A novel electrochemiluminescent cytosensor using dual-target magnetic probe recognition and nanozymes-catalyzed cascade signal amplification for precise phenotypic enumeration of CTCs

doi:10.21203/rs.3.rs-4911090/v1

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A novel electrochemiluminescent cytosensor using dual-target magnetic probe recognition and nanozymes-catalyzed cascade signal amplification for precise phenotypic enumeration of CTCs

https://doi.org/10.21203/rs.3.rs-4911090/v1

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The inability of surgical biopsy to monitor the dynamic evolution of cancer cells hampers its capacity to reflect real-time tumor heterogeneity. Circulating tumor cells (CTCs), as a crucial target in liquid biopsy, offer a novel approach for accurate monitoring of tumors. However, the rarity and complex phenotype resulting from epithelial mesenchymal transition pose challenges for conventional methods such as CellSearch and immunohistochemistry, which have insufficient ability for simultaneous phenotyping and enumeration of CTCs. The enumeration of a single phenotype CTCs is insufficient for accurately assessing disease progression. Herein, we propose a strategy to address this issue by fabricating an electrochemiluminescence cytosensor via the integration of dual-target enrichment and nanozymes-catalyzed cascade signal amplification. The graphene oxide@hollow mesoporous prussian blue/Pt (GO@HMPB/Pt) complex, possessing a large specific surface area and exceptional catalytic activity, is employed for loading a substantial amount of luminol as the signal probe. Dual-target magnetic PPy@Fe₃O₄/Au-antibody/aptamer is utilized for the magnetic capture of both epithelial and interstitial CTCs. Glutathione (GSH) can disrupt Au-S bond on aptamer by a thiol exchange reaction and selectively release a specific subset of phenotypic CTCs, thereby facilitating the efficient capture, accurate classification, and ultrasensitive detection of CTCs in peripheral blood. Using the epithelial MCF-7 and mesenchymal Hela cells as models, the ECL cytosensor demonstrates excellent performance in identifying cells spiked into whole blood. This study presents a novel approach for early detection of metastasis, tracking tumor recurrence, and monitoring therapeutic efficacy.

Electrochemiluminescence cytosensor

dual-target magnetic separation

nanozymes-catalyzed cascade amplification

circulating tumor cells

tumor heterogeneity

Breast cancer is a malignant tumor that poses a significant threat to women's health. Statistics reveal that over 90% of deaths due to cancer are caused by metastasis, which exhibits a strong correlation with the presence of circulating tumor cells (CTCs) ^[1–2]. Moreover, the enumeration of CTCs is a prognostic indicator for adverse progression-free survival (PFS) and overall survival (OS) in breast cancer patients implementing chemotherapy. However, CTCs will lose their epithelial characteristics, such as cell polarity and adherent junctions, transitioning into mesenchymal phenotype with a higher potential for invasion and metastasis, through a process called epithelial-mesenchymal transition (EMT) ^[3–5]. Consequently, relying solely on the enumeration of a single phenotype CTCs is insufficient for accurately assessing disease progression. Real-time monitoring of the amount of CTCs with extremely low concentrations and diverse phenotypes in peripheral blood is of great significance for early diagnosis and metastasis prediction of breast cancer, especially given the current demand for personalized treatment. Therefore, investigating heterogeneity among CTCs has emerged as a prominent field of research.

Currently, the screening approach for CTCs primarily relies on the labeling strategy based on the expression of cell surface proteins, such as cytokeratin, epithelial cell adhesion molecule (EpCAM), N-cadherin, Hsp70, vimentin and so on ^[6–10]. The clinical detection of CTCs still depends on the FDA-cleared CellSearch system, which utilizes magnetic beads modified with EpCAM antibodies combined with fluorescence imaging technology to identify CTCs in the bloodstream ^[11]. However, the limitations of this single-target approach arise from the occurrence of EMT, which hinders its ability to detect EpCAM-negative and mesenchymal CTCs. Therefore, several recent studies profiled multifunctional probe to capture CTCs of all phenotypes using antibody-functionalized microfluidic structure based on different proteins, including EpCAM/CD133, EpCAM/63B6 and oncofetal chondroitin sulfate (ofCS), which are expressed in epithelial, interstitial, and EMT ^[12]. Despite the high throughput capabilities of microfluidic joint fluorescence imaging technology in identifying CTCs, its applicability remains limited due to complex procedure as well as the high cost associated. Furthermore, even though CTCs are extremely rare (only 1 CTC per 10⁹ hematological cells), the presence of monotypic CTCs is far fewer, posing challenges for precise analysis of CTCs heterogeneity ^[13]. Therefore, in addition to efficient capture, selective controlled-release and highly sensitive detection are quite crucial for accurate CTC typing.

Aptamers are a kind of oligonucleotide sequence with particular recognition capabilities. Controlled-release of binding targets can be achieved via aptamer in a nondestructive way based on complementary chain competition. Thiol-modified aptamer can significantly enhance its recognition ability by their rich accumulation on the Au NPs through the Au-S bond, while the Au-S bond can be selectively destroyed via treatment with glutathione (GSH) or other reducing reagents to release the target ^[14–17]. These characteristics render aptamer promising candidates for developing a noninvasive controlled-release probe for CTCs ^[15]. Besides, electrochemiluminescence (ECL) is widely acknowledged as a robust detection method that has been successfully applied in various fields such as medical, environmental and food safety testing due to its inherent advantages of straightforward operation, exceptional sensitivity, rapid analysis speed and cost-effectiveness ^[18–19]. Especially, the introduction of multifunctional nanozyme catalyst with exceptional activity facilitates the rapid decomposition of the luminol co-reactive agent H₂O₂, thereby significantly enhancing the sensitivity of ECL ^[19–20].

In this study, a one-pot synthesis method was used to create conductive polypyrrorole (PPy) hollow nanotubes loaded with Fe₃O₄ and Au NPs (PPy@Fe₃O₄/Au), which exhibits high specific surface area, robust magnetism and facile surface modification capabilities^[21]. The PPy@Fe₃O₄/Au complex serves as a carrier for antibody and aptamer to form double-targeted immune-magnetic probe (PPy@Fe₃O₄/Au-antibody/aptamer), enabling efficient capture epithelial and interstitial CTCs in whole blood. Moreover, the fabricated signal probe involves the integration of graphene oxide sheets (GO) modified with antibodies, hollow mesoporous Prussian blue (HMPB) adsorbed with luminol and Pt NPs synthesis in situ (GO@HMPB/Pt-antibody). This probe functions as both a scaffold for immobilizing the signal molecule luminol and mimicking enzyme that catalyze H₂O₂, significantly enhancing electroluminescence intensity of luminol. The breast cancer cell MCF-7 (EpCAM⁺/N-cadherin⁻) was employed as the epithelial cancer cell model, while the cervical cancer cell line Hela (EpCAM⁻/N-cadherin⁺) served as the mesenchymal cancer cell model. The ECL cytosensor was constructed through a sandwich immune reaction with GO@HMPB/Pt-antibody-luminol modified on the electrode surface to recognize the two-type of CTCs that magnetically separated by PPy@Fe₃O₄/Au-antibody/aptamer dual-target probe, leading to decrease of ECL intensity. Subsequently, the release of epithelial/mesenchymal CTCs that captured by aptamer occurred upon GSH-mediated cleavage of the linker Au-S bond on aptamer, resulting in recover of ECL intensity.

Synthesis of PPy@Fe₃O₄/Au dual-target magnetic probe

Molybdenum trioxide (MoO₃) nanorod template was synthetized according to a reported literature^[21]. Briefly, 1.0 g of (NH₄Mo₇O₂₄)₆⋅4H₂O was dissolved in 12 mL of 11 wt% HNO₃ by ultra-sonication. The clear mixture was transferred and heated at 180°C in a Teflon-lined stainless steel autoclave for 20 h. The resulted product was centrifuged and washed with water as well as ethanol three times, then dried at 60°C for 12 h to obtain MoO₃ nanorods. The as-synthetic MoO₃ (50 mg) and pyrrole monomer (0.1 mL) were dispersed into 45 mL of water by vigorous stirring. Subsequently, 5.0 mL of mixture solution containing HAuCl₄ (10 mg) and FeCl₃ (50 mg) were added to the suspension slowly and reacted for 7 h at room temperature. Then, 5.0 mL of concentrated NH₃⋅H₂O was injected into flask drop by drop within 1.5 h and reacted for another 4 h with N₂. The PPy@Fe₃O₄/Au composites were collected by magnetic separation and washed with water as well as ethanol several times.

The PPy@Fe₃O₄/Au composite was dispersed into PBS (pH 7.4, 10 mM) at a concentration of 1 mg/mL. Subsequently, the carboxyl groups on Fe₃O₄ were activated by EDC and NHS with a concentration ratio of 1:1 for 30 minutes to bind with antibody. Then, 10 µL epithelial antibody of EpCAM (0.5 µg/mL) and mesenchymal aptamer NC3S (5 µM) were added successively to 50 µL of PPy@Fe₃O₄/Au solution and stir at 25°C for 2 h. The PPy@Fe₃O₄/Au-anti-EpCAM/NC3S immunomagnetic nanoprobe was separated by magnetic isolation and re-dispersed in 50 µL PBS. PPy@Fe₃O₄/Au-anti-N-cadherin/SYL3C dual-target magnetic probe with mesenchymal antibody of N-cadherin and epithelial aptamer was synthesized in the same way.

Synthesis of GO@HMPB/Pt-luminol Signal probe

Firstly, PB NPs was synthesized by adding 0.528 g of K₃[Fe(CN)₆]∙3H₂O and 12 g of PVP to 160 mL of HCl (0.01 M), stirring for 1 h at room temperature and then transferring to a Teflon-lined stainless steel autoclave for 20 h at 80℃. Subsequently, the blue PB NPs was obtained after washing with water and ethanol before being vacuum dried. Then, the HMPB was obtained by etching PB NPs (60 mg) with HCl (60 mL, 0.1 M) in the presence of PVP (300 mg) in Teflon-lined stainless steel autoclave for 4 h at 140℃. Secondly, HMPB/Pt-luminol composite was synthesized by employing HMPB (10 mg) as the carrier, sodium borohydride and citric acid (1:1) as reducing agents for in-situ reduction of potassium chloroplatinate (20 mM) at 25℃ for 2 min, followed by electrostatic adsorption of luminol under dark conditions for 12 h. Thirdly, high quality graphene oxide (GO) was prepared by electrochemical exfoliation in 200 mL of solution containing oxalic acid (0.28 mol) and hydrogen peroxide (0.5 mol), using graphite foil (10 mm×10 mm×0.5 mm) as the anode and platinum foil as the cathode with a distance of 2 cm under voltage of 10 V for 0.5 hours. The resulting black GO was activated by a mixture of EDC and NHS (1:1) for amide bond formation to link antibodies. Finally, a mixture of 10 µL GO-anti-EpCAM/anti-N-cadherin (1 mg/mL) and 40 µL HMPB/Pt-luminol composite was incubated and shaken in the dark for 2 h, followed by centrifugation and washing steps to obtain the GO@HMPB-Pt-luminol reporter.

Cell Line Culture and Enrichment

Hela and MCF-7 cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. 7702, Du145, BEAS-2B and Hepg2 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. The culture of the cells was conducted in a 5% CO₂ incubator at 37°C and cells were separated from the medium by centrifugation and diluted with PBS to different concentrations.

The pre-synthesized PPy@Fe₃O₄/Au dual-target magnetic probe was utilized for the capture and magnetic separation of CTCs with different phenotypes. Hela cells and MCF-7 cells were diluted with PBS to various concentrations: 1, 5, 25, 125, 250, 500, 1250, 2500, and 5000 cells/mL. Subsequently, the two diluted cell types were combined at equivalent concentrations and the synthesized PPy@Fe₃O₄/Au dual-target magnetic probe (10 µL) was added to different dilutions of cell solutions (1 mL). Then, the mixture was stirred at room temperature for 0.5 h followed by magnetic separation. Finally, the magnetic probe was washed with a PBS solution and re-suspend in a final volume of PBS buffer solution (10 µL) to accomplish enrichment.

Construction of the ECL Cytosensor and analysis of CTCs in PBS buffer

The ECL cytosensor was constructed by adding GO@HMPB/Pt-luminol reporter onto the surface of clean glassy carbon electrode (GCE) following thorough washing with deionized water and drying with N₂. Subsequent, 7.5 µL of the above magnetic probe with CTCs captured were dropped to the electrode surface and incubated in 37°C incubator for 30 minutes to facilitate the binding of CTCs onto GCE. The prepared cytosensor was immersed in PBS solution containing 2 mM H₂O₂ for ECL testing, with the photomultiplier tube high voltage (PMT) as 600 V and the detect range adjusted to 0.2–0.6 V. After performing full-cell count, 5 µM of GSH was introduced to disrupt the Au-S bond within the aptamer and PPy@ Fe₃O₄/Au, thereby releasing epithelial or mesenchymal CTCs capturing by aptamer.

Phenotypic Counting of CTCs in Whole Blood

The whole blood samples were collected from a healthy volunteer after obtaining informed signed consent and processed within 24 h. The subsequent detection of CTCs in whole blood was performed using the same procedures, except that the PPy@Fe₃O₄/Au dual-target magnetic probe was incubated with whole blood samples containing different concentrations of spiked target CTCs (MCF-7/Hela at a ratio of 1:1, ranging from 2 to 10000 cells/mL).

Principle for Phenotypic Enumeration of CTCs in Blood

The designed strategy of this ECL cytosensor is illustrated in Scheme 1. Initially, PPy@Fe₃O₄/Au nanotubes were synthesized via a one-pot method using MoO₃ nanorods as templates. During this process, the polymerization of pyrrole, formation of Fe₃O₄ as well as Au NPs and removal of MoO₃ templates occurred simultaneously. Generally, the EpCAM and N-cadherin are known to be highly expressed on MCF-7 (epithelial type of CTCs) and Hela cells (mesenchymal type of CTCs), respectively. Moreover, the SYL3C and NC3S aptamers have been reported to exhibit specific recognition towards epithelial and mesenchymal CTCs. Subsequently, the antibody/aptamer combination of anti-EpCAM/NC3S and anti-N-cadherin/SYL3C were immobilized onto PPy@Fe₃O₄/Au due to the abundant carboxyl groups of Fe₃O₄ and Au-S bond (Scheme 1A). In addition, GO@HMPB/Pt-luminol signal probe was obtained as shown in Scheme 1B. The two-dimensional GO, possessing excellent conductivity and large specific surface area, served as a carrier for anti-EpCAM and HMPB/Pt NPs loaded with substantial amounts of luminol. HMPB exhibits a hollow structure and possesses a significant quantity of mesoporous sites suitable for loading Pt NPs and luminol. Scheme 1C depicts the diagram for capturing, magnetically separating and electrochemical phenotypic counting two types of CTCs in whole blood with the nanozymes-catalyzed cascade amplification. Upon incubation with a blood sample, the PPy@Fe₃O₄/Au dual-target capture probe effectively recognizes and captures target MCF-7 and Hela cells. The external magnet facilitates the selective separation of target cells from a large excess of other cells, such as leukocytes and erythrocytes. GO@HMPB/Pt-luminol signal probe conjugated with EpCAM antibody was modified onto the glassy carbon electrode to bind with the enriched CTCs and report the corresponding ECL intensity. Peroxidase-like HMPB and high activity Pt NPs synergistically catalyze the H₂O₂ into reactive oxygen species and enhance the ECL intensity significantly. However, the ECL intensity was attenuated upon the capture of CTCs onto electrode and subsequently restored by cleaving the Au-S bond through GSH to selectively release epithelial or interstitial CTCs.

Characterization of the PPy@Fe ₃ O ₄ /Au and GO@HMPB/Pt Nanocomposites.

The micromorphology and elemental composition of the synthesized nanomaterials were characterized by SEM and TEM firstly. PPy@Fe₃O₄/Au has been synthesized and its magnetic as well as catalytic properties were preliminarily studied in our previous work^[21]. The detailed structure and element composition were further studied with TEM and energy dispersive X-ray spectroscopy (EDS) elemental mapping, as shown in Fig. 1A and Figure S1. TEM images showed a typical tube-like structure uniformly loaded with Fe₃O₄ and Au NPs. The magnified image (insert) indicated slight agglomeration of the Fe₃O₄ and Au NPs due to the anisotropic dipolar interactions. Furthermore, the mapping images corresponding to Fe, Au, C, N and O elements provided additional evidence for the successful formation of PPy@Fe₃O₄/Au.

The TEM image of GO synthesized using the electrochemical exfoliation exhibited a thin lamellar morphology, in accordance with previous literature reports (Fig. 1B). The SEM and TEM images (Fig. 1C, 1D) clearly showed the cubic structure of PB NPs converted to distinct hollow structure of HMPB after etched by hydrochloric acid. The TEM image (Fig. 1E, Figure S2) confirmed that the HMPB and Pt NPs were successfully supported on GO. As shown in Fig. 1F-J and L, high angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and the corresponding elemental mapping were conducted to further confirm the distribution of HMPB and Pt NPs on GO. The high-resolution transmission electron microscopy (HRTEM) image (Fig. 1K) demonstrated that GO@HMPB/Pt possessed a lattice fringe spacing of 0.226 nm, corresponding to the (111) plane of metallic Pt NPs. These above results confirmed the successful preparation of the GO@HMPB/Pt. Meanwhile, the morphology and elemental distribution of the GO@PB/Pt composite were characterized. As depicted in Figure S2, PB exhibited a solid cubic particle while Pt nanoparticles were uniformly dispersed on both the surface of PB and GO.

SERS and FTIR were adopted to characterize the functional groups on GO@ HMPB/Pt. From the SERS spectra (Fig. 2A), the typical D-band (1352 cm^− 1) and G-band (1591 cm^− 1) peaks for GO and the characteristic peak for C ≡ N band at 2163 cm^− 1 of HMPB were observed. The D-band/G-band intensity ratio of GO was significantly lower compared to Hummers' method, indicating that the mild oxidation process results in a reduced presence of D-defects ^[22]. FTIR spectra (Fig. 2B) provide insights into characteristic functional groups including -OH (3273 cm^− 1), C = O (1641 cm^− 1), C-O (1545, 1373, 1045 cm^− 1) and C ≡ N (2079 cm^− 1). Subsequently, the crystal structure of GO@HMPB/Pt was confirmed by XRD in Fig. 2C. The diffraction peaks at 17.4°, 25°, 35°, 39.5°, 50.6°, 54°, 57.3°, 66° and 68° are associated with PB (JCPDS NO.73–0687) ^[23]. The sharp peak at 26.5° of GO ^[24] and two weak peaks at 40.1°and 46.2°correspond to Pt NPs (JCPDS NO. 04-0802) ^[25]were emerged after combination. The XPS spectrum show typical peaks of Pt 4f, C1s, N1s, O1s and Fe2p from 0 to 1000 eV (Figure S3). Figure 2D shows the N 1s spectrum, with 397.04, 398.38, 400.3, and 402.6 eV correspond to C ≡ N, C-N and N-H. The Fe2p spectrum exhibits two contribution, Fe 2p_3/2 and Fe 2p_1/2, and four binding energy peaks were located at 707.8, 712.7, 721.4 and 724.9 eV, which can be assigned to the existence of [Fe(CN)₆]⁴⁻ and Fe³⁺ (Fig. 2E). The detailed spectrum of C1s and Pt4f were shown in Figure S3. All the peak positions confirm the presence of predominantly surface end groups such as -COOH, -OH, and –NH₂ on the GO@HMPB/Pt surface, providing extensive sites for antibody loading. The Brunauer–Emmett–Teller (BET) analysis in Fig. 2F reveals that the surface area of HMPB is approximately 110.4 m²/g, which is larger than PB (49.5 m²/g).

Construction of the ECL Cytosensor and Feasibility Tests

After confirming the successful synthesis of the capture vector and the signal probe, the ECL cytosensor was constructed. As displayed in Fig. 3A, rapid separation of PPy@Fe₃O₄/Au was achieved within 7 s using a magnet, which demonstrated good magnetic properties of PPy@Fe₃O₄/Au. Additionally, the changes observed in Figure S4 regarding the Zeta potential demonstrate the successful fabrication of the PPy@Fe₃O₄/Au dual-target magnetic probe and its proficient capability to capture CTCs. A standard chromogenic system consisting of TMB and H₂O₂ was employed to investigate the peroxidase-like of GO@HMPB/Pt. As shown in Fig. 3B, a typical blue color appears in the GO@HMPB/Pt + TMB + H₂O₂ system, while it appears nearly colorless in control experiments without H₂O₂, TMB or GO@HMPB/Pt. The corresponding UV-vis absorbance spectra of GO@HMPB/Pt + TMB + H₂O₂ system show a prominent peak at 625 nm, which confirms the inherent peroxidase-like activity of GO@HMPB/Pt. Additionally, the superior performance of GO@HMPB/Pt compared to PB, HMPB and HMPB/Pt is verified by the deepest color and strongest absorbance at 625 nm generated due to GO@HMPB/Pt (Figure S5). Additionally, the zeta potential gradually becomes negatively charged, indicating the successful assembly of the cascade amplifying signal molecule (Figure S6). The peroxidase mimics of GO@HMPB/Pt serve not only as a redox mediator to H₂O₂ but also as a scaffold carrier to immobilize luminol to form a solid-state luminophore, which accelerates the generation of reactive oxygen species and decreases the distance between the luminophore and electrode. The loading capacity of HMPB/Pt was determined through UV-vis absorption spectroscopy and the loading capacity of luminol by HMPB/Pt was calculated to be as high as 91.5% (Figure S7).

The technique of electrochemical impedance spectroscopy (EIS) offers comprehensive insights into the modification process of the ECL cytosensor. As illustrated in Fig. 3C, the progressive increase in electron-transfer resistance (Ret) indicates the successful modification of the glassy carbon electrode step-by-step. Especially, the Ret values increased significantly after CTCs captured on GCE. However, when the Au-S bond was cleaved by GSH to release Hela cells, decrease of Ret value was observed. This result indicates that the ECL cytosensor was feasible for simultaneous phenotypic typing and quantification of CTCs. Meanwhile, the universality of prepared cytosensor to epithelial or interstitial cell release was further verified by ECL response performance while adjusting the type of recognition probe. Figure 3D displays the histogram of ECL response during the construction of CTCs cytosensor. The peroxidase-like activity of GO@HMPB/Pt acts as a co-reaction accelerator to cascade amplify signal of luminol (column a). Due to the high impedance of biomolecules, the ECL intensity gradually decreases with the introduction of double-target probes (column b), interstitial Hela (column c) and mixed Hela and MCF-7 (column d). The cleavage of the Au-S bond by GSH resulted in the release of interstitial Hela cells and partial recovery of ECL response, which was consistent with the result of EIS. Meanwhile, the ECL sensor achieved comparable results by replacing the recognition molecules of the double-target magnetic probe with N-Cadherin and SYL3C to release epithelial MCF-7 cells (Figure S8). These above results demonstrate the successful construction of the ECL immunosensor, which possesses a universal capability to capture and release CTCs exhibiting diverse phenotypes.

Phenotypic Counting of CTCs in PBS Buffer

To evaluate the analytical performance of the ECL cytosensor for phenotypic enumeration of CTCs, samples containing different concentrations of Hela, MCF-7 and a mixture of dual cells (1:1) in PBS buffer were analyzed by the ECL sensor. Since the ECL cytosensor was constructed based on specific recognition and electron transfer rates on electrode surface, the concentration of carrier, recognition probe, luminol, GSH and pH value of the electrolyte were optimized prior to investigating the analytical performance of the cytosensor. As seen in Figure S9, the efficient isolation of CTCs can be achieved using 1 mg/mL of PPy@Fe₃O₄/Au. The carrier of GO (0.2 mg/mL), recognition probe of anti-EpCAM (0.5 µg/mL), N-Cadherin antibody (0.5 µg/mL), NC3S (5 µM), luminophor of luminol (0.5 mM), GSH (5 µM) for cleaving Au-S bond and pH = 8 were optimized and selected for the following electrochemical tests. Then, the performance of the ECL sensor on CTCs with a single phenotype was validated. The ECL intensity gradually decreased as the concentration of Hela or MCF-7 cells increased as the capture of cells onto electrode inhibited electron transfer rates at the electrode surface (Fig. 4A, C). The relationship between the peak intensity and the logarithm of Hela or MCF-7 cell concentrations showed linearity within the ranges from 1 to 10000 cells/mL and 1 to 5000 cells/mL, with R² values of 0.995 and 0.999, respectively (Fig. 4B, D). These results validate that the constructed ECL sensor is universally applicable for both enrichment and detection of single-phenotype CTCs. Then, to demonstrate the cell phenotypic counting capacity of ECL sensor, we performed simultaneous detection of Hela and MCF-7 cells as epithelial and mesenchymal models to analyze the EMT phenotype of tumors. As illustrated in Fig. 4E, the ECL intensity exhibited a negative correlation with cell concentration, but showed recovery following cleavage of Au-S bond by GSH. Two distinct linear segments were independently acquired with linear correlation coefficient of 0.993 and 0.998. On the basis of S/N = 3, the calculated limit of detection (LOD) for mixed and epithelial were 2 cells/mL and 1 cells/mL, respectively. Compared to previously reported methods, the ECL sensor presented here not only enables simultaneous detection of multi-phenotype CTCs, but also provides accurate quantification of single-phenotype CTCs with higher sensitivity and lower LOD (Table 1).

Table 1

Comparison of the analytical performance for CTCs detection.
Technique	Target cell	Linear range (cells mL^− 1)	Limit of Detection (cells mL^− 1)	reference
Fluorescence	A549 MCF-7	5–5×10³ 5–5×10³	2 4	^[26]
EIS	MCF-7	1–40	3	^[27]
Square Wave Voltammetry	MCF-7	5–5×10⁵	5	^[28]
Amperometry	MCF-7	10 − 1×10⁴	3	^[29]
ECL	MCF-7	10 − 1×10⁴	8.91	^[30]
ECL	MCF-7	8 − 1×10⁵	2	^[31]
Photoelectrochemistry	MCF-7	50 − 1×10⁷	18	^[32]
ECL	MCF-7 Hela	1–5×10³ 1–1×10⁴	2.48 and 1.8	This work

To investigate the specificity of the established ECL sensor, a range of control compounds including ions (Zn²⁺, Na⁺, K⁺, Mg²⁺, I⁻, Cu²⁺, SO₄²⁻, Cl⁻) at a concentration of 2 mM, molecules (UA, AA, DA, GSH, glucose) at a concentration of 10 mM, proteins (BSA, Protein S, Arg, Gly, Recombinant, MUC1) at a concentration of 5 µM and other cells (HL-7702, DU145, BEAS-2B, Hepg2) at a concentration of 10000 cells/mL were analyzed using the designed sensor (Fig. 5A). Despite the abundance of other compounds in comparison to the blank experiment, no significant decrease in peak intensity was obtained. Additionally, the coexistence of target CTCs with other compounds does not alter the ECL responses, confirming the exceptional interference resistance capability of this sensor. Furthermore, the cycling and long-term stability of ECL sensor were studied. As shown in Fig. 5B, the relative standard deviations (RSDs) for the ECL response of target cells (Hela/MCF-7 = 1:1 with a total 1000 cells/mL) were found to below 3% undergoing 12 cycles. Additionally, the ECL intensity exhibited minimal decline even after a duration of 12 days, indicating good stability of the cytosensor (Figure S10A). Moreover, the reproducibility of the ECL immunosensor was evaluated by preparing six independent electrodes for the determination of 2000 cells/mL target CTCs, and the RSD was calculated to be 0.507% (Figure S10B). In summary, the above results further validate the reliability of the proposed ECL platform for precise phenotypic counting of CTCs.

Phenotypic Enumeration of CTCs in Whole Blood

The capture and phenotypic enumeration of CTCs in human whole blood were further researched to explore the potential clinical applications of the ECL sensor. Different concentrations of CTCs with the ratio of Hela to MCF-7 as 1:1 were spiked into the whole blood that obtained from a healthy volunteer without any pretreatment and the CTCs were then separated by the magnetic PPy@Fe₃O₄/Au dual-target probe. Then, the corresponding ECL response obtained due to the capture and the release of CTCs was recorded for phenotypic enumeration analysis. The analysis results of CTCs in whole blood and PBS agreed well for detection of two types of CTCs (Fig. 6A) and single phenotypic CTCs (Fig. 6B). Although the ECL responses in whole blood exhibit a slight decrease compared to that in PBS buffer at low concentration, the accurate phenotypic enumeration of CTCs can still be effectively achieved, which suggest that our developed ECL sensor exhibits significant potential for application in the precise detection of CTCs in whole blood for personalized diagnosis of cancer.

In summary, we have successfully constructed an ECL cytosensor for the isolation, enumeration, and EMT phenotypic profiling of CTCs in whole blood. A sandwich-type cytosensor was built using PPy@Fe₃O₄-Au-antibody/aptamer to capture and magnetic separation of both epithelial and interstitial CTCs, while GO@HMPB/Pt-antibody/luminol was used as ECL signal probe. The utilization of GSH to cut-off the Au-S bond is subsequently employed to selectively release a specific subset of phenotypic CTCs, facilitating accurate enumeration of phenotypic CTCs. Moreover, the developed cytosensor demonstrates remarkable efficacy in monitoring extremely low concentration of CTCs in whole blood. Given these good properties, the cytosensor holds great potential for cancer screening, tumor progression monitoring, and treatment response evaluation.

Supplementary Information The online version contains supplementary material available at:

Materials, reagents and apparatus; characterization of PPy@Fe₃O₄/Au and GO@PB/Pt; XPS spectra of GO@HMPB/Pt; zeta potential characterization of cells capture processes; optimization of catalytic performance; zeta potential analysis during the preparation of GO@HMPB/Pt-luminol; UV spectrum and calibration plots of different concentration of luminol; the histogram of ECL response during the cytosensor construction; optimization of pH and different compounds concentration during the test; the long-term stability and reproducibility of cytosensor.

Authorship contributions Congcong Shen: Writing – review & editing, Supervision, Funding acquisition. Simin Fan: Writing – original draft, Validation, Data curation. Xiaoqing Li: Writing – original draft, Validation, Data curation. Fanshu Guo: Writing –original draft, Software, Methodology, Data curation. Junru Li: Writing – original draft, Investigation, Data curation. Minghui Yang: Writing – review & editing, Supervision, Funding acquisition.

Data availability Data will be made available on request.

Ethical Approval No approval of research ethics committees was required to accomplish the goals of this study because experimental work did not involve human or animal samples.

Funding The authors thank the support of this work by the National Natural Science Foundation of China (Nos. 22004028, 22174163), the Science and Technology Research Project of Henan province (No. 202102310139).

Conflict of interest The authors declare no competing interest.

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No competing interests reported.

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You are reading this latest preprint version

A novel electrochemiluminescent cytosensor using dual-target magnetic probe recognition and nanozymes-catalyzed cascade signal amplification for precise phenotypic enumeration of CTCs

Status:

Version 1

Abstract

Figures

Introduction

Experimental section

Synthesis of PPy@Fe₃O₄/Au dual-target magnetic probe

Synthesis of GO@HMPB/Pt-luminol Signal probe

Cell Line Culture and Enrichment

Construction of the ECL Cytosensor and analysis of CTCs in PBS buffer

Phenotypic Counting of CTCs in Whole Blood

Results and discussion

Principle for Phenotypic Enumeration of CTCs in Blood

Construction of the ECL Cytosensor and Feasibility Tests

Phenotypic Counting of CTCs in PBS Buffer

Phenotypic Enumeration of CTCs in Whole Blood

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

A novel electrochemiluminescent cytosensor using dual-target magnetic probe recognition and nanozymes-catalyzed cascade signal amplification for precise phenotypic enumeration of CTCs

Status:

Version 1

Abstract

Figures

Introduction

Experimental section

Synthesis of PPy@Fe3O4/Au dual-target magnetic probe

Synthesis of GO@HMPB/Pt-luminol Signal probe

Cell Line Culture and Enrichment

Construction of the ECL Cytosensor and analysis of CTCs in PBS buffer

Phenotypic Counting of CTCs in Whole Blood

Results and discussion

Principle for Phenotypic Enumeration of CTCs in Blood

Construction of the ECL Cytosensor and Feasibility Tests

Phenotypic Counting of CTCs in PBS Buffer

Phenotypic Enumeration of CTCs in Whole Blood

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Synthesis of PPy@Fe₃O₄/Au dual-target magnetic probe