Non-chromogenic peptide substrates for detection of enzymes by means of capacitance changes at metal electrodes

A new type of substrate for enzyme detection has been developed. The substrate is non-chromogenic and is used in an assay method based on electrode adsorption. The rate of change in the electric capacitance of the electrode is monitored and taken as a measure of the substrate adsorption. Substrate adsorption is in turn proportional to substrate bulk concentration and thus subject to changes by enzymes. The new substrate introduces a new concept in enzyme detection: as it is non-chromogenic it may contain appropriate amino acids on both sides of the bond subject to enzymatic cleavage.     

Detection of proteolytic enzymes in agar electrophoresis

An agar substrate containing hyaluronic acid and horse serum for the detection of hyaluronidase activity following electrophoresis was found suitable for the detection of proteolysis. The substrate was refined for the detection of only proteolysis by elimination of the hyaluronic acid and substitution of bovine serum albumin in place of the serum. The refined substrate revealed two bands of proteolytic activity in papain. The refined substrate also revealed weak proteolytic activity in a “chromatographically pure” preparation of hyaluronidase. The method should be adaptable for the detection of multiple electrophoretic forms of several proteolytic enzymes.     

Incorporation of fluorescent enzyme substrates in agarose gel for in situ zymography

The currently available methods for the detection of proteases in tissue sections are characterized by limited substrate specificity and low sensitivity and are also cumbersome. We have developed a novel in situ zymography method that uses a synthetic substrate conjugated to a fluorescent tag for detection of proteases in tissue sections. In the presence of active enzyme, the fluorescent tag is cleaved off from the substrate peptide chain resulting in an approximately 100-fold increase in the fluorescent signal. In order to minimize the diffusion of the fluorescent tag, the substrate is incorporated into 1% agarose prior to overlaying onto the tissue section. This method retains the morphological details of the tissue section, is highly sensitive and specific for the designated peptide sequence, and provides information regarding the functional status of the enzyme. Thus, this method could be used for detection and monitoring of enzymatic activity in tissue sections for a variety of applications.     

The comparative evaluation of spectrophotometric and chemiluminescent detection in an immunoenzyme test of antibodies to the antigens of the bovine leukosis virus]

Comparative evaluation of the sensitivity limit in the detection of antibodies to bovine leukemia virus in the enzyme immunoassay with the use of chemiluminescent and spectrophotometric detection techniques was carried out. In this assay 3-amino-1,4-phthalazinedion was used as chemiluminescent substrate and ortho-phenylenediamine, as chromogenic substrate. The chemiluminescent signal was registered by means of a special luminometer designed at the Institute of Biochemistry (Lithuanian Acad. Sci.). The use of the chemiluminescent substrate permitted the detection of proteins in amounts 2-3 times lower than those detected by the spectrophotometric technique.     

Double-layer fluorescent zymography for processing protease detection

We have developed a novel double-layer zymographic method for the detection of specific processing proteases of a target proprotease using a specific fluorescent substrate. The target processing proteases were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gel was subsequently incubated with the target proenzyme used as the substrate. A cellulose acetate membrane was immersed in 10% glycerol and then soaked in the fluorescent substrate solution. The slab gel of the processing protease was covered with the fluorescent substrate membrane, making a double layer. The double layer was incubated at 37 degrees C, and the released fluorescent band, in which the processing protease was located, was detected using UV light. The advantages of the double-layer fluorescent zymographic method are as follows: (i) the specific detection of target proprotease using a specific substrate, (ii) a relatively rapid and sensitive method, (iii) effective detection using small amounts of crude material, and (iv) wide applications that include the detection of processing proteases and activators for target proteases. Typical examples used for the detection of the processing proteases, such as plasminogen activator, chymotrypsinogen activator, procaspase-3 processing protease and caspase-3 activators, using this new method are described in this article.     

A rapid method for detecting bacterial contamination in the presence of Penicillium and Streptomyces antibiotic fermentations

Contamination in antibiotic fermentation processes results in major economic and process problems. The detection and elimination of contamination is a continual objective for fermentation companies. While efforts continue to eliminate contamination by improving equipment and sterile techniques, it is still imperative to have a rapid method for detecting contamination in laboratory-stage inoculum and seed tanks. This article describes the successful studies leading to the adoption of the BACTEC, an automatic bacterial detection system, as a supplemental detection technique. The BACTEC system detects contamination by incubating samples with a selected(14)C-labeled substrate or substrates. The resulting metabolism of substrate produces (14)C-labeled CO(2) which is then quantified and expressed as a growth index, permitting detection of contamination more rapidly at a much earlier time than is possible with conventional detection techniques that involve Phenol red dextrose broth, streak plates, and microscopic examination techniques.     

Ultrasensitive chemiluminescent and colorigenic detection of DNA, RNA, and proteins in plant molecular biology.

Nonradioactive detection methods for DNA, RNA, and protein analysis have been the subject of research for several years. In this paper the application of the digoxigenin nucleic acid labeling system, in combination with the new alkaline phosphatase substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)-phenyl -1,2-dioxetane, to the special requirements of the analysis of transgenic plants is described. Earlier detection systems lacked the required ultrasensitive limits of detection necessary because of the large genomes found in plant cells. Routine detection of single-copy genes from transgenic plant species requires the detection of bands of picograms of specific DNA, which is easily achieved by employing the AMPPD substrate. Optimal conditions of genomic Southern analysis have been successfully adapted for Northern blotting techniques. Detection of foreign proteins in transgenic plants has proven difficult because of the very small amounts of detectable specific protein. Until now, utilization of biotinylated antibodies in combination with a streptavidin-alkaline phosphatase conjugate has been the most sensitive procedure. By introducing the AMPPD substrate, a further significant enhancement of sensitivity leading to detectable signals in the picogram range can be obtained.     

Evaluation of p-naphtholbenzein-beta-D-galactoside as a substrate for bacterial beta-galactosidase

We describe the synthesis of a new substrate for the detection of beta-galactosidase and evaluate its performance in comparison with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) and cyclohexenoesculetinbeta-D-galactoside (CHE-Gal). Of 206 Enterobacteriaceae strains able to hydrolyze X-Gal, 194 (94.2%) hydrolyzed CHE-Gal and 192 (93.2%) hydrolyzed p-naphtholbenzein-beta-D-galactoside (PNB-Gal). We conclude that PNB-Gal is an effective substrate for the detection of beta-galactosidase.     

Selective glucose detection based on the concept of electrochemical depletion of electroactive species in diffusion layer

A glucose detection approach based on the concept of electrochemical depletion of electroactive species in diffusion layer was established, using scanning electrochemical microscopy (SECM). By controlling the glucose oxidase (GOD) modified electrode (substrate electrode) at a proper potential of electrochemical oxidation of interfering electroactive species, i.e., ascorbic acid (AA), an interference-free microcircumstance was formed in the diffusion layer of the substrate electrode. Consequently, we could successfully sense hydrogen peroxide generated from an enzymatic reaction by locating a Pt ultramicroelectrode (UME) (tip electrode, 5 microm in radius) into the diffusion layer of the substrate electrode. Properties of this interference-removing approach based on electrochemical depletion were systematically investigated. Results showed that the interference-removing efficiency was significantly determined by the tip-substrate distance and substrate potential. When the tip-substrate distance was 11 microm (2.2 times of the tip electrode radius) and the substrate potential was 0.5 V, nearly 90% of AA (0.5 mM) could be depleted within 30s without consumption of H2O2. Under these conditions, 0.1 mM AA showed no influence on the detection of 0.5 mM glucose. The linear range of glucose detection is 0.01-1 mM with a detection limit (DL) of 0.005 mM (correlation coefficient is 0.9948). This research will open a new way for developing selective micro-biosensors.     

Glucose oxidase-mediated polymerization as a platform for dual-mode signal amplification and biodetection

We report the first use of a polymerization-based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual-mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization-based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization-based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)-avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 μg/mL of GOx-avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme-mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non-specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor-beta.     

High-sensitivity protein detection by a new "contact-copy" method using a protein A-neomycin phosphotransferase II fusion protein

A new system for high-sensitivity protein detection by an immunoenzymatic "contact-copy" procedure is described. It is based on two components: (i) a microbiologically produced bifunctional fusion protein of protein A and neomycin phosphotransferase II (protein A-NPT II) in which the protein A moiety acts as a second immunological reagent while NPT II catalyzes the detection reaction and (ii) a novel kanamycin-loaded substrate matrix (kanamycin-cyanuric chloride-activated and sulfanilic acid-derivatized paper) brought into direct contact with a protein-carrying matrix after blot or dot application and initial immunoreaction--the NPT II enzyme reaction with [gamma-32P]ATP as cosubstrate leads to phosphorylation of the substrate kanamycin on the substrate matrix, which is used for further analysis. The contact-copy method has at least the same detection sensitivity as procedures employing 125I-protein A, but allows extremely short exposure times and avoids probe prelabeling. Twenty-five picograms of specific protein blotted from sodium dodecyl sulfate-polyacrylamide gels onto nitrocellulose is detected after 15 min of autoradiography. The limit of detection in dot tests was found to be 10 pg per dot (3 mm2). The method is suitable for quantitative determination of antigens in the range down to 100 pg. Several contact copies of the same original protein-carrying matrix can be produced and used for detection or quantitative analysis without destroying the original matrix.     

Design of helical proteins for real-time endoprotease assays

Proteases play a key role in cellular biology and have become priority targets for new pharmaceuticals. Thus, there is a high demand for specific, sensitive, and quick assays to monitor the activity of endoproteases. We designed affinity-tagged helical proteins with unique protease cleavage sites and thus constructed universal, molecularly defined, and uniform substrates for in vitro detection of IgA endoprotease. The substrate is a 10.5-kDa recombinant helical protein with a high-affinity (His)(6)-tag at the amino-terminal end. Further elements are a unique proteolytic recognition site and a C-terminal helical extension, which is cut off by the protease. Proteolytic action can be monitored in real time using surface plasmon resonance spectroscopy. Femtomole amounts of protease could be reliably and quantitatively detected within a few minutes after the start of the reaction. The detection signal changed linearly with the amount of protease and was independent of the applied sample flow rate. The biochip can be reversibly loaded with the recombinant protease substrate, so that the SPR assay is well-suited for automation. By substituting an HIV protease site for the recognition site of the IgAse, we also obtained a substrate for the quantitative and sensitive detection of HIV-1 endoprotease. Our substrate design is thus generally applicable.     

2D aggregation and selective desorption of nanoparticle probes: a new method to probe DNA mismatches and damages

A 2D colorimetric DNA sensor is reported based on the 2D aggregation of oligonucleotide-modified gold nanoparticle probes resulting from the molecular hybridization between these latest and their complementary single stranded DNA targets. To increase their mobility the nanoparticles are adsorbed on a fluid lipid bilayer, itself supported on a substrate. The hybridization between the target and the mobile nanoparticle probes creates links between the nanoparticles resulting in the formation of nanoparticle aggregates in the plane of the substrate. This aggregation is detected using a new method based on the selective desorption of non-aggregated nanoparticles. The addition of dextran sulfate induces the substitution of non-aggregated gold nanoparticles while aggregated ones are stable on the substrate. We show that this detection method is highly specific and allows the detection of DNA mismatches and damages.     

显色基质鲎试剂法在人血白蛋白热原检测中的应用

为了对显色基质法测定人血白蛋白中的内毒素含量方法进行探讨。以内毒素,鲎试剂及显色基质,在一定的条件下反应释放出对硝基苯胺(PNA),溶液呈现黄色,于波长545nm处比色读数,其产色深浅与内毒素浓度呈线性关系,从而定量测定出检品中内毒素含量。结果表明标准曲线的线性相关系数r≥0.98,人血白蛋白经3.3倍稀释后无干扰作用。显色基质法与家兔法比较,有灵敏、快速、能定量、重复性好的特点,可用于人血白蛋白内毒素含量的测定。     

Improved chemiluminescent western blotting procedure.

A chemiluminescent Western blotting procedure and its application in assays for human transferrin and human immunodeficiency virus-I antibodies are described. The procedure is based on a chemiluminescent substrate, adamantyl 1,2-dioxetane aryl phosphate and alkaline phosphatase-labeled detection antibodies. Different membranes (polyvinylidene fluoride, nitrocellulose, nylon) and a proprietary membrane treatment agent (Nitro-Block) have been studied. This sensitive blotting procedure utilizing AMPPD, a polyclonal rabbit anti-transferrin:goat anti-rabbit IgG-alkaline phosphatase detection complex, and a PVDF membrane blocked with Nitro-Block permits the detection of 125 pg (1.6 fmol) of human transferrin. A novel 1,2-dioxetane substrate, CSPD, has also been evaluated.     

Detection of competitive enzyme inhibition with end point progress curve data

A model for a dimensionless factor, the inhibition detection limit (IDL), which describes the limit of detection of competitive inhibition for end point assays as a function of the proportion of substrate converted into product, has been developed. For a given end point enzymatic assay, the IDL function has a maximum that is dependent on the error structure parameters (four parameters) of the assay, the value of [S]o/K(ms), and the extent of product inhibition (K(ms)/K(mp)). Accordingly, the substrate conversion level that maximized the ability to detect samples with high Ki/[I] ratios was predicted for each member of a population of simulated assays. Furthermore, we identified a consensus substrate conversion level where the probability of a near-optimal robustness and detection limit for all the members of the assay population is maximal. Unlike the optimal substrate conversion level for individual assays, this consensus substrate conversion level was dependent only on [S]o/K(m), K(ms)/K(mp), and whether the signal increases or decreases during the course of the reaction. Consensus substrate conversion levels were beyond the initial velocity range for almost all the analyzed assay populations. It was shown that the IDL factor was a more informative indicator of assay quality than the popular Z' factor.     

Construction of a more sensitive fluorescence sensing material for the detection of vascular endothelial growth factor, a biomarker for angiogenesis, prepared by combining a fluorescent peptide and a nanopillar substrate

We have established a highly sensitive and selective protein detection technology in combination with the nanofabrication technique. A silica nanopillar chip with a 200-nm pitch and 1000-nm height pillar substrate was fabricated by electron beam lithography and deep reactive ion etching method. Fluorescent peptides, with high affinity towards vascular endothelial growth factor (VEGF), were immobilized on nanopillar chip via a self-assembled monolayer made from 3-aminopropyltrimethoxysilane and glutaraldehyde under optimal conditions. The fluorescence intensity of the fluorescent peptide on the nanopillar substrate increased with increasing VEGF concentrations, as determined by a fluorescence spectrophotometer and fluorescent scanning image analysis. The dissociation constant (K(d) value) calculated by the non-linear least square curve fitting method was 6.0 × 10(-9)M, which contributed to the highly sensitive detection of VEGF. The fluorescence intensity of the fluorescent reagent on the nanopillar substrate upon binding to VEGF was higher than that obtained using the flat substrate because the dense and tall nanopillar array increased the virtual protein binding area. The reproducibility tests and lifetime measurement indicate the fluorescent reagent to be a useful biosensor for the detection of VEGF in this system. These experimental results clearly showed that the combination of a fluorescent reagent and a nanopillar substrate may be widely applicable as a convenient method for the detection of VEGF.     

A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase–liquid chromatography–tandem mass spectrometry/multiple reaction monitoring assay

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNTs is achieved by the endopeptidase–mass spectrometry (Endopep–MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type B (BoNT/B) in the Endopep–MS assay. Our strategy was based on reported BoNT/B–substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide led to a 5-fold increased sensitivity of the assay both in buffer and in a clinically relevant human spiked serum.     

Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases.

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.     

Sequential chemiluminescent detection of target DNAs without stripping and reprobing.

We present a simple method for sequential chemiluminescent detections of two different DNA loci on a single Southern blot. First, an enzyme-linked DNA probe for a unique sequence is detected with a horse-radish peroxidase (HRP) substrate followed by the detection of another enzyme-linked DNA probe for a different unique sequence with an alkaline phosphatase (AP) substrate that simultaneously inhibits the chemiluminescence generated by HRP. Such sequential detection steps eliminate the need to strip and reprobe blots and can be performed with no intervening steps.