyrazine-8,9-dicarbonitrile exhibited the highest activity (IC50 = 4.60 μM). DNA-binding experiments suggested that these derivatives bind to DNA through intercalation with intrinsic binding constants K all above 105 M−1. Optical property studies indicated that these compounds have long emission wavelength (λem > 560 nm), high quantum yields in toluene (Φf = 0.59 for 3-(morpholin-4-yl)acenaphtho[1,2-b]pyrazine-8,9-dicarbonitrile), and large Stokes shift (ΔS > 130 nm).

A series of trans-5-substituted-acenaphthene-1,2-diols were obtained in 21–72% yield with 97–100% ee by baker’s yeast-mediated reduction of the corresponding acenaphthylene-1,2-diones, in the presence of DMSO as a co-solvent and under vigorous agitation. The absolute configuration of (−)-trans-5-methoxy-acenaphthene-1,2-diol trans-3b and (−)-trans-5-bromo-acenaphthene-1,2-diol trans-3c was assigned as (S,S) and (−)-trans-5-thiomorpholin-acenaphthene-1,2-diol trans-3d was established as (R,R) by exciton-coupled circular dichroism.trans-(1S,2S)-Acenaphthene-1,2-diolC12H10O2Ee = 97%[α]D20=-24.1 (c 0.28, CHCl3)Source of chirality: asymmetric redutionAbsolute configuration: (1S,2S)trans-(1S,2S)-5-Methoxyacenaphthene-1,2-diolC13H12O3Ee = 100%[α]D20=-24.2 (c 0.26, CHCl3)Source of chirality: asymmetric redutionAbsolute configuration: (1S,2S)trans-(1S,2S)-5-Bromoacenaphthene-1,2-diolC12H9BrO2Ee = 99%[α]D20=+15.1 (c 0.35, EtOAc)Source of chirality: asymmetric redutionAbsolute configuration: (1S,2S)trans-(1R,2R)-5-Thiomorpholinacenaphthene-1,2-diolC16H17NO2SEe = 100%[α]D20=-29.6 (c 0.24, CHCl3)Source of chirality: asymmetric redutionAbsolute configuration: (1R,2R)

A series of cyanonitrovinyl neonicotinoids were designed and synthesized via five steps in about 35% overall yields. All compounds were structurally characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, infrared (IR), and high-resolution mass spectrometry (HRMS), and single-crystal X-ray diffraction analysis of 2-[1-[(6-chloropyridin-3-yl)methyl]-2-imidazolidinylidene]-2-nitroacetonitrile revealed that the double bond is (E)-configured. The preliminary agriculture bioassay indicated that one compound exhibited moderate insecticidal activity against pea aphid.

A new class of organic dyes based on acenaphthopyrazine derivatives, containing pyrazine group as the electron acceptor and o-dicarboxyl acids as the anchoring groups were designed and synthesized for application in dye-sensitized solar cells (DSCs). These dyes have short synthesis routes and are easily adsorbed on the surface of TiO2. Under illumination of simulated AM1.5 solar light (100 mW cm−2), a total solar energy conversion efficiency (η) of 4.04% was obtained for the 3-(diphenylamino)acenaphtho[1,2-b] pyrazine-8,9-dicarboxylic acid (AP-1) in the preliminary tests, in comparison with the conventional N719 dye (η = 7.05%) under the same conditions.

A family of 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carboxylic acid derivatives were synthesized as a result of our efforts to modify a series of acenaphthopyrrole aromatic-heterocycle compounds that proved to be potent anticancer drugs. Among the derivatives, 3d (3-(dimethylamino-propylamino)-8-oxo-8H-acenaphtho-[1,2-b]pyrrole-9-carboxylic acid) and 3g (3-piperidine-8-oxo-8H-acenaphtho-[1,2-b]pyrrole-9-carboxylic acid) showed potential anticancer activity and different action mechanism from our previously reported compounds. UV–vis absorption, circular dichroism and viscosity measurement indicated that effect of both compounds on the advanced DNA conformation was different, although they could bind to DNA in the same way. Cell cycle analysis showed that 3d could induce S-phase arrest followed by apoptosis, while 3g induced apoptosis. The results seem to imply that different action mechanism could contribute to the dissimilitude of biological activities toward 3d and 3g.

A series of 5-alkylamino substituted amonafide analogues were synthesized from naphthalic anhydride by three steps including bromization, amination and CuI/proline catalyzed coupling reaction. The CuI/L-proline catalyzed coupling reaction was first applied to the naphthalimide system. These new amonafide analogues showed potential anticancer activities against HeLa and P388D1 cell lines in vitro, and 4a, 4b, and 4h exhibited better activity than amonafide against HeLa cell under the same experimental conditions. More importantly, the new analogues could avoid the side effect of amonafide due to their structure, in which lacks a primary amine at the 5 position. Moreover, the DNA-binding of the analogues was also investigated.

A series of novel N-substituted angular furoquinolinone derivatives were synthesized and evaluated for their antitumor activities against QGY, K562, HeLa, P388, and A549 cell lines in vitro. The derivatives bearing basic amino side chain showed an improved antitumor activity. Compound 5hN-(2-dimethylamino-ethyl)-2-(4,8,9-trimethyl-2-oxo-2H-furo[2,3-h]quinolin-1-yl)-acetamide exhibited the highest activities against P388 and A549 cell lines, which are evidenced by the IC50 values that are four to five fold lower than that for unsubstituted parent compound. DNA-binding experiments suggested that these derivatives bind to DNA through intercalation.

A novel series of ‘4-1’ pentacyclic naphthalimides, where the chromophore consists of a naphthalimide moiety, fused to an imidazole ring containing an unfused aryl or heteroaryl ring, were synthesized and evaluated for in vitro antitumor activity. In general, the new derivatives showed an improved cytotoxic activity over amonafide. DNA binding experiments supported that this class of compounds behaves as effective DNA-intercalating agents.

A 1,8-naphthalimide-derived fluorogenic probe was reported to label SNAP-tag fusion proteins in living cells. The probe can rapidly label a SNAP-tag and exhibit a fluorescence increase of 36-fold due to the additive effects of environment sensitivity of fluorophores and inhibition of photo-induced electron transfer from O6-benzylguanine to the fluorophore. The labeling of intracellular proteins has been successfully achieved without a wash-out procedure.

A rapid-response fluorescent probe ACDM was developed for the selective and sensitive detection of human albumin (HA) via binding onto a non-drug binding site. ACDM was successfully used to detect trace HA in various biological samples including diluted plasma and cell culture supernatants.

Near-infrared (NIR) fluorescence imaging technology calls for highly bright and photostable emissive materials for long-term and real-time bioimaging and medical diagnosis. Herein, we report that four aminocyanine dyes were covalently encapsulated inside silica nanoparticles by reverse microemulsion using different linkage methods. The fluorescence brightness and photostability of the obtained fluorescent silica nanoparticles (FSNPs) were found to have direct correlation with the used covalent encapsulation methods, especially the number of anchoring sites of the encapsulated dyes. The aminocyanine dye 4-Si contains three anchoring sites for embedding into the silica nanoparticles, and provides FSNP-4 – the FSNP with the best brightness and photostability.

A highly selective long-wavelength fluorescent probe TCFB has been developed for the detection of hCE2. The probe can be used for real-time monitoring of hCE2 activity in complex biological systems.