A Solvent-Mediated Excited-State Intermolecular Proton Transfer Fluorescent Probe for Fe3+ Sensing and Cell Imaging.

Constructing excited-state intermolecular proton transfer (ESIPT-e) fluorophores represents significant challenges due to the harsh requirement of bearing a proton donor-acceptor (D-A) system and their matching proton donating-accepting ability in the same molecule. Herein, we synthesized a new-type ESIPT-e fluorophor (2-APC) using the "four-component one-pot" reaction. By the installing of a cyano-group on pyridine scaffold, the proton donating ability of -NH2 was greatly enhanced, enabling 2-APC to undergo ESIPT-e process. Surprisingly, 2-APC exhibited dual-emissions in protic solvents ethanol and normal fluorescence in aprotic solvents, which is vastly different from that of conventional ESIPT-a dyes. The ESIPT emission can be obviously suppressed by Fe3+ due to the coordination reaction of Fe3+ with the A-D system in 2-APC. From this basis, a highly sensitive and selective method was established using 2-APC as a fluorescent probe, which offers the sensitive detection of Fe3+ ranging from 0 to 13 µM with the detection limit of 7.5 nM. The recovery study of spiked Fe3+ measured by the probe showed satisfactory results (97.2103.4%) with the reasonable RSD ranging from 3.1 to 3.8%. Moreover, 2-APC can also exhibit aggregation-induced effect in poor solvent or solid-state, eliciting strong red fluorescence. 2-APC was also applied to cell-imaging, exhibiting good cell-permeability, biocompatibility and color rendering. This multi-mode emission of 2-APC is significant departure from that of conventional extended p-conjugated systems and ESIPT dyes based on a flat and rigid molecular design. The "one-pot synthesis" strategy for the construction of ESIPT molecules pioneered a new route to achieve tricolor-emissive fluorophores.

Partnered Excited-State Intermolecular Proton Transfer Fluorescence (P-ESIPT) Signaling for Nitrate Sensing and High-Resolution Cell-Imaging.

Nitrite (NO2-) is a common pollutant and is widely present in the environment and in human bodies. The development of a rapid and accurate method for NO2- detection is always a very important task. Herein, we synthesized a partnered excited-state intermolecular proton transfer (ESIPT) fluorophore using the "multi-component one pot" method, and used this as a probe (ESIPT-F) for sensing NO2-. ESIPT-F exhibited bimodal emission in different solvents because of the solvent-mediated ESIPT reaction. The addition of NO2- caused an obvious change in colors and tautomeric fluorescence due to the graft of NO2- into the ESIPT-F molecules. From this basis, highly sensitive and selective analysis of NO2- was developed using tautomeric emission signaling, achieving sensitive detection of NO2- in the concentration range of 0~45 mM with a detection limit of 12.5 nM. More importantly, ESIPT-F showed the ability to anchor proteins and resulted in a recognition-driven "on-off" ESIPT process, enabling it to become a powerful tool for fluorescence imaging of proteins or protein-based subcellular organelles. MTT experimental results revealed that ESIPT-F is low cytotoxic and has good membrane permeability to cells. Thus, ESIPT-F was further employed to image the tunneling nanotube in vitro HEC-1A cells, displaying high-resolution performance.

A solvent-assisted ESIPT fluorescent dye for F-/Ag+ sensing and high-resolution imaging of the cilia in live cells.

A solvent-assisted ESIPT fluorescent dye was synthesized and used as a probe (2-PPN) for the detection of F-/Ag+ and high-resolution imaging of the cilia in live cells. The developed ESIPT fluorophore exhibited strong tautomeric fluorescence in protic solvents and normal emission in aprotic solvents, which is a significant departure from that of conventional intramolecular ESIPT compounds. The H-binding interaction of F- and the chelation of Ag+ with the ESIPT module of 2-PPN resulted in significant tautomeric emission quenching. From this basis, the 2-PPN-based assays for the detection of F- and Ag+ were established. The detection limit for F- and Ag+ sensing is 2.4 nM and 1.5 nM, respectively. The selective experimental results showed that no tautomeric fluorescence change of 2-PPN could be observed in the presence of the other inorganic ions in the same medium, revealing high selectivity of 2-PPN to F- and Ag+. Furthermore, MTT assay experiments proved that the probe 2-PPN exhibited low cytotoxicity and good cell membrane permeability. The probe was also further successfully utilized to image the cilia in vitro MCF7 cells, displaying its high-resolution imaging performance.Graphical abstract.

Highly cysteine-selective fluorescent nanoprobes based on ultrabright and directly synthesized carbon quantum dots.

Strongly green fluorescent carbon dots (CQDs) have been directly synthesized from 2,4-diaminophenylhydrazine and 2-hydroxy-5-methylisophthalaldehyde through a facile solvothermal method. The novel CQDs exhibit high fluorescence quantum yield and excellent water solubility due to the abundant amino and hydroxy groups on their surface. The use of the as-prepared CQDs combined with Cu2+ constructed a "turn-on" switch cysteine-responsive nanoprobe. In the CQDs-Cu2+ assemblies, the binding of Cu2+ to CQDs results in the fluorescence quenching of CQDs by electron transfer mechanism, while the addition of cysteine leads to the fluorescence recovery because of the competitive binding between cysteine and CQDs to Cu2+. The nanoprobes showed high sensitivity to cysteine with the detection limit of 2.6 nmol L-1. The selectivity investigation results demonstrated that the Cu2+-integrated nanoparticles were highly selective toward cysteine over the other amino acids and biologically related metal ions. The proposed nanoprobe was then employed for detecting the recovery of cysteine in rabbit serum and plasma samples and imaging the cysteine in cancer cells, and the recovery was found to be 98.2-104.0%. This "synthesis-modification integration" strategy for the fabrication of CQDs may offer a new sight for the preparation of multifunctional nanostructures and broadening the application of CQDs in bioimaging. Graphical abstract Fluorescent carbon dots (CQDs) were directly synthesized from 2,4-diaminophenylhydrazine and 2-hydroxy-5-methylisophthalaldehyde. CQDs exhibit high fluorescence quantum yield and excellent water solubility due to the abundant amino and hydroxy groups on their surface. The use of CQDs combined with Cu2+ constructed a cysteine-responsive nanoprobe, which showed high sensitivity to cysteine with the detection limit of 2.6 nM.

An acid-free sensing strategy for detecting nitrite using dihydroquinoline-8-carboxylate as a probe.

Nitrite (NO2-) has been identified as a typical pollutant harmful to the human body and heavily assayed in the fields of food safety and water quality control. The mainstream sensing strategies for detecting NO2- depend on Griess reaction or its improved methods which employ Griess reaction to initiate further inter-or intramolecular interaction to generate readout signals. However, a significant drawback of these methods is the use of strongly acidic media. In this study, we designed and synthesized a new NO2--specific fluorescent probe (ethyl 3-cyano-2-hydroxy-5-imino-8-(3-methoxy-3-oxopropyl)-4-(pyridin-2-yl)-5,8-dihydroquinoline-8-carboxylate, DHQC). DHQC exhibited strong green fluorescence in an acetonitrile-PBS (10 mM) mixed system (pH 7.0). In the neutral medium and at room temperature, the fluorescence of DHQC changed from green to blue with the addition of NO2-. The preliminary mechanistic investigation reveals that NO2- can induce the decarboxylation of the probe DHQC. Based on this finding, a high sensitive and selective method for NO2--detection was established, which showed good linearity in a range of 5∼50 µM with a limit detection of 3.5 nM (3σ). Given the unique properties of DHQC, a DHQC-loaded hydrogel bead device was further developed and employed for rapid monitoring of NO2-, exhibiting the advantages of simple preparation, high sensitivity, and fast response compared with traditional sensing reagents. In addition, DHQC was also used as a fluorescent probe for cell-imaging in live cells, exhibiting good cell permeability and biocompatibility. This study proposes a potential strategy for constructing smart fluorimetric probes used for NO2- detection.

Structural basis of molecular recognition between ESCRT-III-like protein Vps60 and AAA-ATPase regulator Vta1 in the multivesicular body pathway.

The AAA-ATPase Vps4 is critical for function of the multivesicular body sorting pathway, which impacts cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. Vps4 activity is stimulated by the interaction between Vta1 and Vps60, but the structural basis for this interaction is unclear. The fragment Vps60(128-186) was reported to display the full activity of Vps60. Vta1 interacts with Vps60 using its N-terminal domain (Vta1NTD). In this work, the structure of Vps60(128-186) in complex with Vta1NTD was determined using NMR techniques, demonstrating a novel recognition mode of the microtubule-interacting and transport (MIT) domain in which Vps60(128-186) interacts with Vta1NTD through helices α4' and α5', extending over Vta1NTD MIT2 domain helices 1-3. The Vps60 binding does not result in Vta1 conformational changes, further revealing the fact that Vps4 ATPase is enhanced by the interaction between Vta1 and Vps60 in an unanticipated manner.

A polystyrene-based ESIPT fluorescent polymeric probe for highly sensitive detection of chromium(vi) ions and protein staining.

A "two-step" preparation method of an excited-state intermolecular proton transfer (ESIPT) fluorescent polymer (f-PP) is reported here. The synthesis of f-PP involves the acetylation of polystyrene and a "multicomponent one pot" reaction. The as-prepared polymer bears a group of ESIPT fluorescent units, enabling it to exhibit high brightness, moderate solubility and ESIPT fluorescence. F-PP gives off tautomeric bright green fluorescence under UV-tamp and the dual-emission could be specifically suppressed by Cr(vi). This phenomenon cannot be elicited by other competing species. On this basis, an ESIPT polymeric probe-based method for the determination of Cr(vi) was developed, offering high sensitivity (19.5 nM) and selectivity. The f-PP was successfully used to detect Cr(vi) in real water samples by standard adding methods, indicating its application feasibility.

Protein-activated and FRET enhanced excited-state intermolecular proton transfer fluorescent probes for high-resolution imaging of cilia and tunneling nanotubes in live cells.

Excited-state intermolecular proton transfer (inter-ESPT) fluorescent probes responsive to specific bioactive molecules should be greatly promising for protein sensing, DNA mutation simulating and cellular process regulating. However, the inter-ESPT molecules are recessive ESPT fluorophores, which need the assistance of other molecules with both hydrogen-bond accepting and donating abilities to turn on the tautomeric fluorescence. Valid design strategies to create powerful inter-ESPT fluorescent probes are poorly developed, particularly for proteins as targets. We recently reported a unique supramolecular strategy to trigger the inter-ESPT process based on the probe-protein recognition by H-bonding and to image protein-based subcellular structures in live cells. Herein, we found that our inter-ESPT probes (inter-ESPT-01) bearing a 2-amino-3-cyanopyridine scaffold can anchor proteins and light up the "invisible" ESPT state, so as to image the proteins or the protein-based subcellular organelles. More importantly, the inter-ESPT emission of inter-ESPT-01 can be significantly enhanced by the FRET process between amino and imino tautomers, endowing the inter-ESPT-01 probes with super-bright tautomeric fluorescence. The expressed proteins Ecallantide and MarTX were selected as the models to light up the inter-ESPT fluorescence of the probes and revealed that the inter-ESPT process can be triggered by the specific probe-protein recognition events. In the use of the super-bright inter-ESPT fluorescence, not only the proteins, but also the protein-based cilia and tunneling nanotubes (TNTs) can be specifically visualized in living cancer cells. Furthermore, such recognition-driven strategy allows us to construct a unique inter-ESPT probe to track and image specific endogenous proteins in live cells, highlighting the potential of inter-ESPT fluorogens as novel intelligent biomaterials.

A fluorescent probe based on modulation of ESIPT signaling for the highly selective detection of N2H4 and cell-imaging.

The broad occurrence of the hydrazine (N2H4) residues in aqueousenvironment is a potential threat to human health. Currently, the mainstream strategy for designing N2H4-specific probes is to functionalize a fluorophore with nucleophilic sites for the reductionreaction with N2H4. In this work, we designed and synthesized an excited-state intermolecular proton transfer (inter-ESPT) fluorescent dye(2-amino-4-(4-methoxyphenyl)-7,8-dihydro-5H-spiro[quinoline-6,2'-[1,3]dioxolane]-3-carbonitrilem, DQN) and used it as a probe to sense N2H4. DQN exhibits blue fluorescence in conventional solvents, which is assigned to its normal emission. In the presence of N2H4, the probe DQN can anchor the N2H4 molecule via hydrogen binding, enabling DQN to undergo inter-ESPT process and light up its tautomeric fluorescence. From this basis, an inter-ESPT-based method for N2H4 detection was established, offering high selectivity and sensitivity (11.5 nM). Furthermore, we demonstrated that the probe DQN can recognize the proteins in living cells, affording cell-imaging. This research provides a promising sensing strategy for monitoring N2H4 in water environments and this inter-ESPT dye is a powerful tool for cell-imaging.

Spectral Properties Echoing the Tautomerism of Milrinone and Its Application to Fe3+ Ion Sensing and Protein Staining.

Knowledge on the spectral properties of the tautomers of milrinone (MLR) in solvents and solid-state, as well as under light conditions is of critical importance from both theoretical and practical points of view. Herein, we investigated the spectral properties of MLR in different conditions using UV-Vis and fluorescence spectroscopies. The experimental results demonstrated that MLR can undergo the tautomerization reaction induced by solvent polarity, light and pH, eliciting four tautomeric structures (enol, keto, anion, and cation forms). The interesting multi-functional groups in MLR enable it to coordinate with metal ions or to recognize gust molecules by H-bonding. In the use of MLR as an excited-state intermolecular proton transfer (inter-ESPT) fluorescent probe, a highly sensitive and selective analysis of Fe3+ was developed, which offered a sensitive detection of Fe3+ with the detection limit of 3.5 nM. More importantly, MLR exhibited the ability of anchoring proteins and led to the recognition-driven turn-on inter-ESPT process, highlighting the potential for the probe to image proteins in electrophoresis gels. The spectral experimental results revealed the possible degradation mechanism, so that we can better understand the side effects of oral preparations. The use of the available drug as an inter-ESPT fluorescent probe is simple and accurate, providing a good method for Fe3+ ion sensing and protein staining.

An Immunosensing System Using Stilbene Glycoside as a Fluorogenic Substrate for an Enzymatic Reaction Model.

A natural product, stilbene glycoside (2,3,5,4'-tetrahydroxydiphenylethylene-2-O-glucoside, TBG), has been evaluated for the first time as a potential substrate for horseradish peroxidase (HRP)-catalyzed fluorogenic reactions. The properties of TBG as a fluorogenic substrate for HRP and its application in a fluorometric enzyme-linked immunosensing system were compared with commercially available substrates such as p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red using Brucella melitensis antibody (BrAb) as a model analyte. The immunosensing body based on HRP-BrAb was constructed by dispersing graphite, BrAg and paraffin wax at room temperature. In a competitive immunoassay procedure, the BrAb competed with HRP-BrAb to react with the immobilized BrAg. In the enzymatic reaction, the binding HRP-BrAb on the sensing body surface can catalyze the polymerization reaction of TBG by H2O2 forming fluorescent dimers and causing an increase in fluorescence intensity. TBG showed comparable ability for HRP detection and its enzyme-linked immunosensing reaction system, in a linear detection ranging of 3.5´10-8~7.6´10-6g/L and with a detection limit of 1.7´10-9 g/L. The immobilized biocomposite surface could be regenerated with excellent reproducibility (RSD=3.8%) by simply polishing with an alumina paper. The proposed immunosensing system has been used to determine the BrAb in rabbit serum samples with satisfactory results.

A specific nanoprobe for cysteine based on nitrogen-rich fluorescent quantum dots combined with Cu2.

As a new member of the carbon quantum-dot family, fluorescent nitrogen-rich quantum dots (NRQDs) were prepared by a mixed solvothermal method using 2-azidoimidazole and aqueous ammonia as reactants. These NRQDs are rich in nitrogen up to 40.2%, which are endowed with high fluorescence quantum yield, good photostability, water-solubility and favourable biocompatibility. We further explored the use of NRQDs combined with Cu2+ as a nanoprobe for sensing fluorescently of cysteine (Cys) in complex biological samples. In this sensing system, the fluorescence is significantly quenched via energy transfer from NRQDs to Cu2+ for the coordination of amino-containing groups with Cu2+. The strong affinity between Cu 2+ and Cys leads to the formation of Cu2+-Cys complexes and cause the detachment of Cu2+ from the surface of NRQDs, thus the fluorescence of NRQDs recover. This nanoprobe allows analysis of Cys by modulating the switch of the fluorescence of NRQDs with a detection limit of 5.3nM. As expected, the proposed NRQDs-Cu2+complex-based nanoprobes were successfully applied for the determination of Cys in human serum and plasma samples with recoveries ranging from 97.2% to 105.7%. The probe ensemble was also successfully applied to imaging of Cys in living cells with satisfactory results, which shows strong potential for clinical diagnosis.

Intrinsically fluorescent and highly functionalized polymer nanoparticles as probes for the detection of zinc and pyrophosphate ions in rabbit serum samples.

Intrinsically fluorescent polymer nanoparticles (F-PNPs) were synthetized from 2-hydroxy-5-methylisophthalaldehyde and melamine by solvothermal method. F-PNPs can emit strong yellow green fluorescence at 542 nm without the conjugation to any external fluorescent agent and surface modification. Owing to the abundant amino and hydroxyl groups on their surface, the F-PNPs possess multiple binding sites, good biocompatibility and excellent water-solubility. Addition of Zn2+ to the F-PNPs solution resulted in a blue shift (Δλ=40 nm) with obvious enhancement in the fluorescence intensity at 502 nm; while there was negligible change in the presence of other metal ions. The subsequent treatment with pyrophosphate (PPi) can cause fluorescence recovery of F-PNPs by pulling the Zn2+ out of the coordination cavity of F-PNPs-Zn2+ nanocomposites. No interference was observed from other anions and nucleotides, making the F-PNPs-Zn2+ ensembles highly sensitive and selective nanoprobes for PPi. The detection limit is 2.75 × 10-8 M/L and 7.63 × 10-8 M/L for Zn2+ and PPi, respectively. The proposed nanoprobes were then used for detecting the recovery of Zn2+ and PPi in rabbit serum samples, which were found to be 99.4-104.2% and 98.6-104.7%, respectively. The present strategy for the fabrication of nanoparticles may offer a new sight for the preparation of polymer nanostructures. The F-FNPs based probes can provide an accurate method for the detection of Zn2+ and PPi in serum samples.

A fluorescence enhancement-based sensor using glycosylated metalloporphyrin as a recognition element for levamisole assay.

A fluorescence sensor based on the supermolecular recognition by glycosylated metalloporphyrin for levamisole (LEV) assay is reported. For the preparation of a LEV-sensitive active material, 5, 10, 15, 20-tetrakis[2-(2, 3, 4, 6-tetraacetyl-beta-D-glucopyranosyl)-1-O-phenyl] porphyrin and its metal complexes were synthesized and used in an optode membrane prepared by including glycosylated metalloporphyrin in chitosan matrice. The immobilized glycosylated metalloporphyrin is shown to be weakly fluorescent as a result of the inhibiting of the electron tansfer by central metal. The fluorescence enhancement of the metalloporphyrin modified optode membrane by LEV is based on the complexation with the central metal moiety of metalloporphyrin and weakening the inhibiting of the electron tansfer for metalloporphyrin. The glycosylated metalloporphyrin/chitosan optode membrane showed excellent selectivity toward LEV with respect to a number of interferents and exhibited stable response. The calibration graph obtained with the proposed sensor was linear over the range of 1.3x10(-5)-3.5x10(-7)ML(-1), with a detection limit of 3.5x10(-7)ML(-1) for LEV. The prepared sensor is applied for the determination of LEV in pharmaceutical preparations and the results agreed with the values obtained by the pharmacopoeia method.

Bacteria-modified amperometric immunosensor for a Brucella melitensis antibody assay.

A novel amperometric immunosensor setup is described which uses horseradish peroxidase (HRP) as a label in conjunction with a current-based Brucella sensor. The Bacteria modified immunosensor was constructed by using a biocomposite formed by dispersing graphite powder into a mixture of Brucella melitensis and silicate polymer gel. The enzyme-labeled antibody can readily diffuse toward the encapsulated antigen (Brucella melitensis), which retains its binding properties, and the association reaction is easily detected at the surface exposed to the solution. The use of an oaminophenol (o-AP) substrate and amperometric detection at -150 mV (vs. SCE) results in a relatively low detection limit of 3.5 ng/ml and a linear detection range of 3.5 ng/ml to 200 ng/ml. Based on an optimized parameter, the prepared sensor was used to detect the Brucella melitensis antibody in serum samples by using a competitive binding assay. The results demonstrate the feasibility of employing the proposed immunosensor for the detection for Brucella melitensis antibody in a clinical analysis.

A novel fluorescence sensor based on covalent immobilization of 3-amino-9-ethylcarbazole by using silver nanoparticles as bridges and carriers.

A novel technique of covalent immobilization of indicator dyes in the preparation of fluorescence sensors is developed. Silver nanoparticles are used as bridges and carriers for anchoring indicator dyes. 3-amino-9-ethylcarbazole (AEC) was employed as an example of indicator dyes with terminal amino groups and covalently immobilized onto the outmost surface of a quartz glass slide. First, the glass slide was functionalized by (3-mercaptopropyl) trimethoxysilane (MPS) to form a thiol-terminated self-assembled monolayer, where silver nanoparticles were strongly bound to the surface through covalent bonding. Then, 16-mercaptohexadecanoic acid (MHDA) was self-assembled to bring carboxylic groups onto the surface of silver nanoparticles. A further activation by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) converted the carboxylic groups into succinimide esters. Finally, the active succinimide esters on the surface of silver nanoparticles were reacted with AEC. Thus, AEC was covalently bound to the glass slide and an AEC-immobilized sensor was obtained. The sensor exhibited very satisfactory reproducibility and reversibility, rapid response and no dye-leaching. Rutin can quench the fluorescence intensity of the sensor and be measured by using the sensor. The linear response of the sensor to rutin covers the range from 2.0 x 10(-6) to 1.5 x 10(-4) molL(-1) with a detection limit of 8.0 x 10(-7) molL(-1). The proposed technique may be feasible to the covalent immobilization of other dyes with primary amino groups.

A selective artemisinin-sensor using metalloporphyrin as a recognition element entrapped in the Au-nanoparticles-chitosan modified electrodes.

An amperometric artemisinin (ARN) sensor based on the supramolecular recognition of glycosylated metalloporphyrin, which is included in the Au-nanoparticles-chitosan film coated on the glass carbon electrodes, was developed. For the improvement of the selectivity of artemisinin detection, 5,10,15,20-tetrakis[2-(2,3,4,6-tetraacetyl-beta-d-glucopyranosyl)-1-O-phenyl]porphyrin (T(o-glu)PPH) metal complex [FeT(o-glu)PPCl] was synthesized and employed as a ARN-sensitive and -selective material in the amperometric sensors. The proposed [FeT(o-glu)PPCl]/Au-nanoparticles modified electrodes showed excellent selectivity and sensitivity toward ARN with respect to a number of interferents and exhibited stable current response, which can be attributed to the coordination of ARN with the [FeT(o-glu)PPCl] in the electrodes. The calibration graph obtained with the proposed sensor was linear over the range of 1.8x10(-7)-1.7x10(-9)moll(-1), with a detection limit of 1.7x10(-9)moll(-1) for ARN. Significant advantages of the proposed procedure over the conventional reductive electrochemical methods are the selective detection and the relatively low applied potential requirement of the ARN-sensor. The prepared sensor is applied for the determination of ARN in plant samples and the results agreed with the values obtained by the pharmacopoeia method.

Approach on quantitative structure-activity relationship for design of a pH neutral carrier containing tertiary amino group.

The quantitative structure-activity relationship (QSAR) for neutral carriers used to prepare hydrogen ion sensors has been studied. A series of synthesized carrier compounds were taken as the training set. Five molecular structure parameters of the compounds were calculated by using CNDO/2 algorithm and used as feature variables in constructing QSAR model. The lower and upper limits of the linear pH response range were taken as the activity measure. The corresponding model equations were derived from the stepwise regression procedure. With the established QSAR model, a new pH carrier, (4-hydroxybenzyl) didodecylamine (XIII) was proposed and synthesized. The PVC membrane pH electrode based on carrier XIII with a wide pH linear response range of 2.0-12.5 was prepared. Having a theoretical Nernstian response slope of 57.2+/-0.3 mV/pH (n=5 at 25 degrees C) without a super-Nernstian phenomenon, the sensor had low resistance, short response time, high selectivity and good reproducibility. Moreover, the sensor was successfully applied to detecting the pH value of serum samples.

Schistosoma japonicum antibody assay by immunosensing with fluorescence detection using 3,3',5,5'-tetramethylbenzidine as substrate.

An immunosensing system for Schistosoma Japonicum antibody (SjAb) assay has been developed which is useful for the diagnosis of schistosomaisis. To circumvent the difficulty of regeneration of the immunosensing device, the sol-gel technique is used which results in a considerable retention of the activity of the encapsulated antigen (SjAb) and easily diffusing into the pores of the polymeric silica matrix. The surface of the immunosensing device prepared can easily be renewed by simply polishing. The regenerated surface serves as a platform for the competitive immuno-reaction of HRP-SjAb and SjAb with SjAg bound at the surface. By using 3, 3', 5, 5'-tetramethylbenzidine (TMB) as the substrate, the amount of HRP-SjAb bound is quantitated fluorimetrically, which is in turn related with the SjAb content. An amplification effect is obtained by using the enzymatic reaction, and an improved detection limit of 4.5 ng ml(-1) is thus realized. The optimum analytical conditions such as pH, amount of the labeled antibody and flow rates of substrate carrier solution were established. The immunosensing procedure shows a pseudo linear response range from 4.5 to 55 ng ml(-1). The proposed procedure has been employed to determine SjAb in serum samples.

Fluorometric enzyme immunosensing system based on a renewable immunoreaction platform for the detection of Schistosoma japonicum antibody.

A fluoroimmunosensing device which was based on ferulic acid (FA)/horseradish peroxidase system for the detection of Schistosoma japonicum antibody (SjAb) has been developed. To circumvent the difficulty of regeneration of immunocomposite surface, a natural chitosan-epoxy resin matrix was used for the immobilization of SjAg. The surface of the immunocomposite layer reacted was easily regenerated by simple polishing. The renewed surface served as a platform for the competitive immuno-reaction of HRP-SjAb and SjAb with SjAg immobilized at the support body surface and for enzymatic reaction. A novel fluorescent substrate ferulic acid for HRP, which is relatively stable toward H(2)O(2), has been adapted in the proposed fluorometric enzyme immunosensing system. FA can been catalyzed to produce a non-fluorescent species. The amount of HRP-SjAb bound to the aforementioned renewable surface layer, which is related to the content of SjAb in samples could be quantitized by measuring the decrease of fluorescence of FA induced by HRP-SjAb. The chitosan incorporated in matrix is favorable for the amplification of this sensing system due to the electrostatic reaction with FA. The proposed method showed a linear response ranging from 45 to 150ngml(-1), with an improved detection limit of 45ngml(-1). The method has been employed to determine SjAb in serum samples.