Analytica Chimica Acta (v.723, #C)

Fabrication process of biofunctionalized nanoparticles and principle of the performed bioassay. First, aptamer 1 and aptamer 2 were immobilized onto the surface of MNPs and the surface of NaY0.78F4:Yb0.2, Tm0.02 UCNPs, and NaY0.28F4:Yb0.70, Er0.02 UCNPs. Next, S. Typhimurium and S. aureus were added and, due to the highly affinity of aptamer to corresponding bacteria, the aptamer 1-MNPs-S. Typhimurium complex bind NaY0.78F4:Yb0.2, Tm0.02 UCNPs modified aptamer 1, and the aptamer 2-MNPs-S. aureus bind NaY0.28F4:Yb0.70, Er0.02 UCNPs modified aptamer 2. Finally, the luminescent signal was effectively amplified with the help of a magnetic field.Display Omitted► Simultaneous detection of two kind of bacteria. ► Bacteria-specific aptamer recognition. ► Dual-color upconversion luminescent nanoparticles labeling. ► Aptamer conjugated-magnetic nanoparticles-based separation and concentration.A sensitive luminescent bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus was developed using aptamer-conjugated magnetic nanoparticles (MNPs) for both recognition and concentration elements and using upconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The bioassay system was fabricated by immobilizing aptamer 1 and aptamer 2 onto the surface of MNPs, which were employed to capture and concentrate S. Typhimurium and S. aureus. NaY0.78F4:Yb0.2,Tm0.02 UCNPs modified aptamer 1 and NaY0.28F4:Yb0.70,Er0.02 UCNPs modified aptamer 2 further were bond onto the captured bacteria surface to form sandwich-type complexes. Under optimal conditions, the correlation between the concentration of S. Typhimurium and the luminescent signal was found to be linear within the range of 101–105  cfu mL−1 (R 2  = 0.9964), and the signal was in the range of 101–105  cfu mL−1 (R 2  = 0.9936) for S. aureus. The limits of detection of the developed method were found to be 5 and 8 cfu mL−1 for S. Typhimurium and S. aureus, respectively. The ability of the bioassay to detect S. Typhimurium and S. aureus in real water samples was also investigated, and the results were compared to the experimental results from the plate-counting methods. Improved by the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, and the different emission lines of Yb/Er- and Yb/Tm-doped NaYF4 UCNPs excited by a 980 nm laser, the present method performs with both high sensitivity and selectivity for the two different types of bacteria.
Keywords: Aptamer; Salmonella Typhimurium; Staphylococcus aureus; Magnetic nanoparticles; Upconversion nanoparticles;

Three replicate injections of a localized region of a LC × LC–DAD data set.Display Omitted► We describe a spectrally based alignment method for LC × LC–DAD data. ► Alignment between injections is not dependent on selection of a reference injection. ► The alignment process allows for non-linear peak shifting between injections. ► Simulated data sets aligned by this method produced % recoveries close to 100%. ► The method was able to align and quantify experimental data with overlapping peaks.Parallel factor analysis was used to quantify the relative concentrations of peaks within four-way comprehensive two dimensional liquid chromatography–diode array detector data sets. Since parallel factor analysis requires that the retention times of peaks between each injection are reproducible, a semi-automated alignment method was developed that utilizes the spectra of the compounds to independently align the peaks without the need for a reference injection. Peak alignment is achieved by shifting the optimized chromatographic component profiles from a three-way parallel factor analysis model applied to each injection. To ensure accurate shifting, components are matched up based on their spectral signature and the position of the peak in both chromatographic dimensions. The degree of shift, for each peak, is determined by calculating the distance between the median data point of the respective dimension (in either the second or first chromatographic dimension) and the maximum data point of the peak furthest from the median. All peaks that were matched to this peak are then aligned to this common retention data point. Target analyte recoveries for four simulated data sets were within 2% of 100% recovery in all cases. Two different experimental data sets were also evaluated. Precision of quantification of two spectrally similar and partially coeluting peaks present in urine was as good as or better than 4%. Good results were also obtained for a challenging analysis of phenytoin in waste water effluent, where the results of the semi-automated alignment method agreed with the reference LC–LC MS/MS method within the precision of the methods.
Keywords: Comprehensive two-dimensional liquid chromatography; Diode array detector; Four-way data; Chromatographic alignment; Parallel factor analysis;

Tucker core consistency for validation of restricted Tucker3 models by Mohsen Kompany-Zareh; Yousef Akhlaghi; Rasmus Bro (18-26).
.Display Omitted► Tucker core consistency (TuckCorCon) parameter is presented for the first time. ► TuckCorCon can be used to evaluate the Tucker3 models with simplified cores. ► TuckCorCon is close to 100 for a good simple core and it is low for a bad one. ► TuckCorCon was tested and approved using simulated and experimental data sets. ► The TuckCorCon parameter helps to decide about the proper size of the core.In Tucker3 analysis of three-way data array obtained from a chemical or biological system, it is sometimes possible to use a priori knowledge about the system to specify what is called a restricted Tucker3 model. Often, the restricted Tucker3 model is characterized by having some elements of the core forced to zero. As a simple example, an F-component PARAFAC model can be seen as a restricted (F, F, F) Tucker3 model in which only superdiagonal elements of the core are allowed to be nonzero. The core consistency diagnostic was previously introduced by Bro and Kiers for determining the proper number of components in PARAFAC analysis. In the current study, this diagnostic is extended to other restricted Tucker3 models to validate the appropriateness of the applied constraints. The new diagnostic is named Tucker core consistency (TuckCorCon). When the dimensionality and the pattern of the restricted core is valid, the simple core of restricted Tucker3 model and a corresponding unrestricted core will be similar and in this case the TuckCorCon will be close to maximum (100%). A simulated chemical equilibrium data set and two experimental data sets were used to evaluate the applicability of the TuckCorCon to decide about the appropriateness of dimensionality and pattern of the core nonzero elements in the restricted Tucker3 models.
Keywords: Three-way data; Restricted Tucker3; Tucker core consistency; Rank deficiency;

Electrochemical detection of a powerful estrogenic endocrine disruptor: Ethinylestradiol in water samples through bioseparation procedure by Noelia A. Martínez; Sirley V. Pereira; Franco A. Bertolino; Rudolf J. Schneider; Germán A. Messina; Julio Raba (27-32).
Display Omitted► We developed an electrochemical method for the determination of ethinylestradiol in water samples. ► Modified magnetic particles were employed for the bioseparation and preconcentration procedures. ► The use of magnetic particles offers high selectivity due to antigen–antibody binding specificity. ► The detection was carried out by using a glassy carbon electrode modified with carbon nanotubes.The synthetic estrogen ethinylestradiol (EE2) is an active component of oral contraceptives (OCs), considered as an endocrine disrupting compound (EDC). It is excreted from humans and released via sewage treatment plant effluents into aquatic environments. EDCs are any environmental pollutant chemical that, once incorporated into an organism, affects the hormonal balance of various species including humans. Its presence in the environment is becoming of great importance in water quality. This paper describes the development of an accurate, sensitive and selective method for capture, preconcentration and determination of EE2 present in water samples using: magnetic particles (MPs) as bioaffinity support for the capture and preconcentration of EE2 and a glassy carbon electrode modified with multi-walled carbon nanotubes (MWCNTs/GCE) as detection system. The capture procedure was based on the principle of immunoaffinity, the EE2 being extracted from the sample using the anti-EE2 antibodies (anti-EE2 Ab) which were previously immobilized on MPs. Subsequently the analyte desorption was done employing a sulfuric acid solution and the determination of the EE2 in the pre-concentrated solution was carried out by square wave voltammetry (SWV).This method can be used to determine EE2 in the range of 0.035–70 ng L−1 with a detection limit (LOD) of 0.01 ng L−1 and R.S.D. < 4.20%. The proposed method has been successfully applied to the determination of EE2 in water samples and it has promising analytical applications for the direct determination of EE2 at trace levels.
Keywords: Magnetic particles; Bioseparation; Ethinylestradiol; Electrochemistry;

Display Omitted► HS-β-CD/AuNPs/hollow nitrogen-doped carbon microspheres hybrids were synthesized. ► A new electrochemical sensor for naphthols was developed based on the prepared hybrids. ► The sensor shows good analytical performance for simultaneous detection of naphthols.Due to awfully harmful to the environment and human health, the qualitative and quantitative determinations of naphthols [1-naphthol (1-NAP) and 2-naphthol (2-NAP)] are of great significance and receive great attention. In this paper, gold nanoparticles (AuNPs)/hollow nitrogen-doped carbon microspheres (HNCMS) hybrids (AuNPs/HNCMS) were prepared and functionalized with thiolated-β-cyclodextrin (HS-β-CD) for the first time, and then applied successfully in sensitive and simultaneous electrochemical detection of naphthols. The results show that the oxidation peak currents of naphthols obtained on the HS-β-CD/AuNPs/HNCMS modified glassy carbon (GC) electrode are much higher than that on the AuNPs/HNCMS/GC, HNCMS/GC and bare GC electrodes. Additionally, compared with other electrochemical sensors developed previously, the proposed electrode results in improved detection limits of about four times for 1-NAP (1.0 nM) and two orders of magnitude for 2-NAP (1.2 nM). The linear response ranges of both 1-NAP and 2-NAP are 2–150 nM.
Keywords: Electrochemical sensors; Naphthol; Hollow nitrogen-doped carbon microspheres; Cyclodextrin; Gold nanoparticles; Environmental analysis;

Display Omitted► Electrospinning CuO microfibers onto electrodes as glucose sensors were studied. ► The CuO microfibers were composed of CuO particles. ► Size of CuO particles relies on percentage content of copper nitrate in precursor. ► Performance of the sensor depends on size of CuO particles.Fluorine tin oxide (FTO) electrode modified by copper oxide microfibers (CuO-MFs) composed of numerous interconnected CuO nanoparticles (CuO-NPs) for nonenzymatic glucose sensor was prepared by electrospinning precursor containing high percentage content of copper nitrate with subsequent calcination. The results of scanning electron microscope (SEM) showed the size of CuO particles composing CuO-MFs depended on the percentage content of copper nitrate in precursor solution. With increasing the percentage content of copper nitrate, the interconnected CuO-NPs would gradually replace the large-size CuO particles to accumulate the CuO-MFs, which have the potential to provide larger surface area and more reaction sites for electrocatalytic activity toward glucose. As a glucose sensor, the CuO-MFs modified FTO electrode prepared by 40 wt.% of copper nitrate exhibited a high sensitivity of 2321 μA mM−1  cm−2 with a low detection limit of 2.2 nM (signal/noise ratio (S/N) = 3). Additionally, the application of the CuO-MFs modified FTO electrode as a glucose sensor for biological samples was demonstrated with satisfactory results.
Keywords: Copper oxide microfibers; Copper oxide nanoparticles; Electrospinning; Nonenzymatic; Glucose sensor;

Display Omitted► The preparation of thermo-sensitive molecular imprinted hydrogel (MIH) for the template BSA. ► Choosing 2-acrylamido-2-methyl-propanosulfonic acid (AMPS), N-isopropylacrylamide (NIPAm) and acrylamide (AAm) as the monomers. ► Temperature, functional monomer and cross-linking degree were optimized respectively. ► The MIH were characterized by FT-IR and SEM, and investigated by different adsorption experiments. ► The selectivity of the MIH was verified by direct adsorption of single reference protein, protein mixture and real sample.A novel temperature-sensitive molecular imprinted hydrogel composed of 2-acrylamido-2-methyl-propanosulfonic acid (AMPS), N-isopropylacrylamide (NIPAm) and acrylamide (AAm) has been prepared by free-radical cross-linking copolymerization in aqueous solution under two different temperatures (25 °C and −20 °C). Bovine serum albumin (BSA, pI 4.9, MW 66.0 kDa) is used as the template protein. The influence of the external temperature stimuli on the affinity of the hydrogels was investigated, and the optimal binding conditions were tested. The adsorption capacity (Q max) and association constant (K) for the specific interaction between the hydrogel and the template protein were determined by Langmuir isotherm plots. Several types of reference protein, which are different in molecular weights and isoelectric points were chosen to investigate the selectivity of the hydrogels. It was shown that the shape memory and the charge effect were the major factors for the recognition. This imprinted hydrogel was used to specifically adsorb the BSA from the protein mixture and real sample, which demonstrated its potential selectivity.
Keywords: Molecular imprinting; Thermosenstive hydrogel; Bovine serum albumin (BSA); Recognition; Protein mixture;

Ion imprinted 3-mercaptopropyltrimethoxysilane (MPTS) coated stir bar for selective extraction of trace Cd(II).Display Omitted► Ion imprinted polymers were proposed as the coating for SBSE for the first time. ► Cd(II) imprinted MPTS-silica coating was prepared by a double-imprinting concept. ► A novel method of SBSE–ICP-MS was developed for the determination of Cd in waters. ► This method is rapid, selective, sensitive and applicable for determining trace Cd(II) in waters.Cd(II) imprinted 3-mercaptopropyltrimethoxysilane (MPTS)-silica coated stir bar was prepared by sol–gel technique combining with a double-imprinting concept for the first time and was employed for stir bar sorptive extraction (SBSE) of trace Cd(II) from water samples followed by inductively coupled plasma mass spectrometry (ICP-MS) detection. A tetramethoxysilane (TMOS) coating was first in situ created on the glass bar surface. Afterward, a sol solution containing MPTS as the functional precursor, ethanol as the solvent and both Cd(II) and surfactant micelles (cetyltrimethylammonium bromide, CTAB) as the template was again coated on the TMOS bar. The structures of the stir bar coating were characterized by FT-IR spectroscopy. Round-bottom vial was used for the extraction of Cd(II) by SBSE to avoid abrasion of stir bar coatings. The factors affecting the extraction of Cd(II) by SBSE such as pH, stirring rate and time, sample/elution volume and interfering ions have been investigated in detail, and the optimized experimental parameters were obtained. Under the optimized conditions, the adsorption capacities of non-imprinted and imprinted coating stir bars were found to be 0.5 μg and 0.8 μg bar−1. The detection limit (3σ) based on three times standard deviations of the method blanks by 7 replicates was 4.40 ng L−1 and the relative standard deviation (RSD) was 3.38% (c  = 1 μg L−1, n  = 7). The proposed method was successfully applied for the analysis of trace Cd(II) in rain water, East Lake and Yangtze River water. To validate the proposed method, certified reference material of GSBZ 50009-88 environmental water was analyzed and the determined value is in a good agreement with the certified value. The developed method is rapid, selective, sensitive and applicable for the analysis of trace Cd(II) in environmental water samples.
Keywords: Stir bar sorptive extraction; Cadmium (II) imprinted 3-mercaptopropyltrimethoxysilane coating; Double-imprinting; Sol–gel technique; Environmental water sample; Inductively coupled plasma mass spectrometry;

Display Omitted► We developed a simple method for the preparation of salivary microvesicles. ► GeLC–MS/MS was used for the proteomic analysis of salivary microvesicles. ► There are 63 proteins were identified and 35 are unique to salivary microvesicles.Microvesicles (MVs) have been shown to affect the physiology of neighboring recipient cells in various ways. They play an important role in tumor progression/metastasis and angiogenesis in cancer and may be useful therapeutic tools, as well as a mechanism of cell-to-cell communication. They have been visioned as an important biomarker or biomarker source for the detection of different diseases. Human saliva is a biological fluid with enormous diagnostic potential, which harbors plenty of salivary MVs. The goal of this study is to investigate the proteomic profiling of MVs in human saliva through a simple preparation procedure by using filtration and centrifugation. Gel electrophoresis was combined with LC–MS/MS (liquid chromatography–mass spectrometry) for the proteomic analysis of MVs. After SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) protein separation, the whole lane was cut into 25 bands, and each band was subjected to in-gel trypsin digestion. The peptides extracted from each band were loaded to LC–MS/MS for protein identification. Through protein database search, 63 proteins were identified for human salivary MVs. Several members of different protein families were identified, including annexin, keratin, actin, immunoglobulin and S100. This study showed that although there was an overlap with the proteins from human saliva and salivary exosomes, salivary MVs contained their own unique proteins. These results will poise human salivary MVs as a non-invasive tool for the early detection of different diseases.
Keywords: Human saliva; Microvesicles; Proteomics; Gel electrophoresis with liquid chromatography-mass spectrometry; Diagnostics;

Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry by Uma K. Aryal; Chiann-Tso Lin; Jong-Seo Kim; Tyler H. Heibeck; Jun Wang; Wei-Jun Qian; Yuehe Lin (68-75).
Display Omitted► Immunoaffinity purification and LC–MS for detecting phosphorylated BChE in plasma. ► Detection in plasma achieved by both MS/MS and accurate mass measurements. ► A potential general approach for the detection of modified BChE in human plasma.Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we developed an immunoaffinity purification and liquid chromatography–mass spectrometry (LC–MS) strategy for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChE polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. Using a purified BChE protein, we initially identified the exact phosphorylation site on the serine residue (S198) with a 108 Da modification by both MS/MS and accurately measured parent ion masses and quantified the extent of phosphorylation on S198 following paraoxon treatment to be >99.9%. Then, the phosphorylated BChE peptide in paraoxon-treated human plasma following immunoaffinity purification was successfully identified based on the accurate measured mass and retention time information initially obtained from the purified BChE protein. Thus, immunoaffinity purification combined with LC–MS represents a viable approach for the detection and quantification of phosphorylated BChE as an exposure biomarker of organophosphates and nerve agents.
Keywords: Butyrylcholinesterase; Organophosphates; Paraoxon; Mass spectrometry; Immunoaffinity purification;

In this paper, [AlQ]3+ had good selectivity with the hole of PVA-C-I and could react with the fluorescein isothiocyanate anion on the outside of hole by electrostatic interaction to form ion-association complex [AlQ]3+·[FITC] 3. Thus, a new coupling technique for the determination of trace Q based on solid substrate room temperature phosphorimetry and poly (vinyl alcohol) complex imprinting was established.Display Omitted► The phosphorimetry-poly (vinyl alcohol)-complex imprinting coupling technique (PVA-C-I-RTP) was exploited. ► The phosphorimetry with the technology of PVA-C-I for the determination of trace quercetin was established. ► This technology could identify quercetin, which could be used to separate, enrich and determine quercetin. ► The mechanism for the determination of quercetin using PVA-C-I-RTP was also discussed. ► This work promoted the research progress and the interpenetration between complex imprinting technique and SSRTP.Al3+ could react with quercetin (Q) to form [AlQ]3+ complex which could be used as a template for the preparation of poly (vinyl alcohol)–[AlQ]3+ complex imprinting (PVA-C-I). The [AlQ]3+ not only had good matching ability and selectivity with the cavity of PVA-C-I, but also could react with the fluorescein isothiocyanate anion (FITC) on the outside of cavity by electrostatic interaction to form ion-association complex [AlQ]3+·[(FITC)]3. The ion-association complex could emit strong and stable room temperature phosphorescence (RTP) on polyamide membrane (PAM) and the ΔI p of the system had linear relationship with the content of Q, showing the highly selective identification of PVA-C-I to Q. Thus, a new coupling technique for the determination of trace Q based on solid substrate room temperature phosphorimetry and poly (vinyl alcohol) complex imprinting (PVA-C-I-SSRTP) was established. The linear range and limit of detection (LOD) of this method were 0.010–2.0 (×10−12  g mL−1) and 2.0 × 10−14  g mL−1, respectively, showing wide linear range and high sensitivity of PVA-C-I-SSRTP. This method was used to determine the content of Q in waste water, and the results are consistent with those by spectrofluorimetry. Meanwhile, the mechanism for the determination of Q using PVA-C-I-SSRTP was also discussed.
Keywords: Quercetin; Aluminum; Poly (vinyl alcohol)–[Al quercetin]3+complex; Solid substrate room temperature phosphorimetry-complex imprinting coupling technique;

Display Omitted► The quantum dots-ssDNA probe was designed for the determination of virus DNA. ► The fluorescence of quantum dots was effectively quenched by carbon nanotubes. ► The addition of target H5N1 DNA restored the quenched fluorescence of quantum dots. ► The proposed method exhibited high sensitivity and good selectivity for H5N1 DNA.In this paper, a simple and sensitive approach for H5N1 DNA detection was described based on the fluorescence resonance energy transfer (FRET) from quantum dots (QDs) to carbon nanotubes (CNTs) in a QDs-ssDNA/oxCNTs system, in which the QDs (CdTe) modified with ssDNA were used as donors. In the initial stage, with the strong interaction between ssDNA and oxCNTs, QDs fluorescence was effectively quenched. Upon the recognition of the target, the effective competitive bindings of it to QDs-ssDNA occurred, which decreased the interactions between the QDs-ssDNA and oxCNTs, leading to the recovery of the QDs fluorescence. The recovered fluorescence of QDs was linearly proportional to the concentration of the target in the range of 0.01–20 μM with a detection limit of 9.39 nM. Moreover, even a single-base mismatched target with the same concentration of target DNA can only recover a limited low fluorescence of QDs, illustrating the good anti-interference performance of this QDs-ssDNA/oxCNTs system. This FRET platform in the QDs-ssDNA/oxCNTs system was facilitated to the simple, sensitive and quantitative detection of virus nucleic acids and could have a wide range of applications in molecular diagnosis.
Keywords: Influenza A virus; Fluorescence resonance energy transfer; Quantum dots; Carbon nanotubes;

A colorimetric and surface-enhanced Raman scattering (SERS) dual-signal sensor for Hg2+ with high sensitivity and selectivity has been developed based on Hg2+-induced anti-aggregation of Bismuthiol II–gold nanoparticles (AuNPs).Display Omitted► A dual-signal sensor for Hg2+ was developed by mixing Bismuthiol II and AuNPs. ► Colorimetric sensing was achieved based on the Hg2+-inhibited aggregation of AuNPs. ► Hot spots were formed with the aggregation of AuNPs, allowing SERS detection. ► The method showed high sensitivity and selectivity.The addition of Bismuthiol II to the gold nanoparticles (AuNPs) solution led to the aggregation of AuNPs with a color change from red to blue. As a result, hot spots were formed and strong surface-enhanced Raman scattering (SERS) signal of Bismuthiol II was observed. However, the Bismuthiol II-induced aggregation of AuNPs could be reversed by Hg2+ in the system, accompanied by a remarkable color change from blue to red. As evidenced by UV–vis and SERS spectroscopy, the variation in absorption band and SERS intensity was strongly dependent on the concentration of Hg2+, suggesting a colorimetric and SERS dual-signal sensor for Hg2+. The sensor had a high sensitivity, low detection limits of 2 nM and 30 nM could be achieved by UV–vis spectroscopy and by SERS spectroscopy, respectively. Other environmentally relevant metal ions did not interfere with the detection of Hg2+. The method was successfully applied to detect Hg2+ in water samples. It was simple, rapid and cost-effective without any modifying or labeling procedure.
Keywords: Bismuthiol II; Gold; Colorimetry; Surface-enhanced Raman scattering; Mercury; Sensor;

Display Omitted► A 3D ordered macroporous self-doped polyaniline/Prussian blue film was prepared. ► In the film, PB revealed good stability in neutral and weak alkalescent solution. ► A novel glucose biosensor was fabricated based on the 3DOM bicomponent film. ► The biosensor showed a linear range of 2–1600 μM and a detection limit of 0.4 μM. ► The biosensor can detect the blood sugar in real samples without any pretreatment.In this paper, a three dimensional ordered macroporous self-doped polyaniline/Prussian blue (3DOM SPAN/PB) bicomponent film was fabricated via the inverted crystal template technique using step-by-step electrodeposition. In this bicomponent film, PB not only acted as a redox mediator, but also presented increased stability in neutral or weak alkaline solution by the protection of SPAN layer on the top. A novel glucose biosensor was fabricated based on the large active surface area and excellent conductivity possessed by the 3DOM SPAN/PB film. The applying experimental conditions of the glucose biosensor have been optimized. Under the optimal conditions, the biosensor showed a wide linear range over three orders of magnitude in glucose concentrations (from 2 to 1600 μM) and a low detection limit of 0.4 μM. Moreover, the biosensor exhibited short response time, high selectivity and excellent operation stability, which can be applied to detect the blood sugar in real samples without any pretreatment.
Keywords: Three dimensional ordered macroporous; Self-doped polyaniline; Prussian blue; Glucose oxidase; Biosensor;

Display Omitted► N,N′-diBoc-dityrosine (DBDY) was conjugated with two isoniazid (INH) molecules. ► Due to the quenching effect of INH, DBDY–(INH)2 lost the fluorescence of DBDY. ► Only papain and chymopapain catalyzed the hydrolysis of DBDY–(INH)2. ► DBDY–(INH)2 can be used as a selective and sensitive assay of papain and chymopapain. N,N′-diBoc-dityrosine (DBDY), which was synthesized by the oxidative C–C coupling of 2 N-Boc-l-tyrosine molecules, was conjugated with two isoniazid (INH) molecules. Due to the quenching effect of INH, DBDY–(INH)2 lacks the fluorescence of DBDY. As such, it was tested for use in the detection of proteases by measuring fluorescence recovery. In this study, serine proteases (chymotrypsin, trypsin, subtilisin, and proteinase K), metalloproteases (thermolysin and carboxypeptidase A, dispase, and collagenase), aspartic proteases (pepsin and aspergillopepsin) and cysteine proteases (papain and chymopapain) were chosen. Reported optimum assay conditions were chosen for each enzyme. Only papain and chymopapain catalyzed the hydrolysis of DBDY–(INH)2 and led to fluorescence recovery, possibly due to their extensive binding sites and the INH-mediated inhibition of metalloproteases and aspartic proteases.
Keywords: Dityrosine; Fluorescence; Protease assay; Papain; Chymopapain;