A dual-functional electrochemical aptasensor was successfully developed using a novel cyclohexanehexone-melem covalent-organic framework (M-HO-COF) for the simultaneous detection of vascular endothelial growth factor 165 (VEGF165) and living osteosarcoma cells (K7M2). The M-HO-COF was synthesized via condensation polymerization between melem and hexaketocyclohexane octahydrate, resulting in a porous nanosheet-like structure with high surface area, extensive π-conjugation, and abundant C=N groups. These features enabled efficient immobilization of a VEGF165-targeted aptamer through weak intermolecular forces, preserving its structural integrity and recognition capability. The fabricated sensor demonstrated exceptional sensitivity, achieving a limit of detection (LOD) of 0.18 fg mL⁻¹ for VEGF165 across a wide dynamic range from 1 fg mL⁻¹ to 10 ng mL⁻¹. When applied to live K7M2 cells, which overexpress VEGF165, the same platform achieved an LOD as low as 49 cells mL⁻¹, showcasing its ability to bridge molecular and cellular diagnostics. The sensor’s performance was monitored via electrochemical impedance spectroscopy (EIS), revealing a progressive increase in charge-transfer resistance (Rct) upon each modification step: bare Au electrode → M-HO-COF/Au → Apt/M-HO-COF/Au → target-bound complex. This behavior confirmed successful construction of the biosensing interface and effective transduction of biological recognition into measurable electrical signals.
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**Structural and Functional Advantages of the M-HO-COF Platform**
The superior performance of the M-HO-COF-based aptasensor stems from its unique nanoarchitecture and chemical functionality. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed a loosely assembled, ultrathin nanosheet morphology with rough surfaces and cotton yarn-like aggregation, indicating high porosity and large accessible surface area. High-resolution TEM (HR-TEM) displayed a thin, amorphous sheet without distinct lattice fringes, consistent with a functional COF lacking long-range crystallinity but possessing intrinsic electronic properties. Energy-dispersive X-ray spectroscopy (EDS) mapping confirmed homogeneous distribution of carbon, nitrogen, and oxygen throughout the matrix. X-ray diffraction (XRD) patterns exhibited peaks at 25.6° and 27.1°, corresponding to the (002) plane of graphitic carbon, suggesting partial ordering. Fourier-transform infrared (FT-IR) spectroscopy identified key absorptions at 1618 cm⁻¹ (C=N stretch), 1460 cm⁻¹ (C–N bending), and 802 cm⁻¹ (heptazine ring vibration), confirming the presence of melem units. X-ray photoelectron spectroscopy (XPS) further verified the existence of C–C, C–N, C–O, N–C=O, pyridinic N, tertiary N, N–H, and oxidized species. Together, these data validate the successful synthesis of M-HO-COF and highlight its rich functional group landscape—C=C, C=N, C=O, and NH₂—which facilitates stable aptamer anchoring and enhances electron transfer efficiency.
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**High Selectivity, Stability, and Reproducibility in Complex Environments**
The selectivity of the aptasensor was rigorously evaluated against various interferents, including PSA, OPN, AFP, EGFR, Mb, BSA, IgG, and their mixtures, all present at 100-fold higher concentrations than VEGF165.Neurofilament heavy polypeptide Antibody Purity & Documentation EIS responses showed negligible signal changes, confirming minimal non-specific binding.p27 Antibody Biological Activity Confocal laser scanning microscopy (CLSM) visualized selective uptake of Cy3-labeled aptamer–COF complexes only in K7M2 cells, not in normal L929 cells, further validating specificity.PMID:35191852 Stability was assessed by storing three sensors bound to K7M2 cells (5×10² cells mL⁻¹) at 4 °C for 15 days. EIS measurements revealed an RSD of only 3.8% over time, with the sensor retaining approximately 112% of its initial response, demonstrating excellent long-term stability. Reproducibility was tested using five independently fabricated electrodes detecting K7M2 cells at 1×10², 5×10², and 1×10³ cells mL⁻¹, yielding RSDs of 4.83%, 3.27%, and 3.70%, respectively—within acceptable limits. These results confirm that the system is robust, reliable, and suitable for practical deployment in clinical settings.
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**Real-World Application in Human Serum and Regenerability Assessment**
To assess real-world applicability, human serum samples were collected with ethical approval and used to validate the sensor’s performance. Samples were filtered, diluted 500-fold in PBS (pH 7.4), and spiked with VEGF165 at levels ranging from 0 to 5×10⁴ pg mL⁻¹. EIS responses were recorded and compared to a calibration curve. The mean apparent recovery was found to be 97.41% with a relative standard deviation of 4.60%, confirming high accuracy and reliability in complex biological matrices. In pure serum, endogenous VEGF165 was detected at 1.49 fg mL⁻¹—ten times below the LOD—indicating no significant overexpression in the sample. Furthermore, the sensor’s regenerability was tested by washing the VEGF165-bound electrode with 0.05 M HCl and rinsing with Milli-Q water. The regenerated sensor successfully detected VEGF165 (1 pg mL⁻¹) for up to seven cycles with minimal signal loss, demonstrating excellent reusability. This feature reduces cost and waste, making the platform ideal for repeated testing in diagnostic workflows.
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**Broader Implications and Future Directions in Biomedical Diagnostics**
This work presents a paradigm shift in biosensing by introducing a bifunctional platform capable of detecting both biomolecules and whole living cells with unprecedented sensitivity and versatility. The M-HO-COF-based aptasensor not only enables early cancer diagnosis through trace-level detection of VEGF165 but also allows direct monitoring of cancer cell presence and activity. Its ability to detect K7M2 cells at 49 cells mL⁻¹ surpasses most existing cytosensors and opens new possibilities for liquid biopsy and metastasis tracking. Future developments will focus on expanding the platform to other cancer types, integrating multiplexed detection capabilities, and miniaturizing the system for point-of-care use. Additionally, the combination of COF materials with electrochemical readout offers potential for wearable devices, real-time monitoring, and closed-loop therapeutic systems. As biomedical sensing evolves toward personalized and precision medicine, this technology sets a new benchmark for next-generation diagnostic tools—offering speed, sensitivity, stability, and sustainability in one integrated solution.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com