A New Method of Extracting Polygonatum sibiricum Polysaccharide with Antioxidant Function: Ultrasound-Assisted Extraction-Deep Eutectic Solvents Method.

Polygonatum sibiricum Polysaccharide (PsP) with antioxidant function is the main active component of Polygonatum sibiricum (P.sibiricum). The currently poor extraction yield and extraction methods of PsP cannot meet the application of that in food industrial production. In this research, an ultrasound-assisted extraction-deep eutectic solvents (UAE-DESs) method, which has never been used in the PsP industry, was first used to extract PsP. The extraction conditions were optimized by the response surface method (RSM). Both the extraction yield and antioxidant function were simultaneously considered during the optimization process. The indicators of PsP's level and antioxidant activity in vitro were used to present the extraction yield of the UAE-DESs method, the purity, and the antioxidant effect of PsP. Under the optimal conditions, which included a liquid-solid ratio of 26:1 (mL:g), extraction temperature of 80 °C, ultrasonic time of 51 min, and ultrasonic power of 82 W, the PsP extraction yield could reach (43.61 ± 0.09)%, which was obviously higher than single DESs (33.81%) and UAE (5.83%), respectively, and the PsP appeared favorably antioxidant function. This research proposed an efficient extraction method for PsP, filled the basic research gap, and further improved the development of PsP as a dietary supplement with antioxidant function in the food industry.

[Conditional optimization and methodological evaluation of liquid protein microarray in detecting cystatin C].

OBJECTIVE: To optimize the detection conditions and evaluate of cystatin C(CysC) by liquid protein microarray. METHODS: CysC was detected by double antibody sandwich method using liquid protein microarray. On the basis of determining the optimal concentration combination of captured antibody and detected antibody, the detection conditions were optimized by determining the biological detection limit and lower detection limit, drawing the S-shaped curve and judging the linear range, and establishing the standard curve and regression equation. Methodsologically evaluate the accuracy, precision, reportable range and analytical specificity of the detection method. RESULTS: The optimal concentration combinations of CycC trapping-detection antibodies were 26.6 µg/mL-1∶800. The lower limits of detection and biologic limits of detection of the CysC were 0.037 and 0.237 ng/mL, respectively. Regression equation were as followes: y=-3.315x~2+283.04x+160.89, R~2=0.9921. The relative bias of CysC which was detected on the liquid protein microarry was 5.81%. The dilution recovery and recovery were 70.35%-84.91%(n=3)and 79.94%-122.41%(n=3)respectively. The correlation coefficient of method ology comparison experiment was r=0.616, P<0.05, and there was no significant difference between the two method(t=0.948, P=0.358); The within-run precision range from 3.54% to 4.03%(n=10); The between-run precision range from 12.07% to 15.05%(D=5, n=3); The reportable range was 0.26-3784.04 ng/mL. The analysis of interference test result showed that the both concentrations of hemoglobin(160.00, 71.11 g/L) had interference to the result of CysC detected on the chip. CONCLUSION: This study completed the optimization of conditions and methodological evaluation of liquid protein microarray in detecting CysC.

The role of Sirtuin 1 and its activators in age-related lung disease.

Aging is a major driving factor in lung diseases. Age-related lung disease is associated with downregulated expression of SIRT1, an NAD+-dependent deacetylase that regulates inflammation and stress resistance. SIRT1 acts by inducing the deacetylation of various substrates and regulates several mechanisms that relate to lung aging, such as genomic instability, lung stem cell exhaustion, mitochondrial dysfunction, telomere shortening, and immune senescence. Chinese herbal medicines have many biological activities, exerting anti-inflammatory, anti-oxidation, anti-tumor, and immune regulatory effects. Recent studies have confirmed that many Chinese herbs have the effect of activating SIRT1. Therefore, we reviewed the mechanism of SIRT1 in age-related lung disease and explored the potential roles of Chinese herbs as SIRT1 activators in the treatment of age-related lung disease.

Au@SiO2 SERS nanotags based lateral flow immunoassay for simultaneous detection of aflatoxin B1 and ochratoxin A.

Agricultural products are frequently contaminated by mycotoxins. Multiplex, ultrasensitive, and rapid determination of mycotoxins is still a challenging problem, which is of great significance to food safety and public health. Herein, a surface-enhanced Raman scattering (SERS) based lateral flow immunoassay (LFA) for the simultaneous on-site determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA) on the same test line (T line) was developed, in this study. In practice, two kinds of Raman reporters 4-mercaptobenzoic acid (4-MBA), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) encoded silica-encapsulated gold nanotags (Au4-MBA@SiO2 and AuDNTB@SiO2) were used as detection markers to identify the two different mycotoxins. Through systematic optimization of the experimental conditions, this biosensor has high sensitivity and multiplexing with the limits of detection (LODs) at 0.24 pg mL-1 for AFB1 and 0.37 pg mL-1 for OTA. These are far below the regulatory limits set by the European Commission, in which the minimum LODs for AFB1 and OTA are 2.0 and 3.0 µg kg-1. In the spiked experiment, the food matrix are corn, rice, and wheat, and the mean recoveries of the two mycotoxins ranged from 91.0% ± 6.3%-104.8% ± 5.6% for AFB1 and 87.0% ± 4.2%-112.0% ± 3.3% for OTA. These results demonstrate that the developed immunoassay has good stability, selectivity, and reliability, which can be used for routine monitoring of mycotoxin contamination.

Construction and Evaluation of a Novel MAP Immunoassay for 9 Nutrition-and-Health-Related Protein Markers Based on Multiplex Liquid Protein Chip Technique.

We attempted to construct and evaluate a novel detection method to realize simultaneous detection based on a multiplex liquid protein chip technique for nine nutrition-and-health-related protein markers to meet the requirement of an accurate, simultaneous and comprehensive analysis of the proteomics of nutrition and health. The lower limits of detection, biological limits of detection and regression equations of serum ferritin (SF), soluble transferrin receptor (sTfR), c-reactive protein (CRP), retinol-binding protein4 (RBP4), apolipoprotein B (ApoB), alpha-fetoprotein (AFP), prealbumin (PA), carcino-embryonic antigen (CEA) and D-Dimmer (D-D) were determined after a series of optimal experiments. Then, the results of the methodological evaluation for this novel method indicated that the accuracies were between 70.12% and 127.07%, the within-run precisions were between 0.85% and 7.31%, the between-run precisions were between 3.53% and 19.07%, the correlation coefficients between this method and other methods were above 0.504 (p < 0.05), and the direct bilirubin (DBIL) of low concentration and the indirect bilirubin (IBIL) of high concentration could not interfere with the detected results of nine indicators. The novel multiplex detection method, which can increase accuracy and improve the ability of comprehensive analysis, can basically meet the requirement of detection and the diagnosis of the proteomics of nutrition and health.

Carbon emission reduction policy with privatization in an oligopoly model.

This study constructs a mixed oligopoly model that includes a public enterprise and two private enterprises. Game theory was adopted to explore the effects of carbon emission reduction policies. In addition, this study analyzes the optimal carbon emission trading prices and privatization decisions. The results show that the proportion of state-owned shares and the equity efficiency gap affect the equilibrium results for different carbon emission policies. Privatization increases the profits of public firms but does not necessarily increase social welfare. Different carbon emission reduction policies have different effects on the equilibrium results. Moreover, the emission reduction target is not completely consistent with the maximum social welfare target and should be comprehensively considered. The government can intervene by setting carbon emission trading prices and making privatization decisions. Full and partial privatization may be optimal decisions.

Ru(bpy)32+ as a photoinduced oxidase mimic for colorimetric detection of biothiols.

We have found that tris (2,2'-bipyridyl) ruthenium (II) (Ru(bpy)32+) possesses a high photo-induced oxidase-like activity and is capable of catalyzing the color reaction of 3,3',5,5'-tetramethylbenzidine (TMB) with dissolved oxygen. Ru(bpy)32+ has a catalytic constant (Kcat) that is twice as high as that of fluorescein, 170 and 275-fold higher than that of 9-mesityl-10-methyl acridine and Eosin Y, respectively. Electron spin resonance spectroscopy (ESR) and radical scavenging experiments have verified the major active radicals involved in the color reaction are •OH. A colorimetric biothiol assay has been successfully developed for the oxidase-like activity of Ru(bpy)32+ can be suppressed by sulfhydryl compounds. A linear dependence between the decrease in absorbance and the logarithm of thiol concentrations can be found ranging from 5.0 to 50 µM, with a detection limit of 1.0 µM. This work reveals a new oxidase mimic with high catalytic activity and will facilitate the utilization of this oxidase mimic in biochemical analysis.

Optimization and Application of A Bionic System of Dynamic Co-Culture with Hepatocytes and Renal Cells Based on Microfluidic Chip Technique in Evaluating Materials of Health Food.

We aimed to explore the optimization and application of a bionic system of dynamic co-culture with hepatocytes and renal cells based on the microfluidic chip technique in evaluating emodin, which might replace the conventionally cytological evaluation technique of health food. After optimal experiments, the improved bionic system was composed of human hepatocellular carcinoma cells (HepG2), human renal glomerular endothelial cells (HRGECs), rat tail collagen type I, and gelatin with optimized concentrations (1.3 mg/mL + 7.5%). The applicability of the bionic system indicated that the growth stability was appropriate (CV: 7.36%), and the cell viability of that gradually decreased with the increasing of emodin concentration from 0−100 µM, which statistic significances were at 50 and 100 µM (p < 0.05), and the stained results of dead/live cells also showed the same trend. The LDH level appeared rising trend after decline between 0 µM and 100 µM emodi, and the level of that at 100 µM emodin was significantly higher than that at 25 µM and 50 µM emodin, respectively. The BUN level continuously and significantly declined with the increasing of emodin concentration (p < 0.05). Our research realized the application of this optimized bionic system in evaluating emodin, and provided a useful platform and reference for further in vitro alternative research with regard to evaluating the efficacies of health food in the future.

Metagenomic insights into the rumen epithelial integrity responses to the vitamin B1 supplement under high-concentrate diets treatments.

Subacute ruminal acidosis (SARA) becomes the most common nutritional metabolic disease in high-yielding dairy cows and later fatting beef cattle because of the increasing consumption of high-concentrate diets in modern feeding patterns. Our previous research found a certain piece of evidence that adding 180 mg thiamine/kg DMI could increase the rumen pH and regulate the structure of the rumen microbial community in vivo. However, there is still limited experimental data on the effects of SARA on thiamine status, the damage to the structure of rumen epithelial cells, and the underlying mechanism of the epithelium alterations. For this purpose, a total of 18 Angus bulls (average 22.0-months-old) with an average live weight of 567.6 ± 27.4 kg were randomly allocated into a control treatment (CON), a high-concentrate diet treatment (HC), and a high-concentrate diet with the vitamin B1 supplement treatment (HCB). All bulls were conducted with a 7-day adjustment period followed by a 60-day-long main feeding procedure. Results indicated that ADFI and ADG significantly decreased in the HC treatment compared with CON (P < 0.05), while significantly increased after the VB1 supplement (P < 0.05). Besides, ruminal acetate content was significantly downregulated while propionate was significantly upregulated under the HC treatment compared with CON (P < 0.05); however, these alterations showed a completely inverse regulatory effect on the VB1 supplement compared with HC (P < 0.05). These changes causatively induced a significant decrease in the A/P ratio in the HC treatment compared with CON and HCB treatments (P < 0.05). Bacterial communities in the HC treatment could be separated from those in CON through PCoA axes 1 and 2. Meanwhile, the VB1 supplement significantly altered the bacterial communities compared with the HC treatment, except for HCB-3. Furthermore, the HC treatment significantly upregulated the expression of JNK, Bax, Caspase-8, Caspase-3, Caspase-9, and Cyt-C compared with CON, while significantly downregulated the expression of Bcl-2. The VB1 supplement showed a complete converse gene expression compared with HC. In conclusion, the VB1 supplement could effectively attenuate the alterations that occurred when exposed to high-concentrate diets, and help promote production performance through increased fermentability.

Bergenin ameliorates airway inflammation and remodeling in asthma by activating SIRT1 in macrophages to regulate the NF-κB pathway.

Airway inflammation and remodeling are critical pathological changes in asthma, and macrophage activation plays a vital role in this process. Sirtuin 1 (SIRT1) reduces airway inflammation by affecting macrophages in asthma. This study aimed to investigate the potential benefit and underlying mechanism of the SIRT1 agonist bergenin as a treatment for asthma. We performed in vivo and in vitro experiments by establishing a Sirt1 fl/fl -LysMcre mouse asthma model and using the alveolar macrophage-like cell line MH-S, respectively. Our results show that Sirt1 fl/fl -LysMcre asthmatic mice exhibited more severe airway inflammation and airway remodeling than wild-type mice. As an activator of SIRT1, bergenin attenuated asthmatic airway pathology and reduced production of interleukins 1ß, IL-5, IL-6, and matrix metalloproteinase 9 (MMP-9) in wild-type asthmatic mice. However, the therapeutic effects of bergenin were significantly attenuated in Sirt1 fl/fl -LysMcre asthmatic mice or following coadministration with the SIRT1 inhibitor EX-527. Further experiments showed that activation of SIRT1 by bergenin deacetylates nuclear factor κB and hinders its nuclear translocation, thereby affecting IL-1ß, IL-5, IL-6, and MMP-9 production by regulating transcriptional activity. Our study suggests that bergenin can improve asthma-induced airway inflammation and remodeling by activating SIRT1 in macrophages.

Serum Creatinine to Cystatin C Ratio is an Effective Indicator for Muscle Strength Decline in Men with Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

Purpose: This study explored the value of the serum creatinine/cystatin C (Cr/CysC) ratio in diagnosing the reduction of muscle strength in men with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Patients and Methods: In this study, we enrolled 72 male patients with AECOPD and 32 male patients with stable chronic obstructive pulmonary disease (COPD). We compared clinical characteristics between the AECOPD and stable COPD groups. Then, we subdivided AECOPD patients into normal muscle strength and low muscle strength groups; we compared the clinical characteristics between these two groups. We analyzed the relationships of serum creatinine (Cr), cystatin C (CysC), and Cr/CysC ratio with clinical characteristics in male AECOPD patients. We also investigated whether the Cr/CysC ratio could aid in the diagnosis of muscle strength decline via receiver operating characteristic curve and binary logistic regression analysis. Results: We found that handgrip strength, Cr/CysC ratio, serum Cr, FEV1, FVC, and FEV1%pred were lower in AECOPD patients than in stable COPD patients. Among AECOPD patients, BMI, weight, FEV1, FVC, FEV1%pred, and Cr/CysC ratio were lower in the low muscle strength group than in the normal muscle strength group; there were more patients with ≥2 acute exacerbations within the past year in the low muscle strength group. The Cr/CysC ratio was correlated with handgrip strength, FEV1, FVC, FEV1%pred, BMI and weight. The area under curve for low handgrip strength was greater for the Cr/CysC ratio than for Cr. Binary logistic regression analysis showed that a Cr/CysC ratio <0.99 was a risk factor for decreased muscle strength in male patients with AECOPD. Conclusion: The Cr/CysC ratio is a useful predictor of muscle strength decline in male AECOPD patients, while a low Cr/CysC ratio is a risk factor for muscle strength decline in male patients with AECOPD.

Bifunctional antibody and copper-based metal-organic framework nanocomposites for colorimetric α-fetoprotein sensing.

Cu2+ are found to greatly reduce the photoinduced oxidase activity of fluorescein and then inhibit the chromogenic reaction catalyzed by fluorescein. A simple colorimetric assay for Cu2+ is established. Based on this, bifunctional nanocomposites of α-fetoprotein (AFP) antibody (Ab) and copper-based metal-organic framework (Ab2@Cu-MOF) are synthesized by the simple self-assembly of AFP Ab2, Cu2+, and 4,4'-dipyridyl: the binding site of AFP Ab2 exposed on the surface of the nanocomposites can specifically recognize AFP antigen; Cu2+ in nanocomposites can inhibit the visible light-induced activity of fluorescein. The structure of Ab2@Cu-MOF disintegrate and Cu2+ is released in an acetate buffer solution. The higher the amount of AFP antigens, the more significant the inhibitory effect. Thus, the Ab2@Cu-MOF immunoassay for AFP determination is established using 3,3',5,5'-tetramethylbenzidine as chromogenic substrate with a detection limit of 35 pg.mL-1. This simple, cheap, and sensitive method sheds substantial light on practical clinical diagnosis. Meanwhile, the mechanism of inhibition is revealed to facilitate the targeted selection of enzyme regulators. Graphical abstract Diagrammatic illustration of Cu2+ detection (part a) and Ab2@Cu-MOF immunoassay for sensing α-fetoprotein based on the synthesized Ab2@Cu-MOF nanocomposites (parts a and b).

A signal amplification strategy for prostate specific antigen detection via releasing oxidase-mimics from coordination nanoparticles by alkaline phosphatase.

A novel signal amplification method for prostate specific antigen (PSA) is developed by freeing fluorescein with photoinduced oxidase-like activity from coordination nanoparticles (CNPs) in the presence of alkaline phosphatase (ALP). CNPs loaded with fluorescein (F@CNPs) are obtained in aqueous solution by self-assembly using Tb3+ as metal ion, guanosine monophosphate (5'-GMP) as ligand, and fluorescein as signal molecule. The F@CNPs display outstanding properties of simple synthesis, low cost, good water solubility, negligible leakage and satisfactory load capacity. Fluorescein is quantitatively encapsulated in CNPs with a binding ratio of 92.72%. Meanwhile, ALP can specifically hydrolyze the phosphate group of 5'-GMP ligand, triggering the destruction of F@CNPs and leakage of fluorescein. Fluorescein, a photoinduced oxidase mimic, can catalyze the oxidation of non-fluorescent Amplex UltraRed (AUR) into fluorescent resorufin under LED lamp. This strategy exhibits good sensitivity for ALP detection. In addition, a new immunoassay for PSA is validated by labelling ALP on PSA antibody. The low detection limit of 0.04 ng mL-1 in detecting PSA is appropriate for PSA detection in real samples. Therefore, the work not only establishes a new strategy for ALP and PSA determination, but also provides a new conception for putting photoinduced oxidase-like fluorescein in practical application.

Myricetin inhibits TNF-α-induced inflammation in A549 cells via the SIRT1/NF-κB pathway.

BACKGROUND: Although myricetin exerts anti-inflammation, anti-cancer, and anti-oxidation effects, the relationship between myricetin and tumor necrosis factor alpha (TNF-α) -stimulated inflammation in A549 cells remains unclear. This study sought to assess whether myricetin has an anti-inflammatory effect on TNF-α-induced A549 cells and clarify the potential mechanisms. METHODS: Cell viability was examined with a Cell Counting Kit-8, and cytokine levels were determined by enzyme-linked immunosorbent assay and reverse transcription-quantitative PCR. Potential mechanisms were further explored by western blotting, immunofluorescence, and SIRT1 activity assays. RESULTS: In A549 cells, TNF-α stimulation upregulated the production of interleukin-6 (IL-6) and interleukin-8 (IL-8). Moreover, TNF-α activated the nuclear factor-κB (NF-κB) pathway, as confirmed by IκB-α degradation, and phosphorylation and nuclear migration of NF-κB p65. However, pretreatment with myricetin significantly attenuated the observed responses triggered by TNF-α. Mechanistically, myricetin strongly increased the deacetylase activity through decreasing phosphorylation, but not expression, of sirtuin-1 (SIRT1) in TNF-α-stimulated A549 cells. Myricetin-mediated SIRT1 activation was further evidenced by the decreased acetylation of NF-κB p65 and p53. Subsequently, all of these concurrent changes were reversed by the addition of salermide (SIRT1 inhibitor), illustrating the critical role of SIRT1 in mediation of anti-inflammatory processes by myricetin. CONCLUSIONS: Myricetin, an enhancer of SIRT1, inhibited TNF-α-induced NF-κB activation in A549 cells, therefore, reducing their inflammatory response. Our findings provide insight for novel therapies for inflammation-related diseases, such as asthma and chronic obstructive pulmonary disease.

New optical method for the determination of ß-galactosidase and α-fetoprotein based on oxidase-like activity of fluorescein.

Fluorescein has been found as an efficient visible-light-induced oxidase mimic and its catalytic performance is group-dependent. Herein, a facile colorimetric strategy for ß-galactosidase (ß-gal) was developed using fluorescein di ß-D-galactopyranoside (FDG) as a probe based on the analyte induced change in oxidase mimicking activity of fluorescein derivatives. FDG doesn't possess any visible-light-induced oxidase activity and can generate fluorescein and fluorescein mono ß-D-galactopyranoside (FMG) in the presence of ß-gal. The in situ generated fluorescein and FMG possess high oxidase-like activities under visible-light illumination and could catalyze the oxidation of 3, 3', 5, 5'-tetramethylbenzidine (TMB) upon short irradiation by light-emitting diode (LED) lamp. Thus, the ß-gal activity can be selectively detected in linear range from 0.10 to 12.9 µg mL-1 with a limit of detection (LOD) of 0.04 µg mL-1. We further integrated with the visual detection of α-fetoprotein antigen (AFP) based on the corresponding colorimetric signal induced by ß-gal-linked colorimetric immunoassay, a LOD of 0.08 ng mL-1 could be achieved. Significantly, our proposed assay provides a facile sensing platform based on the change in enzyme mimicking activity induced by analytes. In addition, this optical method works without complex synthesis procedure and efficiently avoids participation of unstable H2O2 as an oxidant. Therefore, the present work not only shows the excellent assay performance in ß-gal and tumor biomarker detection, but also opens up a new avenue for its application in practical optical sensing.

Competitive method for fluorescent dopamine detection in cerebrospinal fluid based on the peroxidase-like activity of ficin.

Dopamine (DA), a catecholamine neurotransmitter, is considered to be an important indicator for mental diseases detection in the clinic. In this study, a novel fluorescent sensing platform consisting of the ficin-H2O2-tyramine system for determining DA in cerebrospinal fluids (CSF) was established. The proposed method is based on the fact that ficin, a mimetic peroxidase, can catalyze H2O2 decomposition into OH radicals, which can oxidize non-fluorescent tyramine into fluorescent dityramine. When DA was introduced, DA can compete with tyramine for OH and resulting in the oxidation reaction of tyramine inhibited along with the fluorescence intensity of the system decreased, which provides a unique strategy for fluorescence detection of DA. Under optimal conditions, the fluorescence intensity decreased linearly with the DA level over a wide concentration range from 0.05 to 12.0 µM (R2 = 0.995) with a detection limit of 46 nM (3σ/k). More importantly, the proposed sensing approach exhibits high sensitivity, good selectivity and has been successfully applied to DA sensing in complex biological samples, which made it hold great potential for DA determination in chemical and biological analytical applications.

Umbelliferone as a Small Molecular Peroxidase Mimic towards Sensitive Detection of H2O2 and Glucose.

In this work, umbelliferone, a kind of coumarin derivative, was proved to exhibit peroxidase-like activity that could catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide to generate a blue-colored oxide (oxTMB). The catalytic mechanism is similar to that of native enzymes (e.g. horseradish peroxidase, HRP) and nanozymes, which follow the Michaelis-Menten kinetics behavior. Meanwhile, the 7-hydroxyl group of umbelliferone plays a significant role in the peroxidase-like activity. Compared with enzymes and nanozymes, this small molecular mimic enzyme possesses the advantages of low cost, simple molecular structures, small molecular weight and high stability against harsh conditions. Based on the favorable peroxidase mimetic activity of umbelliferone, a convenient, practical and sensitive H2O2 and glucose detection method was successfully established. This work not only opens some new inspirations into seeking for novel molecular enzyme mimetics with excellent catalytic activities, but also provides promising assays for clinical diagnosis.

Fluorescein as a Visible-Light-Induced Oxidase Mimic for Signal-Amplified Colorimetric Assay of Carboxylesterase by an Enzymatic Cascade Reaction.

We have found that fluorescein possesses high visible-light-induced oxidase mimetic activity and could transform colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB) without unstable and destructive H2 O2 under visible-light illumination. Instead, fluorescein uses oxygen as a mild and green electron acceptor, and its activity can be easily controlled by the switching "on/off" of visible light. In addition, the visible-light-induced catalytic mechanism was elucidated in detail and, as the main reactive species h+ and O2.- accounted for TMB oxidation. Based on the fact that fluorescein diacetate (FDA) possessed no activity and generated active fluorescein in situ in the presence of carboxylesterase (CaE), a signal-amplified sensing platform through a cascade reaction for CaE detection was constructed. Our proposed sensing system displayed excellent analytical performance for the detection of CaE in a wide linear range from 0.040 to 20 U L-1 with a low detection limit of 0.013 U L-1 . This work not only changes the conventional concept that fluorescein is generally considered to be photocatalytically inert, but also provides a novel sensing strategy by tailoring the enzyme mimetic activity of fluorescein derivatives with analyte.

Peroxidase-like activity of 2',7'-difluorofluorescein and its application for galactose detection.

The peroxidase-like activity of 2',7'-difluorofluorescein (DFF), was investigated using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate in the presence of H2O2. DFF could catalyze oxidization of TMB by H2O2 to produce a blue colored oxidation product. Effect of various reaction conditions, such as pH, temperature, H2O2 concentration and reaction time on the catalytic activity of DFF was studied. The peroxidase-like activity of DFF was found to follow Michaelis-Menten kinetics, and its catalysis accorded with ping-pong mechanism. The calculated kinetic parameters (Kcat) of DFF catalysis showed higher peroxidase-like activity than fluorescein and 2',7'-dichlorofluorescein (DCF). According to the radical capture and electron spin resonance (ESR) spectroscopy results, we confirmed that hydroxyl radical (•OH) is the active specie of catalytic process. It is known that the oxidation of galactose by galactose oxidase (GAOx) enzyme leads to the formation of H2O2, the H2O2 released in this reaction was consequently quantified using DFF as peroxides mimics and TMB as the chromogen. Thus, a combination of above two reactions was exploited to establish a method for galactose detection. Under the optimum conditions, the linear range of this method was from 10 µM to 20 mM with the detection limit down to 3 µM. Moreover, the developed method was applied to detect galactose in urine samples. Our work will facilitate the utilization of DFF intrinsic peroxidase-like activity in medical diagnostics and biotechnology.