On-site monitoring of serum low-density lipoprotein (LDL) is fundamental for the diagnosis, prevention, and management of cardiovascular disease. Herein, an electrochemical aptasensor was developed for sensitive LDL detection integrating an aptamer-mediated CRISPR-Cas12a system with reduced graphene oxide-MXene-copper (I) oxide nanocomposites (RGO-MXene-Cu2O). A complementary DNA strand (activator) of the LDL aptamer was designed and hybridized with the LDL aptamer. Upon LDL binding with the aptamer, the activator was released and triggered the Cas12a-crRNA complex. This activated complex rapidly cleaved single-stranded DNA (ssDNA) immobilized on the RGO-MXene-Cu2O surface. The cleavage event generated a measurable electrochemical signal via Cu2O redox reactions (recorded by differential pulse voltammetry, DPV), which were positively correlated to LDL concentration. The proposed sensing platform exhibited a linear relationship with LDL concentrations in the range of 0.001-0.4 mu g/mL and log-linear relationship across the range of 0.4-10.0 mu g/mL. The limit of detection was as low as 0.988 ng/mL. When applied to detect human serum samples, the relative errors between the clinical data and the measured results ranged from 0.56% to 4.56%. Moreover, the platform demonstrated outstanding specificity and operational repeatability, underscoring its potential as a robust and highly sensitive diagnostic tool for the early detection of cardiovascular disease.

Elevated levels of low-density lipoprotein (LDL) represent a major contributor to cholesterol accumulation in vascular endothelial cells, promoting atherosclerotic plaque formation and increasing the risk of life-threatening cardiovascular events. To address the urgent need for rapid and reliable LDL monitoring, we developed a ternary nanocomposite (rGO-MoS2-Pt NPs) via the hierarchical assembly of reduced graphene oxide (rGO), molybdenum disulfide (MoS2), and platinum nanoparticles (Pt NPs), which exhibited exceptional peroxidase-like activity. By immobilizing an LDL-specific aptamer (Apt) onto the nanozyme surface, a targeted recognition probe (rGOMoS2-Pt NPs-Apt) was prepared to construct a sandwich-type colorimetric aptasensor. The sensing platform was further integrated with smartphone-based chromatic analysis to facilitate point-of-care serum diagnostics. Under optimized conditions, the aptasensor exhibited a linear response to LDL concentrations ranging from 20 to 190 mu g/mL (R2 = 0.996), with a detection limit as low as 2.034 mu g/mL. When evaluated in human serum matrices, both the colorimetric and smartphone detection modes displayed excellent reproducibility (relative standard deviation of 0.11 %-0.95 % and 0.77 %-1.59 %) and high accuracy (relative errors of 0.29 %-5.26 % and 0.10 %-4.09 %). The nanozyme-powered, smartphone-coupled complementary performance of the two detection methods enhances diagnostic reliability of the biosensing system, implying the potential for cardiovascular risk screening and early clinical assessment.

Hepatocellular carcinoma (HCC) remains one of the most lethal malignancies worldwide, underscoring the urgent demand for early diagnostic strategies to improve patient survival rates. Glypican-3 (GPC3), a highly specific biomarker of HCC, has emerged as a promising target for early detection. Herein, we developed an innovative magnetic colorimetric aptasensor using ferrosoferric oxide@carbon quantum dots (Fe3O4@CQDs) nanocomposites. The Fe3O4@CQDs nanocomposites combine the magnetic properties of Fe3O4 with the unique optical characteristics of CQDs, exhibiting intrinsic peroxidase-like activity. The animo-modified GPC3 aptamer (GPC3(Apt)) was covalently conjugated onto the Fe3O4@CQDs nanocomposites surface via EDC/N-hydroxysuccinimide (NHS) chemistry to form the recognition probe (Fe3O4@CQDs-GPC3(Apt)). Upon specific binding between the aptamer and GPC3, the resulting complexes (Fe3O4@CQDs-GPC3(Apt)-GPC3) modulate the catalytic oxidation of o-phenylenediamine (OPD) by H2O2, generating a yellow oxOPD product whose absorbance intensity correlated with GPC3 concentration. The proposed colorimetric aptasensor demonstrated a detection limit of 3.16 ng/mL and a linear range of 10-200 ng/mL, with high specificity against interfering proteins. It was successfully validated in human serum samples, yielding recoveries between 99.27% and 109.33%. Owing to its operational simplicity and cost-effectiveness, this platform offers transformative potential for point-of-care HCC screening. Beyond advancing GPC3 detection, the work provides a flexible design paradigm that can be adapted for assays targeting other disease biomarkers.

Atherosclerotic cardiovascular disease (ASCVD) remains a leading global health threat, necessitating precise monitoring of low-density lipoprotein (LDL) as a key risk biomarker for assessing ASCVD risk. Herein, an electrochemical/colorimetric dual- channel aptasensor was developed by integrating nitrogen-doped reduced graphene oxide-Hemin-trimanganese tetroxide nanoparticles (NrGO-Hemin-Mn3O4 NPs) with the CRISPR/Cas12a system. The CRISPR/Cas12a system introduces a powerful signal amplification cascade: a single target binding event activates the trans-cleavage of numerous ssDNA probes, translating into a highly amplified electrical and optical response. The NrGO-Hemin-Mn3O4 NPs serves as a conductive redox probe and exhibits superior peroxidase-like activity through the synergistic effect between Hemin and Mn3O4. Mechanistically, surface-bound single-stranded DNA (ssDNA) initially induces steric hindrance, which obstructs electron transfer and suppresses the enzyme-mimicking performance of the NrGO-Hemin-Mn3O4 NPs. Upon the target LDL binding, the released activator DNA triggers the trans-cleavage activity of Cas12a to degrade the ssDNA, thereby restoring both the electroactivity and catalytic performance of the probe. Experimental results demonstrated that the dual-channel aptasensor achieved a wide linear range from 0.01 to 1000 nM with a detection limit of 0.01 nM, demonstrating that CRISPR integration is pivotal for achieving high sensitivity in complex biological matrices. This dual-channel strategy offers a sensitive, intuitive tool for early clinical screening of ASCVD diseases.

Quantitative detection of Golgi protein 73 (GP73), a promising biomarker for hepatocellular carcinoma (HCC), is crucial for early diagnosis. In this study, we developed a fluorescence aptasensor for sensitive and specific detection of GP73 based on fluorescence resonance energy transfer (FRET) between GP73 aptamer-functionalized nitrogen/sulfur co-doped graphene quantum dots (N, S-GQDs-GP73Apt) as the fluorescence donor and molybdenum disulfide@reduced graphene oxide (MoS2@RGO) as the fluorescence acceptor. In the absence of GP73, the fluorescence of N, S-GQDs-GP73Apt was effectively quenched by MoS2@RGO. Upon introduction of GP73, specific binding between GP73 and its aptamer triggered dissociation of the donor-acceptor complex, disrupting FRET and restoring fluorescence. Under optimized conditions, the normalized fluorescence intensity showed a good linear relationship with GP73 concentration ranging from 0.0001 to 100.0 μg/mL, with a low detection limit of 0.019 ng/mL. The aptasensor's performance was further validated using human serum samples with relative error ranging of 0.76%-3.85% and no statistically significant difference via t-test compared with ELISA results, demonstrating its potential as a reliable and cost-effective platform for early HCC diagnosis.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide, and early diagnosis is crucial. Golgi protein 73 (GP73) has emerged as a promising serum biomarker for HCC. However, current detection methods often fail to meet routine screening requirements due to limitations in sensitivity and operational simplicity. To address these challenges, we have developed a novel fluorescent aptamer-based sensor for highly sensitive GP73 detection based on the fluorescence resonance energy transfer (FRET) mechanism between graphitic carbon nitride quantum dots (g-CNQDs) and a copper-based metal–organic framework (Cu-TCPP). g-CNQDs were covalently conjugated with a GP73-specific aptamer to serve as the fluorescent donor, while two-dimensional Cu-TCPP nanosheets acted as the efficient acceptors. Fluorescence was quenched upon donor–acceptor interaction via FRET. In the presence of GP73, aptamer–target binding disrupted FRET interaction, separating the donor from the acceptor and restoring fluorescence in a concentration-dependent manner. Under optimal conditions, the sensor exhibited excellent linearity over a concentration range 1.0–225.0 ng mL⁻¹, with a detection limit as low as 0.907 ng mL⁻¹. Recoveries for spiked human serum samples ranged from 95.96

Hepatocellular carcinoma (HCC) is one of common cancer that seriously endangers human health. Designing methods for early, rapid, and accurate diagnosis of HCC has become the key point. Golgi protein 73 (GP73), a novel potential biomarker for HCC, is crucial for diagnosis and treatment of HCC. In this study, a colorimetric sensor with rapidity, smplicity and high specificity was established for detection of GP73 based on peroxidase-like activity of hemin-reduced graphene oxide--manganese dioxide (H-rGO-MnO2). The H-rGO-MnO2-GP73(Apt1) signal probe was synthesized by carboxyl of H-rGO-MnO2 nanozyme and amination of GP73 aptamer (GP73(Apt1)) though amide reaction. In the presence of GP73, the sulfhydryl-modifed GP73 aptamer (GP73(Apt2)), as the capture probe, and the signal probe both specifically recognized GP73, forming a sandwich structure (GP73Apt2-GP73-H-rGO-MnO2-GP73(Apt1)). This structure could catalyze the oxidation of H2O2 to produce hydroxyl radical (OH), thereby oxidizing the colorless phthalenediamine (OPD) into the yellow 2,3-diaminophenazine (DPA). The quantitative detection of GP73 was achieved by measuring the characteristic absorbance of DPA at 450 nm. In the GP73 concentration range of 10-150 ng/mL, there was a good linear relationship between the DPA absorbance at 450 nm (A 450 nm) and the GP73 concentration under optimal conditions. The linear equation was A (450 nm)= 0.00321CGP73+0.8988, with the correlation coefficient (R-2) of 0.9960 and the detection limit (LOD) of 5.38 ng/mL. The colorimetric sensor was applied to detection of GP73 in human serum samples, with recoveries of 88.4%- 98.8%. This sensor showed high specificity, sensitivity, and stability, and had potential for clinical detection of GP73, providing a new approach for the early diagnosis of HCC.

Early diagnosis of hepatocellular carcinoma (HCC) is crucial for improving patient survival and treatment outcomes and the early detection of biomarkers for HCC is key to achieving this goal. However, conventional detection methods often lack sufficient specificity and sensitivity. In recent years, CRISPR/Cas12a-based biosensing has gained significant attention due to its ease of use and high sensitivity, demonstrating its potential to address the limitations of conventional detection methods. This paper primarily reviews the research progress of CRISPR/Cas12a-based biosensors for HCC detection, introducing their fluorescence, electrochemical, colorimetric, and other detection principles, as well as practical applications in detail. Additionally, the differences in sensitivity, specificity, and detection speed among different types of CRISPR/Cas12a biosensors are comparatively analyzed. Finally, the potential future directions for the development and application of CRISPR/Cas12a technology in clinical settings are explored.

Cardiovascular disease (CVD) remains a significant worldwide health challenge, with mortality rates rising rapidly. Low-density lipoprotein (LDL) is a crucial serum biomarker for the early diagnosis of CVD, which can significantly improve outcomes and reduce mortality. Herein, a label-free electrochemical aptasensor for rapid detection of LDL was developed based on the titanium carbide-carboxymethyl chitosan-hemin (MXene-CMCS-Hemin) nanocomposites as the electrochemical signal probe. Firstly, gold nanoparticles (Au NPs) were electrodeposited onto a screen-printed carbon electrode (SPCE) to form a conductive substrate. Subsequently, the MXene-CMCS-Hemin nanocomposites were anchored onto the Au NPs/SPCE surface. Then LDLApt was immobilized on the surface of MXene-CMCS-Hemin/Au NPs/SPCE to construct the electrochemical aptasensor. When LDL specifically bound with the LDLApt to form LDL-LDLApt complexes, hindering the electron transfer and reducing the Hemin oxidation current, LDL detection can be achieved via differential pulse voltammetry (DPV). Under optimal circumstances, the changes of Hemin's oxidation current showed a good linear response with LDL concentration in the range of 0.1-4.0 μmol/L with a detection limit of 0.095 μmol/L (S/N = 3). The aptasensor demonstrated good performance with the relative errors of 0.60 % to 6.58 % for the direct detection of LDL in human serum samples, which offers a novel tool for the clinical diagnosis of CVD.

Golgi protein 73 (GP73) has emerged as a critical biomarker for the diagnosis of hepatocellular carcinoma (HCC), and its detection is essential for the effective treatment of this disease. In this study, we developed a colorimetric/electrochemical dual-mode aptasensor for GP73 detection using a hemin-reduced graphene oxide‑manganese oxide (H-rGO-Mn3O4) nanozyme and the bifunctional probe, 3,3',5,5'-tetramethylbenzidine (TMB). The GP73-specific aptamer (Apt) was conjugated to the H-rGO-Mn3O4 nanozyme, which was then immobilized on the surface of a gold nanoparticle-modified screen-printed electrode (Au NPs/SPE) through hybridization with a complementary DNA strand (cDNA) attached to the electrode. TMB served as a dual-signal probe for both colorimetric and electrochemical detection. In the presence of GP73, Apt binds specifically to the target, forming a H-rGO-Mn3O4-Apt/GP73 complex structure. This binding event causes the nanozymes to detach from the electrode surface, reducing its peroxidase activity while simultaneously restoring the electrochemical signal. Under optimized conditions, the dual-mode aptasensor exhibited excellent linear responses for GP73 concentrations ranging from 0.0001 to 100 ng/mL, with limits of detection (LOD) of 0.1 pg/mL for both the colorimetric and electrochemical modes. The aptasensor was successfully applied to measure GP73 levels in serum samples, yielding highly consistent results. Its performance was further validated by comparison with the standard ELISA method. Compared to single-signal aptasensors, this dual-mode approach not only improves detection sensitivity but also minimizes the risk of false positives or false negatives, making it a more reliable tool for accurate GP73 detection in clinical diagnostics.

Golgi protein 73 (GP73) is a novel serum marker for hepatocellular carcinoma (HCC) screening and diagnosis. In this study, we developed a sandwich electrochemical sensor for GP73 detection utilizing reduced graphene oxide-palladium nanoparticles-Prussian blue (RGO-Pd-PB) nanomaterial as the core detection element. RGO-Pd-PB nanomaterial exhibited peroxidase-like activity and were conjugated with a GP73-specific aptamer to form the signal recognition probe (RGO-Pd-PB-Apt). A GP73 aptamer was immobilized onto gold nanoparticles modified screen-printed electrode (Au NPs/SPE) to create the capture probe. Upon GP73 binding, a sandwich complex (RGO-Pd-PB-Apt/GP73/Apt/Au NPs/SPE) was formed. This complex catalyzed H2O2-mediated silver deposition, where the resulting silver ion signal intensity quantitatively correlated with GP73 concentration. The sensor demonstrated excellent performance, achieving a detection limit of 0.033 ng ml-1 and a linear detection range of 1.0 to 100.0 ng ml-1. Validation in human serum samples showed strong agreement with enzyme-linked immunosorbent assay results, with relative errors between 1.02% and 7.27% and relative standard deviations ranging from 1.29% to 5.94%. This electrochemical aptamer biosensor demonstrated high specificity, sensitivity, reproducibility, and stability, providing a promising new platform for the early HCC detection. A sandwich-type electrochemical aptasensor for determination of GP73 was constructed.RGO-Pd-PB enhanced silver deposition signal amplification strategy was used.Apt immobilized onto Au NPs/SPE used as capture probe and RGO-Pd-PB-Apt as the recognition probe.The aptasensor responded linearly with GP73 concentration in 1.0-100.0 ng/mL with LOD of 0.033 ng/mL.GP73 aptasensor had high sensitivity, good selectivity and excellent relative error.

Golgi protein 73 (GP73) is highly expressed in hepatocellular carcinoma (HCC) and considered a sensitive serum biomarker for HCC diagnosis. Herein, a label-free electrochemical aptasensor for ultrasensitive detection GP73 based on nitrogen-doped reduced graphene oxide-ferrocene-platinum@palladium nanoparticles (NRGO-Fc-Pt@Pd NPs) was constructed. The NRGO-Fc-Pt@Pd NPs offered excellent conductivity, biocompatibility, and large surface area, serving as both an electrode modifier and a redox probe due to Fc's good electroactivity. GP73 aptamer (GP73Apt) was covalently bound with the NRGO-Fc-Pt@Pd NPs to form the signal probe. When GP73 specifically bound to GP73Apt to form GP73-GP73Apt complex and fell off from the electrode, it leads to an increased Fc(Fe(II)/Fe(III)) peak current in differential pulse voltammetry (DPV) due to the enhancing of the sensing interface's electroactivity. Under optimized conditions, the label-free electrochemical aptasensor demonstrated a linear relationship with GP73 concentration in the range 1.0 ng/mL-300 ng/mL, with R2 of 0.99594 and a limit of detection (LOD) of 0.3524 ng/mL. In addition, the aptasensor showed good specificity, reproducibility, and stability. Furthermore, the developed aptasensor was applied to detect the GP73 in actual serum samples with good recoveries (97.85

Hepatocellular carcinoma (HCC), characterized by poor early diagnosis, exhibits the second-highest lethality rate among malignancies. This clinical challenge underscores the significance of Golgi protein 73 (GP73) as a promising serum biomarker for HCC detection. Herein, a ratiometric fluorescent aptasensor was constructed employing a dual-signal modulation strategy. The system integrated boron, nitrogen co-doped carbon quantum dots (BNCQDs) as the first fluorescent signal (I445) with a functional copper-based metalorganic framework conjugated with GP73-specific aptamer (Cu-MOF-Apt). The latter served dual functions: target recognition and peroxidase-mimetic catalyst for converting o-phenylenediamine (OPD) to 2,3-diaminophenazine (DAP), another fluorescent signal (I560). The peroxidase-like activity of Cu-MOF increased with the increase of GP73Apt attached. In the presence of GP73, target binding induced structural disintegration of Cu-MOF-Apt through GP73-Apt complex formation, thereby suppressing DAP generation. This target-responsive process led to the reduction of the fluorescence intensity of DAP and the increase of the fluorescence intensity of BNCQDs. Under the optimal conditions, the established ratiometric relationship (I445/I560 = 0.0007X + 0.7021, R2 = 0.997) enables quantitative detection of GP73 in the range of 25.00-600.00 ng/mL with the limit of detection (LOD) of 14.06 ng/mL. Clinical validation using serum specimens demonstrated excellent reproducibility (RSD 0.28 %-0.98 %) and recovery rates (99.75 %-107.49 %). The ratiometric fluorescent aptasensor's linear range effectively covers clinically relevant GP73 concentrations in HCC patients, while the robust serum analysis performance confirms its potential for practical diagnostic applications.

Organic-inorganic halide perovskite nanocrystals (PNCs) have been extensively utilized in the field of sensing, attributed to their superior optoelectronic properties, including high photoluminescence quantum yield, tunable band gap, and narrow full width at half maximum. However, the majority of sensors based on PNCs have traditionally relied on a single fluorescence signal emission and its intensity variations, leading to limitations in visualization resolutions. Hence, we developed a rapid, sensitive, and visually discernible dual-emission ratiometric fluorescence sensor for the detection of methylamine (MA) gas that based on the in situ formation of methylamine lead bromide (MAPbBr3) PNCs within europium metal-organic frameworks (Eu-MOFs) in the ambient atmosphere. In this strategy, Eu-MOFs not only served as the protective matrices to enhance the stability of MAPbBr3 PNCs but also as luminescent materials in ratiometric fluorescence sensing, owing to their exceptional luminescent properties. More importantly, benefiting from the excellent gas adsorption properties and protective abilities of MOFs, the proposed sensor demonstrates higher sensitivity, stability, and reproducibility. Experimental results indicated a good linear correlation with MA concentrations ranging from 9.0 to 90.0 ppm, achieving a limit of detection as low as 1.0 ppm. In comparison to other reported methodologies, this approach demonstrates a remarkably short response time of 20 s.

In this paper, a fluorescent aptamer sensor based on magnetic ferroferric oxide@boron, nitrogen-doped graphene quantum dots (Fe3O4@B,N-GQDs) was developed to detect Golgi protein 73 (GP73). GP73 aptamer (GP73Apt) functioned with Fe3O4@B,N-GQDs (Fe3O4@B,N-GQDs-GP73Apt) and was used as the fluorescent donor, while molybdenum disulfide (MoS2) with a large surface served as the fluorescent acceptor. The fluorescence of Fe3O4@B,N-GQDs-GP73Apt was quenched by MoS2 based on the fluorescence resonance energy transfer (FRET) principle. However, in the presence of GP73, Fe3O4@B,N-GQDs-GP73Apt could specifically bind to GP73, forming a Fe3O4@B,N-GQDs-GP73Apt-GP73 complex. This binding event caused Fe3O4@B,N-GQDs-GP73Apt to move away from the surface of MoS2, thus blocking the FRET process and recovering the fluorescence. Under optimal conditions, a linear relationship was established between the fluorescence recovery and the concentration of GP73 within the range of 10-1000 ng/mL (R2 = 0.9918), and the limit of detection was 7.37 ng/mL. Additionally, when the sensor was applied to test actual samples, the recovery rates were in the range of 98.80%-101.18%, and the relative standard deviations were between 0.29%-3.04%. These findings demonstrated the excellent detection performance of the proposed fluorescent aptamer sensor.

Hepatocellular carcinoma (HCC) is a high-mortality malignancy that urgently requires sensitive early-stage biomarkers. Glypican-3 (GPC3), a highly specific protein biomarker for HCC, calls for efficient detection strategies. Herein, we developed a label-free electrochemical aptasensor based on nitrogen-doped reduced graphene oxide-ferrocene-polyaniline (NRGO-Fc-PANI) nanocomposites and GPC3 aptamer for GPC3 detection. The NRGO-Fc-PANI combined the large high surface area and conductivity of NRGO, high electrical conductivity and stability of PANI, and the good redox properties of Fc, which functed as in situ electrochemical redox signal indicator to monitor the electrochemical changes. Upon target binding, the formation of GPC3-aptamer complex fell off from the electrode, exposing the conductive NRGO-Fc-PANI layer and enhancing the differential pulse voltammetry (DPV) response. The aptasensor exhibited a linear detection range of 10.0-100.0 ng·mL-1 (R2 = 0.996) and a low limit of detection (LOD) of 2.88 ng·mL-1. It demonstrated excellent selectivity toward interfering proteins, retained 70.8 % of its initial signal after 11 days of storage, and attained a recovery range of 97.0-109.5 % in human serum samples. By eliminating the need for enzymatic or fluorescent reporters, the aptasensor reduced reagent costs and operational complexity, presenting a clinically promising tool for the early diagnosis of HCC.

Cardiovascular disease (CVD) is the leading cause of death worldwide, particularly in the developing countries. Low-density lipoprotein (LDL), a critical biomarker for the early detection and intervention of CVD, provides valuable diagnostic and prognostic information for CVD. Herein, we developed a dual-signal electrochemical aptasensor for LDL detection based on a peroxidase-like nanozyme composed of reduced graphene oxide@molybdenum disulfide-ferrocenecarboxylic acid (rGO@MoS2-Fc). In the process of electrochemical testing, rGO@MoS2-Fc achieved the synergistic amplification of electrochemical signals both an enhanced Fc oxidation current detected by differential pulse voltammetry (DPV), and an increased hydrogen peroxide (H2O2) decomposition current via amperometric current-time (i-t) analysis. The DPV signal, corresponding to Fc oxidation, exhibited a logarithmic linear response over a broad LDL concentration ranged from 0.001 to 100.0 μg/mL, with a limit of detection (LOD) as low as 0.91 ng/mL. Meanwhile, the i-t signal, arising from H2O2 decomposition, displayed a linear range of 1.0 to 80.0 μg/mL and an LOD of 0.80 μg/mL. This dual-signal strategy exhibited good selectivity, reproducibility, stability and self-calibration capability. Analysis of human serum samples showed that the proposed strategy is expected to be a powerful method for early-stage CVD diagnosis and clinical management.

Low-density lipoprotein (LDL) is a critical biomarker for cardiovascular disease (CVD) risk assessment. Accurate and sensitive monitoring of LDL levels in human serum is crucial for timely diagnosis, targeted intervention, and reduction of mortality. In this paper, we constructed a fluorescent aptamer sensor for LDL determination based on sulfur-doped graphitic carbon nitride quantum dots conjugated with LDL aptamer (S-g-CNQDs-LDLApt) as a fluorescence donor and MXene-supported gold nanoparticles (MXene-Au) as the energy acceptor, based on fluorescence resonance energy transfer (FRET) principle. The sensing mechanism relies on LDL concentration-dependent fluorescence recovery mediated by the disruption of FRET. Under optimal conditions, the fluorescent aptamer sensor exhibited a linear dynamic range of 20.0-100.0 μg/mL (regression equation: Y = 0.00874× - 0.07426, R2 = 0.9900), with a detection limit (LOD) of 0.343 μg/mL. In clinical serum samples, the relative standard deviations (RSD) ranged from 0.48 % to 2.64 %, confirming the sensor's reliability for LDL analysis in biological matrices. The proposed biosensor demonstrates great potential for clinical serum analysis, offering a novel paradigm for point-of-care CVD risk stratification.

BACKGROUND:Golgi protein 73 (GP73) has emerged as a promising serological biomarker for hepatocellular carcinoma (HCC), with its combination with alpha-fetoprotein (AFP) substantially improving diagnostic precision in early-stage HCC detection. To address this clinical need, we constructed an electrochemical dual-aptasensor for parallel quantification of GP73&AFP. RESULTS:GP73 aptamer (GP73Apt) functionalized with reduced graphene oxide-ferrocene-polyaniline (rGO-Fc-PANi) nanocomposites and AFP aptamer (AFPApt) conjugated with nitrogen-doped reduced graphene oxide-cuprous oxide (NrGO-Cu2O) nanostructures were employed as the dual-signal probes. Upon GP73&AFP binding, the GP73Apt-GP73 interaction induced double-helix dissociation of aptamer-complementary DNA (cDNA) complexes, stabilizing the Fc-mediated electrochemical structure and amplifying Fc redox current. Conversely, AFPApt-AFP interaction induced steric hindrance through aptamer-protein complex formation, synergistically combined with dielectric property changes to suppress Cu2O redox signals. Under optimal conditions, the aptasensor demonstrated logarithmic linear ranges of 0.001-100.0 ng/mL for both biomarkers, achieving lower LOD of 0.48 pg/mL (GP73) and 1.77 pg/mL (AFP). Additionally, GP73&AFP were directly measured in human serum samples and demonstrated excellent reproducibility with RSD of 1.15-3.52 % for GP73 and 3.96-5.97 % for AFP. SIGNIFICANCE:This work presents a groundbreaking advancement in HCC diagnostics through the development of a dual-mode electrochemical aptasensor. The system's sensitive simultaneous detection capability, combined with operational stability in complex biological matrices, positions it as a transformative tool for early-stage HCC screening and management.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Early detection of HCC is pivotal for early treatment, thereby improving survival rates. Golgi protein 73 (GP73) is a promising biomarker for early HCC diagnosis, making it crucial to develop a sensitive method to detect GP73. In this study, a novel ratiometric electrochemical aptasensor was fabricated for the sensitive detection of GP73 using Hemin-Cu-based metal-organic framework (Hemin-Cu-MOF) nanocomposites and GP73 aptamers. The Hemin-Cu-MOF nano- composites possessed not only excellent electroactive property with the Hemin and Cu-MOF as dual signal outputs, but also good electroconductivity to amplify the electrochemical signals. The Hemin-Cu-MOF nano- composites with numerous active sites was used to label the GP73 aptamer (AptI) and construct the Hemin-CuMOF-AptI signal probe. Another sulfhydryl-modified GP73 aptamer (AptII) was immobilized on the gold nanoparticles (Au) modified screen-printed carbon electrode (Au/SPCE) through gold-sulfur (Au-S) bonds and used as a capture probe. Upon adding GP73, AptI and AptII specifically recognized GP73 to form the sandwich complex AptII@GP73@Hemin-Cu-MOF-AptI, making the dual signal of Hemin-Cu-MOF change and indicating GP73 level. Under optimal experimental conditions, the ratio of two oxidation peak currents (I Cu-MOF /I Hemin ) was proportional to the GP73 concentration (1.0-300.0 ng/mL), with a detection limit as low as 0.625 ng/mL. Furthermore, the aptasensor was tested in real human serum samples using the standard recovery method, with recoveries ranging from 104.10% to 116.66%. Thus, the proposed aptasensor has great potential in clinical analysis for detecting GP73 and early diagnosing of HCC.