Cardiac Troponin I-Responsive Nanocomposite Materials for Voltammetric Monitoring of Acute Myocardial Infarction

Acute myocardial infarction (AMI) is a severe cardiovascular disease characterized by heart muscle damage due to inadequate blood supply, leading to a life-threatening risk of heart attack. Herein, we report on the design of polyaminophenol-based thin film functional polymers and their thorough optimization by electrochemical, spectroscopic, and microscopic techniques to develop a high-performance point-of-care voltammetric monitoring system. Molecularly imprinted polymer-based cTnI-responsive nanocomposite materials were prepared on an electrode surface by imprinting a specific cTnI epitope, integrating polyaminophenol electrodeposition, along with gold nanoparticles (AuNPs) and graphene quantum dots (GQDs). The characterization techniques, including cyclic and square wave voltammetries, electrochemical impedance spectroscopy, atomic force microscopy, fluorescence microscopy, attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), and contact angle measurements proved the efficient fabrication of the voltammetric monitoring system relying on cTnI-responsive functional thin films. The sensing platform prepared with the optimized nanocomposite composition of AuNPs, GQDs, and molecularly imprinted polymers exhibited very high sensitivity, reproducibility, specificity, and affinity toward cTnI. The sensor showed a storage stability of 30 days, demonstrating great potential for use in early and point-of-care diagnosis of AMI with its 18 min detection time.

For contact angle measurements and fluorescence microscopy, screen-printed gold electrodes (SPGEs; DS220AT, Metrohm, Germany) were employed as flat surfaces.The MIP film on SPGEs was synthesized using the Metrohm DropSens connector with identical parameters to those used for gold wires.

Working electrode preparation
The gold wires underwent two rounds of boiling in a 2.5 M KOH solution for 4 hours and were subsequently stored in concentrated sulfuric acid (H2SO4).Prior to each experiment, the wires were immersed in concentrated nitric acid (HNO3) for 10 min, rinsed with Millipore water, and carefully dried using nitrogen (N2) gas.For AFM samples, gold-coated silicon wafer chips replaced the gold wires as the working electrode.These chips were cleaned by immersion in an acidic piranha solution (sulfuric acid [H2SO4] and hydrogen peroxide [H2O2], x:y ratio, 3:1) for 5 min and then dried under a moderate flow of N2 gas.

Electrochemical measurements
Electrochemical measurements were performed in a 2.0 mL electrochemical cell using a three-electrode system.Gold and platinum wires served as the WE and counter electrode (CE), respectively, while a silver electrode (Ag/AgCl) functioned as the reference electrode (RE).The PalmSens4 workstation (Belltec, Germany) was used for all electrochemical protocols.CV, SWV and EIS measurements were carried out in a solution containing 10 mM K3[Fe(CN)]6 and 0.1 M KCl at room temperature.For CV, a potential range of -0.2 to 0.8 V and a scan rate of 0.05 V s -1 were employed.SWV measurements were performed with potentials ranging from -0.3 to 0.8 V, an amplitude of 0.05 V, and a frequency of 5 or 10 Hz.
Prior to each measurement, the cell was cleaned by rinsing it three times with double-distilled water and dried using a gentle flow of N2 gas.Each measurement was repeated at least three times to ensure reproducibility and reliability of the results.Table S1 shows the final parameters used in electrochemical characterization, electropolymerization, and involved in the template removal process.

Synthesis of gold nanoparticles (AuNPs)
For the synthesis of AuNPs, the strategy described by Piella et al. 1 was employed.In a three-necked round bottom flask, 0.1 mL of tannic acid (2.5 mM) was stirred with 1 mL of potassium carbonate (150 mM) and 150 mL of sodium citrate (2.2 mM) to form a reducing mixture.After the mixture attained 70°C, 1 mL of tetrachloroauric acid (25 mM) was injected.
Within a couple of seconds, the solution turned blackish grey, subsequently orange-red, and the AuNPs were obtained.The resultant particle (∼3.5 nm) concentration was 7 × 10 13 NPs mL -1 . 2  Epitopes are amino acid chains within proteins that are typically found on the protein's surface and are accessible for binding to their respective receptors.For sensor preparation, carefully selected epitopes can be chemically modified and immobilized on the sensor surface prior to the imprinting process.Modifying the epitope with a cysteine group is particularly advantageous, as cysteine contains functional groups like -SH and -NH2 that exhibit a strong affinity for gold, enabling the formation of a SAM 5,6 .Therefore, during the synthesis of the cTnI epitope, L-cysteine was attached to the end of the epitope to facilitate template modification.

Table S2:
The apparent charge transfer rate constant (Kapp) extracted from the EIS data from    supp. (

,Figure S1 :
Figure S1: The strategic mapping for conducting optimization studies, A) Optimization of template concentration, its incubation time, and template removal conditions, B) Optimization of 2-AP monomer as well as nanomaterials concentration (with optimized template and monomer parameters).

Figure S4. A )
Figure S4.A) The optimization of concentration and incubation period of template (Cysepitope) adsorption and B) the optimization of voltage for template removal.

Figure S5 .Figure S6 .
Figure S5.Plausible mechanism of electrooxidation pathway of cysteine for removal of the template.

Figure S8A .
Figure S8A.Optimization results of different concentrations and compositions of GQD and AuNPs sensors, and combinations of their nanocomposite sensors.Each sensor type was examined with 3 replicas.