Color-produing diagnostic systems, reagents and methods
a diagnostic system and dye technology, applied in the field of dye dyeing diagnostic systems, reagents and methods, can solve the problems of inability to meet the poc application of dye dyeing tests, inability to reliably detect the color of dyeing labels,
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example 1
Direct Coupling of Hydrogen Donors with Primary Amino Groups to Carboxylate Modified Particles
[0104]Hydrophilic carboxylate-modified latex particles (CML) with a high density of carboxylic groups and a diameter of 0.2-3.0 uM were washed with water and 0.1M MES buffer, pH 6.3 using centrifugation at 14,000 rpm for 3-5 minutes using siliconized polypropylene tubes. To a final suspension (4% solid bead volume) of washed particles, 20-40 mM solutions of donors, or mixtures of donors with biotin-PEG-amine (or diamino-PEG), in 0.1M MES, pH 6.3-6.8 were added. The concentration of biotin-PEG-amine or diamino-PEG was 10% of that of hydrogen donors, usually as a mixture, 18 mM and 2 mM, correspondingly. The final concentration of particles was 1-2% solid. Solutions of EDAC (1M in 0.1M MES, pH 6.3) and 0.4M NHS were added into tubes to a final concentration of EDAC and NHS in reaction mixtures, 0.1M and 0.04M, respectively. Reactions proceeded with gentle shaking for 60-90 minutes at room tem...
example 2
Coupling of Hydrogen Donors with Carboxylate Particles Through BSA / Polyacrylic Acid or BSA / Polymaleic Acid Spacers
[0105]Washed hydrophilic carboxylate particles prepared as described in Example 1 were first activated with EDAC / NHS in suspension (1-2% solid particle volume) in 0.1M MES, pH 6.3 with 0.1 M EDAC and 0.04 M NHS for 30 min. Activated particles were centrifuged and resuspended in 0.1M MES, pH 6.8 to a concentration of 1% solid. BSA solution in 0.1M MES, pH 6.8 was added to final BSA concentration 5 mg / ml. Coupling reaction proceeded for 60 min with gentle shaking Particles with immobilized BSA were washed several times with PBS-EDTA and finally with 0.1M MES, pH 6.3. BSA particles could be used for direct loading with hydrogen donors or additionally modified with polyacrylic acid or polymaleic anhydride to increase the density of carboxyl groups.
[0106]For loading onto BSA particles, polyacrylic acid (MW 2K, 6K or 15K) was first activated at a concentration of 5% with EDAC / ...
example 3
Synthesis of Water Soluble Polycarboxylic Polymer-Hydrogen Donor Conjugates
[0110]3.1. BSA-EDTA / EGTA-Hydrogen Donors / PEG-Biotin Conjugates.
[0111]Hydrogen donors can be coupled with BSA directly using the EDAC / NHS activation chemistry described for the synthesis of particle-based donors. However, higher loading of hydrogen donors to BSA can be achieved by modifying the amino groups of BSA with a low molecular weight, branched tetra-acetic molecule such as EDTA and EGTA, as described below.
[0112]EDTA and EGTA at 0.25M were partially activated with 50 mM EDAC / 20 mM NHS in 0.1M MES, pH 6.3. At these concentrations, ˜25% of the available carboxyl groups in tetracetic acid compounds are activated. The activation reaction proceeded for 30 minutes and activated EDTA or EGTA were added immediately into 80-100 mg / ml BSA solution in 0.1M MES, pH 6.3 to make reaction mixtures containing BSA at 40-50 mg / ml and a concentration of activated tetra-acetate compound at 125 mM. Reaction mixtures were i...
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