Smart blood spots for whole blood protein analysis

Abstract

A reactor for whole blood sampling integrated with instant protein digestion in a "lab-on-paper" format is introduced here. The sampling reactor was fabricated on commercially available filter paper using 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (HEMA-VDM) polymerization followed by the immobilization of trypsin. Immobilization conditions were investigated with respect to temperature, enzyme amount and immobilization time. The highest reactor efficiency with respect to protein digestion was obtained with 1.25 mg trypsin per reactor immobilized at room temperature for 3 hours. Commercially available cellulose filter papers and DMPK-C cards were modified and immobilized with trypsin prior to whole blood sampling. Filter paper specifications including thickness (180-220 μm), weight (77-92 g m-2) and porosity (11-25 μm) were investigated with respect to performance (digestion efficiency and extraction recovery). From this study, it was found that a medium thickness paper with higher weight and porosity is optimal for reactor efficiency. The reactors were tested and compared with respect to a standard dried blood spot procedure for protein digestion. The most efficient reactors obtained 134 ± 14 and 124 ± 7 high confidence protein groups for freeze thawed and fresh whole blood samples, respectively.

Next-Generation Dried Blood Spot Samplers for Protein Analysis: Describing Trypsin-Modified Smart Sampling Paper

“…Functionalization using HEMA-VDM: For the HEMA-VDM functionalization, the procedure published by Skjaervø et al [29,[36][37][38] was slightly modified and used. In short, the fibers on the paper discs were first silanized by applying 10 µL of freshly prepared silanizing solution (this is a mixture of 2.5 mg DPPH, 330 mg DMF and 172 mg γ-MAPS prepared in a glass vial).…”

“…Prior to injection, clean-up was carried out on 100 µL of the sample using in-housemade solid phase extraction as described by [29]. After drying, the residue was reconstituted using 100 µL 0.1% formic acid and transferred to the injection vial.…”

“…Here, cellulose was first silanized, then functionalized through the polymerization of 2hydroxyethyl methacrylate (HEMA) and 2-vinyl-4,4-dimethylazlactone (VDM), allowing trypsin to bind covalently to paper [29]. Besides this protocol, there are other possibilities to bind proteins covalently to paper [30][31][32][33][34], among which is also the simple KIO 4 -mediated oxidation of cellulose to create two aldehyde groups per cellulose unit, allowing subsequent protein binding.…”

“…The long-term goal of our research is to simplify MS-based protein analysis from dried matrix samples (such as DBS), and we aim to integrate time-consuming procedures within the sampling device for a prompt start to the sample preparation upon sample collection [29,[35][36][37][38].…”

Smart proteolysis samplers for pre‐lab bottom‐up protein analysis – Performance of on‐paper digestion compared to conventional digestion

“…Our group has been working with the concept of smart sampling in combination with MS determination since 2017 [45]. This concept is based on the covalent binding of trypsin to cellulose allowing a DBS sampler to start with the digestion step at the moment of sampling [46][47][48][49]. This smart sampler does not compromise the ease of sampling as the format remains practically unchanged.…”