Rapid capture and labeling of cells on single domain antibodies-functionalized flow cell

Abstract

Current techniques to characterize leukocyte subgroups in blood require long sample preparation times and sizable sample volumes. A simplified method for leukocyte characterization using smaller blood volumes would thus be useful in diagnostic settings. Here we describe a flow system comprised of two functionalized graphene oxide (GO) surfaces that allow the capture of distinct leukocyte populations from small volumes blood using camelid single-domain antibodyfragments (VHHs) as capture agents. We used site-specifically labeled leukocytes to detect and identify cells exposed to fungal challenge. Combining the chemical and optical properties of GO with the versatility of the VHH scaffold in the context of a flow system provides a quick and efficient method for the capture and characterization of functional leukocytes.

Enhanced Osteogenic Differentiation of Stem Cells on Phase-Engineered Graphene Oxide

“…GO solution (4 mg/mL, Sigma-Aldrich) was used as a stock solution, which was diluted with deionized water to 1 mg/mL before the coating of GO. The procedures of GO coating were same as described previously. , In brief, first, cleaned glass coverslips (sonicated for 30 min in acetone, 95% ethanol, and deionized water washing) were subjected to amine-functionalization with 3% 3-aminopropyltriethoxysilane (Sigma-Aldrich) into toluene for 30 min, followed by washing with 95% alcohol and deionized water and then drying by nitrogen gas. The functionalized glass coverslips were immersed in the GO solution for 1 h and placed on a rocker for gentle shaking to allow uniform GO immobilization on its surface.…”

“…Carboxyl groups on GO substrates were activated through the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide coupling reaction followed by immersing in 0.5 mM maleimide−poly(ethylene glycol)-NH 2 (PG2-AMML-400, Nanocs) in PBS for 6 h for carboxyl groups and primary amines bonding covalently. Subsequently, maleimide functional groups of GO substrates were cross-linked by incubating the osteogenic growth peptide with FITC-labeled cysteine CALKRQGRTLYGFGGK(FITC) in a concentration of 20 μg/mL in PBS containing 5 mM tris(2-carboxyethyl)phosphine hydrochloride for 2 h. 27,28 Statistical Analysis. The data were analyzed via one-way analysis of variance using the GraphPad Prism software and represented as the mean ± standard deviation based on at least three independent experiments and eight representative fluorescent images.…”

Broadening the scope of sortagging

“…Furthermore, several examples exist showing the labeling of proteins with uorescent dyes utilizing SML. The C-terminal modication with oligoglycine-functionalized dyes was performed 53,[125][126][127] as well as the N-terminal modication with LPETG-functionalized dyes. [128][129][130] Wu et al demonstrated that protein labeling can be also performed in living Caenorhabditis elegans cells.…”

Current Conjugation Methods for Immunosensors

“…In addition, transpeptidase can also be used to create site specific modification on VHH since a short peptide recognition sequence can be easily incorporated into recombinant expression vectors. The Ploegh group incorporated the sortase recognition motif, LPXTG, at the C terminus of VHHs, which can then be used for installation of a short GGG peptide with a biorthogonal handle ( Figure 4 B) [ 55 , 56 , 68 , 69 , 70 ].…”