Beads stained

Plot the peak positions against the manufacturer s specified antibody binding capacity for each bead in the mixture to create a standard curve. Figure 3 illustrates curves for Simply Cellular beads stained with a PE-labeled antibody in a direct assay (A), and m an indirect assay with a PE-labeled antimouse IgG (B). The increased sensitivity of the indirect assay is evident from the rightward shift of the curve. 

Two step screening was done for the 430,000 library beads. In the first step, the beads stained by lpM NBD-DCA were selected. The fluorescent beads were picked up by a capillary using an inverted fluorescent microscope for observation. The 399 peptide beads obtained were then washed with ethanol. After washing, the beads still fluorescing were excluded since they had bound irreversibly to the NBD-DCA. Thus, only 18 beads were tested in the second step. In the second step, the beads were subjected to a competitive reaction to select only the peptides binding to the DCA moiety of the NBD-DCA. They were suspended in a 5mM DCA solution containing 5% ethanol for 3h and only those five beads for which the fluorescence decreased were selected. Finally, the sequences of the five beads obtained were determined by a standard Edman degradation method. 

The typical shape of the fluorescence signals in scattering media is shown in Fig. 5.55 and Fig. 5.56. The fluorescence of beads stained with IRD38 (Li-Cor, Inc.) embedded in agarose phantoms was recorded by TCSPC. A femtosecond titanium-sapphire laser was used for excitation, and a H7422-50 PMT module for detection. The IRF width of the system is about 300 ps. Figure 5.55 shows the variation in the recorded fluorescence decay data with the pH for beads at the surface of the phantom. 

It is important to verify reproducible staining behavior, so expect to strip and reprobe the beads a few times. A colorless bead after a specificity test should still bind and stain in the presence of the native, biotinylated target in the absence of a competing ligand. Reproducibility is an essential key to successful performance of the bead staining approach. 

A test for secondary amines (e.g. proline) is the Chloranil test (1 drop of a 2% acetaldehyde solution in DMF, followed by one drop of a 2% solution of p-chloranil in DMF, leave for 5 mins). A positive test gives blue stained beads. 

The on-bead assay was conducted according to Scheme 3.19, which shows the chain of events, which leads to a colorimetric response, when an oligosaccharide binds effectively to the B. purpurea lectin. The lectin was covalently linked to biotin, a small molecule with an extremely high affinity for streptavidin. The bead-lectin-biotin conjugates were then exposed to streptavidin, linked to the enzyme alkaline phosphatase. Alkaline phosphatase hydrolyses phosphate esters [e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 110]. When the 5-bromo-4-chloro-3-hydroxyindole (111) is released, in the presence of nitro blue tetrazolium (NBT), it forms a dark purple, insoluble dye, thus staining beads where there was a favorable binding interaction. 

Fig. 2 Chemical structures and typical spectral properties of the common fluorophores that can be used for staining of the polymeric beads...

Fig. 4 Schematic representation of the techniques used for making stained beads (a) staining during polymerization (b) staining via covalent or electrostatic coupling of an indicator to the beads surface (c) staining via swelling (d) preparation of dye-doped beads via precipitation (e) spray-drying (f), grinding...

Polymeric beads obtained via emulsion polymerization, precipitation, etc. can be stained with dyes providing that both have functional groups available [7]. Covalent coupling is mostly preferred but the attachment based on strong electrostatic interactions is also feasible. This method is mostly used to design pH- and ion-sensitive micro- and nanobeads. The dynamic response of such systems can be... 

Gouanve et al. [9] presented another approach to designing copper nanosensors. They prepared cross-linked polystryrene beads (0 14 nm) and functionalized the surface with 1,4,8,11-tetraazacyclotetradecane (Cyclam), which selectively bound copper ions. The core of the beads was stained with a lipophilic fluorescent dye 9,10-diphenylanthracene by swelling. Fluorescence of the dye was quenched in the presence of Cu2+ due to FRET. The particles were suitable for sensing Cu2+ in micromolar concentrations. 

---