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Original Article
:19;
3842025
doi:
10.25259/AJC_384_2025

Design of a label-free mass sensor based on three-dimensional nanomagnetic bead for ultrasensitive detection of cardiac troponin I

, , , , , , , ,
a School of Public Health, Hebei Medical University, Shijiazhuang, PR China
b School of Pharmacy, Hebei Medical University, Shijiazhuang, PR China
c Hebei Key Laboratory of Environment and Human Health, Shijiazhuang, PR China
d Institute of Medicine and Health, Hebei Medical University, Shijiazhuang, PR China
e Hebei Key Laboratory of Forensic Medicine, Hebei Province, Shijiazhuang, P.R. China
† Authors contributed equally to this work and share co-first authorship.

*Corresponding author: E-mail address: 17800872@hebmu.edu.cn (L. Niu)

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

In this research, an innovative and highly sensitive mass sensor was designed for cardiac troponin I (cTnI) detection based on 3D nanomagnetic beads, which do not need to be labelled using signal probes. The proposed sensor was characterized by several technologies, such as transmission electron microscopy (TEM), UV-visible spectrophotometry, atomic force microscopy (AFM), Fourier transform infrared spectrometer (FTIR), scanning electron microscope (SEM), etc. Under optimum conditions, the frequency showed a remarkable linear dependence on cTnI concentration within the interval of 1.0 pg mL-1 to 10.0 ng mL-1, with a detection limit of 0.2 pg mL-1. This biosensor showed ultrasensitivity, time-efficiency, and lower costs, and consequently can be expected to be applied in real life.

Keywords

Aptamer (apt)
Cardiac troponin I
Mass sensitizing solution
Nanomagnetic beads
Quartz crystal microbalance

1. Introduction

Cardiovascular diseases (CVDs) are the principal cause of death in the world [1,2]. With the aging of the population in the future, the morbidity and mortality of CVDs are expected to rise continuously, and the situation of prevention and control is very serious. Among these, acute myocardial infarction (AMI) ranks as one of the most prevalent CVDs in China. In severe instances, it has the potential to directly result in irreversible myocardial injury or necrosis [3]. Up to now, the most commonly used diagnostic modalities for AMI are electrocardiogram (ECG) and routine blood tests, but since only a small percentage of AMI patients exhibit changes in ECG [1,4,5]; the diagnosis is often made clinically by using a variety of cardiac biomarkers in the blood. These include cardiac troponin I (cTnI), cardiac troponin T (cTnT), creatine kinase (CK), myoglobin (Mb), and C-reactive protein (CRP) [6]. Among these indices, cTnI is considered the preferred and important marker for diagnosis because of its strong specificity and sensitivity, and as it appears only in the presence of cardiomyocyte injury [7]. It is, thus, used to monitor the status and prognosis of AMI patients. Previous studies have successfully achieved precise capture and quantitative detection of cTnI in serum, providing reliable technical support for the early diagnosis of myocardial infarction [8,9]. At the present stage, there are several methods for the detection of cTnI, and each has its own advantages and disadvantages, such as enzyme-linked immunosorbent assay (ELISA) [10], radioimmunoassay [11], colorimetric assay [12,13], fluorescence immunoassay [14], and electrochemical immunoluminescence assay [15-17], etc. These methods have a high sensitivity and a wide linear range, but the pre-treatment of samples for these methods is complicated, time-consuming, and the requirements of experimental instruments are high. In addition to the aforementioned methods, studies have also employed electrochemical immunoassay and surface plasmon resonance (SPR) immunoanalysis for the detection of cTnI. Despite their wide linear detection range, these methods exhibit poor stability and low sensitivity, making them less suitable for trace detection [18]. In contrast, quartz crystal microbalance (QCM) possesses the advantages of outstanding sensitivity, convenient operation, rapid detection, and reduced apparatus cost [19].

QCM was initially developed as a mass sensor for gas phase under vacuum, which is characterized by simplicity, real-time response, high sensitivity, and no labeling required for the detection process. As a surface-sensitive analytical technique with high sensitivity and real-time on-line detection, it has been widely used in biomedicine, energy and environment, and food safety [20-22]. Various technological approaches, such as molecular imprinting technology, nanotechnology, aptamer technology, etc., have been combined with QCM. [23-25]. This enhances the mass signal by preparing different materials to modify the QCM electrode/target. Currently, the commonly used magnetic nanoparticles (MNPs) are easy to synthesize and have a mass of their own, which can be used for mass signal amplification to achieve ultrasensitive detection of the target.

MNPs, which are nanoscale magnetic particles, are emerging materials with the advantages of low toxicity, adjustable size, easy synthesis, and low cost compared to other precious metal particles [26,27]. The mass of the MNPs can increase the mass signal. The use of reagents, such as antibodies, aptamers, and others, to modify their surface, can help them couple with cells, nucleic acids, proteins, and other substances. Additionally, nucleic acids aptamer can greatly increase the specificity of the nanomagnetic beads [28].

Nucleic acid aptamers are in vitro screened oligonucleotide fragments that can specifically bind proteins or other small molecules [29] and have short screening cycles, simple synthesis, good stability, high affinity [30,31], and a wide range of target molecules [32]. Nucleic acid aptamers have the property of specific recognition of targets [33-36], which is very similar to the antigen recognition property of antibodies, so they are also called “chemical antibodies.” Compared with antibodies, nucleic acid aptamers have a small molecular weight, good stability, and no immunogenicity [37]. Hence, they are often used as novel molecular recognition tools and widely used in the preparation of bio-nanosensors [38].

In this study, a novel mass-sensitized sensor for three-dimensionally distributed MNPs, constructed using complementary DNA, was designed by using a mass-signaling probe based on the ultrasensitive response of QCM to mass changes. It enabled the presence of a very small amount of the target to cause a sufficient mass change on the electrode surface. The ingenious use of advance pre-amplification strategy greatly improves the detection efficiency greatly improved, and the amplification of mass signals could be realized in a short time. In contrast to alternative approaches, this technique demonstrated excellent sensitivity and convenience, enabling the enhanced identification of cTnI without complex sample preparation.

2. Materials and Methods

2.1. Reagents

FeCl3·6H2O, anhydrous ethanol, hydrogen peroxide, anhydrous sodium acetate, ethylene glycol, sulfuric acid solution, 1,6-Hexanediamine, bovine serum albumin (BSA), streptavidin, glutaraldehyde, and 6-mercaptohexanol (MCH) were obtained from Aladdin Industrial Corporation (Shanghai, China). The cTnI standard was purchased from the Institute of Metrology and Science (Beijing, China). cTnT, Mb, and CRP were purchased from UpingBio Technology (Shenzhen, China). In this study, all substances employed were of analytical grade. The water utilized in all experiments was derived from a Milli-Q ultrapure water system. All the synthetic DNA sequences (apt1, apt2 of cTnI, S1, and S2) used during the experiments were procured from Sangon Biotechnology Co., Ltd (Shanghai, China), and their sequences are presented in Table S1.

Table S1

2.2. Apparatus

In this study, QCM was performed on a CHI440B workstation (CH Instruments Inc., Shanghai, China). The morphological and structural characterization of the mass-sensitizing solution was performed using transmission electron microscopy (TEM; JEOL JEM 2100Plus, Japan), UV-visible spectrophotometry (UV-Vis; TU-1901, China), and a Fourier transform infrared spectrometer (FT-IR; FTIR-650, China). Additionally, characterizations of layer-by-layer modifications on electrode surfaces were performed using atomic force microscopy (AFM; Bruker dimension icon, Germany) and scanning electron microscope (SEM) (Zeiss Supra55, Germany).

2.3. Preparation of mass-sensitizing solution

High-quality ammonia-functionalized magnetic nanospheres were prepared with adjustable particle size by using FeCl3·6H2O as the sole iron source and 1,6-hexanediamine as the ligand [39]. Then, 1.0 g of FeCl3·6H2O and 2.0 g of anhydrous sodium acetate were weighed, and 30.0 mL of ethylene glycol was added and mixed well with magnetic stirring. After adding 2.5 g of 1,6-hexanediamine to the above mixture, the reaction proceeded under agitation at a temperature of 50°C for 15 min. The reacted solution was transferred into a high-pressure reactor at a high temperature of 198°C for 6 h. Upon the completion of the reaction, the black product was collected by magnetic separation, and the upper layer of liquid was discarded. The magnetic beads were washed alternately with hot ultrapure water and anhydrous ethanol thrice and dried under vacuum at 50°C for 12 h to obtain MNPs. The magnetic beads were washed in hot ultrapure water and anhydrous ethanol three times, respectively, then dried under vacuum at a temperature of 50°C for 12 h, resulting in the formation of MNPs.

The desired capture probes were synthesized using the classical glutaraldehyde method [40]. First, 5.0 mg of the synthesized MNPs was weighed out and added to 5.0 mL of 0.01 M phosphate buffer solution (PBS) with a pH of 7.4. The mixture was then sonicated and dispersed for 10 min. Subsequently, 1.25 mL of glutaraldehyde (25% w/v) was added, and the resulting solution was shaken in the dark at 37°C for 2 h. After activation was completed, the enriched MNPs were separated by magnet, then sonicated and washed three times, and then they were resuspended. Afterwards, 0.5 mL of affinity protein (15.0 μM) was added and continuously incubated for overnight at 37°C with shaking. The obtained substance was stored in a sealed box at 4°C. Then, 20.0 μL of biotin-modified apt1 (1.0 μM) was mixed with 380.0 μL of affinity-modified MNPs and shaken at 37°C for 2 h. After the reaction completion, the apt1/MNPs were magnetically separated, washed three times to obtain apt1/MNPs, a specific capture probe for cTnI.

The prepared capture probe apt1/MNPs solution of 190.0 μL was taken from two tubes each, and 10.0 μL of solution containing S1 and 10.0 μL of solution containing S2 (4.0 μM) were added and mixed homogeneously, then incubated at 37°C for 1.5 h with shaking in the dark. The remaining unbound sites on the surface of the magnetic beads were sealed by adding 0.1% BSA after magnetic separation and cleaning, and then immediately resuspended in PBS to obtain apt1/MNPs/S1 and apt1/MNPs/S2 solutions, respectively. The above prepared two tubes of solution were mixed and incubated at 37°C for 1.5 h with shaking in dark, the two single-stranded DNA S1 and S2 bound to the magnetic beads were combined by cross-linking hybridization. Ultimately yielding the desired mass-sensitizing solution apt1/MNPs/S after magnetic separation and washing.

2.4. Modification of QCM electrode and cTnI detection

To ensure that the gold electrode chip surface of the quartz crystal is free of impurities, the chip electrode needs to be cleaned before use. Piranha solution (H2O2:H2SO4 = 1:3) was dropped and coated on the chip surface for 20 min. Subsequently, the sample was treated in ultrasonic ultrapure water and anhydrous ethanol, respectively, for 20 min, followed by drying in a nitrogen atmosphere. The fundamental frequency of the bare electrode was measured to be 7.995 MHz before utilization. Only when the frequency value is stable can the subsequent modification proceed.

The aptamer was immobilized onto a gold chip electrode using a module of continuous adenine bases as described in reference [41]. A 10.0 μL volume of a 1.0 μM apt2 solution was carefully dropped onto the surface of the gold electrode, and the electrode was then incubated at 4°C overnight. Following this incubation, the gold electrode was blocked by treating it with 10.0 μL of a 1.0 MCH solution for 0.5 h.

To the QCM gold chip electrode modified with apt2, 10.0 µL cTnI solution of 0.3 ng mL-1 was added, then incubated at 37°C for 30 min. After that, the mass-sensitizing solution apt1/MNPs/S was drop, which was prepared in advance, and then incubated at 37°C for 80 min, then washed and dried, and then the frequency was detected [42].

The modified apt2 on the QCM electrode was used as a capture probe. When cTnI was presence in the solution, the apt2 could catch it. After the adding of pre-amplified mass-sensitizing solution apt1/MNPs/S, the sensitized probe was bound to the electrode to form a “sandwich” structure. Therefore, the presence of a very small amount of the target can cause a significant mass increase of the electrode and an obvious change in the frequency. The specific experimental method has been shown in Scheme 1.

Schematic diagram of electrode modification and cTnI detection.
Scheme 1.
Schematic diagram of electrode modification and cTnI detection.

3. Results and Discussion

3.1. Characterization of mass-sensitizing solution

The morphology of MNPs synthesized by the one-pot method, as well as MNPs modified with apt1, S1, and S2, were characterized using TEM. As shown in Figure 1(a), the unmodified MNPs showed uniform morphology and a similar size. After modifying the MNPs with apt1, S1, and S2, it can be clearly seen that (Figure 1b) the surface of the MNPs was successfully modified with DNA strands, and the MNPs were connected with each other more tightly due to the effect of S1 and S2 hybridization.

TEM images of (a) bare magnetic nanobeads, (b) magnetic beads after incubation with modified apt1, and single-stranded DNA amplification.
Figure 1.
TEM images of (a) bare magnetic nanobeads, (b) magnetic beads after incubation with modified apt1, and single-stranded DNA amplification.

In the presence of glutaraldehyde, the surface of MNPs can be modified with streptavidin to thereby fix apt1. The binding of MNPs with streptavidin and apt1 was characterized using UV-Vis. Figure S1(a) showed that, after an overnight incubation of streptavidin with MNPs, the UV-Vis spectra were blue-shifted, proving that part of the affinities had bound to the amine-functionalized MNPs. Similarly, a notable red shift appeared in the UV-Vis spectra (Figure S1b) of the self-assembled apt1/MNPs in comparison to MNPs, proving that apt1 had been successfully coupled to the MNPs.

Figure S1

The composition of the mass-sensitizing solution (apt1/MNPs/S) was characterized using FTIR (Figure S2). The characteristic peaks of Fe-O (590 cm-1) and N-H (1600 cm-1) of MNPs can be seen in the figure. During the synthesis of MNPs, 1,6-hexanediamine was used as a ligand, which resulted in a large number of amino groups on the surface of MNPs. The C-N (1190 cm-1) characteristic absorption peaks could prove that the surface of MNPs was successfully modified by apt1 as well as ssDNA.

Figure S2

3.2. Characterization of the QCM aptasensors

The modifications on the surface of the electrode were characterized using AFM, as shown in Figure S3(a). The bare QCM gold electrode could be seen to be relatively thinner and uniform in thickness, while the thickness of the modified electrode increased significantly with the layer-by-layer modification (Figure S3b). The difference between these two graphs proved a successfully modification of apt1/MNPs/S.

Figure S3

The morphology of the layer-by-layer modification of apt2, apt1/MNPs/S, and the distribution of elemental content on the electrodesurface were characterized by SEM and energy-dispersive X-rayspectroscopy (EDS). After modification of apt2, some uniform DNA small molecule structures can be seen on the electrode surface (Figure 2a). When the final mass sensitizing solution was modified on the electrode surface, a large number of MNPs were tightly connected due to the hybridization of S1 and S2 (Figure 2b). The presence of C, O, N, and P elements is also presented in the mapping (Figure 2c), which further proved that apt2 was successfully modified on the surface of the QCM. After the mass sensitizing solution was modified, the presence of C, O, N, P, and Fe elements could be clearly observed (Figure 2d). The EDS spectrum also further confirms the successful modification on the surface of the QCM electrode (Figure2e-f).

SEM images of (a) QCM modified aptamer and (b) mass-sensitizing solution; EDS elemental mappings of (c) C, O, N, and P elements on QCM modified aptamer and (d) C, O, N, P, and Fe elements on QCM modified mass-sensitizing solution; EDS spectrum of (e) QCM modified aptamer and (f) mass-sensitizing solution.
Figure 2.
SEM images of (a) QCM modified aptamer and (b) mass-sensitizing solution; EDS elemental mappings of (c) C, O, N, and P elements on QCM modified aptamer and (d) C, O, N, P, and Fe elements on QCM modified mass-sensitizing solution; EDS spectrum of (e) QCM modified aptamer and (f) mass-sensitizing solution.

3.3. Electrochemical characterization

In this study, the sequential modification of the QCM electrode was investigated using differential pulse voltammetry (DPV) in a supporting solution of PBS (pH=7.0, 0.1 M) with 5 mM potassium ferricyanide solution and 0.1 M KCl. As shown in Figure 3(a), the peak current response value reached the maximum (curve a) this was attributed to the fact that the unmodified QCM electrode was made of gold, which facilitates efficient electron transfer. However, when the electrode surface was functionalized with apt2, the peak current experienced a substantial decline (curve b). This reduction was primarily due to the presence of apt2, which obstructed the electron transfer process. After further modification of MCH to cover the remaining sites, the peak current continued to decrease (curve c). When the modified electrode captured the target cTnI, the peak current decreased again (curve d). After the addition of the sensitizing solution, the peak current was minimized due to the large spatial site resistance of the magnetic bead aptamer complex (curve e).

(a) DPVs and (b) CVs spectra of bare QCM (I), apt2/QCM (II), MCH/apt2/QCM (III), cTnI/MCH/apt2/QCM (IV), and apt1/MNPs/S/cTnI/MCH/apt2/QCM (V).
Figure 3.
(a) DPVs and (b) CVs spectra of bare QCM (I), apt2/QCM (II), MCH/apt2/QCM (III), cTnI/MCH/apt2/QCM (IV), and apt1/MNPs/S/cTnI/MCH/apt2/QCM (V).

Similarly, cyclic voltammetry (CV) can be verified for layer-by-layer modification of the electrode (Figure. 3b) in a 5.0 mM [Fe(CN)6]3−/4− solution with 0.1 M KCl at a scan rate of 100 mV/s. The QCM with bare gold electrode (curve I) exhibited a couple of redox peaks characterized by excellent reversibility. Following the modification of apt2 (curve II), a notable decline in redox peaks became evident. Subsequently, MCH was applied at the electrode surface to fill the remaining gaps (curve III), and the redox peak current kept declining. Following the incorporation of the target cTnI onto the MCH/apt2/QCM sensor, the reduction in redox peak current was observed again, which was attributed to the specific interaction between cTnI and apt2 (curve IV). After modification of the mass-sensitizing solution, the peak current signal continued to decrease due to the binding of cTnI to apt1/MNPs/S, and the magnetic bead aptamer complexes greatly impeded the transfer of electrons across the electrode surface (curve V).

The electrode surface coverage Γ of the bare electrode, and the final modified sensitizer apt1/MNPs/S electrode can be calculated by Eq. (1) [43]:

(1)
Q = n F A Γ

where Q is the value of the charge at the time of detection, while n denotes the count of transferred electrons (n = 1), A is the active surface area of the electrical level of 0.205 cm2, and F is the Faraday constant of 96,485 C mol-1. After calculating, the Γ (bare electrode) was obtained as 19.5 nmol cm-2 and Γ (apt1/MNPs/S) as 17.6 nmol cm-2. The decrease in Γ indicated that due to the layer-by-layer modification, the concentration of electroactive substances on the surface of the electrode was reduced. DPV and CV results were consistent with each other verifying the layer-by-layer modification of the QCM electrode.

Firstly, the electroactive surface area of the modified electrode with the mass-sensitizing solution was evaluated through CV using [Fe(CN)6]3−/4− as the redox indicator. The linear regression equation of I = 7.7349 × ν1/2 + 0.4593 (r = 0.986) is obtained. This indicates that the electrochemical reaction at the electrode surface is controlled by diffusion. According to the Randles-Sevcik equation (Eq. 2) [44]:

(2)
I p = 2 . 69 × 1 0 5 n 3 / 2 D 1 / 2 ν 1 / 2 A C

where I p denotes the peak current reduction of [Fe(CN)6]3−/4−, n represents the electron number that transferred during the redox reaction (n = 1), D stands for the diffusion coefficient of [Fe(CN)6]3−/4− (D = 6.70 × 10-6cm2/s at 25°C), ν denotes the scan rate (V s-1), while A represents the electroactive surface area (cm2) of the modified electrodes, and the concentration of [Fe(CN)6]3−/4−is denoted as C (C = 5.0 × 10-6 mol cm-3). By this means, the calculated electroactive surface area is 0.2833 cm2, which is lower than that of the actual area (0.5102 cm2). It is remarkable that this decreased electroactive surface area is attributed to the magnetic bead aptamer complexes.

3.4. Optimization of the experimental conditions

3.4.1. Influence of the size of MNPs

The size of the magnetic beads is a crucial factor that can affect the sensor’s sensitivity. Therefore, the size of MNPs was optimized in this experiment. According to the literature [45], the size of MNPs is mainly determined by the amount of 1,6-hexanediamine. With the increase of 1,6-hexanediamine dosage, the frequency reached its maximum at 2.5 mg. So, 2.5 mg of 1,6-hexanediamine was used as the best experimental condition for synthesizing MNPs (Figure S4a).

Figure S4

3.4.2. Optimization of the concentration of apt1

The concentration of apt1 showed a significant effect on the capture for cTnI as well as the sensor sensitivity. A suitable concentration of apt1 will fully capture cTnI and firmly bind the whole sensitizing group apt1/MNPs/S to cTnI, causing the enhancement of mass signal and the change of frequency. As shown in Figure S4(b), the value of frequency modulation increased with the rising in apt1 concentration, and stabilized at 1.0 μM of apt1 concentration. Thus, a concentration of 1.0 μM was deemed the most suitable reaction concentration.

3.4.3. Optimization of the incubation time (mass-sensitizing solution with cTnI)

The apt1 in the mass-sensitizing solution apt1/MNPs/S specifically recognized cTnI, which bound the whole apt1/MNPs/S to the electrode by sandwiching, causing a change in the mass signal, which in turn affects the change in frequency. Therefore, the optimal incubation time was studied. With the prolongation of the incubation time, the mass-sensitizing groups bound to the electrode surface increased continuously, and the frequency increased and reached the maximum value at 80 min. Hence, 80 min was deemed the optimal binding time for subsequent experiments (Figure S4c).

3.4.4. Optimization of the incubation time (mass-sensitizing solution for amplification)

The apt1, MNPs, S1, and S2 were mixed under certain conditions for pre-amplification for mass signal amplification. With the extension of the time of amplification, the MNPs were closely connected to each other through the complementary parts of the S1 and S2 chains, and the target could bind enough apt1/MNPs/S for the mass signal to increase significantly. When the amplification incubation time was 1.5 h, the frequency change value reached the maximum. Consequently, 1.5 h was chosen as the optimal time for mass sensitization group amplification incubation (Figure S4d).

3.4.5. Optimization of the ratio of concentration of apt1 to S1 and S2

In addition to the amplification time, the concentration ratio of apt1/MNPs also has a certain effect on the mass sensitizer. Under the appropriate concentration ratio, the complementary parts of S1 and S2 bases will connect enough apt1/MNPs/S1 and apt1/MNPs/S2 in the form of “hand in hand” to ensure that the maximum frequency change value can be obtained after recognizing the target. The optimized apt1 concentration of 1.0 μM was used as the benchmark, and different ratios of S1 and S2 were formulated. When the ratio of apt1 to S1/S2 was 1:4, the frequency change value reached the highest (Figure S4e), and then the frequency almost did not change anymore in the subsequent ratios, so 1:4 was chosen as the optimal concentration ratio.

3.4.6. Optimization of the concentration of apt2

The apt2 concentration of the electrode surface modification directly affects the amount of cTnI bound to the target, which in turn affects the number of mass-sensitizing groups bound, and thus the amount of frequency change. As reflected in Figure S4(f), the detection value was maximized at an apt2 concentration of 1.0 μM. After that, the concentration of apt2 continued to increase. Too large a concentration of apt2 might produce a large spatial site resistance, hindering the binding of cTnI, and thus the value of frequency change gradually decreased. Therefore, 1.0 μM was selected as the optimal concentration of apt2.

3.4.7. Optimization of the incubation time (apt2 with cTnI)

With the extension of time, apt2 could capture more cTnI and thus bind more apt1/MNPs/S. As the incubation time increased, the electrode surface mass kept increasing and reached the maximum at 60 min of incubation time. So, 60 min was chosen as the subsequent binding time of apt2 to cTnI (Figure S4g).

3.5. Analytical performances of the QCM aptasensor

In the QCM aptasensor analysis, there was a good correlation between the signal intensity and the concentration of the target substance. Therefore, under the best optimized conditions, the QCM aptasensor will be utilized to detect different concentrations of cTnI. As reflected in Figures 4(a and b), there is a good linear correlation between the frequency change values and the logarithm of cTnI concentration in the range of 1.0 pg mL-1 to 10.0 ng mL-1. The regression equation (Eq. 3) is

(3)
Δ F = 9 . 481Ln C cTnI + 68 . 228  r = 0. 994 ,   Δ F :   Hz , C cTnI :   ng mL − 1 ,

(a) QCM frequency variation of aptasensor in the presence of 0, 0.001, 0.003, 0.01, 0.05, 0.3, 0.5, 1.0, and 10.0 ng mL-1 cTnI; (b) The corresponding linear relationship between cTnI concentration and QCM frequency variation.
Figure 4.
(a) QCM frequency variation of aptasensor in the presence of 0, 0.001, 0.003, 0.01, 0.05, 0.3, 0.5, 1.0, and 10.0 ng mL-1 cTnI; (b) The corresponding linear relationship between cTnI concentration and QCM frequency variation.

with the limit of detection (LOD) of 0.2 pg mL-1. In contrast to other methods for detecting cTnI (Table S2), the established method demonstrated a broad linear dynamic range and excellent characteristics.

Table S2

3.6. Specificity, reproducibility, stability, and real sample analysis

Selectivity is one important parameter for the evaluation of biosensors. So, cTnT, Mb, and CRP, which often coexist in blood samples, were selected as interferences to examine the specificity of the present sensor, and the results of the assay has been shown in Figure 5(a). This indicates that the proposed method showed an excellent selectivity, and the interferences investigated had no significant effects on the detection of cTnI.

(a) Selectivity of the proposed QCM aptasensor for 0.3 ng mL-1 cTnI from 3.0 ng mL-1 cTnT, 3.0 ng mL-1 Mb, 3.0 ng mL-1 CRP. (b) Repeatability of the QCM aptasensor at the cTnI concentration of 0.3 ng mL-1 in five different electrodes. (c) Stability of the QCM aptasensor at the cTnI concentration of 10.0 ng mL-1 in different days (0, 2, 4, 6 and 8 days).
Figure 5.
(a) Selectivity of the proposed QCM aptasensor for 0.3 ng mL-1 cTnI from 3.0 ng mL-1 cTnT, 3.0 ng mL-1 Mb, 3.0 ng mL-1 CRP. (b) Repeatability of the QCM aptasensor at the cTnI concentration of 0.3 ng mL-1 in five different electrodes. (c) Stability of the QCM aptasensor at the cTnI concentration of 10.0 ng mL-1 in different days (0, 2, 4, 6 and 8 days).

Reproducibility is another important parameter for the evaluation of biosensors. Under the same experimental conditions, five modified sensors were prepared simultaneously, and the same samples were analyzed, and the relative standard deviation of the three parallel determinations turned out to be 2.95% (Figure 5b), After conducting a t-test, there was no significant difference in the test results between the first sensor and the fifth sensor (P=0.49>0.05), indicating that the sensor has a good reproducibility [46,47].

To assess the stability of the sensor for long-term storage, and the sensor was placed at 4°C for storage after measuring the frequency value, and the sensor was tested every two days and at a fixed time in accordance with the periodic measurement method, and it was found that the frequency of the sensor did not change significantly in 8 days, and the RSD was 2.15% (Figure 5c). Furthermore, based on the references [48], we used the t-test method to analyze the results on the first day and the eighth day. The absence of a statistically significant difference (P=0.724>0.05) proved that the sensor had good stability.

In order to study the effectiveness of this mass sensor in detecting cTnI in real samples, human serum samples were compared and analyzed using ELISA and this sensor, respectively. As the shown in Table S3, Using t-test, there was no statistically significant distinction between the two methods (P=0.214>0.1) [49,50]. Using spiked recovery, different concentrations of cTnI (0.05, 0.5, and 1.0 ng mL-1) were added to normal serum samples, and the RSD of the spiked samples were in the range of 2.23%-4.97%, and the recoveries were in the range of 91.33%-106.67%, as shown in Table S4. The findings presented above demonstrate that the designed sensor is capable of effectively detecting cTnI in actual samples and highlighting its broad applicability in various fields.

Table S3

Table S4

4. Conclusions

A pre-amplifiable mass gain-sensitive aptamer sensor was designed and used for the detection of cTnI. The method is easy to use, greatly reduces the assay time, and has good sensitivity, specificity, and reproducibility. Under optimal conditions, the sensor showed an excellent linear range (1.0 pg mL-1-10.0 ng mL-1) and an ultra-low detection limit of 0.2 pg mL-1. The method can be successfully applied to the detection of cTnI in human serum, and the results obtained are satisfactory, which has promising potential for clinical applications.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (No. 82073601) and the National Natural Science Foundation of Hebei (No. H2024206344).

CRediT authorship contribution statement

Xi Ze: Manuscript preparation, Design, Experimental studies. Meng Jiang: Manuscript editing and review, Experimental studies, Data analysis. Kai Kang: Data acquisition, Literature search. Yipeng Wang: Data acquisition, Literature search. Wen Yan: Statistical analysis, Definition of intellectual content. Yan Jin: Statistical analysis, Definition of intellectual content. Jingyi Wang: Statistical analysis, Definition of intellectual content. Kejia Xu: Statistical analysis, Data analysis. Lingmei Niu: Concepts, Design, Manuscript editing and review.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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.

Supplementary data

Supplementary material to this article can be found online at https://dx.doi.org/10.25259/AJC_384_2025.

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