Programmable spherical nucleic acids integrated with MOF-confined copper nanoclusters facilitate electrochemiluminescence detection of prostate-specific biomarkers

Abstract

Alpha-methylacyl-CoA racemase (AMACR) is a promising prostate cancer biomarker due to its high specificity in distinguishing prostate cancer from benign prostatic diseases. However, its low abundance in biological environments presents a significant detection challenge. To address this, we developed an innovative all-in-one s̲pherical n̲ucleic a̲cid (SNA) platform for highly sensitive and selective electrochemiluminescence (ECL) detection of AMACR. The SNAs incorporate two types of ice-cream probes (IC probes), each consisting of interlocked hairpins and circular templates. Specifically, the all-in-one SNAs were elaborately designed to achieve key three functions: (i) the arrangement of IC probes on magnetic nanoparticle interfaces creates a spatially confined environment, promoting rapid interactions, and enhances AMACR conversion efficiency; (ii) the integrated templates and primers within the IC probes facilitate rolling circle amplification (RCA), resulting in exponential signal amplification; and (iii) the products generated through RCA serve as activators for the CRISPR/Cas12a system, remarkably improving its activation efficiency. Upon AMACR activation of the aptamer-prelocked DNA walker, the all-in-one SNAs were specifically driven to initiate RCA, generating exponentially amplified activators to effectively activate the CRISPR/Cas12a system. Additionally, we established a novel ECL nano-complex using a zinc-metal–organic framework loaded with Cu nanoclusters for signal output. This platform demonstrates exceptional sensitivity and specificity for detecting low-abundance biomarkers, offering significant potential for advancing clinical diagnostics.

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1. Introduction

Superior to other prostate biomarkers, alpha-methylacyl-CoA racemase (AMACR) has been distinguished as an emerging candidate for accurately differentiating prostate cancer from benign prostatic diseases.^1,2 However, the ultralow concentration of AMACR in human serum necessitates the development of more sensitive and specificity amplification methods.³ Current methods for detecting AMACR primarily rely on immunohistochemical techniques. Nevertheless, these methods face limitations such as inconsistent results, limited sensitivity, and poor cost-effectiveness.^4,5 To address these issues, biosensing platforms have been developed, offering advantages of high sensitivity, low cost, and rapid analysis.^6,7 Recently, Ying et al. proposed a MoS₂-based electrochemical biosensor for AMACR detection, which generated electrical signal output by detecting the metabolic intermediate of AMACR.⁸ However, this approach measures AMACR activity indirectly, leading to non-specific results due to interference from serum components. Leveraging their unique programmability, specific recognition ability, and high enrichment capacity, spherical nucleic acids (SNAs) have been harnessed to engineer intelligent biosensors in clinical diagnosis and gene regulation.^9,10 Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR) have gained significant traction as the most promising molecular tool in molecular biology and disease diagnosis.¹¹ Considering its efficient trans-cleavage activity, many researchers have combined CRISPR with SNAs to enhance the sensitivity and specificity of biosensing applications.^12,13 In our previous work, we utilized SNAs as nucleic acid converters to convert the AMACR into activators that can effectively activate the trans-cleavage activity of the CRISPR/Cas12a system, enabling a high-specific assay for AMACR.¹⁴ However, the proposed SNAs could only convert the target into activators at a 1 : 1 ratio, resulting in limited activation efficiency of the CRISPR/Cas12a system.¹⁵ As a simple, isothermal, and efficient nucleic acid amplification technique, rolling circle amplification (RCA) can achieve approximately 10³-fold amplification within 1 hour, making it an appealing solution for activator amplification.^16,17 For example, Yan et al. designed the padlock probes programmed with complementary sequences of the CRISPR/Cas12a activator and target miRNAs, which could hybridize with miRNAs to initiate RCA and amplify activators for quickly turning on the CRISPR/Cas12a signal switch.¹⁸ However, the randomly dispersed miRNAs and padlock probes resulted in a low efficiency of RCA. Based on these considerations, we first developed the all-in-one SNAs featuring multifunctional ice-cream probes (IC probes), which integrated enhanced target conversion efficiency, promoted RCA amplification efficiency, and improved CRISPR/Cas12a activation efficiency, realizing highly specific conversion and exponential amplification of AMACR.

By virtue of high sensitivity, good controllability, and low background interference, electrochemiluminescence (ECL) has gained increasing prominence in the field of analytical applications.^19,20 Compared with the conventional ECL luminophores, noble metal nanoclusters (NCs) have become increasingly popular owing to their facile preparation, rich physicochemical properties, and high biocompatibility.^21,22 Among noble metal NCs (such as Au NCs and Ag NCs), Cu NCs are preferred due to their relatively low cost and nontoxic nature.²³ For instance, Zhu et al. synthesized methionine/N-acetyl-L-cysteine templated Cu NCs, which possessed tunable near-infrared region and ECL emission wavelength for sensitive detection of matrix metalloproteinase-2.²⁴ However, the inherent instability of Cu NCs may lead to adverse agglomeration or decomposition, limiting their further application.^25,26 Interestingly, we discovered that the porous nanomaterials with a confined space can restrict nonradiative transitions and enrich electrochemically active substances, enhancing the ECL efficiency.²⁷ Metal–organic frameworks (MOFs), as a classic porous nanomaterial, exhibit remarkable properties such as high specific surface area, good biocompatibility, and functional adjustability.^28,29 Superior to bulk MOFs with few exposed active sites, two-dimensional (2D) MOFs possess greater ductility and more active sites.³⁰ Building upon these merits, we combined the Cu NCs as the ECL emitter with a 2D zinc-metal–organic framework (Zn-MOF) as a porous nanoconfined carrier, developing a novel nanocomplex Cu NCs@Zn-MOF, which exhibits a stable and robust ECL response that has not been previously reported.

In this work, we delicately crafted an all-in-one SNA coupled with the Cu NCs@Zn-MOF-based ECL platform for the sensitive and specific detection of AMACR. In Scheme 1A, two types of IC probes were anchored on gold-magnetic nanoparticles (Au@Fe₃O₄) to form the all-in-one SNAs. Importantly, the IC probes were composed of hairpins modified with the apurinic/apyrimidinic (AP) sites and the circular templates programmed with the complementary sequence of the activator. In the presence of AMACR, the aptamer-prelocked DNA was specifically activated via AMACR–aptamer recognition. The activated DNA walker was subsequently hybridized with the IC probes. Later, these AP sites on the loop of hairpins could be recognized and cleaved by apurinic/apyrimidinic endonuclease 1 (APE1), releasing the hybrids of circular templates and primers, which were collected and purified through magnetic separation. With the assistance of phi29 DNA polymerase, the hybrids of circular templates and primers initiated the RCA reaction, generating abundant activators for rapidly activating the CRISPR/Cas12a system (Scheme 1B). As displayed in Scheme 1C, the initial ECL response of Cu NCs@Zn-MOF was quenched by ferrocene-labeled DNA (Fc-DNA) to obtain the “signal-off” state. Upon activation by AMACR, the activated CRISPR/Cas12a system then cleaved the Fc-DNA on the Cu NCs@Zn-MOF-based ECL platform, thereby restoring the ECL signal for quantitative analysis of AMACR. Therefore, this work offered a prospective method for sensitive and specific detection of low-abundance biomarkers.

Scheme 1 Schematic illustration of the all-in-one SNA-based ECL platform for the detection of AMACR. (A) The formation process of the all-in-one SNAs. (B) AMACR-activated DNA walker-driven all-in-one SNAs to initiate RCA for CRISPR/Cas12a system activation. (C) The proposed ECL biosensing platform for the quantitative determination of AMACR, where “Activator*” denotes the complementary sequence of the activator.

2. Results and discussion

2.1. Morphologies of the Cu NCs, Cu NCs@Zn-MOF and Zn-MOF

The synthesis process of Cu NCs@Zn-MOF is shown in Fig. 1A. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were employed to characterize the Cu NCs, Cu NCs@Zn-MOF, and Zn-MOF. As illustrated in Fig. 1B, the TEM characterization of Cu NCs revealed its favorable dispersity in aqueous solution. According to the TEM characterization, the average diameter of Cu NCs was calculated to be 1.30 ± 0.24 nm (Fig. S2, ESI†). The inset of Fig. 1B further highlights a crystal lattice distance of 0.267 nm, corresponding to the (111) lattice spacing of face-centered Cu.³¹ Furthermore, the SEM characterization studies of Cu NCs@Zn-MOF (Fig. 1C) and Zn-MOF (Fig. 1D) presented similar 2D nanosheet structures and sizes of about 500 nm. Notably, the Cu NCs@Zn-MOF showed numerous particles distributed across its surface, indicating the successful enrichment of Cu NCs compared to the relatively smooth surface of Zn-MOF. X-ray photoelectron spectroscopy (XPS) was utilized to analyze the element composition and characteristic peaks of Cu NCs@Zn-MOF. As shown in Fig. 1E, Cu NCs@Zn-MOF was made up of carbon (C), copper (Cu), oxygen (O), nitrogen (N), and zinc (Zn) elements, which was in accordance with its energy dispersive spectrometry (EDS) mapping images (Fig. 1G). In addition, quantitative element analysis of Cu NCs@Zn-MOF is shown in Table S3 (ESI†). Furthermore, the characteristic peaks of Cu NCs at 933.30 and 953.35 eV (Fig. 1F) could be attributed to 2p_3/2 and 2p_1/2, suggesting the presence of both Cu (0) and Cu(I).³² Additionally, the X-ray diffraction (XRD) of Cu NCs@Zn-MOF nanocomplex exhibited two strong diffraction peaks at 10.5°, 19.3°, and 22.2°, corresponding to the (002), (123), and (033) lattice planes of zeolitic imidazolate frameworks (Fig. S3A, ESI†).³³ Inductively coupled plasma mass spectrometry (FTIR) analysis displayed a blue shift in the C=O stretching vibration and a new peak of 546 cm⁻¹, which confirmed the anchoring of Cu NCs onto the Zn-MOF surface or within its pores via coordination bonding (Fig. S3B, ESI†).³⁴ The inductively coupled plasma mass spectrometry (ICP-MS) analysis further quantified the mass loading ratio of the Cu NCs at 7.68 wt% (Table S2, ESI†), validating the effective integration of Cu NCs into the Zn-MOF framework.

Fig. 1 Synthesis and characterizations of the nanomaterials. (A) Schematic diagram of fabrication for Cu NCs@Zn-MOF. (B) TEM image of the Cu NCs. Inset: The crystalline structure of Cu NCs. (C) SEM image of the Cu NCs@Zn-MOF. (D) SEM image of the Zn-MOF. (E) Full-region XPS spectrum of the Cu NCs@Zn-MOF. (F) Detailed XPS spectra of Cu 2p region. (G) EDS mapping images of the Cu NCs@Zn-MOF.

2.2. Optical and electrochemical characteristics of the Cu NCs@Zn-MOF

To investigate the luminescence properties of Cu NCs@Zn-MOF, fluorescence (FL) and ECL spectra were applied. Under an excitation wavelength of 365 nm, Cu NCs@Zn-MOF exhibited a fluorescence emission at 568 nm (Fig. 2A). In the case of ECL emission, the bare GCE exhibited a typical ECL emission peak at 672 nm (curve c, Fig. 2B), corresponding to the radiation transition of 1(O₂)²*.³⁵ Meanwhile, both Cu NCs@Zn-MOF (curve a, Fig. 2B) and Cu NCs (curve b, Fig. 2B) exhibited a maximum ECL emission peak at 576 nm, which was consistent with the reported literature, indicating that Cu NCs serve as the ECL luminophore in Cu NCs@Zn-MOF.³¹ Furthermore, the three-dimensional (3D) surface image (Fig. 2C) and the heat map image (Fig. 2D) of Cu NCs@Zn-MOF consistently revealed a maximum ECL emission peak at 576 nm. Notably, the maximum ECL emission peak of Cu NCs@Zn-MOF was in close proximity to the FL spectrum peak, suggesting that the ECL emission likely originated from its intrinsic state.³⁶

Fig. 2 Optical and electrochemical characteristics of the Cu NCs@Zn-MOF. (A) FL excitation (a) and emission (b) spectra of the Cu NCs@Zn-MOF. (B) ECL spectra of (a) Cu NCs@Zn-MOF/GCE, (b) Cu NCs/GCE, and (c) bare GCE. (C) 3D surface image and (D) heat map image of the Cu NCs@Zn-MOF. (E) ECL intensities and (F) CV curves of (a) bare GCE, (b) Cu NCs/GCE, (c) Zn-MOF/GCE, and (d) Cu NCs@Zn-MOF/GCE in 10 mM S₂O₈²⁻.

To figure out the improved performances of Cu NCs@Zn-MOF, we conducted ECL and CV measurements across different modified electrodes. Specifically, Cu NCs/GCE exhibited a higher ECL signal (curve b, Fig. 2E) and redox peak current (curve b, Fig. 2F) than those of the bare GCE (curve a in Fig. 3E and F). Meanwhile, a slightly enhanced ECL intensity (curve c, Fig. 2E) and an improved reduction peak current (curve c, Fig. 2F) was obtained for Zn-MOF/GCE. This enhancement is attributed to the pore confinement effect of Zn-MOF, which accelerates S₂O₈²⁻ to generate more active intermediates (SO₄˙⁻). Notably, Cu NCs@Zn-MOF/GCE exhibited the strongest ECL signal (curve d, Fig. 2E) and highest peak current (curve d, Fig. 2F). This superior performance can be ascribed to the following factors: (i) the Zn-MOF provide a large specific surface area and abundant accessible surface sites for enriching Cu NCs, (ii) Zn-MOF with micro–nano structure restrict nonradiative transitions and enrich electrochemically active substances of Cu NCs to improve the ECL efficiency, and (iii) sufficient active groups of Zn-MOF accelerate the reduction of S₂O₈²⁻ to generate more SO₄˙⁻, producing additional excited Cu NCs (Cu NCs*) for enhanced ECL emission. Furthermore, the Cu NCs@Zn-MOF composite demonstrated an ECL response of 13 435 a.u. with a relative standard deviation (RSD) of 1.0% during continuous potential scanning after one month of storage at 4 °C (Fig. S5B, ESI†), suggesting the satisfactory stability of Cu NCs@Zn-MOF.

Fig. 3 PAGE analyses of the DNA assembly and RCA reaction. (A) Hybridization of the DNA walker and aptamer. Lane 1, aptamer; Lane 2, DNA walker; Lane 3, hybrid products of the aptamer and DNA walker. (B) Formation process of the circular templates. Lane 1, locker; Lane 2, padlock; Lane 3, locker + padlock; Lane 4, locker + padlock + T4 ligase; Lane 5, locker + padlock + T4 ligase + exonuclease I and exonuclease III. (C) Construction of the IC probes. Lane 1, circular templates; Lane 2, H1; Lane 3, H2; Lane 4, IC probe1; Lane 5, IC probe2. (D) Verification of RCA reaction. Lane 1, IC probe1; Lane 2, IC probe2; Lane 3, DNA walker; Lane 4, IC probes + DNA walker; Lane 5, IC probes + DNA walker + APE1; Lane 6, IC probes + DNA walker + APE1 + phi29 DNA polymerase.

2.3. Feasibility investigation of all-in-one SNAs for AMACR detection

To assess the hybridization of the DNA walker and aptamer, native polyacrylamide gel electrophoresis (PAGE) analysis was performed, as shown in Fig. 3A. Specifically, lanes 1–3 represented the AMACR aptamer, the DNA walker, and their hybrid products, respectively. Notably, a bright band with a large molecular weight could be clearly seen on the top of lane 3, demonstrating the successful formation of the aptamer-prelocked DNA walker. PAGE analysis was employed to further confirm the formation process of the circular templates, as shown in Fig. 3B. Lanes 1–3 correspond to the locker, padlock, and their hybrid products respectively. After the addition of T4 ligase, no significant change was observed in the electrophoretic bands of lane 4. However, a single and clear band was observed in lane 5 after the digestion of linear by-products with exonuclease I and exonuclease III, confirming the successful formation and purification of circular templates. Additionally, the assembly process of the IC probes is depicted in Fig. 3C. Lanes 1–3 showed three individual bands representing the circular templates, H1, and H2, respectively. Lanes 4–5 displayed the hybrid products formed between the circular templates and H1 and H2, respectively, which showed sights of the brilliant bands on the top, indicating the successful assembly of the IC probes. More importantly, the RCA reaction was validated in Fig. 3D. Lanes 1–3 stood for IC probe1, IC probe2, and DNA walker, respectively. When the DNA walker and IC probes were loaded together, a band with a lower migration rate appeared in lane 4, implying the successful hybridization between the DNA walker and IC probes. In addition, a light band with a small molecular weight emerged at the bottom of lane 5 after incubation with APE1, confirming that APE1 specifically recognized and cleaved the AP sites on the hairpins. Moreover, lane 6 exhibited a highly brilliant band on the top, validating the successful execution of the RCA reaction. The pixel intensity of the bands in the PAGE was quantified by the use of ImageJ software (Fig. S7, ESI†), which provided robust quantitative evidence supporting the feasibility of the DNA assembly and RCA reaction.

2.4. Performance analysis of all-in-one SNAs for AMACR detection

Under the optimal conditions (detailed in Fig. S8, ESI†), we conducted a performance analysis to assess the sensitivity, specificity, and stability of the constructed biosensor by monitoring the ECL intensity in response to AMACR. As the concentration of AMACR increased from 1 × 10⁻⁵ μg mL⁻¹ to 1 μg mL⁻¹, the ECL responses showed a significant increase, as depicted in Fig. 4A. Meanwhile, the linear relationship between the ECL intensity (I) and the logarithmic concentration of AMACR (lg c) can be seen in Fig. 4B, expressed as I = 1981.46 lg c + 7731.0 (R = 0.999), and the detection limit was calculated to be 4 × 10⁻⁶ μg mL⁻¹ (the calculation procedure is provided in Section 2.11 of the ESI†). To assess the specificity of the constructed biosensor, we tested the potential interference substances, including prostate cancer-specific antigen (PSA), mucin1 (MUC1), tyrosine kinase 7 (PTK-7), thrombin (TB), microRNA 141 (miRNA-141), and microRNA 375 (miRNA-375). In the presence of AMACR, a statistically significant ECL difference (****P < 0.0001) was observed both individually and in combination with interfering substances when compared to the signal from interferents alone (Fig. 4C). This result clearly demonstrates the remarkable selectivity of AMACR detection. Moreover, to further evaluate the stability of the biosensing platform, the ECL signal was tested at the AMACR concentration of 1 × 10⁻³ μg mL⁻¹ under 14 continuous scans (Fig. 4D). The relative standard deviation (RSD) of the ECL signal from the proposed sensing platform was calculated to be 1.1%, demonstrating the favorable stability of this biosensor for the AMACR quantification (the comparison of different methods for AMACR detection is exhibited in Table S4, ESI†).

Fig. 4 Assay performances of the developed biosensor. (A) ECL intensities of AMACR at different concentrations: 1 × 10⁻⁵ μg mL⁻¹ (a), 1 × 10⁻⁴ μg mL⁻¹ (b), 1 × 10⁻³ μg mL⁻¹ (c), 1 × 10⁻² μg mL⁻¹ (d), 1 × 10⁻¹ μg mL⁻¹ (e) and 1 μg mL⁻¹ (f). (B) The linear relationship of I_ECL and logarithmic of AMACR concentration (ranging from 1 × 10⁻⁵ μg mL⁻¹ to 1 μg mL⁻¹). (C) Specificity of the constructed assay. The mixture included PSA (10 μg mL⁻¹), MUC1 (10 μg mL⁻¹), PTK-7 (10 μg mL⁻¹), TB (10 μg mL⁻¹), miRNA-141 (10 μg mL⁻¹), miRNA-375 (10 μg mL⁻¹), and AMACR (1 × 10⁻¹ μg mL⁻¹). Data are represented as mean ± SD (n = 3), ****P < 0.0001. (D) Stability of the designed biosensor toward 1 × 10⁻³ μg mL⁻¹ AMACR through 14 cycles of cyclic potential scanning.

2.5. Practical application of the proposed biosensor in real human samples

Recovery experiments were performed to investigate the clinical application of the established biosensor. Specifically, the samples were tested by adding AMACR at different concentrations to diluted human serum (5-fold dilution using PBS). As shown in Table 1, the recoveries of AMACR ranged from 91.20% to 118.58%, with RSDs between 1.4% and 3.0%. These results demonstrate the biosensor's excellent potential for early monitoring of AMACR expression and its clinical diagnostic capabilities in practical sample analysis. Additionally, the AMACR concentration in six clinical serum samples was also tested using our ECL biosensor. The relative errors (E_r) compared to the enzyme-linked immunosorbent assay (ELISA) as a reference ranged from −8.7% to 3.9% (Table 2), demonstrating its good consistency and diagnostic potential for clinical applications.

Table 1 AMACR measurements in human serum samples

Target	Added (μg mL^−1)	Found (μg mL^−1)	Recovery (%)	RSD (%)
AMACR	0.01	0.0912	91.20	2.8
	0.1	0.1047	104.71	3.0
	1	1.1858	118.58	1.4

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Table 2 Comparison between the ELISA and this work for AMACR detection in serum samples

Sample number	ELISA (ng mL^−1)	This work (ng mL^−1)	E r (%)
1	9.77	9.52	−2.56
2	9.82	9.80	−0.20
3	11.86	11.48	−3.20
4	0.91	0.83	−8.7
5	1.21	1.26	3.9
6	1.05	1.06	0.95

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3. Experimental

The materials and reagents utilized in this study are detailed in the experimental section of the ESI.†

3.1. Fabrication of the all-in-one SNAs and aptamer-prelocked-DNA walker

The formation process of circular templates is shown in Fig. S1 (ESI†). Specifically, the padlock and locker were mixed in the ratio 1 : 1 (20 μM, 10 μL) and annealed at 95 °C for 5 min, followed by hybridizing at room temperature for 60 min. Afterward, 0.8 μL of T4 ligase (100 U μL⁻¹) was added into the resultant solution and reacted at 16 °C overnight in 1 × T4 ligase buffer to seal the nicks completely, followed by heating at 70 °C for 10 min to terminate the T4 ligase activity. After that, the reaction system was incubated with exonuclease I (2.5 μL, 10 U μL⁻¹) and exonuclease III (5.0 μL, 10 U μL⁻¹) at 37 °C for 60 min to degrade linear DNA by-products, obtaining purified circular templates. The resultant solution was subsequently kept at 80 °C for 15 min to denature both exonuclease I and exonuclease III. The prepared circular templates were then mixed with hairpin 1 (H1) and hairpin 2 (H2) at a ratio of 2 : 1 : 1 to heat at 95 °C for 5 min and slowly cooled for 1 h to assemble IC probes (divided into IC probe 1 and IC probe 2 according to H1 and H2). Following that, the IC probes were mixed with Au@Fe₃O₄ (40 μL, synthesis and morphology details are provided in Section 1.4. and Fig. S4 of the ESI,† respectively) and kept stirring at 4 °C for 12 h. Finally, the synthetic all-in-one SNAs were collected by magnetic separation and then stored at 4 °C for subsequent use. Also, H1 and H2 were treated with 5 μL of tris (2-carboxyethyl) phosphine (TCEP, 1 M) to reduce sulfhydryl in advance.

The aptamer-prelocked-DNA walker was prepared following the steps. The AMACR aptamer and DNA walker were mixed in a 1 : 1 ratio (10 μL, 20 μM) to heat at 95 °C for 10 min and hybridize at 4 °C for 2.5 h. Then, the resultant solution was incubated with 5 μL of exonuclease I (10 U μL⁻¹) at 37 °C for 1 h to remove excess reactants.

3.2. Preparation of Cu NCs@Zn-MOF

Based on the reported literature, Cu NCs were prepared with slight modifications.³⁷ To begin with, 8 mg L-glutathione was dissolved in 5 mL of deionized water and blended with 5 mL of a 10 mM CuSO₄·5H₂O solution at 25 °C for 5 min. Subsequently, 500 mM NaOH solution was added to the mixture to adjust the pH to 5.0, facilitating the formation of Cu NCs. Afterward, the Cu NCs were collected by centrifugation at 10 000 rpm for 10 min and purified using a filter membrane (MWCO 3500 Da) overnight. Finally, the obtained Cu NCs (4.7 mM) were reserved at 4 °C for further use.

The Cu NCs@Zn-MOF was prepared as follows. Initially, 195 mg (CH₃COO)₂Zn and 85 mg 1,4-benzenedicarboxylic acid (H₂BDC) were dissolved in 7 mL of N,N-dimethylformamide (DMF), respectively. Then, the synthetic Cu NCs (10 mL) were mixed with the (CH₃COO)₂Zn solution and stirred for 10 min at 25 °C. After that, the dissolved H₂BDC was gradually added to the resultant solution under continuous mixing within 15 min to obtain a suspension. This suspension was stirred for an additional day at 25 °C to facilitate the formation of Cu NCs@Zn-MOF. After being washed with DMF and ethanol three times, the product was dispersed in 4 mL DMF and stored at 4 °C for further use. Moreover, Zn-MOF was synthesized in similar procedures without the addition of Cu NCs.³⁸

3.3. Construction of the proposed ECL biosensing platform

To begin with, aluminum oxide power was used to polish the glassy carbon electrode (GCE), then cleaned with ethanol and ultrapure water.³⁹ The pretreated GCE was then modified with 5 μL of Cu NCs@Zn-MOF solution at 37 °C. Subsequently, 5 μL of as-prepared Au nanoparticles (Au NPs, detailed in the Section 1.4 of ESI†) were dripped on Cu NCs@Zn-MOF/GCE and dried at 37 °C. Following that, sulfhydryl-modified Fc-DNA (2 μM, 10 μL) was immobilized on Au NPs/Cu NCs@Zn-MOF/GCE at room temperature overnight, establishing the “signal-off” state. Afterward, 10 μL of 6-mercapto-1-hexanol (MCH, 2 mM) was coated on Fc-DNA/Au NPs/Cu NCs@Zn-MOF/GCE to block the unreacted sites. After each modification step, any excess reagents were washed off the modified GCE with ultrapure water. The successful assembly of the biosensor was confirmed through characterization studies using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), as illustrated in Fig. S6 (ESI†).

3.4. Detection process of all-in-one SNAs for AMACR detection

Initially, AMACR standard solution (500 μg mL⁻¹) was diluted to different concentrations (1 × 10⁻⁵ μg mL⁻¹, 1 × 10⁻⁴ μg mL⁻¹, 1 × 10⁻³ μg mL⁻¹, 1 × 10⁻² μg mL⁻¹, 1 × 10⁻¹ μg mL⁻¹, and 1 μg mL⁻¹) using RNase-free water. The target AMACR with these concentrations was then incubated with the aptamer-prelocked-DNA walker for 2 h at 37 °C. This step facilitated the release of the DNA walker through the specific binding of the AMACR aptamer to AMACR. Following that, the above solution (2 μL) was blended with 4 μL of all-in-one SNAs, 1 μL of APE1 (1 U μL⁻¹), and 1 μL of 10 × NEB buffer 4. This mixture was incubated at 37 °C for 2 h, in which the AP sites on hairpins could be cleaved by APE1, releasing the hybrid of primers and circular templates. After magnetic separation, the hybrid of primers and circular templates was collected and reacted with phi29 DNA polymerase (1 μL, 5 U μL⁻¹) and dNTPs (1 μL, 2.5 mM) in 1 × phi29 reaction buffer at 37 °C for 60 min to perform RCA. Subsequently, the RCA products containing a significant number of DNA activators, were incubated with the Cas12a-crRNA solution, containing LbCas12a (1 μM, 2 μL), crRNA (1 μM, 3 μL), 1 × NEB buffer 2.1 (7 μL), and RNase-free water (10 μL) to react for 60 min at 37 °C to activate the CRISPR/Cas12a system. After that, 10 μL of the resultant solution was dropped on the MCH/Fc-DNA/Au NPs/Cu NCs@Zn-MOF/GCE and incubated at 37 °C for 120 min. This step led to the release of the Fc-DNA away from the GCE, thus achieving efficient signal recovery. Finally, the response of the constructed biosensor was recorded using an MPI-E ECL detector, operating within a potential range of −2 to 0 V and a scan rate of 300 mV s⁻¹, with the voltage of the photomultiplier tube (PMT) set as 700 V.

4. Conclusions

In conclusion, we proposed a highly sensitive and efficient approach for AMACR detection by integrating exquisitely designed all-in-one SNAs with the advanced ECL platform of Cu NCs@Zn-MOF. The all-in-one SNAs specifically responded to the AMACR-activated DNA walker, triggering RCA to generate exponentially amplified activators that rapidly activated the CRISPR/Cas12a assay. Interestingly, the all-in-one SNAs enhanced the target conversion efficiency, RCA amplified efficiency, and activation efficiency of CRISPR/Cas12a, ultimately realizing specific and sensitive assays of AMACR. Furthermore, the innovative nanocomplex synthesized by integrating Cu NCs and the Zn-MOF demonstrated stable and enhanced ECL performance, attributed to its enrichment effect and pore confinement-enhanced properties. These advancements collectively establish a promising foundation for developing highly sensitive biosensors for the detection of low-abundance biomarkers, offering significant potential in bioanalytical applications.

Data availability

All the data supporting the findings of this study are available within the article and its ESI† and can be obtained from the corresponding author upon reasonable request.

Conflicts of 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.

Acknowledgements

Financial support was received from the Science and Health Joint Medical Research Project of Chongqing (2024ZDXM004), the Senior Medical Talents program of Chongqing for Young and Middle-aged, the Kuanren Talents Program of the second affiliated hospital of Chongqing Medical University, and the Program for Youth Innovation in Future Medicine, Chongqing Medical University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb00367a

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