Electrochemical Sensors for Rapid Cardiovascular Disease Diagnostics

1 Electrochemical Sensors for Rapid Cardiovascular Disease Diagnostics Vildan Sankoa, H. Cumhur Tekina,b* aMETU MEMS Center, Ankara, 06530, Türkiye bDepartment of Bioengineering, Izmir Institute of Technology, Izmir, 35430, Türkiye Abstract Cardiovascular diseases (CVDs) remain a leading cause of death, particularly in developing countries, where their incidence continues to rise. Traditional CVD diagnostic methods are often time-consuming and inconvenient, necessitating more efficient alternatives. Rapid and accurate measurement of cardiac biomarkers released into body fluids is critical for early detection, timely intervention, and improved patient outcomes. Electrochemical methods offer a robust solution by enabling rapid, sensitive, selective, and multiplex detection of CVD biomarkers, paving the way for early diagnosis and treatment advancements. This review highlights the performance and potential of electrochemical sensors for detecting specific CVD biomarkers and related organic molecules. It explores electrochemical sensing mechanisms, their evolution, the integration of nanotechnology, and diverse sensing platforms. It also examines emerging technologies such as microfluidic, smartphone-integrated sensors, and microneedleand tattoo-based sensors. Challenges and opportunities in integrating electrochemical sensors into point-of-care (POC) and wearable devices are discussed. Finally, the review compares commercial CVD sensors with existing methods and outlines future directions to advance the field. Keywords: Cardiovascular disease, Cardiovascular healthcare, Cardiac biomarkers, Electrochemical sensing, Health monitoring, Point-of-care testing, Sensing technologies, Wearable sensors. 2 Rapid diagnosis and monitoring of cardiovascular diseases (CVDs), which remain a leading cause of global morbidity and mortality, are of critical importance 1-3. CVDs are commonly grouped into the most common ones: heart failure, which is where the heart cannot pump blood effectively to the rest of the body; acute myocardial infarction (AMI), which occurs when blood clots block blood flow to the heart; heart valve complications, where the heart valves do not work correctly; arrhythmia, where irregular heartbeats are observed; and stroke, where blood flow to the brain is blocked 3, 4. Each of these conditions can significantly impact health and requires prompt medical attention. For CVDs, electrocardiogram (ECG), magnetic resonance imaging (MRI), echocardiogram (ECHO), Doppler ultrasound, and computed tomography (CT) methods are often used for diagnosis, but these methods may vary depending on the patient's symptoms 5-7. Although these diagnostic methods allow healthcare providers to determine the presence and prevalence of cardiovascular diseases and make accurate diagnoses and treatment plans, they have disadvantages such as limited diagnostic possibilities, false positive/negative results, operator dependency, limited vision, high cost, and radiation exposure. Considering that, in most cases, more than one method must be used together for accurate diagnosis, the need to develop alternative diagnostic methods has emerged 8-10. Determining the concentrations of biomarkers secreted, especially in body fluids, for determining various CVDs provides significant advantages regarding early diagnosis. These biomarkers allow sensors to deliver accurate, real-time information about cardiovascular health, facilitating early diagnosis, continuous monitoring, and personalized treatment. The ongoing research and development in this field are poised to significantly enhance the capabilities of electrochemical sensors, making them a cornerstone in managing and treating cardiovascular diseases. These biomarkers can be tested by laboratory-based methods such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and liquid chromatography (LC) 11, 12. However, using biosensor technology for CVD biomarker detection and even combining this technology with electronics and nanotechnology has led to the development of current detection technology 13. Optical and electrochemical-based biosensors are popular, but electrochemical sensors that show high sensitivity even at deficient levels in biological fluids are critical for detecting biomarkers 14, 15. In addition, the simple systems, point-of-care (POC) applicability, low costs, digitalization, stability, multiplexing, and reproducibility make this method advantageous and show that it has good potential for determining CVDs 1, 16. Advances in microfluidics, smartphone-integrated systems, and other sensing technologies are critical for developing more innovative, responsive systems that improve quality of life and operational efficiency. Integrating these sensor platforms with advanced data 3 analytics, machine learning (ML), and the Internet of Things (IoT) expands their potential applications and enables innovations in various areas. The need for greater sensitivity, miniaturization, and integration with digital platforms for real-time monitoring and analysis drives the development of these technologies 17-19. This review comprehensively analyzes the literature designed for electrochemical CVDs across a broad range of biomarkers, tabulating and providing a detailed summary of the key findings in a comparative manner. The included table summarizes the performance metrics, target biomarkers, and clinical potential of these electrochemical sensors reported in the literature. Also, this review offers a detailed examination spanning electrochemical experiments at the laboratory scale to cutting-edge methods for identifying CVD biomarkers. Several recent literature reviews have explored electrochemical or other sensing platforms for cardiovascular applications on biosensors 20-22. While these studies provide valuable insights into material selection and device performance, they lack the level of detail found in a wide range of current sensor technologies, which focus solely on electrochemical methods. Moreover, they offer limited evaluation of future commercialization potential and the practical challenges associated with real-world implementation. In detail, this review includes discussions on sensor mechanisms, the incorporation of nanotechnology, and the latest platforms, such as microfluidics, smartphoneintegrated devices, and wearable sensors. By tackling challenges like miniaturization and innovations driven by artificial intelligence (AI), it connects traditional diagnostic methods with next-generation POC solutions, providing essential insights for the progression of CVD sensor technology. This review also discusses the advantages and limitations of these advanced electrochemical sensors compared to commercial CVD monitoring devices. We conclude by addressing the challenges and opportunities in the clinical area of electrochemical sensing platforms and suggest future directions for their development and application in personalized cardiovascular healthcare. Finally, this review highlights the technological advancements, potential clinical applications, and comparative advantages of these innovative electrochemical sensors over conventional commercial devices, underscoring their role in the future of personalized cardiovascular healthcare. Electrochemical (bio)sensors for CVDs detection CVDs markers Biomarkers are tools that can guide identifying and monitoring high-risk patients with cancer, CVDs, and many other diseases. Regarding CVDs, changes in the concentrations of various biomarkers in body fluids, especially blood, are widely used. Cardiac troponin I (cTnI), 4 cardiac troponin T (cTnT), creatine kinase-MB (CK-MB), tumor necrosis factor-alpha (TNFα), C-reactive protein (CRP), and numerous other biomarkers are preferred for this purpose 11, 23. This section briefly mentions some biomarkers used in electrochemical CVDs sensors. Table 1 provides information about commonly used biomarkers in CVDs, including their specificity, specificity level, and cut-off level values. Although some of these biomarkers have high specificity, some others are weak and can be combined with others for acceptable accuracy and sensitivity. Figure 1 provides a schematic representation of CVD and the detection of biomarkers. It illustrates common cardiovascular disorders such as myocardial infarction, heart failure, arrhythmia, and stroke, alongside various diagnostic tools and methods for detecting biomarkers in bodily fluids. Key cardiac biomarkers, including troponins (cTnI, cTnT), B-type natriuretic peptide (BNP), and inflammatory markers (CRP, TNF-α, IL-6), are highlighted within a circular frame to emphasize their importance in diagnosing and monitoring cardiovascular diseases. Figure 1. A schematic representation of critical cardiovascular biomarkers detected in body fluids for diagnosing and monitoring conditions (Mb; Myoglobin, IL-6; Interleukin-6, IL-1; Interleukin-1, H-FABP; Heart fatty acid binding protein, Lp-PLA2; Lipoprotein-associated phospholipase A2, TNF-α; Tumor necrosis factor-alpha, CRP; C-reactive protein, CK-MP; Creatine kinase myocardial band, MPO; Myeloperoxidase, cTnI; Cardiac troponin I, BNP; Btype natriuretic peptide, cTnT; Cardiac troponin T, NT-proBNP; N-terminal pro-B-type natriuretic peptide). 5 The cTnI and cTnT are more specific biomarkers for detecting myocardial damage than those mentioned. While ECHO measurements are both slow and likely to be inaccurate, accurate detection of cTnI can provide more accurate results 11. In addition, cTnT is utilized as a diagnostic indicator of myocardial injury, particularly in cases of acute myocardial infarction 24. BNP is indicated as the most reliable heart failure biomarker and is presented as crucial for diagnosis and prognosis. Therefore, it has a vital role in detecting CVDs. The N-terminalproBNP (NT-proBNP) biomarker also shows similar efficacy to BNP in diagnosing and evaluating heart failure activity but has more prognostic assessment ability 25. It is stated in the literature that these four biomarkers have a high specificity level for diagnosing CVDs. Table 1. Some (bio)markers are used to diagnose and monitor CVDs. CVD Biomarkers Specificity Specificity Level Cut-off Level Ref Cardiac troponin I (cTnI) Myocardial infarction, heart failure High a0.01-0.1 ng mL-1 4, 26 Cardiac troponin T (cTnT) Myocardial infarction High a0.05-0.1 ng mL-1 4, 26 B-type natriuretic peptide (BNP) Acute coronary syndrome, heart failure High b9-142 pg mL-1 4, 26 N-terminal-proBNP (NT-proBNP) Acute coronary syndrome, heart failure High b0.25-2.0 ng mL−1 4, 26 C-reactive protein (CRP) Inflammation Moderate c10.0 mg mL-1 4, 27 Myeloperoxidase (MPO) Inflammation Moderate a350.0 ng mL-1 4, 27, 28 Creatine kinase-MB (CK-MB) Myocardial infarction Moderate a10.0 ng mL-1 26 Myoglobin (Mb) Myocardial infarction Low a70-200 ng mL-1 4, 26 Interleukin-6 (IL-6) Myocardial infarction Low b>1 pg mL−1, f0.07-1 pM 27-29 Interleukin-1 (IL-1) Promotes the formation of the atherosclerotic plaque Low b>1 pg mL−1, f0.07-1 pM 27-29 Heart fatty acid binding protein (HFABP) Myocardial necrosis Low a> 6.0 ng mL-1 28 Lipoprotein-associated phospholipase A2 (LpPLA2) Marker of inflammation risk predictor for stroke Low a> 200 ng mL-1 27, 28 Tumor necrosis factoralpha (TNF-α) Inflammation Low a> 3.6 pg mL−1, f0.07-1 pM 28, 29 Midregional pro-atrial natriuretic peptide (MR-proANP) Heart failure NR* c0.12 ng mL-1 4 Growth differentiation factor-15 (GDF-15) Heart failure NR* c1200-1800 ng mL-1 4 Soluble suppression of tumorigenicity-2 (sST2) Heart failure, inflammation, NR* a35.0 ng mL-1 4 Low-density lipoprotein (LDL) Cause plaque formation, casual factor in the pathophysiology NR* c>160 mg dL−1 28 miRNA-181 Heart development, cardiomyocytes apoptosis, heart arrhythmia, and heart failure NR* NR** 30 6 miRNA-21 Heart development, cardiomyocytes apoptosis, heart arrhythmia, and heart failure NR* NR** 30 miRNA-499 Myocardial infarction NR* NR** 31, 32 Nitric Oxide (NO) Coronary heart disease, peripheral vascular disease, pulmonary hypertension NR* a<100 µM, d100 pM-5 nM 33-35 Uric Acid Hypertension, atrial fibrillation, heart failure, and coronary artery disease NR* a208-428 μM, e120-400 μM, f24.5-35.7 μM 29, 36, 37 Ascorbic acid Cardiovascular disorders NR* c33 -111 μM, f1-10 μM 29, 38 Cholesterol Cardiovascular disorders, hypertension, atherosclerosis NR* b> 240 mg dL−1, e0.773-211.1 µg dL-1 37, 39 Triglyceride Cardiovascular disorders NR* a150-199 mg dL−1 40 NR*: Not reported NR**: To our knowledge, there is no concentration range given in the literature for miRNA-181, miRNA-21 and miRNA-499. However, it is reported that total RNA in plasma is in the range of 6–300 ng mL-1 and miRNA is a few percent of this value 41, 42. Cut-off values: a; serum samples, b; serum/plasma unspecified, c; plasma samples, d; living cells, e; saliva, f; sweat. CRP is a protein produced by the liver; its concentration in patient plasma increases due to atherosclerosis and inflammation. CRP detection is also critical to prevent the increase in deaths due to acute myocardial infarction 11, 43. CK-MB has moderate specificity for diagnosing acute myocardial infarction and is considered an essential biomarker due to its density in myocardial tissue. While the presence of high-specific biomarkers such as cardiac troponin in serum is limited, the increase in salivary levels of CK-MB after acute myocardial infarction makes this biomarker an essential and non-invasive diagnostic tool 44, 45. Myeloperoxidase (MPO) is also being evaluated as a cardiovascular biomarker with moderate specificity. It can also be found in body fluids in neurodegenerative disorders and rheumatoid arthritis. This biomarker also has a wide concentration range in saliva, facilitating noninvasive POC diagnosis, such as CK-MB 46. The most commonly used biomarkers with low sensitivity in CVDs include myoglobin (Mb), interleukin-6 (IL-6), and TNF-α. Mb is the first biomarker to increase concentration after acute myocardial infarction and can be helpful in early diagnosis 47. IL-6 is a crucial biomarker in relatively low concentrations in body fluids such as serum, urine, sweat, and saliva for disease monitoring. However, its concentration in body fluids increases not only in CVDs but also in rheumatoid arthritis, some types of cancer, asthma, and Alzheimer's disease processes 48. The TNF-α levels produced by lymphocytes and macrophages in blood plasma and saliva are associated with heart failure, and changing concentrations suggest that this biomarker plays a 7 vital role in heart function 49, 50. However, as it is also released in the presence of various diseases, such as IL-6, TNF-α screening may be needed in many diseases 51. The literature reveals that microRNAs (miRNAs) derived from various cells can be preferred as biomarkers for early diagnosis and prognosis of diseases. In addition to their stability and secretion into body fluids, their tissue-specific structures allow them to be used as critical noninvasive biomarkers in diagnosing cardiovascular diseases and are increasingly accepted. The data obtained from studies detecting miRNA-21 and miRNA-181 have been associated with cardiovascular diseases. Fibroblasts in the failing heart exhibited elevated levels of miRNA-21. Additionally, miRNA-181 is highly expressed in exosomes secreted by cardiac macrophages, regulating reperfusion after myocardial infarction. Therefore, both miRNA-21 and miRNA-181 may serve as promising biomarker candidates for the diagnosis of cardiovascular disease 30, 52. Also, it is well-established that specific miRNAs, particularly miRNA-499, exhibit a strong association with acute AMI, as their concentrations in circulating blood increase sharply and rapidly following the onset of an AMI episode 31, 53. Furthermore, the levels of these miRNAs in plasma and serum have been shown to rise rapidly and markedly in the bloodstream of patients as early as within the first hour after an AMI. This rapid elevation highlights their potential as early diagnostic biomarkers for AMI, offering higher sensitivity and specificity than conventional antibody-based methods 54. One of the exciting CVDs markers in recent years is nitric oxide (NO), which has been reported to lead to CVDs such as arteriosclerosis or myocardial infarction due to changes in nitric oxide concentration. However, changes in NO levels are not specific to CVDs but can also be a precursor to many diseases, such as hypertension, Parkinson's, and cancer 55, 56. In a 2021 review, the relationship between uric acid, a product of purine metabolism, and cardiovascular diseases such as hypertension, chronic kidney disease, heart failure, and coronary artery disease was evaluated 57-59. In addition, dopamine has been reported to play an essential role in the central nervous, renal, metabolic, endocrine, and cardiovascular systems, and its simultaneous diagnosis with uric acid has been investigated electrochemically 60. There are also views that irregular concentrations of ascorbic acid in the body, as an essential vitamin, may cause many diseases in humans, such as cardiovascular disease, cataracts, and cancer. Therefore, its detection with molecules such as dopamine and uric acid has been reported in the literature 60-62. Cholesterol, another essential compound in biological systems, is also considered to be relevant for the early diagnosis of cardiovascular disorders, so consuming high amounts of cholesterol-containing foods and determining blood cholesterol levels related to this are also important 39, 63. 8 The presence of NO, uric acid, ascorbic acid, and cholesterol molecules in body fluids at concentrations different than expected somehow triggers cardiovascular diseases or indicates the presence of these diseases. However, as far as we know, they do not have any specificity for diagnosing cardiovascular disease. That is, concentration levels outside the cut-off values given in Table 1 for these molecules do not only indicate the presence of cardiovascular diseases. While it can be a diagnostic method based on the simultaneous detection of more than one analyte, it can allow for more accurate results with a multi-analyte system together with biomarkers with high specificity. Current Applications of Electrochemical Methods in CVDs Detection Most of the biomarkers listed in Table 1 have been utilized in various approaches for CVDs detection using electrochemical biosensor platforms, and numerous research studies related to them are available in the literature. This review focuses on the specificity level of biomarkers and summarizes the current applications of electrochemical methods in CVD detection, categorizing them according to this parameter. Figure 2. A schematic representation of the sensing technology and electrode types used to detect electrochemical CVDs. 9 Figure 2 illustrates different electrochemical sensing methods for detecting CVDs. These methods range from essential platforms like screen-printed and glassy carbon electrodes (GCEs) to more sophisticated systems, including microfluidic-based sensors integrated with smartphones and wearable devices. Microneedle arrays provide a means for minimally invasive detection of biomarkers, while sensors available on the market offer practical solutions ready for use. The figure highlights the progression of electrochemical sensors toward more portable, innovative, and user-friendly technologies aimed at the early diagnosis and management of CVD. Table 2 summarizes the significant advancements in electrochemical sensors associated with CVDs biomarkers, with aptasensors and immunosensors becoming the leading technologies. These sensors incorporate advanced nanomaterials like gold nanoparticles, graphene derivatives, and metal-organic frameworks (MOFs) to enhance sensitivity and specificity. Differential pulse voltammetry (DPV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) are commonly employed and offer dependable detection at clinically relevant levels. Most sensors exhibit low detection limits and broad linear ranges that align favorably with clinical needs. The combination of molecularly imprinted polymers and nanomaterials shows potential for POC applications, though challenges concerning reproducibility and real-world reliability persist. These sensors indicate a move toward practical and effective CVD diagnostics. 16 This review discusses a selection of studies focusing on highly specific cardiovascular biomarkers, which were prioritized to ensure diversity and inclusivity. Furthermore, attention was given to studies that introduced significant functional improvements to biosensors. Antifouling strategies, which are crucial due to the considerable challenges posed by biofouling to electrochemical sensors in complex biological fluids, were particularly emphasized as they significantly enhance selectivity and operational stability. Also, the study on microelectrode array surface regeneration is emphasized, marking a significant breakthrough that enhances sensor reusability, allows for continuous monitoring applications, and promotes cost-effective implementation in clinical diagnostics. Moreover, recent representative examples were included to highlight cutting-edge sensor designs that utilize innovative materials and advanced electrochemical methods. Together, these selected studies offer a comprehensive overview of recent advancements and practical approaches in electrochemical CVDs detection. Qin et al. presented an immunosensor for the electrochemical detection of cTnI in human serum 88. In this study, a gold (Au) electrode was used as the working electrode, and its surface was modified with vertically aligned CAEAEAKAKAEAEAAKAK (CEAK16) peptide and Au nanoparticles (Figure 3). Electrochemical measurements were carried out in [Fe(CN)6]3-/4solution containing 0.1 M KCl with the modified electrode surface immobilized with an antibody. It has been stated that the amino acids in the hydrophilic region of CEAK16 in its amphiphilic structure provide a hydration layer of water molecules, effectively reducing nonspecific adsorption and exhibiting antifouling properties. The calibration curve obtained using DPV for the immunosensor calculated that the linear measurement range was 1.0 fg mL1 to 1.0 μg mL-1, and the limit of detection (LOD) was 0.28 fg mL-1. 17 Figure 3. Schematic mechanisms of electrochemical immunosensor for cTnI detection based on CEAK16 peptide. Adapted with permission from ref 88. Copyright 2024 Elsevier. (AuNPs; Gold nanoparticles, cTnI; Cardiac troponin I). Landim et al. developed an immunosensor for electrochemical cTnT detection with a GCE surface modified with polypyrrole/nanoclay hybrid film 180. The authors stated that the properties of nanoclays on the surface, such as good catalytic properties, large surface area, and relatively low cost, are combined with a conductive polymer and are an effective platform. A system was designed in which the biosensor response was used with 5 mmol L−1 [Fe(CN)6]3−/4− as a redox probe, and the interaction was monitored by the SWV electroanalytical method. It was observed that the responses to the analyte reacted as signal off on the platform where the linear working range was determined as 2.5-125.0 pg mL−1 and the LOD was determined as 0.35 pg mL−1. Cen et al. used AuPdCu alloyed nanowire-like networks to strengthen the GCE electrode's electrochemical signal in the CK-MB immunoassay 45. Also, they performed antibody immobilization on the surface they developed. The CVD biomarker detected by chronoamperometry was measured in pH 7.4 PBS containing H2O2. The study observed that the i-t curves taken by changing the analyte concentration between 0.001 and 2000 ng mL–1 tend to decrease as the concentration increases, and the LOD is 0.88 pg mL–1. It has been 18 reported that the biosensor prepared using this alloy, which exhibits properties such as surface area, high catalytic active site, and biocompatibility, also has good recovery in serum samples. Zhang et al. presented an immunosensor for CVD detection by taking advantage of the signal amplification properties and good electron transfer kinetic properties of Ag-Au bimetallic nanoparticles of BNP 139. Antibody immobilization was achieved by directly incubating the modified electrode surface for 2 h at 4°C. The effect of the components of the sensor was examined by cyclic voltammetry (CV), DPV, and EIS electroanalytical methods, and the analytical performance evaluation was made with DPV in the presence of analyte at concentrations between 0.0001–100 ng mL-1. Electrochemical responses determined by indirect measurement were taken in 5 mM [Fe(CN)6]3-/4. It has been stated that the peak current values obtained from the DPV curves decrease with the analyte concentration, and the LOD value is 0.03 pg mL-1. The authors claim that the bimetallic structure increases the synergistic effect of antigen-antibody binding, resulting in good sensor performance. They consider the sensor they developed promising in detecting and monitoring BNP. Ruankham et al. developed a regenerative impedimetric aptasensor to determine the biomarker NT-proBNP in artificial saliva (Figure 4A) 142. The immobilization of amineterminated aptamer and 6-mercapto-1-hexanol (MCH) was performed for the surface modification process of the pretreated working electrode. The linear measurement range of the study, which is based on a label-free measurement system, is specified as 5.0 × 10−3-1.0 pg mL−1, and the LOD as 5.0 × 10−3 pg mL−1 in measurements made with EIS in 5.0 mM [Fe (CN)6]3−/4− redox probe. Impedimetric measurements taken for analytes at different concentrations show that charge transfer resistance values increase with the increase in NTproBNP concentration. The advantages of the excellent electron transfer provided by the gold surface and the binding affinity of the aptamer structures with the biomarker were highlighted for this biosensor, which exhibited good properties such as long-term stability and high reproducibility. Besides its outstanding sensitivity and reproducibility, a key advantage of this work is the microelectrode array's regeneration capacity. The sensor surface was effectively regenerated by rinsing with a 10.0 mM NaOH as a nucleic acid denaturant. Subsequently, it was re-incubated in a PBS solution with NT-proBNP. The cycle of capturing, releasing, and analyzing could be performed at least four times on the same aptamer-functionalized microelectrode without any notable change in its behavior. The authors demonstrated that the sensor surface can be effectively regenerated and reused with minimal signal loss, highlighting its potential for continuous or repeated monitoring in practical diagnostic applications in noninvasive biofluids such as saliva. 19 Figure 4. Schematic mechanisms of A) Impedimetric aptasensor and the detection of NTproBNP. Reproduced with permission. 142 2023, Elsevier. B) CeO2/MWCNTs-(HRP-Strept)- Biotin-anti-MPO-MPO-anti-MPO-MBs immunosensor for determination of myeloperoxidase. Reproduced with permission. 46 2024, Elsevier. (MCH; 6-mercapto-1-hexanol, NT-ProBNP; N-terminal pro-B-type natriuretic peptide, HRP; Horseradish peroxidase, MWCNTs; Multiwalled carbon nanotubes, MPO; Myeloperoxidase, MB; Magnetic beads). Wang et al. modified the GCE surface with Ti3C2Tx MXene/Au nanocomposite for the aptasensor they designed for the detection of CRP by electrochemical method 110. The MXene 20 structure, which has an accordion-like morphology, has a high surface area, and the Auanchored Ti3C2Tx MXene composite structure contributes to the electrochemical properties by improving conductivity. CV and EIS methods were used for electrochemical characterization of components and aptamer immobilization. The electrode surface was also incubated with the ferrocene carboxylic acid (Fc(COOH))/anti-CRP signal probe, and it was determined that the peak potentials shifted to the cathodic region, and it was announced that a successful sandwichtype biosensor was designed for CRP detection. Linear measurement range and LOD were determined as 0.05-80 ng mL-1 and 0.026 ng mL-1, respectively, by the DPV method within the [Fe(CN)6]3−/4− as a redox probe. The electrochemical responses obtained concerning the analyte concentration were determined as signal-on, and good results were obtained regarding the recovery of CRP in real serum samples. In a study, a screen-printed carbon electrode surface was designed to be selective for the amperometric detection of MPO (Figure 4B) 46. The study was designed as a sandwich-type immunosensor, and a specific capture antibody (anti-MPO) was immobilized on carboxylfunctionalized magnetic beads (cMBs). Then, the system in which biotin-anti-MPO was coupled to cerium dioxide nanoparticles (CeO2NP)/multi-walled carbon nanotubes (MWCNT)- horseradish peroxidase-streptavidin (HRP-Strept) conjugates was constructed. The authors reported H2O2 with hydroquinone (HQ) as a redox mediator for amperometric responses and the change in cathode current resulting from HRP-catalyzed reduction of H2O2 via HQ. Offering a LOD of 0.40 ng mL−1 and a linear measurement range of 1.0 to 100.0 ng mL−1, this immunoassay also showed good recovery in saliva samples collected from volunteers. Diao et al. reported that Hcy and CRP are essential biomarkers for CVD and developed an ePADs for their simultaneous determination (Figure 5) 113. Mentioning the importance of multiple biomarker detection in detecting CVDs, the authors developed eutectic gallium indium (EGaIn) nanoparticles decorated with p-phenylenediamine (PPD) and AuNPs on their surface. Later, biomarker-specific aptamers were immobilized on this developed surface to form the ePADs platform. It has been shown that the nanoparticle system improves the electrochemical conductivity of the electrochemical aptasensor well. The ePADs were manufactured by printing and impregnating the filter paper with wax, and an optically clear poly(ethylene terephthalate) (PET) layer was glued to the back of the filter paper for attachment to the adapter. As a result of measurements made with DPV in pH = 7.40 PBS, it is observed that the peak current value of Hcy, an electroactive molecule, increases with increasing concentration. On the contrary, it was found that the peak current values against MB-labeled aptamer CRP decreased with analyte 21 concentration. The ePAD platform, which was also studied in saliva samples, is considered to have strong potential for non-invasive CVDs, giving similar results to ELISA. Figure 5. Schematic mechanisms of aptasensors on ePADs modified with EGaIn nanoparticles for the simultaneous electrochemical detection of Hcy and CRP. Adapted from ref 113. Copyright 2024 American Chemical Society. (EGain; eEutectic gallium indium, PPD; p-phenylenediamine, Au; Gold nanoparticles, Hcy; Homocysteine, CRP; C-reactive protein, MCH; 6-mercapto-1-hexanol). The reviewed studies highlight the rapid advancements in electrochemical biosensing technologies for CVDs biomarkers. The reported sensors exhibit a wide range of detection limits and linear operating ranges, suggesting their potential applicability in clinical and POC settings. In particular, strategies incorporating nanomaterials and biorecognition elements have effectively enhanced electron transfer efficiency, antifouling properties, and biomarker binding affinity. Moreover, some studies have focused on real sample applications, including serum and saliva, demonstrating the potential of these approaches. Table 2 summarizes the primary electrochemical detection strategies for various CVDs biomarkers and presents a comparative analysis of their analytical performance. When considering the whole table, the most striking observation is that electrochemical biosensors focusing on highly specific cardiac biomarkers, such as cardiac troponins (cTnI and cTnT) and natriuretic peptides (BNP and NT-proBNP), demonstrate superior performance in cardiovascular diagnosis. Among the reviewed studies, 22 the sensors developed for these markers, especially those employing advanced nanomaterials and antifouling strategies, stand out for their high sensitivity and selectivity. The immunosensor created by Qin et al. for detecting cTnI showed remarkable sensitivity, achieving a LOD of 0.28 fg mL-1 in serum. This high sensitivity is mainly due to the antifouling CEAK16 peptidemodified AuNP surface, which enhances selectivity and minimizes nonspecific adsorption 88. Similarly, developed by Zhang et al., the BNP immunosensor, which uses Ag-Au bimetallic nanoparticles, achieved an impressively low LOD of 0.03 pg mL-1, thanks to the synergistic effects of electron transfer and signal amplification from the bimetallic nanostructure 139. On the other hand, suggested by Zhang et al. MPO sensor demonstrates reliable performance in saliva with a LOD of 0.40 ng mL-1 46; however, its specificity as a biomarker for CVD is moderate due to its expression in various inflammatory conditions. Additionally, among the electrochemical biosensors targeting NT-proBNP, several studies are notable for their emphasis on noninvasive detection using saliva as the sample matrix. Efforts to diagnose NT-proBNP are increasingly important given its high clinical specificity in diagnosing heart failure and its increasing role in continuous monitoring. The study by Ruankham et al. introduces a label-free impedimetric aptasensor designed explicitly for artificial saliva. This process allows for multiple measurement cycles while maintaining minimal signal degradation, making it wellsuited for potential long-term monitoring applications 142. Similarly, Kudriavtseva et al., Halima et al., and Ghedir et al. developed NT-proBNP immunosensors that function effectively in synthetic and real human saliva 146, 147, 181. These findings demonstrate that reliable detection can be achieved even in challenging biofluids by optimizing surface chemistry and leveraging the strong binding properties of aptamers or antibodies. Collectively, these instances highlight the potential of NT-proBNP as a valuable biomarker for non-invasive electrochemical diagnostics, demonstrating that saliva-based platforms can achieve high analytical performance and practical usability. Electrochemical Sensing Technologies for CVDs monitoring at POC Quantitative tests of biomarkers present in various biological samples are currently performed in health laboratories. Although these analyses provide sensitive results, they are complex, time-consuming, costly, and require expert analysts, which are important limiting factors. For this reason, sensor technologies have come to the fore, and promising developments have been made in determining analytes, especially in the health field and in measurements carried out in laboratory environments. As a result of these satisfactory developments, attention has now turned to personalized sensor technologies. Personalized healthcare monitoring is a 23 service that can significantly increase the quality of healthcare services and, therefore, patients' quality of life, with its advantages such as diagnostic accuracy and instant monitoring. These technologies, called POC systems, require a multidisciplinary field of study including lab-ona-chip, mobile-based, and wearable technologies. When integrated with the superior properties of electrochemistry, they seem suitable for serving a widespread population and have the potential for commercialization. This section discusses electrochemistry-based POC devices for CVD diagnostics, with an emphasis on microfluidic technologies, as summarized in Table 3. 24 Table 3. Summary of electrochemical microfluidic device studies for CVDs detection. Analytes Biosensor Method Modification Substrate Assay Time Volume Linear Range LOD Real Sample Smartphone Integration Ref cTnI Immunosensor DPV Chemically activated surface PI - - 0.1-2.0 ng mL-1 43.33 pg mL−1 Human serum ✓ 182 cTnI Immunosensor DPV Chemically activated surface PDMS 15 min 0.75 μL 50.0 pg mL− 11.0 μg mL− 1 5.0 pg mL− 1 - X 183 cTnI Mb Immunosensor DPV Cys-Ni3V2O8rGO Glass 6 min 100 μL 0.001−1600 ng mL-1 2.0 pg mL-1 4.7 pg mL-1 Human serum X 184 cTnI Immunosensor EIS MWCNT Paper ~1 min ~2 μL 0.05–50.0 ng mL-1 0.05 ng mL-1 Human serum X 185 cTnI Aptasensor SWV PCN-AuNPs Polyimide 2 min 20 μL 0.0001–1000 ng mL-1 0.01 pg mL-1 Human blood ✓ 186 CRP Immunosensor CA Chemically activated surface PET 15 min 150 μL 0.01 ng mL-1 100 μg mL-1 47 pg mL-1 Serum, plasma, whole blood X 187 CRP Immunosensor CA SALP-AAP Nitrocellulose 10 min 200 μL 0-10000 ng mL-1 13 ng mL-1 Human saliva X 188 CRP Immunosensor CC Chemically activated surface PET 15 min 150 μL 10 ng mL-1– 100 μg mL-1 7.6 pg mL-1 Artificial serum, plasma, whole blood ✓ 189 CRP CTnI Procalcitonin Immunosensor SWV GO Paper - 90 μL 0.001–100 μg mL− 1 0.001–250 ng mL-1 0.5 pg mL-1250 ng mL-1 0.38 ng mL− 1 0.16 pg mL− 1 0.27 pg mL− 1 Serum X 190 CRP Cholesterol K+ Aptasensor Chemical Chemical DPV DPV OCPT GO-ZIF67@AuNPs GO-β-CD Valinomycin PET 5 min 100 μL 0–100 ng mL−1 0–120 μM 10-6–1 M - Sweat ✓ 191 25 Dopamine TNF IL-6 Immunosensor EIS GPEDOT:PSS Paper 20 min 2 μL 12.5–400 μM 0.005–50 ng mL-1 2 pg mL-1–2 μg mL-1 3.4 μM 5.97 pg mL-1 9.55 pg mL-1 Human serum X 192 PI; Polyimide, PDMS; Polydimethylsiloxane, MWCNT; Multi-walled carbon nanotube, Csy; l-cysteine, rGO; reduced graphene oxide, PCN; porous g-C3N4, PET; polietilen tereftalat, SALP; streptavidin-alkaline phosphatase, AAP; ascorbic acid monophosphate, GO; graphene oxide, CC; Chronocoulometry, Au; gold, G-PEDOT:PSS; graphenepoly (3,4-ethylenedioxythiophene) polystyrene sulfonate. 32 Figure 7. Schematic mechanisms of electrochemical lateral flow device. Reproduced with permission. 188 2022, Elsevier. (CRP; C-reactive protein, ALP; Alkaline phosphatase). Another study introduces an electrochemical paper-based analytical device (ePAD) designed for the multiplex detection of CVD biomarkers (CRP, cTnI, and procalcitonin) 190. The microfluidic platform is made from wax-printed cellulose paper, allowing for spatially resolved reagent delivery without capillary flow or external pumps. The device features multiple detection zones, each functionalized with specific capture probes for various CVD biomarkers, facilitating simultaneous analysis in a single assay format. The surface modification of stencil-printed carbon electrodes with GO was realized, significantly enhancing the electrochemical performance by increasing the surface area, improving the conductivity, and providing abundant oxygen-containing functional groups for further biomolecule conjugation. The assay can be completed in about 90 minutes, showcasing its potential for quick testing. Electrochemical measurements and SWV readings enhance sensitivity, allowing for quantitative results. Integrating a paper-based microfluidic design, multiprocessing features, and minimal reagent use makes this ePAD an up-and-coming solution for cost-effective, portable, and rapid CVD diagnostics in clinical environments. Rahman et al. introduce a low-cost, flexible, disposable paper-based electrochemical biosensor featuring multiplex detection capabilities 192. The device incorporates a microfluidic 33 platform built on a conductive polymer-modified graphene paper substrate, facilitating efficient sample delivery and analyte distribution while minimizing reagent consumption. The authors modified the electrode surface of the paper-based biosensor by incorporating a graphene and conductive polymer composite, enhancing electron transfer speed, and creating a stable environment for immobilizing biorecognition elements. In this process, graphene nanoplatelets were mixed into a conductive polymer matrix, polyaniline (PANI) or polypyrrole (PPy), and this mixture was evenly applied to the paper substrate's surface. This choice of composite was made due to its complementary benefits; graphene provides a vast surface area and outstanding electrical conductivity, while the polymer contributes mechanical flexibility and functional groups for additional chemical modifications. Screen-printed carbon electrodes that are functionalized with specific antibodies enable the selective detection of dopamine, TNF-α, and IL-6. The assay shows a response time of about 20 minutes with a low sample volume requirement of 2 µL per channel. The biosensor displayed impressive sensitivity, achieving a LOD of 3.4 μM for dopamine, 5.97 pg mL-1 for TNF-α, and 9.55 pg mL-1 for IL-6. A key advantage of the system is its straightforward fabrication process using low-cost materials and its compatibility with microfluidic integration, significantly enhancing analysis throughput and portability. The platform is particularly promising for rapid, decentralized clinical diagnostics and continuous health monitoring applications. Smartphone-integrated electrochemical sensors for CVDs POC devices are combined with sensors and instrumentation equipment to provide “sample-in-answer-out.” Smartphones are one of the most interesting of these equipment due to their widespread use. In recent years, smartphones have become more widespread with features such as cameras, ports, storage features, Wi-Fi, Bluetooth, and even near-field communication (NFC), and the fact that many of these features can be used in POC testing can also be shown as an advantage. In current studies, smartphone-integrated microfluidics are widely studied to benefit from the developments by combining efficient features of both components 198. Although uncommon, smartphone-integrated electrochemical microfluidic systems have also been developed to detect CVDs. Literature summaries of existing studies in this area, which are observed to need development, are provided below and in Table 3. Dudala et al. developed an integrated electrochemical microfluidic biosensor using laserinduced graphene (LIG) electrodes fabricated on polyimide (PI) substrates and connected with PDMS-based microchannels 182. The main objective was to sensitively and selectively detect cTnI, a well-known biomarker for early diagnosis of AMI on a POC platform. The three- 34 electrode system, consisting of a LIG working electrode, a LIG counter electrode, and an Ag/AgCl modified reference electrode, was biofunctionalized using EDC/NHS to immobilize anti-cTnI antibodies. The flexible LIG substrate and the PDMS channel were integrated through a modified thiol-epoxy bonding strategy to ensure leakage-free bonding while preserving electrode functionality. Electrochemical characterization via CV confirmed a semi-reversible redox process and a significantly increased electroactive surface area due to the porous LIG structure. Biosensor performance was evaluated using DPV and showed a LOD of 45.33 pg mL-1 and a limit of quantification (LOQ) of 151.10 pg mL-1, both within clinically relevant ranges for AMI detection. Interference tests with other cardiac biomarkers (cTnT and Mb) showed high specificity. The system was validated using a 3D-printed peristaltic pump alongside a smartphone-controlled miniature potentiostat, tested in both benchtop and portable settings. This highlights its practical usability for POC applications. Khushaim et al. developed a POC platform using porous graphitic carbon nitride (PCN) decorated with AuNPs to detect cTnI (Figure 8) 186. This synthesized conductive composite structure made positive contributions to the biosensor performance because it increased the electrochemically active surface area of the working electrode surface and served as a good biorecognition unit immobilization matrix. The microfluidic chip was fabricated using PDMS, and the microchannel architecture facilitated homogeneous sample distribution on the modified electrode surface, enhancing antigen-antibody interactions. Laser-induced graphene electrodes were coated with PCN@Au dispersion using the drop-casting method, and then a specific aptamer was immobilized on the surface. As a result of voltammetric measurements taken at 0.1 M KCl containing 5 mM [Fe(CN)6]3−/4−, the linear range and LOD of the biosensor were calculated as 0.0001–1000 ng mL-1 and 0.01 pg mL-1. The assay exhibited a total analysis time of approximately 2 minutes and required only 20 µL of the sample, supporting rapid, lowvolume diagnostics. Then, the authors designed the POC system by integrating a custom-made miniature potentiostat and a smartphone reader into the system. The behavior of this system, which offers a small sample size, user-friendly and low-cost features, in electrochemical measurements, was similar to the results obtained with the potentiostat. This platform emerges as a strong contender for next-generation POC cardiac diagnostics by effectively combining porous g-C3N4 for signal amplification, microfluidics for assay management, and smartphone technology for easy data interpretation 186. 35 Figure 8. Schematic representations of A) Synthetic route for GCN, PCN, and PCN-AuNPs, B) Fabrication of aptasensors and cTnI detections, C) POC aptasensing device for cTnI, i) top view, ii) bottom view, and iii) battery. Reproduced with permission. 186 2022, Elsevier. (GCN; Graphitic carbon nitride, PCN; Porous graphitic carbon nitride, AuNPs; Gold nanoparticles, MCH; Mercaptohexanol, BSA; Bovine serum albumin, cTnI; Cardiac troponin I, DNA; Deoxyribonucleic acid). Boonkaew et al. designed an innovative POC diagnostic platform combining electrochemical sensing, microfluidics, and smartphone-based readout for the rapid and sensitive detection of CRP (Figure 9) 189. The detection mechanism is based on a screen-printed carbon electrode modified with nanobody-functionalized gold nanoparticles, which offer 36 enhanced surface area and specific binding affinity for CRP. A transparent PET film prepared the device, creating fast and delayed channels. In this device, they left an opening for the sample to be loaded onto the electrode. After the biorecognition unit immobilization, the buffer was loaded to remove unbound groups from the surface. Then, measurements were made after the probe reached the electrode in the delayed channel, where the redox probe was used. The surface of the working electrode used was functionalized to be suitable for immobilization by anodic pretreatment and exposure to various chemicals. This device requires a sample volume as low as 150 µL and completes assays in about 15 minutes. It measures the electrochemical response using chronocoulometry (CC). Results are sent wirelessly via near-field communication (NFC) to a smartphone app, which processes the data and shows quantitative results. This integration with smartphones greatly improves portability and user-friendliness, removing the necessity for large equipment. This system, which detected a linear working range of 10 ng mL-1-100 μg mL-1 and a LOD value of 7.6 pg mL-1, behaved specifically against other groups that could be found in serum samples in selectivity studies. Additionally, CRP detection was achieved in samples such as artificial serum, plasma, and whole blood samples without requiring pretreatment steps. 37 Figure 9. Developed platform integrating a microfluidic device, electrode system, and smartphone-based potentiostat for CRP detection. A) Schematic illustration of the platform. b) Overall step-by-step modification on the screen-printed graphene electrodes (SPGEs). C) Procedure for CRP detection using chronocoulometry measurement. Adapted from ref 189. Copyright 2024 American Chemical Society. (NFC; Near-field communication, SPGE; Screen-printed graphene electrodes, PBS; Phosphate-buffered saline, RT; Room temperature, CRP; C-reactive protein). Fu et al. present a wearable ring sensor to continuously monitor biomarkers associated with atherosclerosis-induced cardiovascular disease in sweat (Figure 10) 191. This device combines electrochemical biosensing and microfluidic technology, utilizing screen-printing to produce precise multi-channel electrodes. The sensor integrates an electrochemical detection system that enables non-invasive and real-time on-site monitoring of molecules such as CRP, 38 cholesterol, and potassium ions (K+). The developed device operates by passively collecting sweat, eliminating the need for external stimuli, and utilizing advanced signal-processing techniques to enhance sensitivity and specificity. Large-scale patterned screen-printed electrodes provide high sensitivity and efficient electrochemical detection of trace biomarkers in sweat. Modification GO increased the active sites on the surface of the working electrode and improved electron transfer efficiency. Zeolitic imidazolate framework-67 (ZIF-67) and AuNPs, with an ultra-high surface area and highly ordered porous structure, can carry more gold nanoparticles for aptamer binding. The appropriate aptamer is attached to the composite material via an Au-thiol bond, forming an aptamer sensor with high affinity and specificity for CRP. The aptamer is combined with the electroactive redox molecule methylene blue (MB) to facilitate efficient electrochemical signal transduction and amplification. β-Cyclodextrin (βCD), on the other hand, possesses a hydrophobic structure that allows cholesterol molecules to displace MB, helping to convert concentration signals into current signals. The results demonstrate the feasibility of detecting relevant biomarkers at physiologically significant concentrations, highlighting the potential of this wearable technology for early detection and continuous monitoring of atherosclerosis progression. The collected data were wirelessly transmitted to a smartphone, where they were presented through a custom-designed mobile application. This innovative approach offers a promising alternative to traditional blood-based biomarker assessments, paving the way for personalized healthcare strategies. 39 Figure 10. Schematic mechanisms of flexible wearable ring sensor. A) A microfluidic chip to collect sweat secreted onto the epidermis after iontophoresis. The mechanism of in situ microfluidic sweat B) CRP analysis, C) cholesterol analysis, and D) K+ analysis. Adapted with permission from ref 191. Copyright 2025 Elsevier. (CRP; C-reactive protein, ZIF; Zeolitic imidazolate framework, AuNPs; Gold nanoparticles, MB; Methylene blue, β-CD; βCyclodextrin). Microneedle-based electrochemical CVDs sensors Platforms that include microneedles and are classified as minimally invasive allow the collection of interstitial fluid from the skin, thus enabling transdermal drug delivery or biomarker detection applications. Microneedles can provide long-term monitoring for the diagnosis and follow-up of various diseases because they are easy and painless to apply 199. The reason microneedles are called minimally invasive is that they interact with the dermis less than traditional needles. Microneedle-based sensors that can provide POC health monitoring in the diagnosis of target biomarkers in sensor designs and thus enable tracking of clinical information can also be used in wearable sensor technologies 200. This method, which has become quite popular in recent years, has been used to detect analytes such as glucose and serotonin, but it is 40 quite new in wearable electrochemical sensors 201-203. Especially when looking at microneedlebased electrochemical CVDs sensors, it is observed that there are minimal studies. The existing studies are summarized below, and it is noteworthy that almost all focus on electrolyte monitoring, such as Na+ and K+. Li et al. developed a microneedle sensor for the electrochemical detection of cholesterol, one of the CVDs biomarkers, in human biofluid (Figure 11) 204. The design of the microneedle sensor incorporated platinum and silver wires into pyramidal microneedles containing a microgap opening. In addition, this platform, which was designed as an enzymatic sensor, also included the cholesterol oxidase (ChOx) enzyme in the system, which was immobilized on the platinum transducer surface with the help of bovine serum albumin and Nafion. This developed enzymatic cholesterol sensor provides linear measurement between 1-20 μM and 0.5 μM. The electrochemical detection mechanism in this microneedle sensor is based on the catalysis of cholesterol by ChOx immobilized on the surface and detecting the resulting H2O2. Electroanalytical measurements were also performed using chronoamperometry in artificial interstitial fluid and skin-mimicking phantom gel. It also exhibited good selectivity against molecules such as ascorbic acid and uric acid that can be present in the same environment. 41 Figure 11. Schematic illustration and reaction mechanisms of minimally invasive wearable microneedle sensor for detecting cholesterol. Adapted from ref 204. Copyright 2023 Royal Society of Chemistry. (WE; Working electrode, RE; Reference electrode, CE; Counter electrode, ChOx; Cholesterol oxidase). Gao et al. developed a flexible microneedle electrode-based biosensor and detected glucose, uric acid, and cholesterol (Figure 12A) 205. All three analytes were determined using the enzymatic method; glucose oxidase, uricase, and cholesterol oxidase enzymes were used, respectively. Analytes were detected by an amperometric method using enzymes immobilized with glutaraldehyde on the surface of the microneedle electrode array (MEA). Before immobilization, magnetorheological drawing lithography (MRDL) of microneedles on the flexible substrate and sputter coating of Au/Ti film on its surface were performed. Then, the surface was modified with PANI nanofibers and Pt nanoparticles (NPs) to improve detection sensitivity and increase conductivity. The determined linear working ranges were calculated as 2-12 mM, 0.1-1.2 mM, and 1-12 mM for glucose, uric acid, and cholesterol, respectively. Additionally, LOD values are described as 260 μM, 4 μM, and 440 μM for the same sequence. 48 Conclusions and future perspectives Electrochemical sensor technologies are promising for evaluating biomarkers in healthy individuals and those with diseases, facilitating more accurate and personalized diagnostic approaches. By determining baseline biomarker levels in healthy subjects, these sensors can more effectively detect deviations, which is crucial for the early identification and monitoring of diseases. This approach enhances the accuracy of diagnostics and deepens our understanding of biomarker behavior, ultimately leading to improved management of CVDs. Electrochemical sensing platforms have emerged as a transformative technology for the early detection and monitoring of CVDs. The ability of these platforms to detect multiple biomarkers provides a significant advance over traditional diagnostic methods. In addition to their analytical strengths, electrochemical sensors offer distinct benefits that support personalized medicine. Their compact size, low energy use, and seamless integration with wearable and implantable gadgets facilitate ongoing, real-time patient monitoring. Additionally, their ability to identify specific biomarker profiles enables personalized risk assessment, early detection, and customized treatment plans 234, 235. These qualities make electrochemical sensing technologies especially useful for managing cardiovascular diseases in a patient-focused, data-driven way. These sensors are well-suited to the increasing demand for POC diagnostics in healthcare, offering high sensitivity, specificity, rapid response times, and potential for miniaturization. In particular, integrating noble metal nanoparticles, transition metal oxides, and carbon-based nanomaterials has enhanced the signal amplification and accuracy of electrochemical sensors. Despite these advances, several challenges that limit the full-scale adoption of electrochemical biosensors in clinical settings still need to be addressed. Issues such as long-term stability, reproducibility, and selectivity in complex biological environments must be addressed. Developing multiplexed sensors that simultaneously detect multiple cardiovascular biomarkers will provide more comprehensive diagnostic data and improve patient outcomes. Integrating electrochemical sensors with wearable technology presents exciting possibilities for continuous cardiovascular monitoring. Ongoing tracking of cardiovascular biomarkers can revolutionize the management of CVDs through immediate diagnosis and tailored care. These advances are expected to revolutionize personalized healthcare by allowing real-time health monitoring and early intervention, thus significantly reducing the burden of CVDs worldwide. Nonetheless, ongoing monitoring with electrochemical sensors encounters various issues that hinder its practicality. For instance, prolonged exposure to analytes can lead to sensor saturation, which diminishes accuracy, and surface fouling caused by biomolecules may necessitate frequent regeneration. Maintaining long-term stability is vital, particularly in 49 wearable or implantable applications. Furthermore, the significant amounts of data produced require the development of reliable algorithms for real-time analysis. Tackling these issues will be crucial for the progress of continuous monitoring technologies. Continued interdisciplinary collaboration between bioengineers, healthcare professionals, and regulatory bodies will be essential in overcoming the current challenges and realizing the full potential of electrochemical sensing platforms in CVD detection. The integration of electrochemical sensors with telemedicine has the potential to revolutionize modern healthcare. These sensors enable patients to be continuously monitored at home or remotely, allowing for early detection of cardiovascular risks. Combined with telemedicine, these sensors make it possible to immediately detect conditions requiring urgent intervention, especially heart attacks or heart failure. The ability to simultaneously monitor and analyze cardiac biomarkers, along with basic patient parameters such as heart rhythm and blood pressure, may enable healthcare professionals to remotely monitor patients and provide more effective interventions. This integration not only increases patient comfort but also reduces the burden on the healthcare system. It is especially critical for patients living in rural areas with difficult healthcare access. At the same time, by using cloud-based data management with telemedicine systems, large amounts of patient data can be stored and analyzed to make more accurate diagnoses and treatment plans. Thus, combining electrochemical sensors and telemedicine systems is an essential step toward personalized medicine. The use of AI and ML algorithms has grown to enhance signal interpretation and diagnostic precision in electrochemical sensing platforms 236, 237. Support vector machines and principal component analysis, for instance, have been used to classify voltammetric signals and differentiate target from non-target responses in complex biological samples 238. Additionally, convolutional neural networks have been utilized to analyze raw electrochemical data for the detection of cardiac biomarkers, yielding improved sensitivity and reduced false positives 239. Consequently, AI-driven signal processing can significantly enhance the performance and clinical applicability of advanced electrochemical sensors. In the future, integrating AI with real-time electrochemical sensing could open new possibilities for early diagnosis, risk assessment, and personalized treatment options in cardiovascular healthcare. Multiple electrochemical sensors are essential to enhance diagnostic assessment accuracy, reliability, and scope. Unlike traditional methods that rely on single-biomarker detection, multiple sensors allow for simultaneous detection of cardiovascular biomarkers, such as cardiac troponins, BNP, and LDL. This multi-target approach enables a more comprehensive analysis 50 of cardiovascular health, allowing for earlier detection of heart disease and more personalized treatment plans. Metabolite detection is critical in electrochemical CVDs monitoring, providing real-time insights into the metabolic processes underpinning heart health. Metabolites such as glucose, uric acid, and cholesterol are closely linked to cardiovascular function, and their levels can indicate the presence of metabolic dysfunctions that contribute to CVDs. Electrochemical sensors that detect these metabolites offer a rapid, sensitive, and non-invasive means of assessing a patient’s cardiovascular status. This allows for more precise and timely interventions, especially in high-risk patients. Additionally, tracking metabolite changes over time provides valuable information on disease progression and the effectiveness of therapeutic interventions. With continuous advancements in sensor technology, metabolite detection is becoming an indispensable tool in personalized cardiovascular care, enhancing both early detection and long-term management of CVDs. Wearable CVDs biomarker sensors have focused predominantly on K⁺ and Na⁺ due to concentration limits preventing the detection of biomarkers in body fluids other than blood. Monitoring ions such as K⁺ and Na⁺ is crucial in electrochemical CVDs monitoring due to their direct involvement in maintaining the heart's electrical activity and overall function. These ions play vital roles in regulating cell membrane potentials, particularly in cardiac cells, where proper ion exchange is necessary for heart muscle contraction and the propagation of electrical signals that maintain heart rhythm. Electrochemical sensors designed to monitor these ions offer a rapid and sensitive way to detect electrolyte imbalances, providing valuable insights into heart health in acute and chronic settings. However, there are limitations in ion monitoring using electrochemical sensors. One major challenge is the selectivity of the sensors in complex biological fluids, where multiple ions and other interfering species are present. This can affect the accuracy of the measurements. Changes in K⁺ and Na⁺ concentrations are not only associated with CVDs. Many diseases, including renal dysfunction, may cause imbalances in these electrolytes. Therefore, it can be said that changes in K⁺ and Na⁺ levels do not specifically indicate CVDs but also many other systemic disorders. Diet, water consumption, medication use, and K⁺ and Na⁺ levels can be affected. Therefore, it is essential to consider different variables when monitoring the levels of these electrolytes. Additionally, the body's dynamic range of ion concentrations can vary significantly, requiring sensors to be highly adaptable to different physiological conditions. Despite these challenges, advances in sensor technology, such as ion-selective electrodes and nanomaterials, are helping to improve the sensitivity and selectivity of ion detection in CVDs monitoring. 51 Recent studies have also demonstrated that electrochemical biosensors can achieve the sensitivity required to detect low-abundance cardiac biomarkers in non-invasive samples 46, 142, 188, 191. For example, NT-proBNP, CRP, and MPO have been successfully detected in saliva with detection limits in the low picogram per milliliter range 46, 142. Meanwhile, wearable devices have enabled real-time sweat monitoring of CRP and cholesterol 191. These findings confirm that, with proper electrode modifications, signal amplification techniques, and innovative material integration, electrochemical biosensors can function effectively even in environments with low concentrations. Although current research on microneedleand tattoobased electrochemical sensors primarily focuses on electrolyte detection due to their higher levels in interstitial fluid and sweat, there is a significant and expanding opportunity to adapt these platforms for identifying biomarkers specific to CVD. Incorporating advanced surface chemistries, nanomaterials, and antifouling strategies can enhance sensitivity and specificity, even for low-abundance protein and nucleic acid biomarkers. Additionally, integrating these wearable sensors with continuous monitoring systems and data analysis tools could enable realtime, non-invasive, and personalized cardiovascular diagnostics. Future research should focus on translating these technologies to clinically relevant targets such as cardiac troponins, natriuretic peptides, and miRNAs found in alternative biofluids. A significant challenge in developing electrochemical sensors for continuous monitoring is ensuring effective regeneration of the sensing surface. Over time, the accumulation of target analytes and nonspecific binding can compromise sensor performance, resulting in signal shift, reduced sensitivity, and a shorter operational lifespan. Without a reliable regeneration mechanism, sensors must be frequently replaced or recalibrated, hindering their practicality for real-time, long-term monitoring. Consequently, it is vital to develop efficient and gentle regeneration protocols, such as such as chemical washing 142, electrochemical cleaning 240, magnetic separation 241, and re-functionalization 242 that restore the sensor surface without affecting its selectivity and stability. Specifically, regeneration methods need careful optimization for biosensors that utilize biological recognition elements to preserve biomolecular activity while enabling repeated use. Addressing this issue is crucial for developing robust, low-maintenance, and cost-effective continuous sensing platforms, particularly for applications in wearable devices, implantable systems, and point-of-care diagnostics. To sum up, electrochemical sensor technologies provide advanced solutions for diagnosing CVDs, particularly regarding early detection and ongoing monitoring. Nonetheless, most current systems remain confined to laboratory or professional environments. Upcoming 52 research should prioritize the identification of highly specific biomarkers in non-invasive body fluids like sweat, saliva, and interstitial fluid. For example, examining biomarkers such as miRNA-499, miRNA-21, and NT-proBNP in these fluids will be essential for creating nextgeneration sensors 30, 31, 52, 142. Currently, no CVD diagnostic devices are designed for home use, creating a strong demand for highly sensitive portable, user-friendly sensor systems with low detection limits. In this regard, integrated platforms that merge microfluidics, AI-driven data analytics, and smartphone interfaces are gaining attention. Additionally, these systems could serve diagnostic applications and preventive healthcare. Continuous monitoring sensors can detect abrupt changes in biomarker levels, potentially helping to prevent acute events like heart attacks 191. Also, while real-time medication dose adjustments based on biomarker levels are not yet standard in cardiovascular care, continuously monitoring of essential markers can enhance clinical decision-making by offering timely insights into disease progression and treatment response. Acknowledgements This work is supported by the Scientific and Technological Research Council of Türkiye (TUBITAK) under grand no. 22AG032 and OrChESTRA (Organ-on-a-Chip Focused Strategic Partnership) project, which has received funding from the European Union’s Horizon Europe’s research and innovation program under Grant Agreement No. 101079473. 53 REFERENCES (1) Zhong, S.; Chen, L.; Shi, X.; Chen, G.; Sun, D.; Zhang, L. Recent advances in electrochemical aptasensors for detecting cardiac biomarkers: A review. Microchem. J. 2023, 193, 109063. (2) Dong, T.; Zhu, W.; Yang, Z.; Matos Pires, N. M.; Lin, Q.; Jing, W.; Zhao, L.; Wei, X.; Jiang, Z. 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