Graphene Oxide Nanosheets Modified with Single‐Domain Antibodies for Rapid and Efficient Capture of Cells

Abstract

Peripheral blood can provide valuable information on an individual’s immune status. Cell-based assays typically target leukocytes and their products. Characterization of leukocytes from whole blood requires their separation from the far more numerous red blood cells.[1] Current methods to classify leukocytes, such as recovery on antibody-coated beads or fluorescence-activated cell sorting require long sample preparation times and relatively large sample volumes.[2] A simple method that enables the characterization of cells from a small peripheral whole blood sample could overcome limitations of current analytical techniques. We describe the development of a simple graphene oxide surface coated with single domain antibody fragments. This format allows quick and efficient capture of distinct WBC subpopulations from small samples (~30 μL) of whole blood in a geometry that does not require any specialized equipment such as cell sorters or microfluidic devices.

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

“…We synthesized the probe used for VHH labeling, GGGK-[TAMRA]-K-N 3 , with an N terminal amine and a C terminal CONH 2 following standard solid phase synthesis protocols (Chen et al, 2015). Calcium-independent heptamutant Sortase A (Li et al, 2015) (10 μM final concentration) and probe (1 mM final concentration) were added to the VHH (200 μM final concentration) in PBS at 20 °C for 2 h. Ni-NTA (0.5 ml, Qiagen) was then added to the reaction mixture and mixed for 20 min to remove sortase and any unreacted VHH.…”

“…Slides were coupled with 0.5 mM poly(ethylene glycol) bis(amine) (MW 3000) at 25 °C, followed by installation of dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester (DBCO-NHS ester) in PBS for 6 h. Slides were incubated with 50 μM VHH7 or VHH DC13 labeled with peptide GGGK-TAMRA-K-N 3 in PBS for 12 h. The VHH7 and VHH DC13-GO functionalized nanosubstrates were assembled with another glass slide to form a chamber with two different coating surfaces (Agrawal et al, 2008; Chen et al, 2015). …”

“…Since a large percentage of the VHH surface is involved in binding interactions, it is essential to create a uniform orientation using site-specific modifications (Trilling et al, 2013, 2014) to improve the biosensor’s performance. To achieve consistency in orientation, we previously used a combination of sortase-mediated trans-peptidation reactions and ‘click’ chemistry to site-specifically link VHHs with linkers coated onto GO (Agrawal et al, 2008; Chen et al, 2015). The use of these techniques allowed for quick and efficient capture of a distinct leukocyte subpopulation from small volumes of blood.…”

30 years of advances in functionalization of carbon nanomaterials for biomedical applications: a practical review

“…We then conjugated VHH7 nanobodies (single-chain only fragments of conventional antibodies, which are specific to murine Class-II MHC 1 cells) to the DBCO handle. Using this chemistry, we were able to show highly efficient, very specific cell capture 35 of white blood cell populations from small volumes (;30 lL) of murine whole blood [ Fig. 8(g)], using a relatively inexpensive device (without the need for cell sorting or microfluidic apparatus), under ambient conditions.…”

“…We discuss examples of the Belcher group's work [Figs. 4(a)-4(c), 7(a), 7(b) and 8(g)] on the use of a biological template (M13 bacteriophage), as a scaffold for noncovalent functionalization of carbon nanotubes for detection of cancers 32,33 and infectious diseases, 34 and also the use of physical or chemical modification to functionalize graphene oxide (GO) for biosensing applications 35 involving cell capture from whole blood; using the same metrics described above. The goal of this Review is to provide an objective, quantitative (wherever possible) comparison of these parameters, to help researchers make an informed decision while choosing a functionalization technique for their desired application.…”