Facile Comparison of Nonspecific Adsorptions of Proteins on Magnetic Submicron Particles with Alkaline Phosphatases as the Probes

Abstract

The soluble form of Escherichia coli alkaline phosphatase (ECAP), commercialized calf intestinal alkaline phosphatase (CIAP) and biotinylated CIAP (Biotin-AP) were tested as probes for nonspecific adsorptions of proteins on micro/nano materials represented by magnetic submicron particles (MSP). The nonspecific adsorptions of three candidate probes were evaluated by directly measuring the activities of the adsorbed probes with 4-nitrophenylphosphate. The activities of those three candidate probes exhibited reasonable resistance to detergents, but the presences of > 0.1% glycerol in the adsorption systems greatly reduced their nonspecific adsorptions on MSPs. The nonspecific adsorptions of ECAP and Biotin-AP on some MSPs were significant and saturable but became negligible after such MSPs were coated with small zwitterions, bovine serum albumin and/or streptavidin. Only MSPs applicable to chemiluminescence immunoassays showed negligible nonspecific adsorptions of Biotin-AP and ECAP, but all of the tested MSPs exhibited negligible nonspecific adsorptions of CIAP. Therefore, Biotin-AP and ECAP were suitable probes for nonspecific adsorptions of proteins on micro/nano materials.

Ampholytic ion-exchange magnetic beads: a promising tool for selecting short fragments in circulating cell-free DNA analysis

“…The denaturation reagents that were used lysed the cells and introduced genomic contamination. To test the effects of protein denaturation on the yields of plasma cfDNA with MSP-ZEWBs, guanidine hydrochloride and isothiocyanoguanidine were employed, as the adsorption of purified plasmids on MSP-ZEWBs are susceptible to common detergents but reasonably resistant to monovalent anions ( 21 , 25 ). The intensity of DNA ladder was decreased sharply in presence of denaturations reagent ( Figure 3A ).…”

“…Such differences may have led to the controversy regarding the best method for liquid biopsy of cancers through the analysis of plasma cfDNA ( 2 , 15 , 33 , 34 ). Notably, use of MSP-ZEWBs coated with abundant small zwitterions showed negligible nonspecific adsorption of proteins ( 23 , 25 ); indeed, the extraction of plasma cfDNA with MSP-ZEWBs required no protein denaturation and was resistant to white blood cells remaining in the plasma.…”

“…Preliminary studies found that MSP-ZEWBs readily adsorbed purified plasmids at a pH of approximately 4.0 in the absence of proteins and that the adsorbed plasmids were easily eluted at a pH of approximately 8.9. Notably, MSP-ZEWBs showed negligible nonspecific adsorptions of proteins ( 25 ), indicating that the adsorption of plasma cfDNA with MSP-ZEWBs at a pH of approximately 4.0 may need no protein denaturation and be resistant to cells remaining in plasma. For comparison of plasma cfDNA yields based on the quantification of a DNA sequence in plasma cfDNA via quantitative polymerase-chain-reaction (qPCR), a coding fragment in an exon of the Alu gene found in common blood cells is a favorable model to examined the interference of remaining blood cells in plasmas ( 26 , 27 ).…”