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Membrane strip biosensor system for point-of-care testing
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A technology of biosensors and membrane strips, applied in the field of biosensors, can solve problems such as difficulties
Inactive Publication Date: 2006-05-31
BIODIGIT LAB CORP
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Therefore, for field testing, it is clearly difficult to use these complex, multi-step methods
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Embodiment 1
[0100] Example 1: Synthesis of antibody-colloidal gold conjugates
[0101] The acidity and antibody concentration of various reaction solutions were measured according to the standard protocol, and then the conjugates were synthesized under the optimal conditions (references: S.H.Paek et al., 1999, Anal.Lett., Vol.32, 335-360 ).
[0102] Briefly, dialyzed HBsAg-specific antibody solution (100 mg / ml, 0.8 ml) in neutral pH buffer was added to gold solution (8 ml), adjusted to pH 9.0, and reacted for 30 minutes. Then, 1 ml of a 5% casein solution (casein-PB) prepared by dissolving casein in 10 mM phosphate buffer (pH 7.4, PB) was added to the solution and reacted for 30 minutes. After the reaction solution was centrifuged at 15,000 rpm for 45 minutes, the supernatant was removed. Casein-PB was added to the remaining gold precipitate and the final volume of the conjugate was adjusted to 0.2 ml, stored at 4°C until use.
Embodiment 2
[0103] Example 2 Synthesis of antibody-enzyme conjugates
[0104] Conjugation between the analyte-specific antibody and the enzyme is performed by a chemical reaction using a cross-linking agent. After reacting the antibody with a 20-fold molar excess of SMCC for 4 hours at 4°C, the excess SMCC was removed by Sephadex G-15 gel chromatography, and the antibody was directly conjugated with the activated enzyme as described below. For enzyme activation, protein solutions were reacted in 5 mM EDTA-containing PB with 20-fold molar excess of SPDP for 1 hour at room temperature. To introduce sulfhydryl groups on the molecule, DTT (final concentration 10 mM) was added to the reaction mixture and reacted at 37° C. for another 2 hours. Excess reagents were removed on Sephadex G-15 gel columns. Two activation reagents, antibody and enzyme, were mixed at a molar ratio of 1:10 and reacted overnight at 4°C. Purification of these synthesized antibody-enzyme conjugates was performed using ...
Embodiment 3
[0105] Example 3 Signal generating shims with immobilized antibodies
[0106]Nitrocellulose (NC) membranes optimized for migration efficiency and pore size were used as signal generating spacers. An NC membrane (pore size: 12 mm, Millipore) was used for antibody immobilization. Physical adsorption and chemical methods can be used as immobilization methods, and the appropriate method is finally selected by considering the convenience and reproducibility of the method according to the results of the experiment. Antibodies were immobilized at predetermined positions on NC membrane strips (7 x 25 mm) by physical adsorption. 1 mg / ml antibody (1.5 ml) diluted with PB containing 140 mM NaCl was spotted on one position (10 mm from the bottom) of the membrane using a microdispenser, followed by reaction at room temperature for 1 hour. The strips with immobilized antibodies were soaked in 0.5% casein (pH 7.6, casein-Tris) dissolved in 100 mM Tris buffer for 1 hour to block the residua...
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Abstract
The present invention relates to a biosensor for point-of-care testing (POCT) whose detection sensitivity was remarkably improved by introducing to membrane strip chromatographic assay system a successive cross-flow procedure for immune reaction and enzymatic reaction. The present invention relates to a membrane strip biosensor system that comprises (a) a membrane pad ( 10 ) for sample application, (b) a membrane pad ( 20 ) for release of detection binding component, wherein the membrane pad ( 20 ) contains label-linked binding component for detection in a dry state, (c) a signal generation membrane pad ( 30 ) with immobilized binding component for capture, (d) a membrane pad ( 40 ) for absorption of vertical flow medium, (e) a membrane pad ( 50 ) for the supply of substrate solution for enzyme, (f) a membrane pad ( 60 ) for absorption of horizontal flow medium and (g) substrate solution, wherein the system has a cross-arrangement of two groups of the membrane pads, (I) one group of vertically arranged pads, wherein the pad ( 10 ) is partially superimposed and fixed in length at the end of the pad ( 20 ), and the pad ( 20 ) and the pad ( 40 ) are partially superimposed and fixed in length at the both ends of the signal generating membrane pad ( 30 ), respectively; and (II) the other group of horizontally arranged pads, wherein the pad ( 50 ) and pad ( 60 ) are, at the time of signal generation, partially superimposed and fixed at the both lateral sides of the signal generation membrane pad ( 30 ), respectively.
Description
technical field [0001] The present invention relates to a biosensor for Point-of-care Testing (POCT) by incorporating a sequential cross-flow procedure of immune and other reactions for signal generation The introduction into the membrane strip chromatography detection system can significantly improve the analytical performance of the biosensor. technical background [0002] The determination of low concentrations of disease markers (metabolites, proteins, cells, etc.) in body fluids is usually achieved by using biological reactions such as enzyme reactions and antigen-antibody binding. Determination of analytes in complex media is possible because enzymes and antibodies have very high reaction specificity for selectively recognizing their reactive ligands and high reaction efficiency. It is very important to seek the development of a diagnostic system based on these response characteristics for early diagnosis of diseases and adequate treatment of diseases at an early stag...
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