Detail publikace

Microfluidic tool coupled with electrochemical assay for detection of lactoferrin isolated by antibody-modified paramagnetic beads

ZÍTKA, O. KŘÍŽKOVÁ, S. SKALIČKOVÁ, S. FIALOVÁ, D. ADAM, V. KIZEK, R.

Originální název

Microfluidic tool coupled with electrochemical assay for detection of lactoferrin isolated by antibody-modified paramagnetic beads

Typ

článek v časopise ve Web of Science, Jimp

Jazyk

angličtina

Originální abstrakt

Lactoferrin (LF) is approximately 80kDa iron-binding protein, which is important part of saliva and other body fluids. Due to its ability to bind metal ions, it has many biologically important functions. In this study, a method for the isolation of LF from a biological sample using robotically prepared antibody-modified paramagnetic particles was developed using robotic pipetting station. The method consisted of the following optimised steps. ProteinG was bound on the paramagnetic particles, on which goat antibody (10g) was linked. LF was subsequently added to microtitration plate, which had affinity to goat antibody and the interaction lasted for 30min. We found that the highest signals were obtained using the combination of goat antibody 1:3000, murine antibody 1:5000 and conjugate 1:1500. Horseradish peroxidase reducing 3,3,5,5-tetramethylbenzidine (TMB) was linked to the merged complex. The resulted product of this reaction was subsequently analysed spectrometrically with detection limit (3 S/N) as 5 ng/mL. In addition, we also determined TMB by stopped flow injection analysis with electrochemical detection. The limit of detection (3 S/N) was estimated as 0.1g/mL. To compare spectrometric and electrochemical approach for detection of TMB, calibration range of bead-LF-antibodies complex was prepared and was determined using a least-squares correlation with coefficient R-2 higher than 0.95, indicating a very good agreement of the results obtained.

Klíčová slova

Electrochemistry; ELISA; Lactoferrin; Magnetic particles; Stopped flow injection analysis

Autoři

ZÍTKA, O.; KŘÍŽKOVÁ, S.; SKALIČKOVÁ, S.; FIALOVÁ, D.; ADAM, V.; KIZEK, R.

Vydáno

1. 7. 2013

ISSN

0173-0835

Periodikum

Electrophoresis

Ročník

34

Číslo

14

Stát

Spolková republika Německo

Strany od

2120

Strany do

2128

Strany počet

9

BibTex

@article{BUT145194,
  author="Ondřej {Zítka} and Soňa {Křížková} and Sylvie {Skaličková} and Dana {Fialová} and Vojtěch {Adam} and René {Kizek}",
  title="Microfluidic tool coupled with electrochemical assay for detection of lactoferrin isolated by antibody-modified paramagnetic beads",
  journal="Electrophoresis",
  year="2013",
  volume="34",
  number="14",
  pages="2120--2128",
  doi="10.1002/elps.201200631",
  issn="0173-0835"
}