A multiwalled carbon nanotube/dihydropyran composite film electrode for insulin detection in a microphysiometer chamber

Abstract

We have developed a multiwalled carbon nanotube/dihydropyran (MWCNT/DHP) composite sensor for the electrochemical detection of insulin in a microfluidic device. This sensor has been employed for physiological measurements of secreted insulin from pancreatic islets in a Cytosensor previously modified to be a multianalyte microphysiometer (MAMP). When compared with other established electrochemical insulin sensors, the MWCNT/DHP composite film sensor presented improved resistance to fluidic shear forces, while achieving enhanced electrode kinetics. In addition, the preparation of the composite film is straightforward and facile with a self-polymerizing monomer, DHP, used to add mechanical stability to the film. The sensor film was able to detect insulin concentrations as low as 1muM in the MAMP during calibration experiments. The MWCNT/DHP composite sensor has been successfully used for the direct detection of insulin secreted by islets in the microphysiometer.

Fig. 1

Modified sensor head showing 1 mm glassy carbon electrode, 2 mm platinum counter electrode, fluid inlet and outlet, and counter electrode for light addressable potentiometric sensor (LAPS).

Fig. 2

Cyclic voltammograms in 50 mM phosphate buffer (pH 7.4, 0.02% v/v Tween-80) and 72.5 µM insulin solutions in the same buffer at a 3.0 mm GCE coated with each film type vs. Ag/AgCl (2 M KCl). Scan rate: 50 mV/s. (A) MWCNT cast from DMF; (B) MWCNT/dihexadecyl phosphate electrode; (C) MWCNT/DHP electrode.

Fig. 3

(A–C) Optical microscope images of (A) MWCNT cast from DMF, (B) MWCNT/dihexadecyl phosphate, and (C) MWCNT/DHP films on 1 mm GCE of sensor head prior to testing in MAMP. These particular sensor films (A–C) were subjected to 4.5 hours of 180 s cycles with a flow rate of 20 µL/min for 140 s and a stop flow period of 40 s in the MAMP, and their condition recorded through optical microscope images (D–F): (D) MWCNT cast from DMF (film (A) after 4.5 hours of testing in the MAMP), (E) MWCNT/dihexadecyl phosphate (film (B) after 4.5 hours of testing in the MAMP), and (F) MWCNT/DHP (film (C) after 4.5 hours of testing in the MAMP).

Fig. 4

Linear sweep voltammograms of 300 µM insulin in 50 mM phosphate buffer (pH 7.4, 0.02% v/v Tween-80) and of blank buffer at a 1 mm GCE on the modified sensor head coated with MWCNT/DHP composite film vs. Ag/AgCl (2 M KCl) in the MAMP. Scan rate: 50 mV/s.

Fig. 5

Insulin calibration curves of MWCNT/DHP films at + 0.88 V vs. Ag/AgCl (2 M KCl). Insulin was added as a solution in 50 mM phosphate buffer (pH 7.4, 0.02% v/v Tween-80). Buffer was also added as a control. (A) Calibration curves of three different films on a 3 mm GCEs, performed under moderate stirring with each point showing the current increase for that concentration. Each data point on the graph represents twenty seconds or 200 collected data points. (B) Calibration curve of a MWCNT/DHP film on a 1 mm GCE of a modified sensor head in the MAMP microfluidic chamber under diffusion control. Each data point on the graph represents at least five flow – stop flow cycles, comprising 700 collected data points.

Fig. 6

Typical flow – stop flow amperometric current profile of a MWCNT/DHP sensor film in MAMP at + 0.88 V vs. Ag/AgCl (2M KCl) showing the pump on/off periods, peak current (i_p) and peristaltic pump clamping spike. This measurement was performed in low buffered HBSS media with 16.7 mM glucose in the presence of islets: 180 s cycle with a flow rate of 20 µL/min for 140 s and a stop flow period of 40 s and sample volume of 3 µL.

Fig. 7

Normalized stop flow peak current response for MWCNT/DHP film on the 1 mm GCE of the sensor head in microfluidic chamber of MAMP upon stimulation with low and high glucose with and without islets. Measurements were performed in HBSS with 180 s cycle with a flow rate of 20 µL/min for 140 s and a stop flow period of 40 s and sample volume of 3 µL. □: No islets low glucose; ■: No islets high glucose; ○: Islets low glucose; ●: Islets high glucose. INSET: Amperometric response of MWCNT/DHP composite film at + 0.88 V vs. Ag/AgCl (2M KCl) with islets present. All data for this figure was collected with the same sensor, same coating, and same microfluidic chamber to reduce variability.