Electrochemical detection of glutamate and histamine using redox-labeled stimuli-responsive polymer as a synthetic target receptor

Abstract

Glutamate (Glu) and histamine (His) are two major neurotransmitters that play many critical roles in brain physiological functions and neurological disorders. Therefore, specific and sensitive monitoring of Glu and His is essential in the diagnosis and treatment of various mental health and neurodegenerative disorders. Both being non-electroactive species, direct electrochemical detection of Glu and His has been challenging. Herein, we report a stimuli-responsive polymer-based biosensor for the electrochemical detection of Glu and His. The polymer-based target receptors consist of a linear chain stimuli-responsive templated polymer hybrid that is labeled with an osmium-based redox-active reporter molecules to elicit conformation-dependent electrochemical responses. The polymers are then attached to a gold electrode to implement an electrochemical sensor. The cyclic voltammetry (CV) and square-wave voltammetry (SWV) results confirmed the polymers' conformational changes due to the specific target (i.e., Glu and His) recognition and the corresponding electrochemical detection capabilities. The voltammetry results indicate that this biosensor can be used as a 'signal-on' and 'signal-off' sensors for the detection of Glu and His concentrations, respectively. The developed biosensor also showed excellent regeneration capability by fully recovering the initial current signal after rinsing with deionized water. To further validate the polymer's utility as a target bioreceptor, the surface plasmon resonance (SPR) technique was used to characterize the binding affinity between the designed polymers and the target chemical. The SPR results exhibited the equilibrium dissociation constants (K_D) of 2.40 μM and 1.54 μM for the polymer-Glu and polymer-His interactions, respectively. The results obtained this work strongly suggest that the proposed sensing technology could potentially be used as a platform for monitoring non-electroactive neurochemicals from animal models.

Keywords: Electrochemical biosensor; Glutamate; Histamine; Neurotransmitters; SPR; Templated polymers.

Figure 1.

Schematic illustration of the proposed target-selective biosensing platform. (A) a linear-chain stimuli-responsive polymer is templated with a target molecule during RAFT polymerization to create target-specific affinity; (B) After polymer synthesis and template removal, the redox-active molecules (osmium complex, Os(bpy)₂Cl) are attached to the vinylpyridine (VP) in the polymer backbone; (C) When the redox-labeled polymers are grafted to the electrode, the target binding event induces a conformational change of the polymer from an extended to a folded state resulting in a higher electron transfer rate compared to an unbound polymer chian due to the relative position of the redox labels with respect to the electrode; (D) Applying voltammetry allows for target concentration-dependent sensor response due to the shape-changing behavior of the polymer-based target receptors.

Figure 2.

H NMR spectra of the synthesized poly(NIPAM-VP-MAA) copolymer.

Figure 3.

Characterizing the molecular compositions of the stimuli-responsive polymer, Poly(NIPAM-VP-MAA), the osmium complex, Os(bpy)₂Cl₂, and the stimuli-responsive polymer labeled with osmium complexes, Poly(NIPAM-VP-MAA)-g-Os(bpy)₂Cl using (A) FTIR and (B) UV-vis spectra.

Figure 4.

The CV (A) and SWV (B) of the modified electrode with different concentrations of poly(NIPAM-VP-MAA)-g-Os(bpy)₂Cl.

Figure 5.

Electrochemical sensing results using the polymer-modified electrode. CV (A) and SWV (B) plots when the sensor electrode was exposed to 0.5 – 10 μM of N-mGlu; (C) calibration curve for N-mGlu detection using the peak current values from SWV; CV (D) and SWV (E) plots when the sensor electrode was exposed to 0.5 – 10 μM of L-Glu; (F) calibration curve for L-Glu detection using the peak current values from SWV. The error bars represent 1 standard deviation (n = 3). All experiments were performed with 10 mM NaClO₄ electrolyte.

Figure 6.

Characterization of the chemical selectivity for the developed biosensor. The cyclic voltammetry (CV) responses of the polymer-modified electrode under exposure to serotonin (A); dopamine (B); GABA (C); and histamine (D). The square-wave voltammetry (SWV) responses (E) and its calibration curve (F) for histamine detection show the ‘signal-off’ behavior of the biosensor. The electrolyte used was 10 mM NaClO₄.

Figure 7.

Characterization of the binding kinetics of the polymer-based target receptors templated with N-mGlu. The figure shows the real-time association/dissociation curves and the corresponding equilibrium binding affinity plot for poly(NIPAM-VP-MAA) with SPR chip functionalized with N-mGlu (A & B); poly(NIPAM-VP-MAA)-g-Os(bpy)₂Cl₂ with SPR chip functionalized with N-mGlu (C & D); and poly(NIPAM-VP-MAA)-g-Os(bpy)₂Cl₂ with SPR chip functionalized with histamine (E & F). The calibration curves were obtained based on the “Affinity/EC50 model” of curve fitting of the SPR sensorgrams.