A sensitive method for the detection of proteins by high-efficiency fluorescence quenching

A successful method for the detection of electron transfer proteins such as cytochrome c, hemoglobin and myoglobin has been developed based on the fluorescence quenching of semiconductor nanocrystals. High sensitivity and a good linear relationship are obtained.     

Chemoenzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-d-erythritol: a substrate for IspE

Enantiomerically pure 2-C-methyl-d-erythritol 4-phosphate 1 (MEP) is synthesized from 1,2-O-isopropylidene-α-d-xylofuranose via facile benzylation in good yield. Subsequently, 1 is used for enzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-d-erythritol 2 (CDP-ME) using 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (IspD). The chemoenzymatically synthesized 2 can be used as substrate for assay of IspE and for high throughput screening to identify IspE inhibitors.     

Chemoenzymatic synthesis of sialylated oligosaccharides for their evaluation in a polysialyltransferase assay

A series of sialylated β-d-Gal-(1→3)-α-d-GalNAc-octyl containing oligosaccharides representative of those found on mucin type complex O-glycans were synthesized by a chemoenzymatic approach for use in the kinetic characterization of recently cloned polysialyltransferases. Enzymatic incorporation of N-acetylneuraminic acid (sialic acid) into the synthetic acceptors was accomplished by 2,3-(N) and (O)-sialyltransferases to give the target compounds 6-10 in a practical yield.     

Casein-resorufon, a new substrate for a highly sensitive protease assay

Conclusion It has been shown that casein-resorufin can be used as a very sensitive and easily to assay general protease substrate.

---

Casein-Resorufin, ein neues Substrat für einen hochempfindlichen Protease-Assay

     

Chemoenzymatic synthesis of prodigiosin analogues--exploring the substrate specificity of PigC

Analogues of prodigiosin, a tripyrrolic pigment produced by Serratia species with potent immunosuppressive and anticancer activities, have been produced by feeding synthetic analogues of the normal precursor MBC to mutants of Serratia sp. ATCC 39006 or to engineered strains of Escherichia coli; in this way it has been shown that the prodigiosin synthesising enzyme, PigC, has a relaxed substrate-specificity.     

Target-mediated consecutive endonuclease reactions for specific and sensitive homogeneous fluorescence assay of O-methylguanine-DNA methyltransferase

O⁶-Methylguanine-DNA methyltransferase (MGMT) is one of the most important DNA-repair enzymes. Herein, a simple, sensitive and selective homogeneous fluorescence assay strategy is developed for the detection of MGMT on the basis of target-mediated two consecutive endonuclease reactions. The activity assay of MGMT is firstly accomplished using a hairpin-structured DNA substrate to offer a specific recognition site on the substrate DNA for restriction endonuclease PvuII, and thus to initiate the first endonuclease reaction. The product which activates the second endonuclease reaction allows an efficient amplification approach to create an abundance of fluorescence signal reporters. The first endonuclease reaction offers the method high specificity and the second one furnishes the assay improved sensitivity. The results reveal that the MGMT assay strategy shows dynamic responses in the concentration range from 1 to 120 ng mL⁻¹ with a detection limit of 0.5 ng mL⁻¹. By simply altering the alkylated bases, this strategy can also be extended for the detection of other alkyltransferases. Therefore, the developed strategy might provide an intrinsically convenient, sensitive and specific platform for alkyltransferase activate assay and related biochemical studies due to its label-free, homogeneous, and fluorescence-based detection format.     

TNT及RDX蒸气对基于芘氧敏感膜荧光猝灭的研究

制备了以芘为敏感材料,聚苯乙烯(PS)以及聚氯乙烯(PVC)粉末为支持体系的两种荧光猝灭氧敏感膜.PS以及PVC氧敏感膜的最大激发波长分别为349 nm和355 nm,最大发射波长分别为399 nm和398 nm.分子氧对PS以及PVC氧敏感膜的荧光均有猝灭作用.同时,实验发现2,4,6-三硝基甲苯(TNT)和环三亚甲基三硝基胺(RDX)对这两种氧敏感膜的荧光也有一定的猝灭.     

ESIPT-mediated photocycloadditions of 3-hydroxyquinolinones: development of a fluorescence quenching assay for reaction screening

Irradiation of 1,2-dimethyl-3-hydroxyquinolinone (DMQ) leads to excited state intramolecular proton transfer (ESIPT) generating a 3-oxidoquinolinium species which undergoes [3 + 2] photocycloaddition with dipolarophiles. A parallel, fluorescence quenching assay using a microplate format has been developed to evaluate fluorescence quenching of this species with a range of dipolarophiles.     

Chemoenzymatic synthesis of l-tyrosine derivative for a transketolase assay

We have prepared an l-tyrosine derivative bearing a d-threo ketose moiety by a convenient chemoenzymatic route. This compound is of potential interest for developing stereospecific assays for enzymes catalyzing C-C bond cleavage such as transketolase. We showed in vitro by analytical studies (LC/MS and ³¹P NMR) that this compound can release l-tyrosine in the presence of wild type TK extract and bovine serum albumin. This assay is the first step towards a mutant TK selection test that could be developed for yeast cells auxotrophic for l-tyrosine.     

A sensitive method for the detection of catecholamine based on fluorescence quenching of CdSe nanocrystals

A sensitive method for the detection of catecholamine based on the fluorescence quenching of CdSe nanocrystals was developed. The sodium citrate-protected CdSe nanocrystals were synthesized in water solution. The fluorescence quenching of CdSe nanocrystals by dopamine, uric acid, ascorbic acid and catechol was studied; the results showed that all of these four kinds of compounds could quench the fluorescence of nanocrystals, and the quenching constant was 6.3 × 10⁴, 2.57 × 10³, 2.14 × 10³ and 1.168 × 10³, respectively. The order of sensitivity for the biosensor was: dopamine > lactic acid > ascorbic acid > catechol. This method shows good selectivity for dopamine, the detection limit reaches 5.8 × 10⁻⁸ M.     

beta-galactosidase assay using capillary electrophoresis laser-induced fluorescence detection and resorufin-beta-D-galactopyranoside as substrate

beta-Galactosidase was incubated for 60 min with the fluorogenic substrate resorufin-beta-D-galactopyranoside, which is converted by the action of the enzyme into resorufin and galactose. A 160 pL aliquot of reaction mixture was analyzed by capillary electrophoresis utilizing laser-induced fluorescence detection. Based on the detection of the resorufin formed, the limit of detection of beta-galactosidase was 1.5 x 10(-15) M or 900 molecules of enzyme in a 1 microL sample.     

Optimization of the structure-switching aptamer-based fluorescence polarization assay for the sensitive tyrosinamide sensing

In this paper, a structure-switching aptamer assay based on a fluorescence polarization (FP) signal transduction approach and dedicated to the L-tyrosinamide sensing was described and optimized. A fluorescently labelled complementary strand (CS) of the aptamer central region was used as a probe. The effects of critical parameters such as buffer composition and pH, temperature, aptamer:CS stoichiometry, nature of the dye (Fluorescein (F) or Texas Red (TR)) and length of the CS (15-, 12-, 9- and 6-mer) on the assay analytical performances were evaluated. Under optimized experimental conditions (10 mM Tris-HCl, 5 mM MgCl(2) and 25 mM NaCl, pH 7.5 temperature of 22°C and stoichiometry 1:1), the results showed that, for a 12-mer CS, the F dye moderately increased the method sensitivity in comparison to the TR label. The F labelled 9-mer CS, however, did not allow the hybrid formation with the functional nucleic acid, thus emphasizing the importance of the nature of the fluorophore. In contrast, the same 9-mer CS labelled with the TR dye was able to effectively associate with the aptamer and was easily displaced upon target binding as demonstrated by a significant improvement of the sensitivity and a detection limit of 250 nM, comparable to those reported with direct aptasensing methods. The present study demonstrates that not only the CS length but also the nature of the dye played a preponderant role in the performance of the structure-switching aptamer assay, highlighting the importance of interdependently controlling these two factors for an optimal FP-based sensing platform.     

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.     

A strand displacement signal amplification-assisted fluorescence assay for sensitive detection of microRNA

MicroRNA is a vital biomarker because of its abnormal expression in the emergence and development of diseases, especially in cancers. Herein, a label-free fluorescent sensing platform is proposed for detecting microRNA-21, coupled with the cascade toehold-mediated strand displacement reaction and magnetic beads. Target microRNA-21 acts as an initiator to trigger the cascade toehold-mediated strand displacement reaction and it outputs double-stranded DNA. After magnetic separation, the double-stranded DNA is intercalated by SYBR Green I, resulting in an amplified fluorescent signal. Under the optimal conditions, a wide linear range (0.5–60 nmol/L) and low limits of detection (0.19 nmol/L) are exhibited. What's more, the biosensor shows great specificity and reliability between microRNA-21 and other microRNAs involved in cancer (microRNA-34a, microRNA-155, microRNA-10b, and let-7a). Owing to the properties of fabulous sensitivity, high selectivity, and simplicity of operator, the proposed method paves a promising way for microRNA-21 detection in cancer diagnosis and biological research.     

Hapten heterology for a specific and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus insecticide fenthion

Five haptens with different spacer-arm attachment sites on the structure of the organophosphorus insecticide fenthion were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and three haptens containing all or most of the structure of fenthion were conjugated with bovine serum albumin (BSA) for the immunogen. Six polyclonal antisera were raised against the three BSA conjugates, and 30 antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for fenthion. The study revealed the best combination with high sensitivity (I₅₀ of 0.08 ng mL⁻¹) and high assay specificity, which indicated that when structural difference between the analyte and an immunizing hapten is less than that between a coating hapten and the immunizing hapten, a high sensitive enzyme-linked immunosorbent assay (ELISA) in the heterologous system may stand a good chance to be developed. The immunity results showed that heterology in the hapten spacer-arm attachment site of the immunogen could achieve a remarkable improvement in the quantity, sensitivity, and/or specificity of antibody, and that the moiety of an analyte, which is the same as the moiety near/on the immunizing spacer-arm hapten attachment site, contributes greatly to the interaction of antibody and hapten.