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. 2019 Oct 1:142:111522.
doi: 10.1016/j.bios.2019.111522. Epub 2019 Jul 17.

A flow-through microfluidic system for the detection of circulating melanoma cells

Mylamparambil Udayan Anu Prathap  1 , Edgardo Castro-Pérez  2 , José A Jiménez-Torres  3 , Vijaysaradhi Setaluri  4 , Sundaram Gunasekaran  5
Affiliations

A flow-through microfluidic system for the detection of circulating melanoma cells

Mylamparambil Udayan Anu Prathap et al. Biosens Bioelectron. .

Abstract

We report the fabrication of polyaniline nanofiber (PANI)-modified screen-printed electrode (PANI/SPE) incorporated in a poly-dimethylsiloxane (PDMS) microfluidic channel for the detection of circulating tumor cells. We employed this device to detect melanoma skin cancer cells through specific immunogenic binding of cell surface biomarker melanocortin 1 receptor (MC1R) to anti-MC1R antibody. The antibody-functionalized PANI/SPE was used in batch-continuous flow-through fashion. An aqueous cell suspension of ferri/ferrocyanide at a flow rate of 1.5 mL/min was passed over the immunosensor, which allowed for continuous electrochemical measurements. The sensor performed exceptionally well affording an ultralow limit of quantification of 1 melanoma cell/mL, both in buffer and when mixed with peripheral blood mononuclear cells, and the response was log-linear over the range of 10-9000 melanoma cells/10 mL.

Keywords: Electrochemical immunosensor; Melanoma cells; Microfluidics; Polyaniline nanofibers.

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Conflict of interest statement

Conflicts of interest

There are no conflicts to declare.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
(a) Microfluidic device components and assembly sequence. (b) Scheme for MC1RAb-PANI/SPE electrochemical immunoassay for melanoma cell detection. (c) The assembled microfluidic device.
Fig. 2
Fig. 2
Nyquist plot for the MC1R-Ab-PANI/SPE electrode recorded in the frequency range of 0.1 Hz to 10 kHz at a standard potential of +0.15 V, using a sinusoidal potential perturbation of 5 mV amplitude in 0.1 M KCl solution containing 5 mM [Fe(CN)6]3−/4−, with different concentrations of (a) SK-MEL-2 cells and (b) HEK-293 cells. (c) Bode plot for the electrode recorded in the frequency range of 0.1 Hz to 10 kHz at a standard potential of +0.154 V, using a sinusoidal potential perturbation of 5 mV amplitude in 0.1 M KCl solution containing 5 mM [Fe(CN)6]3−/4−. (d) Single frequency impedance profiling results of 1000 SK-MEL-2 cells.
Fig. 3
Fig. 3
(a) Plot of charge (Q) versus time curves in the absence and presence of 1500 SK-MEL-2 cells in 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl solution (10 mL). The dotted lines represent linear fit to determine the intercept at t = 0. ΔQ is the charge difference in the absence and presence of 1500 SK-MEL-2 cells. (b) Bode plot and (c) plot of logarithm frequency vs. phase angle. (d) DPV curve for SK-MEL-2 cells (10 cells/10 mL) at MC1R-Ab-PANI/SPE.
Fig. 4
Fig. 4
(a) DPV curves with different SK-MEL-2 cell loads from 0 to 9000 cells/10 mL at MC1R-Ab-PANI/SPE (200 ng Ab loading). (b) Peak current ratio versus SK-MEL-2 cell concentration. Calibration curves of peak current ratio vs logarithm of SK-MEL-2 cell load: (c) 10 to 1000/10 mL and (d) 2000 to 9000/10 mL.
Fig. 5
Fig. 5
Immunofluorescence staining of HEK293 and SK-MEL-2 cells with MC1R and GAPDH. (Top) HEK-293 cells show DAPI staining and GAPDH expression but do not exhibit MC1R expression. (Bottom) SK-MEL-2 cells show DAPI staining and expression of both MC1R and GAPDH.
Fig. 6
Fig. 6
Electrode binding assay with GFP-Tagged Cells. (a) SK-MEL-2 (left) and HEK293 (right) cells expressing PGK-GFP reporter. (b) Binding assay of SKMEL2-GFP tagged cells on PANI-MC1R electrode (Top). Binding assay of HEK-293-GFP tagged cells on PANI-MC1R electrode (Bottom).
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