Development of a Novel Insulin Sensor for Clinical Decision-Making

Abstract

Background: Clinical decision support systems that incorporate information from frequent insulin measurements to enhance individualized diabetes management remain an unmet goal. The development of a disposable insulin strip for fast decentralized point-of-care detection replacing the current centralized lab-based methods used in clinical practice would be highly desirable to improve the establishment of individual insulin absorption patterns and algorithm modeling processes.

Methods: We carried out the development and optimization of a novel decentralized disposable insulin electrochemical sensor focusing on obtaining high analytical and operational performance toward achieving a true point-of-care insulin testing device for clinical on-site application.

Results: Our novel insulin immunosensor demonstrated an attractive performance and efficient user-friendly operation by providing high sensitivity capability to detect endogenous and analog insulin with a limit of detection of 30.2 pM (4.3 µiU/mL), rapid time-to-result, stability toward remote site application, and scalable low-cost fabrication with an estimated cost-of-goods for disposable consumables of below $5, capable of near real-time insulin detection in a microliter (≤10 µL) sample droplet of undiluted serum within 30 minutes.

Conclusions: The results obtained in the optimization and characterization of our novel insulin sensor illustrate its suitability for its potential application in remote clinical environments for frequent insulin monitoring. Future work will test the insulin sensor in a clinical research setting to assess its efficacy in individuals with type 1 diabetes.

Keywords: immunosensor; insulin measurement; point-of-care; type 1 diabetes.

Figure 1.

Schematic of the novel insulin sensor chip showing the sensor fabrication and electrochemical assay workflow and operation. (a) Images of the chips array and the single sensor comprising two gold-sputtered electrodes, one employed as working electrode (WE) and one as joint reference/counter (RE/CE) electrode. (b) Schematic of the insulin immunosensor and amperometric transduction principle: HRP-tagged sandwich immunoassay and the electrochemical reaction of the H₂O₂/HRP/TMB redox probe that generates a reduction current. (c) Schematic of the insulin analysis protocol, with direct one-step sandwich-type immunoassay for insulin antibodies/antigen binding in a serum sample droplet and representative chronoamperometric measurement for timely insulin levels tracking. Abbreviations: HRP, horseradish peroxidase; DAb, detector antibody; CAb, capture antibody; TMB, 3,3′,5,5′-tetramethylbenzidine; SAM, self-assembled monolayer.

Figure 2.

Results of optimization experiments for critical insulin immunosensor preparation and assay parameters in serum. Chip assays were evaluated in triplicate; error bars were estimated as standard deviation of the three replicates. (a) Immunoreagents involved in the developed one-step insulin assay. Chronoamperometric signals obtained from evaluated parameters of a novel insulin assay using human serum (San Diego Blood Bank) as a negative control or blank (labeled “B” and shown with gray bars) and human serum spiked with 1 nM of recombinant human insulin standard (Sigma-Aldrich) as a positive control or signal (labeled “S” and shown with blue bars). Graphs show change in signal-to-blank (S/B) ratio (red lines) with different assay parameters: (b) Number of incubation steps; (c) sample incubation time; (d) HRP-DAb concentration. Abbreviations: HRP, horseradish peroxidase; DAb, detector antibody; CAb, capture antibody.

Figure 3.

Electrochemical performance and characterization of the developed immunosensor using insulin standards spiked into buffer and serum. (A) Schematic of the homemade sputtered sensor chip for insulin determination along with the immunoreaction and electrochemical transduction processes involved. Chronoamperograms and calibration plots obtained for increasing insulin concentrations in buffer (B) from 0 (a) to 400 pM (i) with 50-pM increments, and in serum (C) from 0 (a) to 1000 pM (e) with 250-pM increments, and (D) from 0 (a) to 400 pM (e) with 100-pM increments. Abbreviations: HRP, horseradish peroxidase; TMB, 3,3′,5,5′-tetramethylbenzidine.

Figure 4.

Reproducibility and stability of the insulin immunosensor. (a) Image of a disposable insulin strip for decentralized POC testing; reproducibility of the insulin immunoassay for the detection of (b) 200-pM insulin standards spiked in buffer and (c) 100-pM insulin standards spiked in serum, using a six-electrode batch for each matrix; (d) amperometric responses obtained in the measurement of 200-pM insulin standards spiked in buffer on each day of test over a 30-day period after fabrication and storage at 4°C; (e) amperometric responses and resulted calibration slope values (red lines) obtained in the measurement of 0- (black bars), 200- (blue bars), and 400-pM (green bars) insulin standards spiked in buffer on each day of test over a 24-hour period after fabrication and storage at room temperature; (f) amperometric responses and resulted signal-to-blank (S/B) values (red lines) obtained in the measurement of 0- (blank, black bars) and 200-pM (signal, blue bars) insulin standards spiked in buffer on each day of test over a seven-day period after fabrication and ordinary mailing shipping transit. Error bars in (d-f) were estimated as the standard deviation of three replicates. Abbreviations: POC, point-of-care; RT, room temperature

Figure 5.

Immunosensor response toward insulin analog using insulin standards spiked into buffer and serum. Chip assays were evaluated in triplicate; error bars were estimated as standard deviation of the three replicates. (a) Schematic of the insulin peptide along with fractions of the amino acids sequences corresponding to human insulin and insulin aspart. Chronoamperograms and signal results obtained in the measurement of (b) buffer solutions and (c) serum samples nonspiked (black lines and bars), and spiked with 200 pM of human insulin “HI” (Sigma-Aldrich) (red lines and bars), 200 pM of insulin aspart analog “NI” (Novolog) (blue lines and bars), and 200 pM of both human insulin and insulin aspart (yellow lines and bars).