Noncovalent Dual-Locked Near-Infrared Fluorescent Probe for Precise Imaging of Tumor via Hypoxia/Glutathione Activation.

Stimulus-responsive fluorescent probes have broad applications in the early detection and treatment of tumors and thus promote the personalized treatment of tumors and improve patient survival. Among the repertoires of probes, dual-locked near-infrared (NIR) fluorescent probes are of great significance due to their improved specificity and multiplex detection in tumor imaging but remain to be explored. In this work, a facile noncovalent strategy for constructing dual-locked probes was proposed. A glutathione (GSH)-activatable single-locked probe CySS (first lock) was preloaded into a hypoxia-responsive molecular container CF3C4A (second lock) through a host-guest interaction to form the dual-locked probe CF3C4A-CySS. Under physiological conditions, CF3C4A-CySS binds strongly to avoid undesired leakage in normal tissues. We have proven that CF3C4A-CySS can be activated and "turn on" its NIR fluorescent signal under the dual key stimulation of hypoxia and GSH in the tumor microenvironment, which enables precise tumor imaging with enhanced accuracy and specificity. Both in vitro and in vivo results indicated the superiority of CF3C4A-CySS in tumor imaging. This work not only provides an effective tool for tumor imaging but also proposes a promising strategy for dual-locked imaging agent construction.

Rapid and Selective 19F NMR-Based Sensors for Fingerprint Identification of Ribose.

Ribose plays an important role in the process of life. Excessive ribose in the human cerebrospinal fluid or urine can be used as an early diagnostic marker of leukoencephalopathy. Fluorinated phenylboronic acid combined with 19F NMR spectroscopy was a powerful method for molecular recognition. However, phenylboronic acid-based sensors for selective detection of ribose are rarely reported in the literature. In this study, the rapid and highly selective recognition of ribose was studied by 19F NMR and 2-fluorophenylboric acid. It was found that 2-fluoro-phenylboric acid was an appropriate 19F NMR-based sensor molecule for the determination of ribose under physiological conditions with high selectivity and robust anti-interference ability. When 2-fluorophenylboric acid was used for the detection of ribose in human urine without any sample pretreatment, a limit of detection of 78 µM was obtained at room temperature under given 19F NMR experimental conditions (400 MHz, 512 scans, ca. 12 min), which can well meet the needs of practical application.

Antibacterial activity and action mechanism of flavonoids against phytopathogenic bacteria.

As the most difficult to control in plant disease, phytopathogenic bacteria cause huge losses to agricultural products and economy worldwide. However, the commercially available bactericides are few and enhance pathogen resistance. To alleviate this situation, 50 flavonoids were evaluated for their antibacterial activities and mechanism of action against two intractable plant bacterial pathogens. The results of bioassays showed that most of the flavonoids exhibited moderate inhibitory effects against Xanthomonas oryzae (Xo) and Xanthomonas axonopodis pv citri (Xac). Remarkably, kaempferol showed excellent antibacterial activity against Xo in vitro (EC50 = 15.91 µg/mL) and quercetin showed the best antibacterial activity against Xac in vitro (EC50 = 14.83 µg/mL), which was better than thiodiazole copper (EC50 values against Xo and Xac were 16.79 µg/mL, 59.13 µg/mL, respectively). Subsequently, in vivo antibacterial activity assay further demonstrated kaempferol exhibited a stronger control effect on bacterial infections than thiodiazole copper. Then, the preliminary antibacterial mechanism of kaempferol was investigated by ultrastructural observations, transcriptomic, qRT-PCR analysis and biochemical index determination. These results showed that kaempferol mainly exerted bacteriostatic effects at the molecular level by affecting bacterial energy metabolism, reducing pathogenicity, and leading to disruption of cellular integrity, leakage of contents and cell death eventually.

Three-dimensional tree-like branched TiO2 nanorods for the highly selective enrichment and determination of lead.

Branched titanium dioxide nanorods (B-TiO2 NRs) grown on fluorine-doped tin oxide glass (FTO) were developed, which can be used as a solid-phase extractant for preconcentration and determination of trace Pb(II) combined with inductively coupled plasma optical emission spectrometry (ICP-OES). The B-TiO2 NR-based glass substrate displayed excellent adsorptive selectivity and capacity for Pb(II); the maximum adsorption capacity was found to be 168.4 mg⋅g-1 PB(II) at pH = 5.0. It proved that the primary extraction mechanism was attributed to soft acid/soft base interactions to form complexes for chemisorption. Investigating the adsorption kinetics and isotherms indicated that the pseudo-second-order and Langmuir models can better describe Pb(II) adsorption on the B-TiO2 NRs. The proposed method presented good linearity from 0.01 to 5 mg⋅L-1 with a correlation coefficient (R2) of 0.9989 and a low limit of detection (LOD) of 2.2 µg⋅L-1 for Pb(II) under optimal conditions. The method was successfully applied to Pb(II) determination in foodstuffs with desirable recoveries from 93.18 to 108.1% and good precision with an RSD of less than 12.2%. This work provides a new strategy for selective extraction and determination of Pb(II) in complicated matrix samples.

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.

Noncovalent Theranostic Prodrug for Hypoxia-Activated Drug Delivery and Real-Time Tracking.

Real-time tracking of hypoxia-activated prodrugs (HAPs) delivery and the release process is of great significance for innovative medical treatments and drug development. Existing theranostic methods for HAPs activation imaging are based on the covalent approach, which suffered from complicated molecular design and tedious synthesis. In this work, a facile noncovalent strategy for constructing an hypoxia-activated theranostic prodrug has been proposed. An hypoxia-activated prodrug, NMAC4A, has been synthesized and bound with an NIR fluorophore CyNH2 through host-guest interaction to form the theranostic prodrug NMAC4A-CyNH2. Interestingly, the NIR fluorescence signal of CyNH2 can be effectively "turned off" after the formation of the stable theranostic prodrug NMAC4A-CyNH2. Because of the selective response to a tumor hypoxic microenvironment, NMAC4A-CyNH2 can realize the tumor-targeted drug delivery, accompanied by its NIR fluorescence "turn on". The synchronization of drug release and fluorescence "turn on" properties of NMAC4A-CyNH2 in an hypoxic microenvironment makes the fluorescence signal an effective tool for a precise tracing of the drug release process. Notably, NMAC4A-CyNH2 has been successfully applied to real-time image tracking of the drug delivery in vitro and in vivo. More importantly, the biodistribution of the theranostic prodrug's metabolites in a tumor and some major tissues have been mapped by mass spectrometry imaging at the molecular level, which further validated the effectiveness of NMAC4A-CyNH2 as a tumor-targeted drug delivery platform and NIR probe. This work will not only provide a promising tool for an hypoxia-activated drug delivery and real-time image tracking but also propose an effective design strategy for noncovalent theranostic prodrug construction.

A Bisboronic Acid Sensor for Ultra-High Selective Glucose Assay by 19F NMR Spectroscopy.

Glucose is a significant analyte both in biology and biomedical science, it is of great importance to selectively detect glucose both in body fluids and complex mixture. In this study, a simple 19F NMR based sensor was synthesized easily, which exhibited a high selectivity and robust anti-interference ability toward glucose detection both in a mixture containing up to 10 saccharides and human urine samples without any pretreatment. Combined with this sensor system, glucose could be well detected in human urine samples and the limit of detection was 0.41 mM by using a 400 MHz NMR spectrometer with 128 scans (ca. 4 min). This method had a potential for specific detection of glucose in complex mixture and diagnosis of diabetes mellitus related diseases in body fluid.

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.

Narjatamolide, an Unusual Homoguaiane Sesquiterpene Lactone from Nardostachys jatamansi.

Narjatamolide (1), an unusual homoguaiane sesquiterpene lactone, was isolated from the roots and rhizomes of Nardostachys jatamansi DC. It represents the new carbon skeleton of a homoguaiane sesquiterpenoid possessing an additional acetate unit spiro-fused with C-4 and C-15 to form a cyclopropane ring. The structure of 1 was elucidated by extensive spectroscopic analyses, and the absolute configuration was confirmed by the electronic circular dichroism (ECD) calculations and X-ray single-crystal diffraction analysis. Compound 1 showed antiproliferative effects against BEL-7402 cell lines with an IC50 value of 5.67 ± 1.43 µM, and the mechanism study showed that 1 induces cell cycle of BEL-7402 cell lines arrest at G2/M phase.

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.

Precisely Traceable Drug Delivery of Azoreductase-Responsive Prodrug for Colon Targeting via Multimodal Imaging.

We report the development of an azoreductase-responsive prodrug AP-N═N-Cy in which the precursor compound AP, a readily available podophyllotoxin derivative, is linked with a NIR fluorophore (Cy) via a multifunctional azobenzene group. This type of azo-based prodrug can serve as not only an azoreductase-responsive NIR probe to real-time tracking of the drug delivery process but also a delivery platform for an anticancer compound (AdP). We have shown that cleavage of the multifunctional azobenzene group in AP-N═N-Cy only occurred in the presence of azoreductase, which specifically secretes in the colon, resulting in direct release of AdP through an in situ modification of a phenylamino group on the precursor AP. Moreover, introduction of the azobenzene group endows the prodrug with an unique fluorescence "off-on" property and served as a switch to "turn on" the fluorescence of Cy as consequence of a self-elimination reaction with breakage of an azo bond. Such a prodrug can be administered orally and exhibit high stability and low toxicity before arriving at the colon. In view of the synchronism of drug release and the fluorescence turn-on process, the fluorescence imaging method was utilized to precisely trace drug delivery in vitro, ex vivo, and in vivo. Distinguishingly, the biodistribution of AdP and Cy in various tissues was further precisely mapped at the molecular level using imaging mass spectrometry. To the best of our knowledge, this is the first time that the in vivo real-time precise tracking of the colon-specific drug release and biodistribution was reported via a multimodal imaging method.

Bungsteroid A: One Unusual C34 Pentacyclic Steroid Analogue from Zanthoxylum bungeanum Maxim.

Bungsteroid A (1), possessing an unreported carbon skeleton, was isolated from the pericarps of Zanthoxylum bungeanum Maxim. It represents the first carbon skeleton of a C34 steroid analogue featuring a unique 6/6/6/6/5-fused pentacyclic skeleton, which has been determined by spectroscopic methods, quantum-chemical 13C NMR, ECD calculations, and calculations of optical rotations. Bungsteroid A showed the antiproliferative effects against HepG2, MCF-7, and HeLa cell lines with the IC50 values of 56.3 ± 1.1, 64.2 ± 0.9, and 74.2 ± 1.3 µM, respectively.

PEGylated NALC-functionalized gold nanoparticles for colorimetric discrimination of chiral tyrosine.

In this work, acid and matrix-tolerant multifunctionalized gold nanoparticles (AuNPs) with an integrated chiral selector towards tyrosine (Tyr) and polyethylenglycol (PEG) chains were developed for visual chiral discrimination of Tyr in biological samples under acid conditions. In brief, AuNPs multifunctionalized with N-acetyl-l-cysteine (NALC) and PEG (PEG/NALC-AuNPs) were prepared via a simple strategy. In the presence of l-Tyr, the color of PEG/NALC-AuNP solution changed from red to gray, while no obvious color change was observed with the introduction of d-Tyr, which indicated that the introduction of PEG onto the surface of AuNPs has no effect on the chiral recognition between l-Tyr and NALC. A computer-aided molecular model was used to clarify the chiral recognition mechanism between NALC and Tyr enantiomers and to further guide the optimization of sensitivity. The resultant PEG/NALC-AuNP sensor presented a significantly improved stability under acid and alkali conditions compared with conventional NALC-AuNPs, resulting in a wider dynamic range (500 nM-100 µM) and a 50 times reduced detection limit by simply adjusting the pH of the sensor system under acid conditions (pH 2-2.5). More importantly, the PEG/NALC-AuNPs can realize the visual chiral discrimination of Tyr enantiomers in biological samples due to their significantly improved long-term stability and reduced interaction towards non-target species.

Further sesquiterpenoids from the rhizomes of Homalomena occulta and their anti-inflammatory activity.

The rhizomes of Homalomena occulta are called Qian-nian-jian in Traditional Chinese Medicine (TCM), which is widely consumed in China owing to its health benefits for the treatment of rheumatoid arthritis and for strengthening tendons and bones. A phytochemical investigation on this famous TCM yielded 19 sesquiterpenoids (1-19) with various carbocyclic skeletons including isodaucane (2, 8, and 9), guaiane (3), eudesmane (4 and 10-15), oppositane (5, 16, and 17), and aromadendrane (18 and 19) types. The structures of new compounds, Homalomenins A-E (1-5), were determined by diverse spectroscopic data. Compound 1 possessed a rare sesquiterpenoid skeleton and compound 5 represented the first example of 1,4-oxa-oppositane sesquiterpenoid. These isolates were evaluated for their inhibitory effects on COX-2 mRNA, COX-2 protein expression, and prostaglandin E2 (PGE2) production in Raw264.7 cells, which demonstrated that compounds 5, 18, 19 showed potent anti-inflammatory activity by suppressing LPS-induced COX-2 expression and PGE2 production in a dose-dependent manner.

Sodium(I)-doped graphitic carbon nitride with appropriate interlayer distance as a highly selective sorbent for strontium(II) prior to its determination by ICP-OES.

Multilayered and porous sodium-doped graphitic carbon nitride (GCN-Na) was prepared and employed to the solid-phase extraction of Sr(II). The sorbent exhibits high adsorption capacity and excellent selectivity for Sr(II). This is due to its small interplanar stacking distance caused by doping with Na(I) which matches the size of Sr(II) better than blank GCN. An original solid-phase extraction method based on GCN-Na coupled with ICP-OES was established for Sr(II), the calibration plots are linear ranging from 0.05-10 mg·kg-1 with the correlation coefficients (R2) above 0.999, the limits of detection are in the range of 0.57-1.52 µg·kg-1 and the preconcentration factor of 80 is achieved using 48 mL sample. It was successfully applied in the extraction and detection of trace Sr(II) in tap water, rice and sea fish. Graphical abstractA multilayer porous sodium(I) doped graphitic carbon nitride nanosheet (GCN-Na) was synthesized and exhibited excellent adsorption capability and selectivity for Sr(II).

Solid/liquid phase microextraction of five bisphenol-type endocrine disrupting chemicals by using a hollow fiber reinforced with graphene oxide nanoribbons, and determination by HPLC-PDA.

A novel solid/liquid phase microextraction (SLPME) technique was developed and applied to the simultaneous determination of five bisphenol-type endocrine disrupting chemicals (EDCs) including bisphenol A, bisphenol S, bisphenol F, bisphenol B and bisphenol AF. The method is based on the use of a graphene oxide nanoribbon-reinforced hollow fiber (GONRs-HF) and 1-octanol. GONRs were longitudinally unzipped from multiwalled carbon nanotubes (MWCNTs) by means of chemical oxidation and carefully characterized. GONRs were dispersed in ultrapure water with the assistance of surfactant and then immobilized into the wall pores of HF. Compared with CNTs, the hydrophilicity of GONRs was improved, and a uniform dispersion was formed. This simplifies the preparation process and reduces waste of materials. The pores and lumen of GONRs-HF were filled with 1-octanol, and then the whole assembly was used for the extraction of the analytes. In comparison with different extraction modes, GONRs-HF-SLPME exhibits a more effective extraction performance for five bisphenol-type EDCs. Response surface methodology was used to optimize experimental parameters affecting the extraction efficiency. The enrichment factors are in the range from 76 to 127 with good inter-fiber and batch-to-batch reproducibility. The method shows good linearity with determination coefficients (R2) higher than 0.9985. The limits of detection are in the range of 0.1-0.4 µg L-1. Recoveries from spiked real samples are between 83% and 114% with relative standard deviations between 1.6% and 7.8%. Graphical abstract Schematic presentation of the preparation of graphene oxide nanoribbons (GONRs) with multiwalled carbon nanotubes (MWCNTs), immobilization of GONRs into the pores of hollow fiber (HF), and application for solid/liquid phase microextraction (SLPME) of five bisphenol-type endocrine disrupting chemicals in three kinds of real samples.

Regulatory role of microRNA-320a in the proliferation, migration, invasion, and apoptosis of trophoblasts and endothelial cells by targeting estrogen-related receptor γ.

Recently, microRNAs (miRNAs) have been demonstrated to participate in many physiological and biological processes, especially by acting as circulating biomarkers or modulators in cell differentiation. Therefore, the aim of the current study was to clarify whether microRNA-320a (miR-320a) regulates the proliferation, migration, invasion, and apoptosis of trophoblasts and endothelial cells. In this study, miR-320a mimics and inhibitors were transfected into HTR.8/SVneo cells and human umbilical vein endothelial cells (HUVECs) using liposomes. Subsequently, the expression of miR-320a and estrogen-related receptor γ (ERRγ) mRNA was detected by a reverse transcription quantitative polymerase chain reaction, whereas the protein expression of ERRγ, vascular endothelial growth factor (VEGF), angiogenin 1 (Ang-1), human 3beta-hydroxysteroid dehydrogenase type 1 (HSD3B1), and human chorionic gonadotropin (HCG) was detected by western blot analysis. Furthermore, the proliferation, invasion/migration, and apoptosis of cells were analyzed by the cell counting kit-8 assay, transwell assay, and flow cytometry, respectively. The results showed that overexpression of miR-320a decreased the optical density (OD) values and the proliferation rate of HTR.8/SVneo cells and HUVECs, while inhibiting the expression of VEGF, Ang-1, HSD3B1, and HCG in these cells. Furthermore, miR-320a reduced the ability of cell invasion and migration, while increasing the rate of cell apoptosis. After cotransfecting the cells with miR-320a and ERRγ small (or short) interfering RNA (siRNA), the decreased ERRγ expression led to inhibited proliferation, migration, and invasion, but increased apoptosis of HTR.8/SVneo cells and HUVECs. Our results further revealed that miR-320a induced the apoptosis of trophoblasts and endothelial cells while inhibiting their proliferation, migration, and invasion by decreasing the expression of ERRγ and by indirectly suppressing the expression of VEGF, Ang-1, HSD3B1, and HCG.

Quality evaluation of six bioactive constituents in goji berry based on capillary electrophoresis field amplified sample stacking.

Goji berry, fruits of the plant Lycium barbarum L., has long been used as traditional medicine and functional food in China. In this work, a simple and easy-operation on-line concentration capillary electrophoresis (CE) for detection flavonoids in goji berry was developed by coupling of field amplified sample stacking (FASS) with an electroosmotic (EOF) pump driving water removal process. Due to the EOF pump and electrokinetic injection showing different influence on the concentration, the analytes injection condition should be systemically studied. Thereafter, the verification of the analytes injection conditions was achieved using response surface experimental design. Under the optimum conditions, 86-271 folds sensitivity enhancement upon normal capillary zone electrophoresis (CZE, 50 mbar × 5 s) were achieved for six flavonoids, and the detection limits ranged from 0.35 to 1.82 ng/mL; the LOQ ranged from 1.20 to 6.01 ng/mL. Eventually, the proposed method was applied to detect flavonoids in 30 goji berry samples from different habitats of China; and the results indicated that the flavonoids were rich in the eluent of 30-60% methanol, which provided a reference for extraction of goji berry flavonoids.

Magnetic boronate modified molecularly imprinted polymers on magnetite microspheres modified with porous TiO2 (Fe3O4@pTiO2@MIP) with enhanced adsorption capacity for glycoproteins and with wide operational pH range.

Boronate-affinity based molecularly imprinted polymers (MIPs) are beset by the unsatisfied adsorption capacity and narrow working pH ranges. A magnetic molecularly imprinted polymer containing phenylboronic acid groups was placed on the surface of Fe3O4 (magnetite) microspheres coated with porous TiO2 (Fe3O4@pTiO2@MIP). In contrast to its silica analog (Fe3O4@SiO2@MIP), the flowerlike Fe3O4@pTiO2 offers more binding sites for templates. Thus, the adsorption capacity of the Fe3O4@pTiO2@MIP is strongly enhanced. The strong electron-withdrawing effects of Ti(IV) enable the boronic acid of the MIP to have better affinity for glycoproteins at a wide pH range from 6.0 to 9.0. Consequently, the Fe3O4@pTiO2@MIP exhibits higher adsorption for glycoproteins than Fe3O4@SiO2@MIP in both basic and acidic medium. The Fe3O4@pTiO2@MIPs were eluted with 5% acetic acid aqueous solution containing 30% acetonitrile, and the eluate was analyzed by MALDI-TOF MS. The method was applied to the selective extraction and quantitation of horseradish peroxidase (HRP) in spiked fetal bovine serum (FBS). The linear range is 0.40-10 µg·mL-1 with the limit of detection of 0.31 µg·mL-1. In our perception, this work has a wide scope in that is paves the way to a more widespread application of boronate affinity based MIPs for analysis of glycoproteins and related glyco compounds even at moderately acidic pH values. Graphical abstract Schematic presentation of the magnetic boronate modified molecularly imprinted polymer on magnetic spheres modified with porous TiO2 (Fe3O4@pTiO2@MIP). It was applied to extract glycoprotein in spiked both basic fetal bovine serum (FBS) and acidic urine samples prior to quantitation by MALDI-TOF mass spectrometry.

Kinetics and inhibition study of tyrosinase by pressure mediated microanalysis.

In the present study, pressure mediated microanalysis (PMMA), a fast, convenient and efficient capillary electrophoresis (CE) method was developed for studying enzyme kinetics of tyrosinase and inhibition kinetics of kojic acid, a model inhibitor of tyrosinase. The enzymatic reaction conditions and CE conditions were optimized in order to obtain high enzyme activity and short analysis time. By PMMA, only the product could be detected at 475 nm, and no voltage was applied to separate the product from the reaction mixture thus greatly simplifying the optimization procedure. The spectrophotometric assay and electrophoretically mediated microanalysis (EMMA) were also performed to validate the developed method. With the present method, the Michaelis-Menten constant (Km) was calculated to be 1.347 mM for tyrosinase. The inhibition constant of kojic acid to free tyrosinase (KI) and kojic acid to tyrosinase/L-DOPA complex (KIS) were calculated to be 36.64 and 74.35 µM, respectively, and the half-maximal inhibitory concentration (IC50) was determined to be 46.64 µM for kojic acid. The developed method is fast and convenient for studying enzyme kinetics, inhibition kinetics and further screening enzyme inhibitors.