An electrochemical aptasensor based on catalytic hairpin self-assembly and co-amplification of AuPd/Fe-MOF and Au/Cu2O for ultrasensitive detection of Cd2+

Cadmium (Cd), a typical heavy metal not essential for the human body, can cause harm to the liver and kidneys upon exposure. Hence, rapid cadmium detection in the environment is of utmost importance. This research presents an effective approach for detecting divalent cadmium ions (Cd2+) using a sensitive dual hairpin (HP) electrochemical aptasensor. The aptasensor incorporates AuPd/Fe-MOF as signal labels and Au/Cu2O as the substrate material, and the rapid detection of divalent cadmium ions (Cd2+) was based on catalytic hairpin self-assembly (CHA), as the recognition strategy. Au/Cu2O increased the specific surface area of the electrode and provided abundant sites for capturing the complementary deoxyribonucleic acid (CDNA) probe. The electrochemical signal is then amplified through synergistic catalytic hydrogen peroxide with AuPd/Fe-MOF, thereby enhancing the aptasensor's sensitivity. Moreover, the dual HP design effectively reduces the likelihood of non-specific capture and minimizes false positives. In this study, various analytical techniques were utilized to characterize the material and evaluate the aptasensor's performance. The morphological characteristics of the material were observed using scanning electron microscopy (SEM). Energy-dispersive spectroscopy (EDS) and X-ray diffraction (XRD) were employed to analyze the elemental composition and distribution of the material, respectively. The electrochemical behavior of the sensor was studied using cyclic voltammetry (CV), while electrochemical impedance spectroscopy (EIS) helped understand the aptasensor's assembly process. Furthermore, the aptasensor's performance was assessed using differential pulse voltammetry (DPV). Under the optimized experimental conditions, the constructed sensor demonstrated effective detection of Cd2+ in the concentration range of 10–4–10 µM, with a remarkably low detection limit of 2.27 × 10–5 µM. The feasibility of the sensor was validated by successfully detecting Cd2+ in real water samples. .


Introduction
Over the years, human activities have caused an increase in heavy metal pollution, with elements like cadmium (Cd) returning to natural environments like water, atmosphere, and soil in more active forms due to various industries' development, such as smelting, construction, and chemical production.Cadmium, being a typical heavy metal not needed by the human body, has a long biological half-life of 10-30 years (Rafati Rahimzadeh et al. 2017).When Cd enters the human body, it accumulates in organs like the liver and kidneys, leading to chronic poisoning.The International Agency for Research on Cancer (IARC) has classified Cd as a human carcinogen.Additionally, Cd can accumulate in crops like rice (Jin et al. 2021), posing health concerns as seen in incidents like the Japan Toyama pain disease.In China, approximately 1.3 × 10 -4 hectares of arable land produce around 5.0 × 10 -4 tons of Cd-contaminated rice annually (Gu and Zhou 2002;Nordberg 2009).Cd pollution is also a significant problem in Japan and Korea, threatening food and drinking water safety (Kwon et al. 2022;Masumoto et al. 2022).
Monitoring heavy metal pollution is crucial for effective management, and detection technology plays a key role in this process.Traditional methods for detecting Cd 2+ include inductively coupled plasma mass spectrometry (ICPMS) (Wang et al. 2022), atomic emission spectrometry (AES) (Gondal et al. 2020), laser-induced breakdown spectrometry (LIBS) (Ren et al. 2022), and atomic absorption spectrometry (AAS) (Ibourki et al. 2021).These methods offer good detection performance and stability but are often complex, time-consuming, and require large amounts of reagents and complex instruments for sample pretreatment before on-line detection.Hence, there is a critical need to develop new detection techniques that are fast, sensitive, specific, and easy to operate to enhance the prevention and control of heavy metal pollution, including Cd 2+ .
Electrochemical aptasensors are composed of electrodes, recognition molecules, and nanomaterials (Guo et al. 2021).Their purpose is to detect targets by measuring changes in electrochemical signals.Gopalakrishnan et al. prepared an electrochemical sensing platform based on cadmium oxide (CdO) nanoparticle modified disposable screen printed carbon electrode (SPCE), which utilized the electrocatalytic activity of cadmium oxide to ascorbic acid (AA), achieving non-enzymatic detection of AA.The detection limit was as low as 53.5 nM (Gopalakrishnan et al. 2018).Unlike electrochemical sensors which are purely composed of nanomaterials and electrodes, aptasensors utilize a recognition strategy based on nucleic acid aptamers (Apts) combined with nanocomposites possessing advantageous properties such as catalytic and electrical conductivity.The flexibility of the recognition strategy allows for a wide range of customization, leading to diverse recognition mechanisms.In contrast to antibody biosensors (Cardoso et al. 2022), which rely on antigen-antibody interactions for target recognition, Apts offer advantages like easier modification, greater design options, diversity, and enhanced stability (Kedzierski et al. 2012).This makes them capable of recognizing various toxic substances without concerns about inactivation and denaturation.Moreover, aptamers can be synthesized and screened with minimal batch variation, facilitating high-volume production.Catalytic hairpin self-assembly (CHA) serves as a non-enzymatic nucleic acid cyclic amplification strategy widely used for signal transduction and amplification reactions in aptasensors (Si et al. 2020).It has been effectively applied in various aptasensors.For instance, Min's group proposed an isothermal amplification system based on dual catalytic hairpin self-assembly and silver nanoclusters with chameleon DNA templates for circRNA detection, achieving an experimental detection limit as low as 1 pM (Yang et al. 2022).The hairpin (HP) self-assembly process involves strict base complementary pairing, forming a loop after initiation which enables efficient amplification.
In the twenty-first century, nanomaterials have gained significant attention from researchers across various fields, such as biology, electromagnetism, medicine, and the environment (Khan et al. 2019).These unique materials possess distinct structural characteristics and physicochemical properties.Modified nanocomposites have demonstrated improved biocompatibility, electrical conductivity, and excellent catalytic properties, making them highly promising for biosensor applications.Precious metal nanomaterials like Au, Ag, Pt, Pd, and others are particularly suitable for electrochemical sensors due to their outstanding electrical conductivity and biocompatibility (John et al. 2021).Additionally, they can bind to nucleic acid chains through specific chemical bonds.For instance, gold nanoparticles can form Au-S bonds with sulfhydryl groups in nucleic acids, providing attachment sites for nucleic acid chains (Hua et al. 2021).
Cu 2 O, a semiconductor material, exhibits good conductivity and excellent biocompatibility, making it widely applicable in electrochemistry and biomedicine.The small size and high surface area of Cu 2 O nanomaterials contribute to their excellent catalytic performance, enabling the catalysis of various substrates, including hydrogen peroxide (H 2 O 2 ) (Kumar-Krishnan et al. 2016;Nair et al. 2018) and generating oxidation-reduction signals.For instance, Jin et al. developed a core-shell nanoparticle composite by combining Cu 2 O and Pt nanoparticles and using modified electrodes to detect dopamine in the presence of ascorbic acid and urea, achieving a detection limit of 3 nM (Jin et al. 2016).In addition, different morphologies of Cu 2 O nanoparticles often show different properties.It has been reported that regular octahedral Cu 2 O has better electrocatalytic activity than cubic Cu 2 O (Zhong et al. 2014).
Metal-organic frameworks (MOFs) are highly customizable coordination polymer materials, formed by metal ions connecting with organic ligands to create a unique 3D spatial configuration.MOFs possess attributes like high porosity, a large specific surface area (Wang et al. 2021), and easy modifiability.Notably, iron-metal organic frameworks (Fe-MOFs) exhibit peroxidase-like properties, enabling the catalysis of H 2 O 2 and generating a clean redox signal with only water and oxygen as byproducts, causing no secondary pollution to the environment.Researchers have successfully employed DNAfunctionalized porphyrin Fe-MOF as a probe and H 2 O 2 as a signal substance in constructing an electrochemical aptasensor for lead detection, achieving a low detection limit of 0.034 nM (Cui et al. 2015).However, Fe-MOF alone lacks significant electrical conductivity, often falling short of the high sensitivity required in electrochemical sensors.Balla's group combined Fe 2 O 3 with reduced graphene oxide (rGO), carbon nanotubes (CNTs), and polypyrrole (PPy) to construct a novel electrode modification material (rGO@CNT@Fe 2 O 3 / PPy) for the detection of Pb 2+ .PPy is able to chelate Pb 2+ , the magnetic properties of Fe 2 O 3 allowed the adsorption of more Pb 2+ , and the presence of rGO and CNTs improved the conductivity of the electrode.The sensor platform enables trace detection of Pb 2+ in the range of 0.02 to 0.26 μM, with detection limits as low as 0.1 nM.However, aptamer sensors often need to consider providing suitable binding sites for nucleic acid chains (Fall et al. 2021).To address this, combining Fe-MOF with noble metal materials has proven to be a viable solution, because nucleic acid chains can often bind to precious metal nanoparticles through sulfhydryl groups.For instance, researchers modified Fe-MOF with Au nanoparticles to amplify the redox signal and constructed a sensor for rotenone detection in foodstuffs, reaching a low detection limit of approximately 0.217 fM (Chun et al. 2022).
Taking advantage of the aforementioned benefits, we prepared Cu 2 O nanoparticles with ortho-octahedral morphology and modified them with Au nanoparticles to synthesize Au/ Cu 2 O nanocomposites, serving as the substrate for the sensor.This modification increases the electrode surface area, enhances electron transfer efficiency, improves catalytic performance, and provides numerous attachment sites for the complementary nucleic acid chain CDNA.Additionally, we prepared Fe-MOF as a signal label material and modified it with Au and Pd to create an AuPd/Fe-MOF composite.This composite functions as a signal label with hairpin-type complementary nucleic acid chains, catalyzes H 2 O 2 to generate electrochemical signals, and synergistically amplifies signals with Au/Cu 2 O.The detection of Cd 2+ is accomplished through the aptamer of the target, which initiates the CHA process.Moreover, the dual HP chain design prevents non-specific binding of signal labels and reduces the likelihood of false positive results.The constructed sensor exhibits exceptional detection performance in tap, lake, and potable water samples.To the best of our knowledge, this is the first time that both of these composite materials (Au/Cu 2 O and AuPd/Fe-MOF) have been simultaneously used and combined with the CHA strategy for detecting Cd 2+ , offering a novel approach for Cd 2+ detection in aqueous environments.

Synthesis of Au/Cu 2 O
Au/Cu 2 O was synthesized based on a previous report with some modifications.Initially, 0.0045 g of Cu(NO 3 ) 2 •3H 2 O and 0.042 g of PVP (K10) were mixed with 15 mL of isopropanol and stirred in a water bath at 60 °C for 10 min until fully dissolved.Then, 1 mL of NaOH (0.012 g/mL) was added dropwise, followed by the addition of 85 µL of N 2 H 2 •H 2 O (35% by mass) drop by drop to the mixed solution after stirring for 10 min.The solution was further stirred for 10 min at room temperature, and Cu 2 O was collected by centrifugation at 10,000 rpm.The obtained Cu 2 O was sequentially washed with deionized water and anhydrous ethanol and then dried under vacuum at room temperature for 12 h.
Subsequently, the previously prepared Cu 2 O was uniformly dispersed in 3.5 mL of deionized water, and 250 µL of HAuCl 4 (5 mM) was mixed with stirring for 5 min.Finally, the prepared Au/Cu 2 O was washed with anhydrous ethanol and deionized water and dispersed in anhydrous ethanol for further use.

Synthesis of Fe-MOF and AuPd/Fe-MOF
Fe-MOF and AuPd/Fe-MOF were synthesized based on a previous report with some adjustments (Taylor-Pashow et al. 2009).Initially, 0.185 g of FeCl 3 •6H 2 O and 0.125 g of 2-aminoterephthalic acid were dissolved in 15 mL of N, N-dimethylformamide (DMF).Subsequently, 200 mL of glacial acetic acid was added to the solution, and the mixture was stirred and kept in an oil bath at 120 °C for 4 h to allow for crystallization.Afterward, the solution was cooled to room temperature, and the material was collected by centrifugation at 8000 rpm.The collected material was washed with DMF and anhydrous ethanol in sequence and then dried under vacuum for 12 h to obtain Fe-MOF.
To prepare AuPd/Fe-MOF, 0.5 mg of the dry solid Fe-MOF was dissolved in 1 mL of ultrapure water.Then, 0.5 mL of HAuCl 4 •4H 2 O (1%) and 0.5 mL of K 2 PdCl 6 (1%) were added to the solution, followed by sonication for 20 min.Next, 2 mL of NaBH 4 (0.1 M) was added dropwise to the solution, which was then magnetically stirred for 30 min.Finally, the obtained AuPd/Fe-MOF was washed with ultrapure water and anhydrous ethanol in sequence and dispersed into 1 mL of ultrapure water.

Electrode pretreatment
Before assembling the aptasensor, the bare gold electrode (AuE) needs to be thoroughly cleaned.A clean electrode surface is essential to maintain the order and integrity of the self-assembled interface (Feng et al. 2011), which is crucial for the feasibility of the sensing strategy.Therefore, ensuring cleanliness is of utmost importance.The first step involves polishing the bare gold electrode to achieve a mirror surface using 0.05 µM aluminum powder.Subsequently, the polished electrodes are subjected to ultrasonic treatment in anhydrous ethanol and water for 10 min each to remove any remaining aluminum powder.Next, the electrode working interface is immersed in piranha solution for 10 min, followed by rinsing with ultrapure water and drying with nitrogen gas.

Construction of aptasensor
The construction process of the aptasensor is presented in Scheme 1.Initially, 5 µL of Au/Cu 2 O suspension was carefully added dropwise onto the electrode surface and then baked under an infrared lamp.After cooling to room temperature, 10 µL of CDNA was added dropwise and incubated in a constant temperature and humidity chamber for 120 min at 37 °C.The binding of CDNA to the electrode was facilitated through Au-S bonding.Subsequently, 5 µL of MCH was applied to the electrode as a blocker for 1 h to minimize the likelihood of non-specific binding during the subsequent steps (Szymczyk et al. 2022).Any remaining MCH was washed away with PBS, and 10 µL of AuPd/Fe-MOF-HP was added and incubated for 100 min at 37 °C.Finally, the AuE/Au/Cu 2 O/CDNA/AuPd/Fe-MOF-HP structure was exposed to 10 µL of Apt and 5 µL of various concentrations of Cd 2+ standard solution for 120 min at 37 °C.The response current within the range of 0.5-0.2V was measured using the DPV method in a PBS solution containing H 2 O 2 .

Sensing strategy
The sensing principle of the aptasensor is illustrated in Scheme 1. Firstly, Au/Cu 2 O is deposited onto the gold electrode as the substrate material, enhancing electron transfer efficiency and providing numerous sites for CDNA attachment (Zhang et al. 2020).Next, CDNA is immobilized on the electrode surface through the Au-S bond, serving to capture Apts and signal labels.Subsequently, the signal labels in the HP state are introduced into the system.Since CDNA itself forms an HP, the signal labels cannot efficiently bind to the electrode to some extent, thus, preventing false positives when the CHA is not activated.The HP DNA has a unique structure where complementary base sequences remain hidden and unexposed, making it challenging for the two strands to bind via complementary base pairing (Bikard et al. 2010).When Apt is added to the MCH/CDNA/AuE system, the hairpin (HP) self-assembly process is immediately triggered.The Apt acts as a promoter, opening the CDNA hairpin, causing CDNA to change from an HP to a straight chain, and exposing the complementary sequence with the HP.At this point, due to the stronger affinity between the HP and CDNA, CDNA acts as a key, opening the hairpin, and the AuPd/Fe-MOF-HP competes with the Apt for binding to the electrode, displacing Apt and allowing it to return to the system in a free state.This process continues to open CDNA in the HP state, leading to continuous CHA.As a result, more AuPd/Fe-MOF binds to the electrode, catalyzing H 2 O 2 and generating a stronger redox signal.However, when both Apt and the target Cd 2+ are present simultaneously, Apt's role as the initiator of the HP self-assembly process is lost.Apt preferentially binds to Cd 2+ and undergoes a conformational change, folding into an HP shape instead of opening CDNA (Yoshizumi et al. 2008;Zhu et al. 2017).Consequently, the HP self-assembly process cannot proceed, and the HP-AuPd/ Fe-MOF remains free in the system, unable to effectively bind to the electrode, leading to a significant reduction in the current value.This enables the successful detection of Cd 2+ .

Characterization for Au/Cu 2 O and AuPd/Fe-MOF
Scanning electron microscopy (SEM) was employed to examine the structure and morphology of the synthesized nanocomposites.Figure 1A shows that the Cu 2 O nanoparticles have an ortho-octahedral shape with an average diameter of about 400 nm.Upon loading with gold nanoparticles, the ortho-octahedral morphology remains intact, but the surface becomes rougher.This morphological feature bears a high resemblance to the previously reported study (Long et al. 2019).Additionally, EDS mapping (Fig. 1D) reveals distinct Cu and O elements, along with uniformly distributed Au elements.XRD analysis (Fig. S1 in the supplementary material) displays characteristic peaks of Cu 2 O at 29.5°, 36.4°,42.2°, 61.3°, 73.5°, and 77.3°, corresponding to crystal planes 110, 111, 200, 220, 311, and 222, respectively.After Au loading on Cu 2 O, distinct peaks of Au appear at 38.1°, 44.3°, 64.5°, and 81.7°.These results confirm the successful preparation of Au/Cu 2 O.
Figure S2 in the supplementary material illustrates that Fe-MOF exhibits a star-shaped structure with an average Scheme 1 Schematic illustration of the designed aptasensor particle size of about 350 nm.Following the modification of Au and Pd nanoparticles on Fe-MOF, AuPd/Fe-MOF (Fig. 1B) shows a rougher surface.EDS analysis confirms the presence of Au, Pd, Fe, C, and O elements, signifying the successful synthesis of AuPd/Fe-MOF.
Furthermore, the electrochemical properties of Au/ Cu 2 O in synergy with AuPd/Fe-MOF were investigated using cyclic voltammetry (CV), as depicted in Fig. 2A.The composite modified electrode exhibits a larger peak current compared to the bare electrode.The catalytic process was explored by varying the scan rate (10 -200 mV/s).It was observed that the redox current gradually increased with the scan rate, and the surface process was quasi-reversible and diffusion-controlled.The peak anodic current and peak cathodic current were found to be linearly proportional to the square root of the scan rate, with regression equations of I pa = 58.57+ 0.35v 1/2 (R 2 = 0.991) and I pc = − 58.06 + 0.16v 1/2 (R 2 = 0.989).

Feasibility of the aptasensor
The first step in our analysis is to assess the feasibility.As an electrochemical testing tool, Electrochemical impedance spectroscopy (EIS) is widely used by researchers to characterize the electrode modification process due to its stability and high sensitivity.In this study, we measured the .06,0.08, 0.10, 0.12, 0.16, 0.18 and 0.20 V/s).B The linear relationship between peak current and square root of scan rates impedance changes at each stage of electrode modification in a 5.0 mM K 3 Fe(CN) 6 solution to validate the feasibility of the aptasensor.Figure 3 illustrates the impedance spectra.The bare gold electrode (curve a) exhibited good electrical conductivity and a low charge transfer resistance (Ret).Upon loading Au/Cu 2 O onto the electrode (curve b), the electron transfer efficiency on the electrode surface improved, and the system entered the diffusion process without forming a prominent arc during charge transfer, resulting in a low Ret value.However, when CDNA was loaded onto the electrode (curve c), the Ret value significantly increased to 510.9 ohms due to the presence of biomolecules impeding electron transfer on the electrode surface.Next, MCH was introduced into the system as a sealer (curve d), which closed the non-specific binding sites on the electrode, leading to a further increase in impedance (Ret = 757.4ohms).Subsequently, AuPd/Fe-MOF-HP was added to the electrode alone (curve e), resulting in a slight increase in impedance (Ret = 840.5 ohms).This can be attributed to the nucleic acid being in the HP state, which is more stable and less prone to dehybridization compared to a straight double-stranded strand.Consequently, it is difficult for the nucleic acid in the HP state to pair with another HP in the same HP state as CDNA, even if there is a base sequence complementary to the HP, before the CDNA hairpin is opened.As a result, most of the added AuPd/Fe-MOF-HP tends to detach from the electrode, while only a small portion adheres to the electrode due to electrostatic forces.This leads to a reduction in electron transfer efficiency.However, upon adding Apt as a promoter to AuPd/Fe-MOF-HP/MCH/CDNA/AuE (curve f), the HP self-assembly process is initiated.Consequently, the initially free AuPd/Fe-MOF-HP effectively binds to the electrode, leading to a significant increase in the density of nucleic acid chains on the electrode surface and a notable enhancement in the impedance value (Ret = 1910 ohms).In the presence of both Apt and Cd 2+ in the system (curve g), the HP self-assembly process is hindered, and the free AuPd/ Fe-MOF-HP fails to bind effectively to the electrode.As a result, the impedance value is notably lower compared to the system without Cd 2+ .However, the impedance value of 1224 ohms is still significantly higher than that of the AuPd/Fe-MOF-HP/MCH/CDNA/AuE system.This is attributed to the presence of not only free AuPd/Fe-MOF-HP on the electrode but also a substantial amount of Apt.After binding with the target, Apt undergoes conformational changes, which further increases the impedance value.The obtained EIS results are in line with expectations, indicating the feasibility of the aptasensor for subsequent studies.

Optimization of experimental conditions
To enhance the performance of the developed sensor, four experimental conditions were optimized: the pH of the PBS solution, CDNA concentration, CHA incubation time, and Apt concentration.Figure 4 shows the optimization of the four experimental conditions, the vertical coordinates of the main figures are the peak current, and the embedded figures are the DPV response figures.The Aptasensor's core relies on various nucleic acid chains, such as Apts, and operating these functionalized biomolecules at the appropriate pH level is crucial for optimal performance.Different pH values of the PBS solution were tested (ranging from 6.0 to 7.5) and it was found that the current response increased until it peaked at pH 7.5, beyond which it decreased (Fig. 4A).Therefore, a pH of 7.5 was selected for the PBS solution.The impact of CDNA concentration was investigated further, which acted as a capture probe in the sensor (Fig. 4B).The current response increased steadily with CDNA concentration from 0.1 to 1.5 µM, reaching the highest value at 1.5 µM.Beyond this concentration, the current response declined.Consequently, a CDNA concentration of 1.5 µM was chosen as the optimal level.
The core process of our aptasensor is the CHA, where the aptamer of the target Cd 2+ plays a crucial role.We observed that the current response reached its peak at an Apt concentration of 1.0 µM and decreased significantly with further increases (Fig. 4C).This decline was due to an excessive amount of Apts, leading to densely packed nucleic acid chains on the electrode surface and reduced electron transfer efficiency.Hence, the optimal Apt concentration was found to be 1.0 µM, and it was essential not to exceed this concentration.Additionally, the incubation time for the CHA process was investigated (Fig. 4D).The current response peaked at an incubation time of 100 min, and there were no significant changes in the current when the time was further extended.Accordingly, we set 100 min as the optimal incubation time for subsequent experiments.

Performance of the electrochemical aptasensor
Using the best experimental conditions, the performance of the aptasensor was assessed via differential pulse voltammetry.Figure 5A presents the variations in the DPV peak for the aptasensor in response to varying concentrations of Cd 2+ .As anticipated, there is a consistent reduction in the DPV peak as the Cd 2+ concentration rises.This suggests a smooth progression of the CHA process, a distinct aptasensor signal, and successful detection.After computational adjustments, a standard curve was established with the regression equation I = − 1.657 lgC Cd 2+ + 8.984 (R 2 = 0.964), as depicted in Fig. 5B.The detection limit (LOD) for this study was calculated as three times the mean standard deviation of the blank sample's results, divided by the slope, resulting in a value of 2.27 × 10 -5 µM.Compared with some reported sensors to detect Cd 2+ , the aptasensor has a wider detection range and a lower LOD (Table 1).

Selectivity, reproducibility and stability of the aptasensor
The assessment of sensor performance relies on selectivity, an essential indicator.To determine the selectivity of the developed sensor, Mn 2+ , Zn 2+ , and Pb 2+ were chosen as interfering substances.As depicted in Fig. 6A, when detecting the target Cd 2+ , the sensor's current values showed significant attenuation compared to the other interferents.Conversely, the current values of the interferents did not exhibit notable differences, affirming the excellent selectivity of the sensor.
Reproducibility was also investigated in this study (see Fig. S4 in the supplementary material).Five electrodes were employed under identical conditions to construct the sensor, yielding a relative standard deviation of test results of less than 3%.This finding indicates the outstanding reproducibility of the sensor.Furthermore, the prepared sensor was stored at 4 °C and subjected to multiple tests over a fifteen-day period.After this duration, the current value still reached approximately 90% of the initial current, demonstrating excellent stability (Fig. 6B).

Analysis of real samples
An effective sensing strategy should be applicable for detecting real environmental samples.To assess the performance of the developed aptasensor in practical applications, we selected natural lake water, retail potable water, and tap water commonly used in the laboratory.These environmental samples underwent centrifugation and filtration through a 0.22 µm filter to eliminate suspended particles before spiking them for detection.The RSD ranged from 2.1 to 5.1%, and the recoveries fell within the range of 94.3-105.4%.These findings indicate that the constructed sensor exhibits good accuracy when utilized for actual sample detection (Table 2).

Conclusion
In conclusion, we successfully developed a sensitive electrochemical aptasensor for detecting Cd 2+ .The sensor utilizes CHA as the core recognition mechanism, AuPd/Fe-MOF as the signal labels, and Au/Cu 2 O as the substrate.The dual HP design effectively prevents non-specific binding, reducing the chances of false positives and enhancing the sensor's stability.This is also the first time that both of these composite materials (Au/Cu 2 O and AuPd/ Fe-MOF) have been simultaneously used and combined with the CHA strategy for detecting Cd 2+ .The constructed aptasensor demonstrated efficient detection of Cd 2+ within the range of 1 × 10 -4 -8.9 µM, with a low detection limit of 2.27 × 10 -5 µM.Compared with various Cd 2+ sensors reported, the sensor platform has a wider linear detection range and a lower detection limit.It exhibited remarkable selectivity, reproducibility, and stability.The application of electrochemical aptasensors for detecting heavy metal ions  is gaining increasing attention as a rapid detection method.Moreover, exploring the construction of electrochemical aptasensors for the simultaneous detection of multiple heavy metals holds promising potential as a powerful tool in environmental detection.4.48 × 10 -1 100.7 3.5 Lake water 8.9 × 10 -2 9.07 × 10 -2 101.9 3.1 2.67 × 10 -1 2.68 × 10 -1 100.3 3.4 4.45 × 10 -1 4.42 × 10 -1 99.32 4.7

Fig. 1
Fig. 1 SEM images of A Au/Cu 2 O and B AuPd/Fe-MOF.C Mapping of Au/Cu 2 O. D EDS of AuPd/Fe-MOF

Fig. 4 Fig. 5 A
Fig. 4 Optimization of the experimental conditions: A pH value of PBS, B The concentration of CDNA, C The concentration of Apt, D The duration of the CHA.The data describes the average results of three experiments.(The vertical coordinates of the main figures are the peak current, and the embedded figures are the DPV response figures) Fig. 6 Selectivity (A) and Stability (B) of the aptasensor for Cd 2+ detection

Table 1
Comparison of different analytical methods for detecting Cd 2+

Table 2
Analytical results of electrochemical aptasensor for detection of Cd 2+ in real samples