Analytical and Bioanalytical Chemistry

, Volume 405, Issue 12, pp 4017–4026

Superior performance of liposomes over enzymatic amplification in a high-throughput assay for myoglobin in human serum

  • Katie A. Edwards
  • Katherine J. Meyers
  • Barbara Leonard
  • Antje J. Baeumner
Research Paper

DOI: 10.1007/s00216-013-6807-3

Cite this article as:
Edwards, K.A., Meyers, K.J., Leonard, B. et al. Anal Bioanal Chem (2013) 405: 4017. doi:10.1007/s00216-013-6807-3

Abstract

Myoglobin is one of several cardiac markers which become elevated in the blood following an acute myocardial infarction and can aid in the diagnosis of a heart attack. Here, a sandwich immunoassay for myoglobin was developed, including a thorough optimization of fluorescent dye-encapsulating liposomes versus enzymatic amplification (alkaline phosphatase and horseradish peroxidase) at each step. The optimized microtiter plate-based assay was capable of detecting as low as 11.3 pg/mL myoglobin and was successfully applied for the quantification of myoglobin in human serum. In comparison to enzymatic approaches, the liposomes demonstrated lower limits of detection, significantly reduced limits of quantification, improved signal discrimination through substantial signal enhancement, and reduced assay time. Liposomes were stable and functional at ambient temperatures for over 400 days. Finally, ease of use was greater due to lack of reliance on additional reagents, non-time-based signal enhancement, and excellent photostability. Optimal conditions identified for enzymatic approaches can also be used for liposome amplification, which makes substitution of these liposomes into existing assays straightforward. Thus, the extensive studies carried out here suggest that liposomes may be incorporated into formats currently utilizing enzymatic enhanced fluorescence with a potential for increased performance on various levels.

Sandwich immunoassay for the cardiac marker myoglobin. Excellent performance of fluorescent dye-encapsulating liposomes for signal enhancement versus enzymes using commercially available fluorescent substrates was demonstrated.

Keywords

Sandwich immunoassay Liposomes Myoglobin Enzymes Fluorescence 

Abbreviations

AMI

Acute myocardial infarction

AUR

Amplex UltraRed

DPPC

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine

DPPG

1,2-Dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], sodium salt

EDC

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

FDP

Fluorescein diphosphate

HSS

HEPES–saline–sucrose

LOQ

Limit of quantitation

OG

n-Octyl-β-d-glucopyranoside

PBS

Phosphate-buffered saline

SRB

Sulforhodamine B

Supplementary material

216_2013_6807_MOESM1_ESM.pdf (1.1 mb)
ESM 1(PDF 1.08 MB)

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Katie A. Edwards
    • 1
  • Katherine J. Meyers
    • 1
  • Barbara Leonard
    • 1
  • Antje J. Baeumner
    • 1
  1. 1.Department of Biological and Environmental EngineeringCornell UniversityIthacaUSA

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