Preliminary development of a DNA aptamer-magnetic bead capture electrochemiluminescence sandwich assay for brain natriuretic peptide

Abstract

Fifty-two candidate DNA aptamer sequences were selected for binding to the cardiovascular biomarker B-type or brain natriuretic peptide (BNP). Candidate aptamers were screened to rank their relative affinities against BNP by an aptamer-based ELISA-like aptamer microplate assay (ELASA). The highest affinity aptamers from ELASA screening were also paired in all possible combinations and screened for electrochemiluminescence (ECL) assay potential in capture aptamer-magnetic bead and ruthenium trisbipyridine (Ru(bpy)32+)-reporter aptamer sandwich formats. The top ECL sandwich combinations utilized the same aptamer pair in either capture or reporting roles with nanogram to low picogram per mL levels of detection even in 50% human serum. ECL assay sensitivity and linearity even in 50% human serum suggest that the aptamer-based assay is at least comparable to other reported immunoassays for BNP.

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“…The peptides were dissolved and diluted up to 500 µg/ mL in sterile phosphate buffered saline (PBS, pH 7.2) conjugated to Dynal TM (Life Technologies, Inc., Carlsbad, CA) tosyl-M280 (2.8 µm diameter) MBs at a ratio of 40 µg of peptide per 100 µL of stock tosyl-MBs by virtue of having primary amines in their structures. Nine rounds of MB-SELEX DNA aptamer development were conducted according to previously published protocols [1,2,12,13] except that the aptamers were either selected in 10% pooled human AB serum (Mediatech Inc., Cat. No.…”

“…After each round of SELEX, the presence of a 100 bp aptamer PCR product in a 2% agarose gel stained with ethidium bromide was used to verify the SELEX process and enable progression to the next round. All DNA SELEX templates, primers as previously described [1,2,12,13], and biotinylated candidate aptamers were synthesized at Integrated DNA Technologies, Inc. (IDT; Coralville, IA).…”

“…Check for updates 2 known as AA-3 and GHRP-6 (abbreviated G6 herein) whose structures and MWs are shown in Figure 1, because they had developed successful high-affinity and highly specific aptamers to several other physiologically active peptides previously [12,13]. The authors further sought to demonstrate the utility of the lead GHRP aptamers when attached to magnetic microbeads (MBs) to pull down, purify and enrich [14,15] their cognate analytes from buffer, human serum and urine with validation by gel electrophoresis and mass spectrometry (MS) as reported herein.…”

A Review of Electrogenerated Chemiluminescent Biosensors for Assays in Biological Matrices

“…Immunoassay; triply amplified at a glassy carbon-Au-graphene electrode Swine serum 0.40 pg/ml 1 pg/ml-50 ng/ml [39] Brain natriuretic peptide DNA aptamer-magnetic bead capture ECL sandwich assay 50% human serum Low pg/ml [40] AMP: Antimicrobial peptide; ECL: Electrogenerated chemiluminescence; PIGE: Paraffin-impregnated graphite electrode; SWNT: Single-walled carbon nanotube.…”

“…Therefore, high-sensitivity assays for this biomarker are needed for diagnosis. Preliminary work was reported for a DNA aptamer-magnetic bead capture ECL sandwich assay [40]. In this sandwich-type assay, capture-aptamer magnetic beads conjugated with BNP, which in turn hybridized with reporter Ru(bpy) 3 2+ -aptamer, which provided an ECL signal in the presence of TPrA.…”