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Original Article
ARTICLE IN PRESS
doi:
10.25259/AJC_583_2025

Electrochemical biosensors to detect cardiac troponin I with heart failure using nanocomposites of Poly(β-Cyclodextrin)-graphene quantum dots on modified glassy carbon electrode: Fabrication and characterizations

, , , , , , , , ,
aJiangxi Provincial People’s Hospital, The First Affiliated Hospital of Nanchang Medical College, Nanchang, China
bThe First Affiliated Hospital of Soochow University, Suzhou, China.
cDepartment of Mechanical Engineering, Lloyd Institute of Engineering & Technology, Knowledge Park II, Greater Noida, Uttar Pradesh, India.
dCentre for Research Impact and Outcome, Chitkara University Institute of Engineering and Technology, Chitkara University, Rajpura, Punjab, India
eElectrical Engineering Department, College of Engineering, King Khalid University, Abha, Saudi Arabia.

*Corresponding author: E-mail address: yang.dongmei211@163.com (D. Yang)

Received: , Accepted: ,
© 2026 Arabian Journal of Chemistry - Published by Scientific Scholar
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

Rapid and precise quantification of cardiac troponin I (cTnI) is critical for early diagnosis of cardiac events; this study introduces a novel electrochemical aptasensor leveraging a graphene quantum dots/poly(β-cyclodextrin) nanocomposite for sensitive and selective detection of cTnI in serum samples. The aptasensor was fabricated by immobilization 5’-amine modified troponin I aptamer on electrodeposited nanocomposite of graphene quantum dots and poly(β-cyclodextrin) on glassy carbon electrode (apt/pβ-CD/GQD/GCE). To prevent non-specific binding, the aptamer-modified electrode was treated with 6-mercaptohexanol (MCH). The structural and morphological analyses indicated to successful electrodeposition of pβ-CD/GQD on the glassy carbon electrode (GCE) surface. Electrochemical analyses using electrochemical impedance spectroscopy (EIS) techniques, cyclic voltammetry (CV), and differential pulse voltammetry (DPV) indicated a sensitive and selective performance of the aptasensor with a wide linear detection range from 10-5 ng.mL-1 to 10-3 ng.mL-1 and a low limit of detection (2.03 fg.mL-1). The sensor showed excellent reproducibility with a relative standard deviation (RSD) of 3.79% for repeated measurements and retained over 94% of its initial response after 55 days, demonstrating outstanding stability. This detection range covers ultra-low to clinically relevant concentrations of cTnI, enabling early diagnosis of cardiac events—particularly important since basal cTnI levels in healthy individuals typically fall below 0.04 ng.mL-1. The aptasensor was validated using serum samples from a healthy individual, achieving recoveries between 90% and 99% with low RSD (3.42% to 4.66%); future work will explore its application to pathological samples. The low-cost and easy-to-fabricate aptasensor provides a favorable alternative for valid and precise cTnI quantification, potentially enhancing the efficiency of early diagnosis of cardiac disease and coronary heart disease.

Keywords

Aptamer
β-cyclodextrin
Cardiac troponin I
Electrochemical aptasensor
Graphene quantum dots

1. Introduction

Actin, the fine filaments of striated muscle, and tropomyosin are both attached to troponin, a complex of three regulatory proteins. Troponin T (TnT), Troponin I (TnI), and Troponin C (TnC) are the three subunits that make up troponin. Each subunit has a unique function. TnT is the tropomyosin binding protein, TnC binds to calcium and is responsible for binding calcium to activate muscle contraction, and TnI prevents myosin Mg+2-ATPase from being activated by actin [1]. Since cardiac TnC and skeletal muscle TnC share all of their amino acid similarities, cardiac TnC is not utilized as a cardiac marker. The cytoplasm contains trace amounts of both TnT and TnI. When a heart muscle is injured, as happens during a heart attack, both TnT & TnI are released. The quantity of TnT & TnI in the blood will increase with the degree of heart injury [2]. A crucial biomarker that allows for noninvasive identification of myocardial damage in a variety of cardiovascular conditions, particularly in people who have coronary heart disease who exhibit acute chest discomfort, is the circulating cardiac Tn levels [3]. Troponin is leaking into your bloodstream from injured cardiac muscle cells when your cTnI levels are greater than the reference range. Troponin levels typically fall between 0 and 0.04 ng/ml. A crucial regulator of cardiac muscle relaxation and contraction, cardiac TnI (cTnI) is extremely unique to the heart and remains elevated for a longer period of time than creatinine kinase-myocardial band (MB) [4].

Determination of cTnI biomarker level is studied using various methods, including enzyme-linked immunosorbent assay [5], colorimetry [6], surface-enhanced Raman scattering [7], chromatography [8], mass spectrometry [9], chemiluminescence [10], and electrochemical sensors [11]. Among these, electrochemical sensors and biosensors are the powerful tools for detecting cTnI biomarkers because of their high sensitivity, specificity, and long-term stability in complex biological samples like blood samples [12-13]. Between the electrochemical sensors, aptamer-based sensors have been exhibited several advantages over traditional diagnostic techniques for cTnI biomarker detection [14-15]. Aptamers are small nucleic acids in single-stranded form that bind to target molecules specifically and with a high affinity [16]. They offer features such as chemical stability, facile synthesis, and low immunogenicity. They can have conformational changes upon target binding, which makes them to have high selectivity and sensitivity and an ideal recognition element in electrochemical biosensors. Aptamers are the molecular recognition component used in Aptasensors, which are novel biosensing devices. The selective binding with aptamers to the target molecules is the basis for aptasensor function [15]. Aptasensors are a sensitive tool that can distinguish trace amounts of troponin in biological fluids, which is crucial for the early recognition of heart disease [17]. New techniques for the detection of cTnI have centered on electrochemical biosensors, which consist of nanomaterials (graphene quantum dots (GQDs) [18], carbon nanotubes [19], and metal-organic frameworks [14] to improve sensitivity and selectivity. Aptamer mediated sensors are becoming popular because they have high affinity and it is able to detect ultra-low cTnI concentration for the early detection [20]. Such advancements have led to fast, low-cost, point of care compatible tools, which are indicative of the current state of the art of cTnI biosensing and provided the motivation for the current aptasensor design. The cTnI is a specific biomarker of myocardial injury, which is exclusively expressed in the myocardium and not in the skeletal muscle; therefore, it is the gold standard for acute myocardial infarction diagnosis [21,22]. The developed sensor exhibits an enhanced specificity by utilizing a high-affinity aptamer immobilized on a carbon-nanostructure surface with reduced non-specific binding, promoting the accuracy of detection of the target in complex samples.

Modification of the electrochemical aptasensors with nanomaterials plays a significant role in enhancing signal and sensitivity, particularly graphene oxide, carbon nanotubes, and quantum dots [17,23,19]. These nanostructures provide high surface area for aptasensor immobilization, excellent conductivity, biocompatibility, and the ability to facilitate electron transfer kinetics. GQDs can be readily functionalized with various biocompatible molecules, which reduce their toxicity and improve their interaction with biological systems [24]. This modification minimizes non-specific binding and enhances their integration within biological environments. Furthermore, high surface of GQDs and abundant electroactive sites allow for high loading of aptamers, increasing sensitivity [25,18]. GQDs were selected for their unique electronic and physicochemical properties, such as large surface area, adjustable optical and electrical properties, and numerous functional groups for further surface modification [26]. In addition to the excellent water solubility of GQDs and lower toxicity compared with carbon nanotubes, GQDs also have better photostability than CNTs, and these characteristics provide the specific binding sites for immobilizing the aptamer and the effective route for electron transfer, resulting in enhanced sensitivity and stability of electrochemical biosensors. Modification of GQDs to use as detectable probes. Moreover, β-cyclodextrin (β-CD) also shows a hydrophobic cavity that can encapsulate and selectively bind specific molecules, promoting the selectivity of the aptasensor [27]. Studies have been indicated that β-CD can act as a scaffold to render aptamers immobilized onto the electrode surface, improving their orientation and accessibility to target molecules [28-29]. GQDs present high conductivity and plenty of electroactive sites for aptamer immobilization, and the hydrophobic cavity of β-cyclodextrin can selectively recognize molecules; both of them simultaneously enhance the amplification of signals and specificity of sensing. Its low-cost and simple fabrication through common materials makes scalable fabrication and practical clinical use for rapid detection of cardiac troponin I readily achievable.

Based on this, an electrochemical aptasensor by using a nanocomposite of GQDs and poly-β-cyclodextrin (pβ-CD) was developed for the sensitive and selective determination of cTnI in the human serum. The integrated GQDs/pβ-CD has a high surface area and good conductivity, and meanwhile, the hydrophobic cavity effectively facilitates the immobilization of the aptamer and the molecular recognition. This sensor is characterized by enhanced electron transfer, minimized nonspecific binding, and excellent analytical performance in comparison with the previous sensors. This aptasensor demonstrates a broad linear range, low detection limit, and excellent stability, holding great potential for rapid and accurate clinical diagnosis of myocardial damage.

This paper not only presents a new aptasensor system with improved sensitivity and selectivity for cTnI determination, but it also demonstrates its promising characteristics for rapid, low-cost, and point-of-care (POC) clinical testing. Its facile synthesis and reliable detection capability make this sensor a promising candidate for rapid detection and early diagnosis of cardiovascular diseases, thus solving the current detectability dilemma of cardiac biomarkers.

2. Materials and Methods

2.1. Chemical

Glucose (C6H12O6, ≥99.5%), Isopropyl Alcohol (C3H8O, ≥99.5%), ferricyanide/potassium chloride (Fe(CN)63−/4−, 99%), amine-polyethylene glycol (PEG)-hydroxyl (NH2-PEG12-OH), β-cyclodextrin (99%), 6-mercaptohexanol (MCH), 1-pyrenecarboxylic acid(97%, py-COOH), and phosphate buffer saline(PBS) were provided from Sigma-Aldrich. Ethylenediamine (C2H4(NH2)2, ≥99.0%), β-Cyclodextrin (C42H70O35, ≥98.0%), and aluminum oxide (Al2O3, ≥99.0%) were purchased from Merck (Germany). Hydrochloric acid (HCl, ≥36.5-38.0%) and ethanol (C2H5OH, ≥99.5%) were sourced from Fisher Scientific (USA). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was provided by Calbiochem (Milan, Italy), and N-hydroxysuccinimide (NHS) was purchased from Fluka (Milan, Italy). The 5’-amine improved troponin I aptamer(5’-NH2-TTT-TTTCGT GCA GTACGC CAACCT TTC TCATGC GCT GCC CCT CTTA-3’) was acquired from Integrated DNA Technologies (Leuven, Belgium). Milli-Q water was used to prepare each solution.

2.2. Synthesis of GQDs

For the synthesis of GQDs, 10 g of glucose was added to 150 mL of deionized water in a round-bottom flask [30]. Then, the mixture was sonicated for 10 min to complete the dissolution of the glucose. After that, 3 mL of concentrated HCl and 5 mL of ethylenediamine were added to the flask. To remove the excess heat and reaction byproducts, a condenser was attached to the flask, which was equipped with a pump containing isopropyl alcohol. A cryogenic cooling system was used to maintain the temperature of the reaction mixture at -10°C. Subsequently, the mixture was magnetically stirred at 110°C by a heating mantle for 100 min to ensure the complete reaction. Then, the resulting mixture was dialyzed in 3500 MWCO dialysis tubing with a molecular weight cutoff of 2000 Da for 2 days. The products were obtained by concentration of dilute solution through rotary evaporation (RE202, Yamato Scientific, Japan) for 12 h intervals, and the water was changed to remove of impurities. For additional characterization and use, the resulting GQDs were freeze-dried and kept in a refrigerator.

2.3. Preparation of modified electrode

Before the modification, a GCE was polished by 0.5 μm alumina slurry. Then, the polished electrode was washed successively with ethanol and deionized water for three times to remove any impurities. The electrodeposition method was used for the preparation of poly(β-cyclodextrin)/GQD/GCE (pβ-CD/GQD/GCE) [31]. For this purpose, a homogeneous mixture was prepared using 6 mL of GQD, 7.5 mg of β-CD, and 24 mL of 0.1 M PBS. Subsequently, the mixture was sonicated for 40 min to achieve a homogeneous β-cyclodextrin/GQD mixture solution. The electrodeposition of pβ-CD/GQD on the GCE surface was conducted on cyclic-voltammetry(CV) between -0.9 to 0.9 V at a 50mV/s scan rate for 6 min in the β-CD/GQD mixture solution. Then, the as-prepared electrode is washed with deionized water and then was dried at room temperature. For comparison purposes, GQD/GCE and pβ-CD/GCE were prepared using the same method in the absence of β-CD and GQD, respectively.

For the fabrication of troponin sensitive aptasensor (aptamer/pβ-CD/GQD/GCE), the pβ-CD/GQD/GCE is immersed in a mix of 1 mM 1- pyrenecarboxylic-acid and 1 mM pyrene-PEG with equal volume ratio for 30 min at room temperature. Then, the electrode surface was activated by carboxyl groups through immersion in a 0.1M PBS (pH 7.4) containing 15.0 mM EDC and 15.0 mM NHS for 25 min. After that, 5’-amine modified troponin I aptamer was immobilized on electrode surface (apt/pβ-CD/GQD/GCE) by covalent connection of 5’-NH2-modified aptamer via incubation in 10 µL of 20 µM PBS (pH 7.4) containing 5’-NH2-modified aptamer for 45 min. β-Cyclodextrin can be utilized as an efficient platform for modification of aptamers on the manipulation of because of its amphiphilic properties and lots of hydroxyl moieties, which can lead to firm and oriented immobilization of aptamers on the electrode surface. This enhanced immobilization also benefits target approach and the effective electron transfer, and then promotes sensitivity and selectivity of the electrochemical aptasensor. Finally, to inhibit the non-specific activity location on the electrode surface, 10μL of 1mM MCH solution was applied to the apt/pβ-CD/GQD/GCE surface (MCH/apt/pβ-CD/GQD/GCE) over 50 min after the apt/pβ-CD/GQD/GCE was cleaned three times using PBS to get rid of the unbound aptamers. Pyrene anchors aptamers on carbon surfaces via π–π stacking [32], while MCH blocks nonspecific sites [33], improving sensor selectivity. Both are standard in electrochemical biosensors.

2.4. Characterization of synthesized nanomaterial and electrochemical studies

Field Emission-scanning electron microscopy (FE-SEM, Zeiss Gemini 560) was used for morphological characterization of products. X-ray diffraction pattern (XRD, Rigaku DMAX-RA, Japan) with Ni-filtered Cu Kα radiation (λ = 1.5406 Å) was used for investigation the structure of products. An ESCA Ulvac-PHI 1600 photoelectron spectrometer from physical electronics was used to perform X-ray photoelectron spectroscopy (XPS) using Al Kα radiation photon energy. Bare and modified GCEs were electrochemically measured at a scan rate of 50 mV/s via cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) techniques in 0.1M PBS with 5.0 mM [Fe(CN)6]3−/4−. An Autolab Potentiostat/Galvanostat (Eco Chemie, Netherlands) was used for all electrochemical tests. DPV measurements were conducted on a 0.05 s pulse width and a pulse amplitude of 50 mV. Diagnostic Kit for Cardiac Troponin I (MP Biomedicals, USA) was used for verifying the aptasensor in prepared real samples. The proposed sensor possessed good stability; the CV, DPV, and EIS signals fluctuated about 5% during 10 successive measurements. Moreover, it was able to recover over 90% of its initial response when exposed-F to MoS2 powder for 2 weeks at 4°C, indicating excellent reproducibility and operational stability. The impedance measurements for each step of electrode modification were done on three electrodes, which were prepared independently. The data are reported in terms of average ± SD, and the Nyquist plots are indicated with error bars. One-way analysis of variance (ANOVA) and Tukey’s post-hoc test were used to determine statistical differences between modification steps, where p < 0.05 was considered significant.

2.5. Real samples preparation

For preparation of the real samples, blood serum samples were acquired from a young healthy volunteer aged 28 years old. The samples were centrifuged at 3000 rpm for 15 min and filtered. Then, the filtered samples were used to prepare 0.1 M PBS with 5.0 mM [Fe(CN)6]3−/4−.

3. Results and Discussions

3.1. Morphological and structural characterizations

The synthesis of the prepared materials was characterized using Field emission scanning electron microscopy (FESEM). Figures 1(a-c) display FESEM images of the pβ-CD, GQDs, and the pβ-CD/GQD nanocomposite modified on GCE, respectively. A sample of pβ-CD shows a compact globular morphology, which is probably due to the physical agglomeration or an aggregation of the polymer chains, with an average particle size of ca. 190 nm. As can be seen from the FESEM image of GQDs (Fig. The surface morphology of pβ-CD/GQD composite-modified GCE appears to be spherical, indicating the formation of very small GQDs with the pβ-CD nanoparticles (Figure 1a).

FESEM image of (a) pβ-CD, (b) GQDs, and (c) pβ-CD/GQD modified GCE surface. (d) XRD patterns of GQDs, pβ-CD, and pβ-CD/GQDs.
Figure 1.
FESEM image of (a) pβ-CD, (b) GQDs, and (c) pβ-CD/GQD modified GCE surface. (d) XRD patterns of GQDs, pβ-CD, and pβ-CD/GQDs.

XRD analysis was applied for investigation the structure of products. Figure 1(d) shows the XRD patterns of GQDs, pβ-CD and pβ-CD/GQDs. XRD pattern of GQDs shows the diffraction peaks at 2θ = 23.48° and 43.51° assigned to diffraction peaks of the graphene structure with (002) and (100) planes, respectively [34]. The peak (002) indicates to the parallel and azimuthal orientation of the aromatic and carbonized structure and the absence of γ-bands linked to amorphous and aliphatic structures [34]. The peak (100) is corresponded to the graphitic and hexagonal carbons [34]. The diffraction peak at 43.51° (2θ > 30°) corresponds to the (100) plane of graphitic carbon and confirms the crystalline nature of the GQDs [35]. No additional peaks indicative of impurities or secondary phases were observed, confirming the high purity of the synthesized materials. As is shown in the XRD pattern of pβ-CD, there is a broad peak at around 2θ =18.46° indicated to amorphous structure. For the pβ-CD/GQDs, the peak (002) of GQDs at 2θ =23.48° has shifted to 23.52° with remarkable decrease in peak intensity. This decrease in the peak intensity can be related to exfoliation of GQDs layers upon electro-polymerization. Moreover, one broad shoulder appears at about 2θ = 18.5° which is the characteristic peak of pβ-CD, demonstrating to the presence pβ-CD in the electrodeposited nanocomposite. Therefore, the XRD results demonstrate that the GQDs were well uniformly dispersed in the pβ-CD matrix, and the semicrystalline structure of pβ-CD is slightly affected by the incorporation of GQDs.

Figure 2(a) exhibits that XPS full scan study of pβ-CD, GQD, and pβ-CD/GQD nanocomposites shows C 1s and O 1s peaks at around 284 and 532 eV, respectively. Figures 2(b-d) show the XPS high resolution spectrum and deconvoluted C 1s peak of GQD, pβ-CD, and pβ-CD/GQD nanocomposites, respectively, where the differences in spectra indicate changes in carbon species among GQD, pβ-CD, and pβ-CD/GQD. The C 1s spectrum of pβ-CD exhibits three peaks at binding energies of approximately 284.8, 286.0, and 288.1 eV, which are attributed to C–C/C–H (sp3 carbon), C–O, and O–C–O groups, respectively, reflecting the carbon moiety’s carboxylic and epoxic environments [36]. No peaks corresponding to metal–carbon (M–C) bonds were observed, confirming the absence of metallic impurities in the samples. The graphitic and carboxylic groups are linked to two distinctive peaks in C1s spectrum of GQD, which are located at binding energies of approximately 284.2 eV for sp2 C=C bonds and 288.2 eV for carboxylic C=O groups [37]. Following the electro-polymerization of pβ-CD, the C 1s spectra of pβ-CD/GQD show peaks from both β-CD and GQD. The peaks at binding energies near 284.2 eV and 284.8 eV correspond to sp2 C=C and sp3 C–C/C–H bonds in the graphitic structure of GQDs, respectively [38]. The peaks at binding energies of 285.2 and 288.1 eV are attributed to the C–O and O–C–O bands of hydroxyl linkage of pβ-CD [39]. These adjusted contributions are in line with the relevant XPS literature values and prove the successful electrodeposition of the pβ-CD/GQD nanocomposite on the GCE surface, corresponding to the anticipated chemical states of carbon groups in the composite. Furthermore, the lack of peaks related to metal–carbon bonds indicates the high purity of the synthesized structures and eliminates the presence of metallic element contaminations.

(a) XPS full scan survey of GQD, pβ-CD, and pβ-CD/GQD nanocomposites, and XPS high resolution spectrum and deconvoluted C 1s peak of (b) GQD, (c) pβ-CD, and (d) pβ-CD/GQD nanocomposites.
Figure 2.
(a) XPS full scan survey of GQD, pβ-CD, and pβ-CD/GQD nanocomposites, and XPS high resolution spectrum and deconvoluted C 1s peak of (b) GQD, (c) pβ-CD, and (d) pβ-CD/GQD nanocomposites.

The IR spectrum of GQDs, pβ-CD/GQD, and MCH/apt/pβ-CD/GQD is shown in Figure 3. The FT-IR spectrum of GQDs exhibited different types of chemically active functional groups, including hydroxy, carbonyl, and epoxy/ether groups. These groups also have probable effects on the solubility, chemical stability, and high fluorescence of GQDs. The broad peak assigned to the OH group was detected at 3445.5 cm⁻1 in the FT-IR spectrum [40]. Also, the strong peaks in the range 1720 cm-1-1635 cm-1, in the characteristic peaks of C=O and С=C, respectively are presented [41-42]. For pβ-CD/GQD, the characteristic peak of β-CD at 1030 cm-1, which associated with the stretching vibrations of C-O-C is observed [43]. Furthermore, the C=O and C=C stretching from GQDs are moved towards 1715 cm⁻1, 1636 cm⁻1, respectively. This may be ascribed to the H-bonding between pβ-CD and GQDs [43]. Firstly, the FT-IR spectrum of MCH/apt/p-CD/GQD displays the characteristic peaks of –NH and –N–C=O and N–C=O stretching vibration at 3020 cm-1, 1649 cm-1, 1562 cm-1, and 1125 cm-1, which proved the successful immobilization of aptamer on the p-CD/GQD [44]. Furthermore, a peak at 3316 cm-1 is attributed to the hydroxyl group in the MCH molecules interconnected with the GQDs [45]. The remaining NH2 band at 1570 cm-1 is ascribed to the primary oleylamine being present together with MCH [45].

FT-IR spectrum of GQDs, pβ-CD/GQD, and MCH/apt/pβ-CD/GQD.
Figure 3.
FT-IR spectrum of GQDs, pβ-CD/GQD, and MCH/apt/pβ-CD/GQD.

3.2. Electrochemical studies

Figures 4(a-c) show the CV and EIS measurements using bare and modified-GCEs into 0.1 M PBS with 5.0 mM [Fe(CN)6]3-/4- as redox probe to study the modification procedures of modified-GCEs. In Figure 4(a), CV measurements for pβ-CD/GQD/GCE as well as GQD/GCE electrode were conducted at a sweep rate of 50 mV/s in 0.1 M PBS with 5.0 mM [Fe(CN)6]3−/4−. As seen, the electro-polymerization of the β-CD layer on GCE resulted in an decrease in peak current compared to the bare GCE because of the β-CD nanostructures hinders electron transfer between the electrode and the analyte due to their insulating nature and dense polymeric network, which limits charge transfer kinetics [46]. Moreover, the peak current in CV curve of GQD/GCE is remarkably increased compared to the bare GCE because of the excellent conductivity of GQD, which facilitate the charge transfer between the redox ions and the electrode. This increase in peak current is attributed to the large surface area, abundant sp2 C=C graphitic domains, and conductive properties of GQD, which collectively improve electron mobility and reduce charge transfer resistance [47]. As observed, electrodeposition of pβ-CD/GQD significantly enhance the peak current toward all electrodes, demonstrating that the modified electrode that was produced responds sensitively because of the great conductivity of synergetic combination of pβ-CD and GQD. The pβ-CD/GQD composite electrodeposition on GCE ensures an unhindered electron transport path by providing a greater surface area as well as a continuous micro-surface, and a balanced presence of conductive sp2 carbon and functional oxygenated groups that contribute to both electron transfer and aptamer immobilization [48]. The thiol-functionalized an aptamer probes fixed the pβ-CD/GQD/GCE surface by forming an amide coupling between the aptamer’s thymine nucleotide and its 5’-NH2 group and the carboxylic acid functionalities of py-COOH [12]. In contrast to covalent linkage, the sp2 graphite carbon network of GQD is unaffected when COOH functions are introduced onto pβ-CD/GQD via contacts, hydrophobic attraction, or hydrogen bonding. This preserves the conductive graphitic framework while enabling stable aptamer immobilization through hydrophilic carboxyl groups. It is suitable for creating platforms for electrochemical sensing. The hydrophilic COOH enables covalent bonding of amine-terminated aptamer, while the lipophilic pyrene portion clings to pβ-CD/GQD efficiently without disturbing C–C,C=C, C–O, and O–C–O functional group of the graphitic structure in GQD and pβ-CD [12]. Subsequently, aptamer modified electrode surface is blocked with MCH to prevent non-specific binding [49]. The aptamers are able to bind to specific target molecules (cTnI). The insulating layer MCH causes hindrance of electron transfer efficiency and leads to further reduced peak current, which confirms effective blocking of nonspecific adsorption sites and enhances sensor selectivity [50]. The MCH/apt/pβ-CD/GQD/GCE is incubated with the target for 45 min to show that the electrochemical aptasensor was feasible. When cTnI is present, the aptamer is firmly attached to the bound protein molecule, blocking [Fe(CN)6]3−/[Fe(CN)6]4− from reaching electrode surface. In this configuration, aptamers undergo conformational change in the presence of the molecule analyte blocks the electron transfer [51]. Thus, the presence on the target molecules was indicated by a drop in the redox peak current. The degree of decrease in current is directly proportional to the target concentration, allowing for quantitative detection. The addition of cTnI to the aptamer-modified electrode leads to a dramatic drop in the CV peak current, and the signal change is much larger than the background noise. This is concentration-dependent reduction and reproducible, which can be used for sensitive and selective determination of cTnI at the clinically important levels. The ΔI have a value higher than the noise level, indicating a statistically significant sensor response when the target is bound.

(a) CV curve at a sweep rate of 50 mV/s in 0.1 M PBS with 5.0 mM [Fe(CN)6]3−/4−, (b) The obtained peak current (Ip) versus square root of the scan rate (√v) for CV measurements at scan rates ranging from 20 to 100 mV/s in 0.1 M PBS with 5.0 mM [Fe(CN)6]3−/4−, and (c) Nyquist plots of GCE and modified GCEs in 0.1 M PBS with 5.0 mM [Fe(CN)6]3−/4−.
Figure 4.
(a) CV curve at a sweep rate of 50 mV/s in 0.1 M PBS with 5.0 mM [Fe(CN)6]3−/4−, (b) The obtained peak current (Ip) versus square root of the scan rate (√v) for CV measurements at scan rates ranging from 20 to 100 mV/s in 0.1 M PBS with 5.0 mM [Fe(CN)6]3−/4−, and (c) Nyquist plots of GCE and modified GCEs in 0.1 M PBS with 5.0 mM [Fe(CN)6]3−/4−.

CV measurements for pβ-CD/GQD/GCE as well as GQD/GCE electrode were conducted at scan rates ranging from 20 to 100 mV/s in 0.1 M PBS with 5.0 mM [Fe(CN)6]3−/4− in order to examine the electrochemical-active surface of modified electrode. The obtained peak current (Ip) versus square root of the scan rate (√v) is presented in Figure 4(b). The Randles-Sevcik Equation (1) can be used to calculate the manufactured electrode’s electrochemically active area [52].

(1)
I p = 2.69 × 10 5 n 3 2 A D 1 2 v 1 2 C

where n presents the amount of electrons transferred (n=1), Ip presents the peak current (A), [Fe(CN)6]3-/4-(7.6×10-6 cm2/s) has a diffusion coefficient of D, a reactant concentration of C (5×10−6 mol/cm3), and a sweep rate (V/s) and A is the electrochemically active area of the electrode (cm2). The electrochemically active areas for electrodes are presented in Table 1. Figure 4(b) shows that the peak current exhibited a linear relationship with the square root of the scan rate, confirming diffusion-controlled kinetics and justifying the use of this equation for calculating the electrochemically active surface area. As seen, the electrochemically active areas for GCE, pβ-CD/GCE, GQD/GCE, and pβ-CD/GQD/GCE are 0.070, 0.064, 0.136, and 0.129 cm2, respectively. More active sites for aptamer immobilization and target binding due to the larger electrochemical surface area of GQD/GCE and pβ-CD/GQD/GCE electrodes afford improved electron transfer and sensor sensitivity. Compared to the pristine GCE, such superiorities as expanded active surface area facilitate the efficient interaction between them and cTnI molecules, which could cater to higher detection sensitivity and excellent analytical performance.

Table 1. A comprehensive examination of the impedance data based on the relevant EIS results matched by the equivalent circuit.
Electrodes A (cm2) Rs (Ω) Rct (Ω) C (µF) W (mS.sec2)
GCE 0.070 18.52 1041.1 65.33 0.681
pβ-CD/GCE 0.064 50.63 1198.5 6.92 0.014
GQD/GCE 0.136 48.92 239.2 0.48 1.499
pβ-CD/GQD/GCE 0.129 49.53 241.3 0.12 1.641
apt/pβ-CD/GQD/GCE 0.105 24.28 438.3 2.93 0.694
MCH/apt/pβ-CD/GQD/GCE 0.081 27.57 495.5 9.51 0.997
cTnI/MCH/apt/pβ-CD/GQD/GCE 0.058 25.62 589.3 8.53 0.087

The electron transfer capability of the modified electrodes was investigated using EIS analyses. Figure 4(c) shows the Nyquist plots and Randles model equivalent circuit. The equivalent circuit is involved solution resistance (Rs), charge transfer resistance (Rct), Warburg resistance (ZW), and double-layer capacitance (Cdl). The comparable circuit that offers a thorough examination of the impedance values fits the relevant EIS results, which are shown in Table 1. The transfer of electrons limited process is indicated by the diameter in the semicircular part that appears in the high-frequency zone of Nyquist plots. The electron transfer resistance values that equal to the semicircle diameter [53]. Accordingly, a smaller semicircle radius represents a lower Rct, indicating faster electron transfer. As observed from Figure 4(c), GCE shows an Rct value of 1041.1 Ω. After the modification of GQD, the impedance value increased slightly to 1198.5 Ω, because of β-CD layer hindered the electron transfer and made the interfacial charge transfer difficult [46]. Further electrodeposition of the GQD contributed to a lower Rct value of 241.2 Ω than that GCE, indicating improved electron transfer. As a conductor, the GQD layer makes it easier for electrons to move between an electrode and the solution of electrolyte. Moreover, for the electrodeposition of pβ-CD/GQD, the impedance spectra of pβ-CD/GQD/GCE exhibits an Rct value of 239.3 Ω, indicating that pβ-CD/GQD/GCE with great surface-area and conductivity may offer an effective electron transfer pathway and significantly increase the rate of electron-transfer between electrode’s surface and [Fe(CN)6]3−/4− solution. With the addition of GQD, the aptamer can capture target protein because of its wide surface area, which increases the number of active sites accessible for analyte interaction, and its abundance of anchoring sites. The semicircle diameter rose significantly to 438.3 Ω when a self-assembling monolayer on thiol-terminated aptamer immobilized onto the pβ-CD/GQD/GCE surface (apt/pβ-CD/GQD/GCE). This was due to the aptamer’s insulating properties. With a Rct value of 495.5 Ω, the semicircle diameter was further increased by blocking the non-specific binding sites when MCH was successively adjusted on the electrode (MCH/apt/pβ-CD/GQD/GCE) [54]. Following the MCH/apt/pβ-CD/GQD/GCE aptasensor recognition reaction with 0.1 ng/mL cTnI for 45 min (cTnI/MCH/apt/pβ-CD/GQD/GCE), the impedance increased greatly to 589.3 Ω, because of the immune complex formation. The binding of cTnI to the aptamer leads to a conformational change, which restricts the electron transfer. Thus, these impedimetric results are in agreement with the CV results, illustrating the successful modification of the electrode and the efficient detection of cTnI.

3.3. Optimization of experimental parameters

Experimental conditions included electrodeposition time for β-CD and GQDs, the incubation time for the aptamer, the incubation time of blocking agent (MCH), and the incubation time for target were meticulously optimized to obtain efficient performance of the electrochemical aptasensor. Figures 5(a-d) show the obtained ΔI (I0-I) as signal for determination 0.1 ng/mL cTnI, I0 is the CV peak current for MCH/apt/pβ-CD/GQD/GCE before incubation in cTnI solution, and I is the CV peak current after incubation in cTnI solution (cTnI/MCH/apt/pβ-CD/GQD/GCE). The incubation time of the blocking agent MCH was also tunned by testing its influence on the electrochemical performance of the sensor. With the increase of the MCH immobilization time, the peak current decreased slowly, and the charge transfer resistance increased gradually, which was attributed to an efficient blocking of the nonspecific binding sites. An appropriate incubation time was also chosen at which a high surface coverage was achieved to avoid nonspecific adsorption but the electron transfer was also kept at an acceptable level to result in a high sensitivity and selectivity for this sensor.

Effect of (a) electrodeposition time for β-CD and GQDs, (b) the incubation time for the aptamer, (c) the MCH incubation time, and (d) the incubation time for target on obtained ΔI for determination 0.1 ng.mL-1 cTnI.
Figure 5.
Effect of (a) electrodeposition time for β-CD and GQDs, (b) the incubation time for the aptamer, (c) the MCH incubation time, and (d) the incubation time for target on obtained ΔI for determination 0.1 ng.mL-1 cTnI.

Figure 5(a) shows the resulting ΔI for fabricated MCH/apt/pβ-CD/GQD/GCE under different electrodeposition times from 2 to 10 min. As the electrodeposition time increased, the obtained ΔI is increased, reflecting an increase in the amount of β-CD/GQD electrodeposition. However, beyond 6 min, the peak current plateaued, demonstrating to saturation, aggregation of nanostructures, reducing the effective surface area, and decreasing the current. Thus, 6 min is selected as the optimal electrodeposition time for β-CD and GQDs on GCE. Reducing or extending the duration may result in incomplete surface modification or thicker layer formation, which could restrain electron transfer and sensor sensitivity. An electrodeposition time of 6 min was found to be an ideal value for good surface coverage and effective electron transfer, and the enhanced sensor performance was thus obtained.

The incubation times for aptamer, MCH, and cTnI were also optimized. Figure 5(b) exhibits that the aptamer immobilization reached a plateau after 45 min. Thus, 45 min was chosen as the optimal aptamer incubation time. Similarly, Figure 5(c) shows that a 50 min incubation time for MCH effectively blocked non-specific binding sites. Finally, a 45 min incubation time for cTnI (Figure 5d) was found to be appropriate for optimal signal generation.

3.4. Detection mechanism and calibration curve

The performance of the MCH/apt/pβ-CD/GQD/GCE biosensor for cTnI detection was evaluated upon the optimized condition. Electrochemical measurements were conducted on triplicate for each cTnI concentration using DPV. Figure 6(a) shows with increasing cTnI concentration, the DPV peak current decreased. It is because of the exact binding of cTnI to the aptamer inhibits the reaction between redox ions and pβ-CD/GQD. These results into a reduction in the electrochemical signal, forming the basis of the detection method. The linear relationship involving ΔI and the logarithmic value of the cTnI concentration at 10-5 to 103 ng.mL-1 is shown in the calibration curve in Figure 6(b). The linear-regression-Equation (2) is stated as:

(2)
Δ I µ A   =  25 . 2131  +  4 . 86285 log C  ng . mL − 1   R 2 = 00. 99978

(a) Obtained DPV for different cTnI concentration (b) The calibration curve. Results foe study (c) reproducibility for four separately fabricated aptasensor, (d) Stability of aptasensor over a 50-day period, and (e) selectivity of the aptasensor for determination 0.1 ng.mL-1 cTnI (n = 4).
Figure 6.
(a) Obtained DPV for different cTnI concentration (b) The calibration curve. Results foe study (c) reproducibility for four separately fabricated aptasensor, (d) Stability of aptasensor over a 50-day period, and (e) selectivity of the aptasensor for determination 0.1 ng.mL-1 cTnI (n = 4).

Thus, the MCH/apt/pβ-CD/GQD/GCE can be a highly desirable aptasensor for cTnI determination. The limit of detection (LOD) of aptasensor is determined using the formula 3.3σ/S, where σ presents referred as standard-deviation of the response, and S presents slope of the calibration curve. The LOD for MCH/apt/pβ-CD/GQD/GCE is found to be 0.9 pg.mL-1. The attained LOD of 0.9 pg.mL-1 is highly superior to traditional sensors, such as antibody-based and single-component nanomaterial ones. The synergistic conductivity and molecular recognition of the graphene quantum dots and poly(β-cyclodextrin) endows the aptasensor with the ultra-sensitivity for cTnI detection, which results in this aptasensor ranking among the finest cTnI detection techniques. Table 2 [11-14, 18, 19, 23, 25, 55-62] presents the comparison between the reported electrochemical cTnI Aptasensors in this work and other reported in literatures, reflecting effectiveness of the proposed MCH/apt/pβ-CD/GQD/GCE.

Table 2. A comparison between our system with reported electrochemical sensor for detection of troponin.
Methods Reagents LR (ng.mL-1) LOD (pg.mL-1) References
DPV MCH/apt/pβ-CD/GQD/GCE 10-5 to 103 0.9 This work
EIS ZnSnO3 perovskite nanomaterials 10-6 to 103 1.87×10-4 [11]
DPV Nitrogen-doped porous reduced graphene oxide electrode 0.01 to 0.1 1  [12]
CV Molecularly Imprinted Polymer 0.009 to 0.8 9 [55]
SWV Cysteine epitope/ortho-aminophenol/GQDs/ gold nanoparticles (Au NPs) 5×10-4 to 10 0.5 [18]
DPV Horseradish peroxidase/antibody/Au organic framework 0.005 to 10 1.7 [56]
CV Bind anti-cTnT monoclonal antibodies/Carboxylated carbon nanotubes/polyethyleneimine 0.1 to 10 33 [13]
EIS GQD/polyimide substrate 1 to 100  1  [57]
DPV Molecularly imprinted polymer based polymethylene blue/Multi-walled carbon nanotubes (MWCNTs)/screen printed carbon electrode 10-3 to 0.008 0.04 [19]
DPV Silver nanoparticles/MoS2/reduced graphene oxide 3×10-4 to 0.2 0.27 [58]
CV Zeolitic imidazolate framework-67@prussian blue analogue --- 3.1×10-4 [14]
SWV Dopamine/Au NPs/GQDs/molecularly imprinted polymer 0.01 to 20 10  [59]
DPV Peptide/antibodies/PtPd@ zirconium nitride functionalized covalent organic frameworks 10-5 to 50 0.0033 [60]
CV Acetic acid functionalized GQDs/Au electrode 0.17 to 3 20 [23]
Chronoamperometry Nitrogen-doped ordered mesoporous carbon/Au NPs nanocomposites 10-5 to 100 0.0024 [61]
DPV Biomimetic nano-molecularly imprinted polymer/graphene screen-printed electrode 0.02 to 0.09  8  [62]
DPV GQD and Polyamidoamine nanohybrid/Au electrode 10-6 to 10 0.02 [25]

(SWV: square wave voltammetry)

3.5. Stability, reproducibility, and anti-interference studies

The stability and reproducibility of the aptasensor were evaluated using four separately fabricated aptasensor over 50 consecutive days. Figure 6(c) shows the excellent intra-assay reproducibility due to the almost identical responses to 0.1 ng.mL-1 cTnI with RSD of 3.79%. The aptasensor were stored in 4°C, and their performance was studied over time. Figure 6(d) shows that after 50 days, the aptasensor retained around 94.93% of their initial response, indicating appropriate long-term stability. The aptasensor maintained more than 94% of its initial response after 55 days, a performance well consistent with previously reported benchmarks of biosensor stability and exceeding operational lifetimes typical of clinical diagnostics applications. The reusability of the aptasensor was initially assessed by washing with buffer to strip cTnI. The sensor partially recovered; the response was approximately 92% of the original level after a regeneration cycle. Nevertheless, with increasing regeneration, signal loss has provided gradually with a performance of about 85% after three cycles, in all probability for the reason of functioning of aptamers. Thus, the sensor is primarily appropriate for single or limited usage to guarantee consistent sensitive sensitivity.

The selectivity of the aptasensor was examined for determination 0.1 ng.mL-1 cTnI in existence of 5ng/mL of various interfering agents such as urea (UA), hemoglobin (HB), myoglobin (MB), bovine serum albumin (BSA), glucose (Glu), and dopamine (DP). Figure 6(e) shows that the aptasensor response is negligible to the interfering agents compared to response to cTnI, reflecting good selectivity of aptasensor. Further measurements in mixed solutions with interferents also confirmed the Aptasensors’ excellent anti-interference performance, with no remarkable changes in the current after incubation with the mixtures compared to the target cTnI alone. The specificity of the sensor was also assessed in human serum samples containing possible interferents. The combined use of β-cyclodextrin, graphene quantum dots, and MCH blocking greatly reduced nonspecific adsorption, which facilitated the accurate and sensitive detection of cTnI in complex biological samples. Therefore, the developed MCH/apt/pβ-CD/GQD/GCE aptasensor shows substantial analytical performance that makes it a favorable candidate for the rapid, selective, and reliable detection of cTnI for application in clinical diagnostics.

In contrast to conventional immunosensors, the sensitivity and stability of our electrochemical aptasensor have been improved, and the detection limit is decreased due to the high specificity of the aptamer and the enhanced conductivity of graphene quantum dots and β-cyclodextrin. Ab In the clinic, the simple, rapid, and reliable detection of cTnI can be achieved using an inactive, cost-effective, and recyclable sensor, with low cost of manufacture and excellent stability.

3.6. Determination of cTnI in real samples

To evaluate the Aptasensors’ performance in a complex biological matrix, various concentrations of cTnI were spiked in prepared human serum, and the optical properties of the MCH/apt/pβ-CD/GQD/GCE aptasensor were monitored. Table 3 shows analytical results of measurements of cTnI level in a prepared real sample of human serum by the standard addition method after the addition four concentrations of cTnI (0.1, 1, 5, and 15 ng.mL-1) in designed MCH/apt/pβ-CD/GQD/GCE aptasensor. Tables 3 exhibits that for the spiked samples, recovery (90.00% to 99.00%) and RSD (3.42% to 4.66%) values imply to precision and validness of the proposed method in monitoring cTnI in human serum, reflecting its potential for useful applications in clinical diagnostics. The results of the studies of real samples with the Diagnostic Kit for Cardiac Troponin I are also summarized in Table 3. As can be seen, the consistency of the results across all samples emphasizes the reliability of the procedure and indicates the robustness of the MCH/apt/pβ-CD/GQD/GCE aptasensor in a complex matrix. The established aptasensor was tested with sera from a healthy donor and pathological samples of patients with heart disease will be used to study clinical application in the future. In the present investigation, we employed serum samples from a serum collection of one healthy donor and added known concentrations of cTnI for the analytical verification. Although these results indicate good reliability and accuracy of the sensor in a complex biological matrix, additional testing will be performed with clinical samples from cardiac patients in future work to confirm the sensor’s effectiveness in actual clinical assessment.

Table 3. The results for determination of cTnI in real samples of human serum samples by using MCH/apt/pβ-CD/GQD/GCE aptasensor and Troponin I diagnostic Kit. (n =4)
Added (ng.mL-1) Aptasensors
 Troponin I diagnostic Kit
Found (ng.mL-1) Recovery (%) RSD (%) Found (ng.mL-1) Recovery (%) RSD (%) Relative difference (%)
0.00 0.00 --- 3.42 0.00 --- 4.33 0.0000
0.10 0.09 90.00 3.79 0.09 90.00 4.81 0.0000
1.00 0.97 97.00 3.52 0.96 96.00 3.85 -1.0416
5.00 4.89 97.80 4.66 4.91 98.20 4.11 0.4073
15.00 14.85 99.00 4.42 14.93 99.53 3.37 0.5358

In addition, the ease and robustness of the fabrication procedures for the aptasensor, as well as its stability and reproducibility, confirm its possible translation into practical manufacturing and POC devices. Both the electrodeposition process and the aptamer immobilization protocol are amenable to high-throughput manufacturing, which allows cost-effective production at large scale. With these superior characteristics of the sensor and high sensitivity and specific response in mixed samples, it has shown great potential for sensitive and rapid determination of cardiac markers in clinical diagnosis.

4. Conclusions

In view of the urgent requirement for ultrasensitive and specific detection of cTnI to solve the shortcomings of traditional diagnostic methods including low sensitivity, complicated fabrication process, and poor stability, an electrochemical aptasensor based on GQDs and pβ-CD with good performances for electron transfer, aptamer immobilization, and nonspecific binding suppression was prepared in this work. Current study was developed to design a sensitive and selective electrochemical aptasensor based on GQDs and β-CD, and application for quantification of cTnI level in human serum samples. The aptasensor was designed based on immobilization of a 5’-amine modified troponin I aptamer on an electrodeposited pβ-CD/GQD nanocomposite on GCE. Comprehensive optimization of experimental parameters, including electrodeposition time, aptamer immobilization time, MCH blocking time, and target incubation time, remarkably improved the aptasensor’s performance and reproducibility. The findings exhibited wide linear detection range from 10-5 to 103 ng.mL-1, low LOD (0.9 pg.mL-1), substantial anti-interference performance, and significant repeatability and stability over 50 days. The aptasensors’ excellent analytical performance and low-cost fabrication method, makes it a valuable tool for quick and accurate quantification of cTnI in clinical diagnostics and many cardiovascular diseases. This work is expected to add knowledge to the field by facilitating an easy, low-cost, and ultrasensitive platform, which offered a solution to overcome the challenges of current sensors. Nevertheless, there are several limitations that need to be addressed such as the application to clinical samples of patients with cardiac pathology so as to fully demonstrate the diagnostic potential of the sensor. Moreover, long-term stability in different storage conditions and reproducibility in large-scale production also need to be studied. Further studies will investigate the feasibility of using the aptasensors for point-of-care diagnostics, utilization of the aptasensors in/back of portable equipment, and performance of the aptasensors in real complex samples, such as multiplex detection of cardiac biomarkers for improved diagnosis accuracy.

Acknowledgment

The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through Large Research Project under grant number RGP2/585/45.

CRediT authorship contribution statement

Xin Zhou, Sicong Jiang, Huangtao Sun, Aimin Xie, Zhihong Qiu, Guansen You, Yuxuan Xingu, Dongmei Yang: Conceptualization, formal analysis, Xin Zhou, Sicong Jiang, Huangtao Sun, Aimin Xie, Zhihong Qiu, Guansen You, Yuxuan Xingu, Dongmei Yang, Shubham Sharma: Investigation, Xin Zhou, Sicong Jiang, Huangtao Sun, Aimin Xie, Zhihong Qiu, Guansen You, Yuxuan Xingu, Dongmei Yang: Writing—original draft preparation, Xin Zhou, Sicong Jiang, Huangtao Sun, Aimin Xie, Zhihong Qiu, Guansen You, Yuxuan Xingu, Dongmei Yang: Writing—review and editing, Shubham Sharma, Mohamed Abbas: Supervision, Mohamed Abbas: project administration, Mohamed Abbas: Funding acquisition, Dongmei Yang, Mohamed Abbas. All authors have read and agreed to the published version of the manuscript.

Declaration of competing interest

There are no conflicts of interest.

Data availability

The raw data can be obtained on request from the corresponding authors, Xin Zhou and Dongmei Yang. The data used to interpret the findings are available from the corresponding authors, Xin Zhou and Dongmei Yang. All the characterizations, analysis, testing’s related work and testings’ have solely been responsible by corresponding authors, Xin Zhou and Dongmei Yang. Additionally, the raw data can be obtained on request from the corresponding authors, Xin Zhou and Dongmei Yang. Any sort of queries in context with the research ethics, integrity and other related unscrupulous issues must be handled and responsible solely by the corresponding authors, Xin Zhou and Dongmei Yang.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript, and no images were manipulated using AI.

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