Competitive method-based electrochemiluminescent assay with protein-nucleotide conversion for ratio detection to efficiently monitor the drug resistance of cancer cells - PubMed (original) (raw)

. 2016 Dec 1;7(12):7094-7100.

doi: 10.1039/c6sc02801b. Epub 2016 Aug 4.

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Competitive method-based electrochemiluminescent assay with protein-nucleotide conversion for ratio detection to efficiently monitor the drug resistance of cancer cells

Wen-Bin Liang et al. Chem Sci. 2016.

Abstract

A simple and highly-efficient approach to monitor the expression of P-glycoprotein (P-gp) in cells was urgently needed to demonstrate the drug resistance of cancer cells. Herein, a competitive method-based electrochemiluminescent (ECL) assay with a single ECL indicator was proposed for the first time to efficiently estimate the concentration ratio of two proteins. By converting the different proteins to partially coincident nucleotide sequences via a sandwich type immunoassay on magnetic beads, the concentration ratio related ECL signals could be obtained via competitive nucleotide hybridization on an electrode surface. This method could thoroughly overcome the limitations of simultaneous ECL assays via multiple ECL indicators with inevitable cross reactions. At the same time, rolling circle amplification was employed to improve the detection performances, especially the detection limit and sensitivity. With P-gp and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a model, the proposed ECL assay was successfully employed to monitor the drug resistance of cancer cells. Compared with conventional technologies, improved sensitivity and accuracy were achieved with a correlation coefficient of 0.9928 and a detection limit of 0.52%. Success in the establishment of the competitive method-based ECL assay offered an efficient strategy to demonstrate the concentration ratio of two proteins and a potential approach for detecting other proteins and nucleotide sequences, revealing a new avenue for ultrasensitive biomolecule diagnostics, especially in cell function research.

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Figures

Scheme 1

Scheme 1. Schematic diagrams of the competitive method-based ECL assay to demonstrate the concentration ratio of P-gp and GAPDH as a model. (A) Schematic diagrams of the sandwich type immunoassay on magnetic beads to convert the different proteins to partially coincident nucleotide sequences; (B) schematic diagrams of the competitive reaction on the sensor surface to obtain the concentration ratio related ECL signals.

Fig. 1

Fig. 1. Characterization of biosensors with different fabrications based on CV (A) and EIS (B); curve a, bare GCE; curve b, AuNPs/GCE; curve c, CNS(A)/AuNPs/GCE; curve d, HT/CNS(A)/AuNPs/GCE. Inset, the equivalent circuit with model impedance data of EIS responses.

Fig. 2

Fig. 2. (A) CV (red line) and ECL–potential (blue line) results of the biosensor in PBS with 25 mM TPrA as coreactant (MCF-7/ADR : MCF-7 = 1 : 2); (B) ECL–time signals of the competitive method-based ECL assay for MCF-7 cells (black line) and MCF-7 cells with MCF-7/ADR cells (MCF-7/ADR : MCF-7 = 1 : 100) (red line).

Fig. 3

Fig. 3. (A) ECL–time responses of the as-proposed competitive method-based ECL assay for different ratios of MCF-7/ADR with MCF-7; (B) calibration curve for the relationship between the ECL intensity and the logarithm of the ratio of MCF-7/ADR : MCF-7.

Fig. 4

Fig. 4. (A) Calibration curve for the relationship between the western blotting responses and the ratio of MCF-7/ADR with MCF-7 (insert, the image of the western blotting results of MCF-7/ADR with MCF-7 with ratios of 9 : 1, 8 : 2, and 5 : 5, respectively); (B) calibration curve for the relationship between the immunofluorescence results with confocal laser scanning microscopy and the ratio of MCF-7/ADR with MCF-7 (insert, the image obtained by confocal laser scanning microscopy of MCF-7/ADR with MCF-7 with ratios of 9.9 : 0.1, and 5 : 5, respectively).

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