20 results about "Fluorogenic Substrate" patented technology

The application discloses a method and application of a Cas12a sensing system based on an sgRNA blocking strategy for single nucleotide polymorphism typing. The method comprises the following steps: pre-annealing sgRNA and blocking chains to form a complex; obtaining a single-stranded DNA target by using an asymmetric recombinase polymerase amplification; constructing a detection system comprising a Cas12a protein, the complex, a target and a fluorescent substrate; activating the trans-cleavage activity of Cas12a through a strand displacement reaction to generate a fluorescent signal, so as to realize SNP typing. The method combines the sgRNA blocking strategy with the Cas12a system for the first time, does not need to depend on a PAM sequence, has the advantages of high universality, high specificity, simple operation, rapidness, low cost and the like, and is suitable for ApoE gene typing and other SNP related detection.

The invention discloses a fluorescent substrate 7-HC-P-E, a preparation method thereof and application of the fluorescent substrate 7-HC-P-E in detection of the active concentration of alkaline phosphatase (ALP). The fluorescent substrate can be catalytically hydrolyzed by ALP under an alkaline condition to release 7-hydroxycoumarin (7HC) with a strong fluorescent signal, so that high-sensitivity and high-specificity detection on the enzyme activity is realized. The invention further provides a synthesis route and an optimization process of the fluorescent substrate, the operation is simple and convenient, the product purity is high, and the fluorescent substrate is suitable for multiple fields such as biological analysis, medical diagnosis and environmental monitoring.

The application discloses a screening method for activity and sensitivity of recombinant factor B for endotoxin detection. The method comprises the following steps: adding a series of endotoxin standard solution with different concentrations in an endotoxin-free 96-well plate, and adding endotoxin-free water as a control; adding an activity detection reagent, wherein the activity detection reagent comprises a buffer, a mixed solution of recombinant factor C and a to-be-detected recombinant factor B, and a fluorescent substrate; reading a zero-point fluorescence value, and then placing the 96-well plate in a 37 DEG C incubator for incubation; reading the fluorescence value again, calculating a fluorescence intensity change value Delta RFU, and determining the result. The method realizes independent and direct evaluation of the activity and sensitivity of the recombinant factor B through a 'two-factor' (only containing the recombinant factor C and the recombinant factor B) detection system, and completely gets rid of the dependence on downstream coagulase activity. The method can help screening of the recombinant factor B with high activity and sensitivity, and the recombinant factor B can be used in a recombinant limulus reagent for endotoxin detection.

The invention discloses a method for detecting protease activity in yeast for making hard liquor. The detection method comprises the following steps: selecting carboxyl magnetic beads according to the characteristics of a Daqu sample, coupling protein to the magnetic beads to obtain a magnetic bead adsorption enzyme sample, marking casein by utilizing fluorescein isothiocyanate to prepare neutral and alkaline fluorescent substrates, and establishing a linear relation curve of enzyme activity and fluorescence intensity by utilizing neutral and alkaline protease standard substances. And finally, after a fluorescent substrate is hydrolyzed by protease to generate a soluble fluorescent peptide fragment, determining a fluorescence intensity value under a specific wavelength and substituting the fluorescence intensity value into the standard curve, so that the protease activity of the yeast for making hard liquor can be accurately determined. According to the detection method, the protein compound is adsorbed and separated through the magnetic beads, so that impurity interference in the yeast crude enzyme is effectively removed, the accuracy of enzyme activity determination is remarkably improved, and the protease activity of the yeast can be reliably determined.

The present application relates to the technical field of water quality monitoring, and discloses a water sample pollution inspection method guided by microorganism detection, comprising: applying a pulsed electric field to a biochemical response system to reduce the mass transfer resistance of the cell membrane of an indicator microorganism by using a physical pore-forming effect; injecting a fluorescent substrate and synchronously collecting fluorescence emission intensity and backscattering light intensity; correcting the amplitude of the fluorescence emission intensity by using the backscattering light intensity to eliminate physical shielding interference and obtain a pre-steady-state fluorescence emission sequence; intercepting a characteristic value of the change rate of the pre-steady-state fluorescence emission sequence to calculate an intrinsic catalytic turnover index representing the specific catalytic activity of intracellular enzymes, and determining the pollution degree of the water sample to be tested according to the offset of the index relative to a preset reference value; and the present application can quickly identify pollutants with transmembrane mass transfer disorders and eliminate the influence of endogenous activity fluctuations of microorganisms on the detection result by means of the cooperation of electric pore-forming intervention and calculation logic.

The present disclosure relates to a novel fluorescent substrate, which is a compound or isomer of formula (I), and its use. A high-sensitivity detection reagent combination and detection method are also provided, which comprises the fluorescent substrate compound or isomer of formula (I) and an enzyme-labeled antibody. The fluorescent substrate and detection method can also be widely used for detecting cells with low expression of specific antigens. Compared with the traditional fluorescent secondary antibody detection method and the TSA-FITC combination method, the detection efficiency of the method of the present disclosure is higher, and the detection sensitivity and stability are higher.

This invention discloses the selective detection of a fluorescent substrate and its application in detecting S-COMT activity, belonging to the field of analytical detection. The selective fluorescent substrate is 3-carboxy / methyldaphne (3A / M-DPN), which can be specifically catalyzed by S-COMT to generate an 8-O-methylated product, producing a strong fluorescence signal at 520 nm, and the fluorescence intensity shows a good linear relationship with S-COMT activity. The substrate of this invention has a high affinity for S-COMT (…). K m =1.41 μM) is much higher than that of membrane-bound MB-COMT ( K m =45.31 μM), catalytic efficiency ( V max / K m It is 280 times more potent than other substrates and possesses advantages such as high selectivity, high sensitivity, and ease of operation. This substrate can be used to distinguish the catalytic contribution of S-COMT from MB-COMT, quantitatively assess the S-COMT activity in various in vitro biological samples, and provide an important tool for S-COMT subtype functional studies, monitoring of S-COMT activity under pathological conditions, and screening of drugs targeting S-COMT.

The present invention discloses a method for detecting glyphosate residues in coffee by fluorescence and chemiluminescence dual-mode, belonging to the technical field of chemical analysis and detection. The method uses succinic acid as a ligand and Ce 4+ as the central ion, synthesizes the Suc-Ce complex by microwave method, and further obtains the Suc-Ce-OH nanozyme through alkali treatment. The Suc-Ce-OH nanozyme has excellent phosphatase-like activity. Alkali treatment can effectively remove surface coordinated water molecules and expose more Ce-O active sites, doubling its hydrolysis efficiency for the fluorescent substrate 4-MUP and the chemiluminescent substrate AMPPD. Based on the specific inhibitory effect of glyphosate on the activity of the phosphatase-like nanozyme, the present invention establishes a dual-mode detection method for glyphosate by fluorescence and chemiluminescence, with detection limits of 0.05 μg / mL and 0.01 μg / mL respectively. It is used to detect glyphosate residues in coffee samples, featuring strong specificity, simple operation, and high sensitivity.

The invention discloses a method for screening activity and sensitivity of a recombinant factor B for endotoxin detection. The method comprises the following steps: adding a series of endotoxin standard substance solutions with different concentrations into an endotoxin-free 96-well plate, and taking endotoxin-free water as a contrast; adding an activity detection reagent, wherein the activity detection reagent comprises a buffer solution, a mixed solution of a recombinant C factor and a recombinant B factor to be detected, and a fluorescent substrate; reading a zero fluorescence value, and then placing the 96-well plate in an incubator at 37 DEG C for incubation; reading the fluorescence value again, and calculating a fluorescence intensity change value delta RFU; and judging a result. According to the method, independent and direct evaluation on the activity and sensitivity of the recombinant factor B is realized through a detection system of'two factors' (only containing the recombinant factor C and the recombinant factor B), and the dependence on the activity of the downstream clotting zymogen is thoroughly eliminated. The method can help to screen the recombinant factor B with high activity and sensitivity, and is used in a recombinant tachypleus amebocyte lysate for endotoxin detection.

The invention discloses an immune cell population function evaluation and screening method based on digital microfluidics, which comprises the following steps: loading a cell suspension containing immune cells to an active matrix digital micro-fluidic chip, and controlling a chip electrode array to carry out on-chip segmentation and dilution on the cell suspension to form a plurality of cell droplets; segmenting a non-specific stimulant or a specific antigen injected into the chip, mixing the segmented non-specific stimulant or specific antigen with the cell droplets in proportion, and performing stimulation incubation; mixing the magnetic beads coated with the cytokine / immunoreaction secretion capture antibody and the enzyme-labeled cytokine / immunoreaction secretion detection antibody, and then mixing and incubating with the cell droplets; carrying out magnetic enrichment on the magnetic beads, and adding fluorescent substrate liquid drops for reaction; fluorescence intensity detection is carried out on the magnetic bead liquid drops, and positive and negative signals are identified, so that negative, strong-positive or weak-positive cell liquid drops after stimulation incubation are distinguished; positioning specific immune response positive cell liquid drops, screening and recycling, and / or continuously diluting and dispersing to complete specific immune cell screening.

The invention relates to the technical field of nucleic acid molecule detection, and discloses a multiple nucleic acid detection kit based on CRISPR-Cas12a, the multiple nucleic acid detection kit comprises a plurality of fluorescence coding microspheres, and each fluorescence coding microsphere comprises a lipid membrane coated solid phase carrier microsphere, a lipid membrane coated solid phase carrier microsphere, a lipid membrane coated solid phase carrier microsphere and a lipid membrane coated solid phase carrier microsphere, the Cas12a / crRNA compound is embedded into the lipid membrane through hydrophobic interaction, and the 3'end of the crRNA is covalently connected with a cholesterol group; the fluorescent substrate probe is embedded into the lipid membrane through hydrophobic interaction and is single-stranded DNA, the 3'end of the fluorescent substrate probe is covalently connected with a cholesterol group, and the 5 'end of the fluorescent substrate probe is covalently connected with a fluorescent group. Different crRNA / Cas12a compounds and fluorescent substrate probes are jointly anchored on the surfaces of the microspheres with different optical codes, each microsphere corresponds to one specific target, multiple nucleic acid targets can be simultaneously detected in the same reaction system, and the detection flux and efficiency are remarkably improved.

The invention discloses an intracellular glycosylated RNA (Ribonucleic Acid) analysis method for modifying a retardation nucleic acid hybridization technology. Analysis detection and in-situ imaging of intracellular glycosylated RNA (glycoRNAs) are realized on the basis of structural transformation of a target nucleic acid mediated fluorescent substrate nucleic acid chain. Specifically, a substrate chain with toehold is designed according to a sequence of microRNA-21 and a glycosylation modification site of the microRNA-21, and the sequence of the substrate chain contains a specific sequence of a Pepper aptamer capable of being combined with a fluorescent small molecule dye HBC. And the sequence design of the substrate meets the condition that when the substrate is hybridized with glyco-microRNA-21 to a glycosylation modification site, the structure of the generated hybridized compound can just generate an 'ON' fluorescence signal. The hybridization of the substrate sequence and the microRNA-21 is full hybridization, and the formed hybridization product cannot generate a fluorescence signal. According to the invention, the detection of low-abundance glycoRNAs under the interference of a large number of non-glycosylated RNAs is realized, and the intracellular in-situ imaging of the glycoRNAs is realized.

The application discloses a compound, a composition and application, and claims the compound shown in formula (I) or a salt thereof. The compound provided by the application is an enzyme responsive fluorescent substrate specially designed for sulfotransferase SULT1A1, and precise recognition and specific catalytic response to SULT1A1 are realized from the molecular structure level. The compound has a low fluorescent background itself, the sulfonated fluorophore in the catalytic reaction product has the characteristics of near-infrared emission and large Stokes shift, can effectively avoid the background interference of excitation light on fluorescent detection, and is excellent in specificity, stability and linearity. Therefore, the compound or the salt thereof and the composition containing the compound or the salt thereof have the prospect of being developed into a reagent, a kit or a microplate for in vitro quantitative or qualitative detection of SULT1A1 activity, in situ imaging and positioning of SULT1A1 in cells or in vivo, and high-throughput screening of SULT1A1 inhibitors or activators.

The present invention provides a fluorescence-based screening method for aryl hydrocarbon receptor (AhR) modulators and its application. The detection principle is to quantitatively evaluate the regulatory effect of small molecules on AhR by biochemically detecting the activity of cytochrome P450 1A1 (CYP1A1) downstream of AhR. To this end, the present invention first developed a class of specific fluorescent substrates suitable for CYP1A1 functional imaging analysis in living cells. Its general structural formula is shown in formula (1): This type of substrate can be specifically catalyzed by intracellular CYP1A1 under physiological conditions to undergo a dechloroethylation reaction and produce a single fluorescent product HDBI. The activity of CYP1A1 in living cells / living tissues can be quantitatively detected by detecting the amount of fluorescent product HDBI generated per unit time, thereby indirectly evaluating the agonist activity of AhR. Based on this type of fluorescent substrate, a living cell-based CYP1A1 functional detection method is constructed, which can be used for high-throughput screening and evaluation of the regulatory effect of compounds on the AhR-CYP1A1 axis, and further evaluate the regulatory effect of compounds on the CYP1A1 upstream gene AhR.

The invention discloses an anti-dextran interference endotoxin detection method and system, and belongs to the technical field of detection and analysis of bioactive substances, and the method comprises the following steps: obtaining a dynamic fluorescence signal of a reaction mixture after a sample to be detected is mixed with a specific reaction system containing a recombinant C factor enzyme solution and a fluorescent substrate, and generating a dynamic baseline model; carrying out feedback incubation control on the reaction mixture according to the dynamic baseline model and the functional model for indicating the standard dynamic behavior, generating actual dynamic data, and carrying out multi-dimensional signal fusion calculation in combination with the dynamic baseline model to generate a fusion fluorescence signal difference value; and performing self-verification concentration judgment according to the fused fluorescence signal difference value and the curve family for indicating the relationship between the endotoxin and the fluorescence response, and generating the endotoxin concentration of the sample. By adopting a dynamic baseline model, feedback incubation control, multi-dimensional signal fusion calculation and self-verification concentration judgment, non-specific interference such as glucan can be eliminated, and the accuracy and reliability of an endotoxin quantitative result are ensured.

The invention provides a urine kidney injury molecule-1 protein immunofluorescence detection reagent detection method and a urine kidney injury molecule-1 protein immunofluorescence detection reagent detection device, which are applied to the technical field of biomedical detection, a sample is pretreated to obtain a diluted urine sample, then the pH range of the diluted urine sample is adjusted, and then a compound is formed by using specific recognition of an antibody to an antigen. The immune complex is captured by using a capture antibody fixed in a detection area in advance, so that the immune complex is enriched in the detection area, and a detection signal is improved. Dropwise adding a fluorescent substrate into the detection area, and finally detecting the fluorescence intensity of the detection area by using a portable fluorescence detector to obtain the concentration of the urine kidney injury molecule-1 protein in the urine sample so as to realize quantitative detection. Therefore, rapid, simple and quantitative detection of the urine kidney injury molecule-1 protein in the urine sample is realized.

Methods of reducing background noise in signal-generating digital assays, such as fluorescent digital immunoassays, using a colorant are disclosed. The methods utilize a binding member that specifically binds to an analyte in a biological sample. The binding member is conjugated to a signal generating compound, e.g., an enzyme, which is dissociated from the binding member. A colorant is added with a signal generating substrate, e.g., a fluorogenic or chromogenic substrate for the enzyme, or after a signal generating substrate to reduce background noise in a fluorescent digital immunoassay. The signal generated between the signal generating compound and the signal generating substrate is detected and correlated to the presence and / or concentration of the analyte.

The invention belongs to the field of biomedical engineering, and particularly relates to a device for monitoring the growth condition of bacterial colonies in a culture dish in real time based on a multi-angle high-definition camera. A detection module of the device is integrated with three 48,000,000-pixel high-definition cameras which are symmetrically arranged in a one-vertical and two-horizontal manner, and images of a culture dish are acquired from multiple angles. And the image processing system carries an RAFT-Stereo algorithm, adopts a three-camera parallax array and closed-loop calibration strategy, and calculates internal and external parameters of the cameras through OpenCV stereoCalibrate to realize accurate analysis of the bacterial colony thickness. The device can be used for monitoring the size, spatial density, hypha network distribution and three-dimensional morphological parameters of bacterial colonies in the culture dish in real time and with high precision, as well as multi-strain competition dynamics, blue-white spot screening and metabolic activity based on a chromogenic or fluorescent substrate culture medium. The method breaks through the limitation of traditional two-dimensional monitoring, significantly improves the accuracy and repeatability of bacterial colony monitoring, and provides comprehensive and accurate data support for microbiological research.

The invention relates to the field of biomolecule detection methods, in particular to magnetic gold nanoparticles and a green preparation method and application thereof. The preparation method comprises the following steps: adding water into tea leaves and heating; filtering and collecting filtrate; preparing a thiol modified Fe2O3 suspension liquid; preparing magnetic gold nanoparticle turbid liquid; the application method comprises the following steps: adding the capture antibody into magnetic gold nanoparticle turbid liquid; adsorbing with a magnet, and removing supernate; adding confining liquid; washing is performed; adding biomolecules to be detected; adsorbing with a magnet, and removing supernate; adding a detection antibody; adsorbing with a magnet, and removing supernate; streptavidin marked with enzyme is added; adsorbing with a magnet, and removing supernate; adding a chemical fluorescent substrate; and adding a stop solution, and detecting the absorbance of the solution. The method not only solves the problems of more impurities, larger particle size change and complicated centrifugal treatment in ELISA (enzyme-linked immuno sorbent assay) detection of the non-magnetic gold nanoparticles prepared by the existing method, but also can be further applied to the future chip ELISA biomolecular detection.

The invention discloses an ADP-ribosylated derivative and a preparation method and application thereof, the ADP-ribosylated derivative comprises two types, the I type derivative is an ADPr-fluorescent probe which is synthesized by directly connecting ADPr and a hydroxyl fluorophore through an O-glucosidic bond; the type II derivative is a triazole derivative which is chemically synthesized by ADPr-N and a specific alkynyl compound through click. The invention also specifically discloses a preparation method of the ADP-ribosylated derivative. The prepared I type derivative is a high-performance fluorescent substrate of ARH3; in-vitro enzyme activity detection proves that various II-type derivatives have remarkable inhibitory activity on ARH3, can be used as ARH3 inhibitors for researching life processes related to ADP-ribosylation, and have application potential in ARH3-mediated disease intervention.