Triple Sensing Modes for Triggered ß-Galactosidase Activity Assays Based on Kaempferol-Deduced Silicon Nanoparticles and Biological Imaging of MCF-7 Breast Cancer Cells.

ß-Galactosidase (ß-Gala) is an essential biomarker enzyme for early detection of breast tumors and cellular senescence. Creating an accurate way to monitor ß-Gala activity is critical for biological research and early cancer detection. This work used fluorometric, colorimetric, and paper-based color sensing approaches to determine ß-Gala activity effectively. Via the sensing performance, the catalytic activity of ß-Gala resulted in silicon nanoparticles (SiNPs), fluorescent indicators obtained via a one-pot hydrothermal process. As a standard enzymatic hydrolysis product of the substrate, kaempferol 3-O-ß-d-galactopyranoside (KOßDG) caused the fluorometric signal to be attenuated on kaempferol-silicon nanoparticles (K-SiNPs). The sensing methods demonstrated a satisfactory linear response in sensing ß-Gala and a low detection limit. The findings showed the low limit of detection (LOD) as 0.00057 and 0.098 U/mL for fluorometric and colorimetric, respectively. The designed probe was then used to evaluate the catalytic activity of ß-Gala in yogurt and human serum, with recoveries ranging from 98.33 to 107.9%. The designed sensing approach was also applied to biological sample analysis. In contrast, breast cancer cells (MCF-7) were used as a model to test the in vitro toxicity and molecular fluorescence imaging potential of K-SiNPs. Hence, our fluorescent K-SiNPs can be used in the clinic to diagnose breast cellular carcinoma, since they can accurately measure the presence of invasive ductal carcinoma in serologic tests.

A novel "Turn-on" fluorometric assays triggered reaction for ß-glucosidase activity based on quercetin derived silicon nanoparticles and its potential use for cell imaging.

ß-Glucosidase (ß-Gluco) is an enzyme that is crucial to numerous diseases, including cancer, and in sector of industries, it is used in the manufacturing of food. Measuring its enzymatic activity is critical for biomedical studies and other activities. Herein, we have developed a novel and precise fluorescent sensing method for measuring ß-Gluco activity based on the production of yellow-green fluorescent quercetin-silicon nanoparticles (Q-SiNPs) produced from quercetin (QN) as a reducing agent and 3-[2-(2-aminoethyl amino) ethylamino] propyl-trimethoxy silane (AEEA) as a silane molecule. ß-Gluco hydrolyzed quercetin-3-O-ß-d-glucopyranoside (QO-ß-DG) to produce QN, which was then used to produce Q-SiNPs. Reaction parameters, including temperature, time, buffer, pH, and probe concentration, were carefully tuned in this study. Subsequently, the fluorescence intensity was performed, showing good linearity (R2 = 0.989), a broad linear dynamic range between 0.5 and 12 U L-1, and a limit of detection (LOD) as low as 0.428 U L-1, which was proven by fluorescence measurements. Most importantly, various parameters were detected and characterized with or without ß-Gluco. The designed probe was successively used to assess ß-Gluco activity in human serum and moldy bread. However, the mathematical findings revealed recoveries for human serum ranging from 99.3 to 101.66% and for moldy bread from 100.11 to 102.5%. Additionally, Q-SiNPs were well suited to being incubated in vitro with L929 and SiHa living cells, and after using an Olympus microscope, imaging showed good fluorescence cell images, and their viability evinced minimal cytotoxicity of 77% for L929 and 88% for SiHa. The developed fluorescence biosensor showed promise for general use in diagnostic tests. Therefore, due to this outstanding sensing modality, we anticipate that this research can provide a novel schematic project for creating simple nanostructures with a suitable plan and a green synthetic option for enzyme activity and cell imaging.

Green-emitting functionalized silicon nanoparticles as an "off-on" fluorescence bio-probe for the sensitive and selective detection of mercury (II) and 3-mercaptopropionic acid.

Herein, we developed a class of functionalized silicon nanoparticles (F-SiNPs) bio-probes named thiol-conjugated F-SiNPs. They combine excellent biocompatibility with small dimensions (<10 nm) and biological usefulness with sustained and robust fluorescence (3.32% photoluminescent quantum yield). Identifying 3-Mercaptopropionic acid (3-MPA), which lowers the quantity of gamma-aminobutyric acid in the brain, and mercury (Hg2+) was a crucially important step since their excessive levels are a sign of several disorders. Using F-SiNPs as a fluorescent bio-probe, we provided an "off-on" technique for sensitively and selectively determining Hg2+ and 3-MPA in this study. The 3-(2-aminoethylamino) propyl (dimethoxymethylsilane) and basic fuchsin as precursors were hydrothermally treated to produce the F-SiNPs exhibiting green fluorescence. Our results suggest that Hg2+ reduced the fluorescence of F-SiNPs because of strong ionic interactions and metal-ligand binding among many thiols and carboxyl groupings at the surface of Hg2+ and F-SiNPs. Additionally, the resultants demonstrated that after being quenched by Hg2+, the produced F-SiNPs led to the distinctive "off-on" response to 3-MPA. Moreover, the method could detect Hg2+ and 3-MPA with limits of detection of 0.065 µM and 0.017 µM, respectively. The technique employed is quick, easy, affordable, and environmentally friendly. The sensing platform has successfully determined Hg2+ and 3-MPA in urine, water, and human serum samples.

Tabersonine Inhibits the Lipopolysaccharide-Induced Neuroinflammatory Response in BV2 Microglia Cells via the NF-κB Signaling Pathway.

The occurrence and development of neurodegenerative diseases is related to a variety of physiological and pathological changes. Neuroinflammation is one of the major factors that induces and aggravates neurodegenerative diseases. The most important manifestation of neuroinflammation is the activation of microglia. Therefore, inhibiting the abnormal activation of microglia is an important way to alleviate the occurrence of neuroinflammatory diseases. In this research, the inhibitory effect of tabersonine (Tab) on neuroinflammation was evaluated by establishing the BV2 neuroinflammation model induced by lipopolysaccharide (LPS). It was found that Tab significantly inhibited the production and expression of nitric oxide (NO), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and reactive oxygen species (ROS) in BV-2 cells stimulated by LPS. In addition, Tab can also inhibit the activation of nuclear factor-κB (NF-κB) induced by LPS, thus regulating inflammatory mediators such as inducible nitric oxide synthase (iNOS). These results indicated that Tab regulated the release of inflammatory mediators such as NO, IL-1ß, TNF-α, and IL-6 by inhibiting NF-κB signaling pathway, and exerting its anti-neuroinflammatory effect. This is the first report regarding the inhibition on LPS-induced neuroinflammation in BV2 microglia cells of Tab, which indicated the drug development potential of Tab for the treatment of neurodegenerative diseases.

Fluorometric assay based on the in situ formation of silicon nanoparticles for the determination of ß-glucuronidase.

As a prodrug-converting enzyme, ß-glucuronidase (ß-GCase) is a lysosomal enzyme participating in the release of glucose from glucopyranosyl glycoside. In this work, for the first time, we have developed an analytical method exhibiting fluorometric signals for straightforward determination of ß-GCase using silicon nanoparticles (Si NPs). Via hydrothermal treatment, in the water bath of 70 °C for 50 min, dopamine (DA) reacts with (3-[2-(2-aminoethylamino) ethylamino] propyltrimethoxysilane) (AEEA) to produce green fluorescent Si NPs. Enlightened by such easy reaction and ß-GCase-triggered specific hydrolysis of dopamine-4-ß-D-glucuronide (DA-GCU) into DA, we have designed an analytical method for ß-GCase sensing through the production of Si NPs. Therefore, through the designed sensing platform, ß-GCase activity was monitored, and the limit of detection (LOD) for this study was 0.02 U/L. Furthermore, the feasibility of the method was assessed by measuring ß-GCase activity in human serum where recoveries and RSD were in the ranges 99-104% and 1.37-3.44, respectively.

Screening of prolyl hydroxylase 2 inhibitors based on quantitative strategy of peptides.

Prolyl hydroxylase 2 (PHD2) is a key oxygen receptor regulating oxygen homeostasis in human body, and it is one of the important targets for drug research and development of hypoxia related diseases. In PHD2 enzymatic reaction, the structure of substrate (HIF-1α556-574) and product (hydroxylated HIF-1α) peptide only differ from one oxygen atom (MW>2000), which makes it a great challenge to separate them accurately and efficiently. In this work, the direct separation and detection of HIF-1α and hydroxylated HIF-1α has been firstly reported based on micellar electrokinetic chromatography (MEKC). Under optimized conditions, the intraday RSD of peak area and apparent electrophoretic mobility of hydroxylated HIF-1α were 1.87% and 0.81% respectively, and the interday RSD were 2.01% and 1.03% respectively. The LOD and LOQ of the MEKC method were 10 µM and 50 µM respectively, and the recoveries was 98.42-105.38%. Subsequently, the feasibility and accuracy of MEKC method to screen PHD2 inhibitors were confirmed by using roxadustat, and the IC50 (10.36 µM) and inhibitor type (competitive) were consistent with literature. Finally, the method was used to screen the PHD2 inhibitory activity of five traditional Chinese medicines (TCMs). The present work not only overcomes the difficulties of direct quantitative detection of hydroxylated HIF-1α, but also provides technical support for exploring and discovering new drug leads for hypoxia-related diseases from complex matrix such as TCMs.

A highly sensitive colorimetric sensing platform based on silver nanocomposites for alkaline phosphatase.

Alkaline phosphatase (ALP) plays significant roles in regulating intracellular processes and is an important biomarker connected to several diseases. In this work, one facile and sensitive sensing platform based on CQD-silver nanocomposites (CQD-silver NPs) for colorimetric detection of alkaline phosphatase (ALP) was introduced. ALP triggers the removal of the phosphate group of ascorbic acid 2-phosphate (AA2P), which is then transformed into ascorbic acid (AA). The as-obtained AA can easily cause significant aggregation of monodispersed NPs and cause the system color to turn from bright yellow to gray. Based on the color change of the ratio of 490 nm/630 nm, ALP was sensitively and selectively detected. Under the optimum, the established method showed linearity for ALP in the range of 0.1-50 U L-1 and the detection limit was low at 0.035 U L-1, and it was subjected to ALP inhibitor screening from goji berry extract. These results indicated that the colorimetric system can be used as a simple tool for visual and fast evaluation of ALP activity as well as providing an alternative to screen ALP inhibitors.

Screening prolyl hydroxylase domain 2 inhibitory activity of traditional Chinese medicine by CZE-UV.

Prolyl hydroxylase domain 2 (PHD2) is a key enzyme regulating the expression of hypoxia inducible factor (HIF). Its inhibitors can improve the expression of HIF and downstream genes, which can treat hypoxia-related diseases. Therefore, the establishment of a reliable PHD2 inhibitors screening method is of great significance for the drug development of hypoxia-related diseases. In this work, an accurate, rapid, and simple screening method for PHD2 inhibitors was introduced by capillary zone electrophoresis (CZE). In order to improve the detection sensitivity, the derivative reaction of α-ketoglutaric acid (α-OG) and 1,2-diaminobenzene (OPD) was used to enhance the UV absorption of α-OG (the substrate in the enzymatic reaction). The CZE method selected 20 mM Na2 B4 O7 buffer (pH 9.0) as the separation buffer, +25 kV as the separation voltage, 25°C as the cartridge temperature, and 210 nm as the detection wavelength. Under this condition, the analysis of a single sample can be realized within 9 min. Compared with the existing reported methods, the present work can directly screen the PHD2 inhibitory activity of traditional Chinese medicine (TCM) extracts, which is of significance for the target-purification of bioactive individual compounds from TCMs. Under the optimal conditions, the PHD2 inhibitor screening platform was successfully established, and it was found that 70% methanol/water extracts of Astragali Radix and Codonopsis pilosula had good PHD2 inhibitory activity. Furthermore, the present work provides a novel approach for screening the PHD2 inhibitory activity of TCM extracts and the discovery of anti-hypoxia bioactive compounds.

α-Glucosidase-Triggered Reaction for Fluorometric and Colorimetric Assays Based on the Formation of Silicon-Containing Nanoparticles.

Designing analytical approaches for enzymatic activity monitoring with high sensitivity and selectivity is of critical value for the diagnosis of diseases and biomedical studies. In this study, we have created a facile one-step synthetic route to prepare orange-red color and yellow fluorescent silicon-containing nanoparticles (Si CNPs) by mixing 3(2-aminoethylamino) propyl (dimethoxymethylsilane) and hydroquinone (HQ) in an aqueous solution. Inspired by the HQ-regulated facile synthetic step and the generation of HQ from α-glucosidase (α-Glu)-catalyzed hydrolysis of 4-hydroxyphenyl-α-d-glucopyranosyl (4-HPαDG), we have designed a straightforward colorimetric and fluorometric α-Glu activity assay using a commercially available 4-HPαDG as the α-Glu substrate. Fluorescent and colorimetric assays for α-Glu activity measurement have been thereby established and exhibited detection limits as low as 0.0032 and 0.0046 U/mL, respectively. Under single excitation at 370 nm, the prepared Si CNPs emitted yellow fluorescence at 520 nm and exhibited an absorbance peak at 390 nm. In addition, the proposed approach reveals various advantages including easy operation, time-saving, and good anti-interference ability. Hence, it could improve the progress of fluorometric and colorimetric enzymatic activity assays with high sensitivity and simplicity. Moreover, the proposed approach was applied for α-Glu inhibitor screening, and its feasibility in real samples was measured by detecting the α-Glu activity in human serum samples.

Synthesis of orange-emissive silicon nanoparticles as "off-on" fluorescence probe for sensitive and selective detection of l-methionine and copper.

Fluorescent silicon nanoparticles (Si NPs) are of great interest as they are free of heavy ions. However, most of Si NPs exhibit blue or green emission, while orange or red-emitting Si NPs are required for an extensive range of applications. Copper ion (Cu2+) and l-methionine (L-Met) detection is critically valuable point since their abnormal level is an indicator of various diseases. In this work, we illustrate an "off-on" method for sensitively and selectively determination of Cu2+ and L-Met using Si NPs as fluorescent probe. The Si NPs emitting orange fluorescence with the quantum yield of 2.23% were prepared via one and easy step of hydrothermal treatment of 3(2-aminoethylamino) propyl (dimethoxymethylsilane) (AEAPDMMS) and 2-aminophenol as precursors. The fluorescence of Si NPs was quenched in the presence of Cu2+ due to the strong metal-ligand coordination and electrostatic interactions between the large amount of amino and hydroxyl groups on the surface of Si NPs and Cu2+. Surprisingly, the resulted non-fluorescent Si NPs-Cu2+ complex displayed a fluorescence "turn-on" toward L-Met, due to the competitive coordination of Cu2+ between L-Met and Si NPs which leads to the unique "off-on" response to L-Met after the release of free Si NPs. The as-proposed approach is fast, simple, low cost and environmental-friendly. More importantly, it has been applied in the determination of Cu2+ and L-Met in water and urine samples, respectively with satisfactory recoveries. Furthermore, the approach could detect Cu2+ and L-Met with detection limit of 0.012 µM and 0.07 µM, which are lower than the level of Cu2+ in drinking water and of L-Met in human urine sample (maximum ~20 µM and ~5.9 µM, respectively).

An online target and rapid screening method for α-glucosidase inhibitors based on capillary electrophoresis.

Screening enzymatic active compounds is one of the important fields in drug research. α-Glucosidase can hydrolyze carbohydrates to monosaccharides after meals and lead to the rise of blood glucose levels in human body. Thus, the inhibition of α-glucosidase activity is an effective approach for the diabetes treatment. In this work, we developed a new method to simultaneously screen multiple bioactive compounds within a single CE running. The affect factors on the method performance, including injection, mixing, incubation, separation and detection, were carefully analyzed and discussed. Under the optimum, the mixture consisting of two internal standards (DMSO and 4-nitrophenol) and five compounds (lyoniresinol, hydroxytyrosol, rutin, kaempferol, and quercetin) was simultaneously screened, and kaempferol and quercetin showed stronger activity and this conclusion was also supported by offline assay. Furthermore, molecular docking was employed for investigating its interaction mechanism. Eventually, the established method has been applied to screen potential α-glucosidase inhibitors from an extract of Lycium barbarum and the peak area of rutin, taxifolin, quercetin, and chlorogenic acid in L. barbarum samples changed before and after the enzymatic reaction, confirming that these four compounds had potential inhibitory activities, which was consistent with the literature data. The present work provides a promising method for the target and rapid discovery of bioactive compounds from a plant extract or mixture.

Fluorescence "turn-on" of silicon-containing nanoparticles for the determination of resorcinol.

A fluorescent nanosensor based on silicon-containing nanoparticles (Si CNPs) with green fluorescence (FL) was prepared by one-step method. The prepared Si CNPs emitted green FL at 470 nm under the excitation at 350 nm. The FL signal of Si CNPs reveals an obvious enhancement in the presence of resorcinol (RC), due to the passivation of surface trap states of Si CNPs via the binding of OH group of RC with the NH group of Si CNPs, which allowed the formation of new radiative electron-hole recombination centers. This was confirmed by some analytical experiments performed on zeta potential, FL lifetime steady state, and the FTIR spectra. Most importantly, this nanosensor could selectively determine RC with high sensitivity and without interference from hydroquinone (HQ) and catechol (CT) as RC isomers. RC was detected in the linear range 0.05-40 µM, with a detection limit of 0.012 µM. The synthesized nanosensor was applied to the determination of RC in fresh fruit juice and water samples. The collected results confirmed the feasibility of our approach with high accuracy.

Target triggered fluorescence "turn-off" of silicon nanoparticles for cobalt detection and cell imaging with high sensitivity and selectivity.

Cobalt ions (Co2+) are among heavy metals ions which cause pollution in environment because of their toxicity and improper degradation. In this work, a new fluorescent approach based on silicon nanoparticles (Si NPs) was designed for Co2+ detection. The fluorescent Si NPs were prepared by mixing 3-aminopropyl trimethoxysilane (APTES) and basic fuchsin, and under the excitation of 400 nm, they emitted green fluorescence at 515 nm. The prepared Si NPs were highly soluble in water, stable to salt and pH, and their fluorescence emission was extremely constant, with the quantum yield of 2.28%. The detailed mechanism studies showed that Co2+ effectively quenched the fluorescence of Si NPs by forming static complex. After optimizing the reaction parameters, a good linear relationship for Co2+ was observed from 0.2 to 60 µM, and the limit of detection was 0.14 µM that is lower than the guideline announced by Department of Environmental Protection for drinking water (1.7 µM). The preparation method of Si NPs was cheap, rapid and simple, and the fluorescent approach was applied to determine Co2+ in Yellow river water, drinking water, and industrial wastewater. Moreover, the Si NPs has good response to exogenous Co2+ in HepG2 cell imaging.

Yellow-emissive carbon dots as a fluorescent probe for chromium(VI).

The authors describe a one-step method for the preparation of yellow fluorescent carbon dots (CDs) starting from 4-aminoacetanilide hydrochloride and 4-acetamidobenzaldehyde. The CDs have excitation/emission peaks at 470/550 nm, good water solubility, salt-tolerance and photostability. Their fluorescence is quenched by hexavalent chromium [Cr(VI)] via static quenching. Fluorescence intensity drops linearly in the 1 to 400 µM Cr(VI) concentration range, and the limit of detection is 0.13 µM. This method is selective for Cr(VI) over potential metal ion interferences and was successfully applied to the detection of Cr(VI) in spiked water and biological tissue samples. Recoveries from spiked samples ranged from 97.7% to 103.8%. Graphical abstract Schematic presentation of (a) the preparation of the CD fluorescent probe and (b), the principle of Cr(VI) determination.

Synthesis of orange-red emissive carbon dots for fluorometric enzymatic determination of glucose.

The authors describe a method for the preparation of orange-red emissive carbon dots (CDs) with excitation/emission peaks at 520/582 nm. The CDs were hydrothermally prepared by a one-pot strategy from trimesic acid and 4-aminoacetanilide. The fluorescence of the CDs is strongly quenched by hydrogen peroxide. The oxidation of glucose by glucose oxidase (GOx) produces H2O2 that quenches the fluorescence via static quenching. Based on this phenomenon, a fluorometric method was established for the determination of glucose. Under the optimum conditions, response is linear in the 0.5 to 100 µM glucose concentration range, with a 0.33 µM limit of detection. The method is selective for glucose over its analogues and was successfully applied to the determination of glucose in diluted human serum and in urine from diabetics and healthy individuals. Recoveries from spiked samples range from 98.7 to 102.5%. Graphical abstract (a) One-step synthetic strategy of the CDs; (b) Schematic illustration of the CDs for glucose detection.