Advancements in electrochemical immunosensors towards point-of-care detection of cardiac biomarkers

Abstract

Cardiovascular disease remains the leading cause of death worldwide, with mortality rates increasing annually. This underscores the urgent need for accurate diagnostic and monitoring tools. Electrochemical detection has emerged as a promising method for swiftly and precisely measuring specific biomarkers in bodily fluids. This approach is not only cost-effective and efficient compared to traditional clinical methods, but it can also be tailored to detect individual biomarkers, which makes it particularly well-suited for point-of-care (POC) applications. The ability to conduct testing at the point of care is crucial for timely interventions and personalized disease management, empowering healthcare providers to tailor treatment plans based on real-time biomarker data. Thanks to recent advancements in nanomaterials, we've seen significant progress in electrochemical detection, leading to the development of specialized rapid immunoassay systems. These systems utilize specific antibodies to target molecules, expanding the range of detectable biomarkers. This innovation has the potential to revolutionize the diagnosis and treatment of cardiovascular diseases by enhancing detection sensitivity and specificity. Ultimately, these advancements aim to improve patient outcomes by enabling earlier diagnosis, more precise monitoring, and personalized therapeutic interventions, which will contribute to more effective management of cardiovascular health globally.

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Introduction

Cardiovascular diseases, also known as CVDs, are conditions that affect the heart and its intricate network of blood vessels. This umbrella term includes various conditions such as coronary artery disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis, and pulmonary embolism.¹ In 2020, about 32% of all global deaths were linked to cardiovascular diseases, resulting in an estimated 523 million cases and 19 million deaths due to cardiovascular complications. These statistics indicate an 18.7% increase in the number of cardiovascular diseases since 2010, highlighting the growing severity of these conditions.² The statistics regarding cardiovascular diseases are emphasized to highlight the seriousness and impact of these heart-related conditions on the global population. The risk factors for cardiovascular disease include an individual's physical and genetic traits as well as their lifestyle habits. Common risk factors include pre-existing conditions such as type 1 and type 2 diabetes, and vascular hypertension. These conditions can lead to blood clot formation and arterial damage. Diagnosis and treatment of cardiovascular diseases have become more challenging due to their complexity. Ideal cardiac biomarkers should be specific to the cardiovascular system, sensitive enough to detect cardiac damage, assist in early and late prognosis, detectable only during disease progression, measure disease severity, differentiate between reversible and non-reversible damage, remain stable under long-term conditions, and be affordable and efficient for detection.³ Some common biomarkers tested in cardiovascular studies include cardiac troponin I & T, myoglobin, C-reactive protein (CRP), heart-type fatty acid binding protein (H-FABP), creatine kinase-myoglobin binding (CK-MB), and D-dimer.⁴ Detection of these specific circulating biomarkers can assist in prognosis and diagnosis, enabling patients to receive prompt treatment strategies before their illness becomes potentially fatal.

Conventional methods for detecting cardiovascular disease involve various assays and sensors, such as ELISAs, LFIAs, CIAs, and electrochemical biosensors. ELISAs (enzyme-linked immunosorbent assays) use antigen–antibody interactions to accurately detect biomarkers. These methods are often effective and offer a wide range of biomarkers for detection. A new method, specifically for cardiac markers, is the blood-based ELISA for secretoneurin, which can accurately measure certain heart diseases and provide personalized disease management for patients. However, this innovative method is unsuitable for rapid assessment due to the need for a large number of patient samples for the test, and it can take weeks for results to be available.⁵ Rapid lateral flow immunoassays are another option that uses antigen–antibody interactions for detection. However, they focus on a smaller number of markers, particularly a few for cardiac biomarkers. This specialized test method has advantages in terms of time, specificity, prediction, and diagnosis. The specificity of cardiac immunoassays stands out as a feature that enhances their usefulness in clinical settings and for point-of-care treatments. These assays are specifically designed for cardiac-specific biomarkers, which minimizes the risk of false-positive or false-negative results and enables highly accurate and reliable diagnostic strategies. Consequently, healthcare providers can rely on the accuracy and precision of cardiac immunoassay results to confidently diagnose heart disease and tailor treatment plans to the patient's needs.⁶ Chromatographic immunoassays (CIAs) are a type of LFIAs that use the flow of a sample to separate biomarker components based on antibody–antigen interactions. In this method, the sample containing the analyte of interest is applied to a special pad designed to test for specific antigens associated with cardiovascular diseases (CVDs). As the sample moves through the pad, it separates within a strip. This movement allows the antigens in the sample to bind to specific lines on the test strip. Once the sample has traveled down the strip, clinicians can measure each biomarker, offering a rapid and more isolated method of biomarker detection.⁷

A biosensor is a device that uses a biological recognition element, such as antibodies, enzymes, aptamers, oligonucleotides, membrane receptors, or whole cells, to detect a transduction element in close proximity and provide analytical information autonomously.⁸ In the detection of cardiac biomarkers, specific antibodies are used to target molecules such as troponins, CK-MB, C-reactive protein, and many other diagnostic markers. Analyzing the interactions of these biomolecules is crucial for the development of biosensor technologies, as various transduction methods are employed to convert these biomolecular recognition interactions into quantitative results.⁹ The commonly studied transduction methods include electrochemical methods, which use components to accurately detect the electrical charge of biomarkers; mechanical methods, which study the frequency shift as a function of mass; and optical methods, which measure the change in refractive index through biochemical interactions with the active layer.¹⁰

Electrochemical transduction methods have been extensively studied for detecting cardiac biomarkers. There are several main categories of electrochemical biosensors, including amperometric, potentiometric, and impedimetric biosensors. The choice of electrochemical biosensor for a specific study depends heavily on the analyte of interest and the intended application of the results. Amperometric biosensors measure the current produced during reduction and oxidation reactions and use Clark oxygen electrodes for quantification.¹¹ Biosensors in this category convert biological recognition events caused by electroactive species produced at the sensor surface. This allows for the quantification of the analyte in the selected fluid. Amperometric sensors offer an affordable option for point-of-care devices using methods such as cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry.¹² Potentiometric biosensors measure the potential difference between a working electrode and a reference electrode near zero current, providing insights into ionic activity during electrochemical reactions.¹³ Examples include pH sensors, gas sensors, and ion-selective sensors. Impedimetric biosensors are the most recently explored type of electrochemical sensors, which allow for label-free detection and rapid analysis of a wide range of analytes, spanning from small molecular sizes to larger cellular sizes.¹⁴

Nanotechnology has enabled the miniaturization and enhancement of biosensors, making them suitable for point-of-care diagnostic testing. Unlike large and centralized laboratory equipment, these portable devices can be used for direct testing at a patient's bedside, using user-friendly tools. These point-of-care biosensors are attractive because they offer increased sensitivity, allowing for lower detection limits compared to conventional methods such as ELISA assays and other automated platforms used in central laboratories.¹⁵ The impressive capabilities of biosensors offer opportunities to improve current standards in clinical analysis and diagnostic applications without the need for time-consuming processing steps. By addressing the limitations of traditional detection methods, such as high cost and time consumption, point-of-care biosensors have the potential to revolutionize the rapid detection of potentially life-threatening diseases. They can prove to be fast, reliable, and accurate real-time determinants (Fig. 1).¹⁶

Fig. 1 Advantage of electrochemical biosensors for rapid and accurate detection of biomarkers (created using https://Biorender.com).

Various conventional biosensors can detect inflammatory biomarkers, including electrochemical techniques like voltammetry, amperometry, and impedance measurement. These methods are adaptable based on the interface material and the detected biomarker type. Immunoassays are particularly efficient in detecting biomarkers using antibodies as capture materials, making them highly valuable in sensor research. Researchers reported the use of electrochemical biosensors to detect biomarkers such as myoglobin,¹⁷ troponin I,¹⁸ troponin T,¹⁹ thrombin,²⁰ creatine kinase-MB,²¹ and many other cardiac biomarkers.²² Electrochemical biosensors are attractive point-of-care devices used as rapid detection methods due to their accuracy, and ability to detect a wide range of analytes.

Role of cardiac biomarkers and their electrochemical detection

It's crucial to quickly assess patients with nonspecific cardiovascular symptoms to start effective treatment and management plans. This is especially important for high-risk patients who may have potentially fatal acute coronary syndrome. Cardiac biomarkers are measurable proteins that show up in the bloodstream when there is damage to the cardiovascular system. In clinical practice, these biomarkers are often measured to determine the presence and progression of cardiovascular damage. Doctors routinely use biomarker measurements along with echocardiography (ECG) and radio diagnostics to make more accurate diagnostic decisions.²³ Both electrocardiography and myocardial enzyme measurement are the current diagnostic pillars in acute myocardial infarction (AMI) and play an important role in clinical assessment. However, ECG alone is often insufficient for the diagnosis of coronary pathologies, as ST elevation can be non-specific.^24,25 So rapid and specific detection is critical for effective triage and treatment of patients with suspected coronary pathology.²⁶ Several electrochemical immunosensors have been developed using antibody proteins to detect cardiac molecules. To enhance the affinity of electrodes for antibodies, metallic and non-metallic nanostructures like nanoparticles, nanocomposites, nanotubes, and nanochannels are utilized. The combination of antibodies with nanomaterials has been demonstrated to produce electrochemical immunosensors with broad linear detection ranges, selectivity, specificity, and low detection limit.²² For many molecules that can be expensive and inaccessible to detect clinically, electrochemical immunosensors represent a promising opportunity for a cost-effective and rapid alternative that improves the detection and treatment of cardiovascular disease.

Cardiac biomarkers play a critical role in the detection of cardiovascular pathologies (Fig. 2) when the patient's medical history and/or ECG are inconclusive. The use of the levels has its limitations due to variations in time of presentation, infarct size, selection criteria, diagnostic threshold, and kinetic factors of the biomarkers.²⁷ Current conventional tools are not well suited for rapid clinical diagnostic methods due to the time and resources required to perform the tests.²⁸ These factors make these standard tests inefficient for making decisions about acute cardiac pathologies. Electrochemical sensors are useful in the detection of biomarkers for cardiac disease because they can rapidly detect and analyze the presence and concentration of biomarkers, which improves diagnostic capabilities and enables early and accurate point-of-care diagnosis.²⁹ Electrochemical immunosensors are widely recognized as a popular and emerging analytical tool due to their simplicity and cost-effectiveness, high sensitivity and specificity, and ease of miniaturization, making these sensors excellent point-of-care medical devices.²⁸Fig. 3 depicts the current clinical evaluation methods for cardiovascular diseases as well as the role of cardiac biomarkers in clinical interventions.

Fig. 2 Different types of cardiac biomarkers and their connection to cardiovascular diseases (created using https://Biorender.com).

Fig. 3 Schematic representation of the role of cardiac biomarker detection (created using https://Biorender.com).

Troponins

Cardiac troponins are used for primary and secondary prevention, diagnosis, prognosis, and treatment of various cardiovascular pathologies, including but not limited to acute myocardial infarction, heart failure, ischemic heart disease, acute coronary syndromes, and cardiomyopathies.³⁰ Troponin is critical in muscle contraction as it links changes in intracellular calcium concentration to the generation of contractile force.³¹ These markers are genetically encoded to be released specifically during cardiovascular injury and are thus unique to the myocardium as they regulate calcium-mediated contraction of cardiac and skeletal muscles in the contractile apparatus.

The cardiac troponin complex is a component of the thin filaments of skeletal and cardiac muscle and consists of three subunits. Cardiac troponin T (cTnT), troponin C (cTnC) and troponin I (cTnI) are cardiac regulatory protein isoforms that function in the control of calcium-mediated interactions between actin and myosin. These filaments act as molecular motors that shorten the sarcomeres, leading to contraction of the myocyte during excitation-contraction coupling and causing contraction and relaxation of the myocardium.³²

The isoforms of cardiac troponin T and I are the most specific and sensitive indicators for the diagnosis of AMI. These cardiac regulatory proteins are involved in calcium-mediated interaction between actin and myosin and are specifically encoded, making them unique to the myocardium.³³ Although there is little evidence of expression of these markers outside the myocardium, their presence is not specific for AMI diagnosis, as the markers are elevated in blood serum in other conditions that result in cardiomyocyte death or damage from non-ischemic causes.³⁴ Both cTnI and cTnT are undetectable in patients without myocardial damage, which emphasizes their importance for diagnostic measurement. Despite the high specificity of cTnT and cTnI, they are no longer considered the only gold standard biomarkers for the detection of AMI, as elevations are also observed in non-ischemic causes of cardiomyocyte death in addition to ischemic necrosis.³⁴

Elevated troponin levels are detectable in serum within 4–8 hours after the onset of clinical symptoms and reach their highest concentration within 12–24 hours. Troponin levels remain elevated for 3–10 days after an AMI, giving them a greater diagnostic range than other biomarkers. The physiologic range of troponin in the blood is 0.0 to 0.04 ng mL⁻¹, with heart injury suspected at levels between 0.04 and 0.39 ng mL⁻¹³⁵ and have the highest diagnostic and prognostic value in clinical practice for the accurate identification of high-risk patients with acute coronary syndromes.³⁶ Measurement of cardiac troponin levels using a fully automated standard assay remains superior to other clinically available inflammatory biomarkers.²⁴ Being highly prospective biomarkers for utilization in AMI, many different immunosensors have been reported for cTnT and cTnI, aiming to improve the accessibility, sensitivity, selectivity, and reliability of their electrochemical detection. A nanohybrid film consisting of pyrrole-2-carboxylic (COOH–Py) and aminated nano clay (NH₂–NCY) was employed for the label-free detection of cTnT.³⁷ This was done by immobilizing cTnT antibodies onto the nanostructure with glutaraldehyde (GA) and blocking the remaining sites with glycine (Gly). It demonstrated performance in both phosphate-buffered saline (PBS) and human serum, with a linear range of 2.5 to 125 pg mL⁻¹ and 1.0 to 10 pg mL⁻¹ and a limit of detection (LOD) of 0.70 pg mL⁻¹ and 0.35 pg mL⁻¹ respectively. Another modified stencil-printed carbon electrodes (SPCEs) with graphene oxide (GO) to create an electrochemical paper-based biosensor for cTnI and CRP detection in human serum.³⁸ This utilized EDC/NHS coupling (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide) to cross-link cTnI antibody to the electrode surface, followed by bovine serum albumin (BSA) to reduce/eliminate non-specific binding. This delivered a wide linear range from 0.001 to 250 ng mL⁻¹ and an LOD of 0.16 pg mL⁻¹ with high sensitivity. Being capable of simultaneous detection of three important CVD biomarkers, multiplex biosensors represent a solution of interest to the lack of specificity biomarkers have to one specific disease.

Based on indium tin oxide (ITO) coated electrodes, a highly selective biosensor was reported for cTnI in spiked blood plasma.³⁹ Surface functionalization was achieved through EDC/NHS coupling and cTnI antibody linkage with BSA preventing nonspecific bindings. This electrochemical immunosensor had a high specificity for cTnI over cTnT, with a range of 0.01 to 100.0 ng mL⁻¹ with an LOD of 0.033 pg mL⁻¹. Strong differentiation between the two troponin isoforms potentially provides clinical insight into AMI patients' progression and health. Being highly reproducible, the biosensor can rapidly provide results within 15 minutes.

Immunoassay using a nanoporous nylon membrane and gold electrodes with antibody crosslinked was used for the accurate detection of troponin within 15 minutes.⁴⁰ Polyimide-based flexible electrode strip sensor for the quantification of cTnT was reported by Nandhinee et al. This reported sensor utilized only 20 μL of sample volume and can detect cTnT in human serum samples with a lower detection limit of 1 pg mL⁻¹.⁴¹ ZnO nanostructures were utilized for developing immunoassays for simultaneous analysis of cTnT and cTnI. As shown in Fig. 4A, the multiplexed array sensor panel was immobilized with anti-cTnT and anti-cTnI antibodies using a crosslinker, and measurements were performed using a potentiostat with a limit of detection of 1 pg mL⁻¹.⁴² Additionally, a number of developed platforms demonstrated the detection of both cTnT and cTnI. An electrochemical immunosensor was developed utilizing simple EDC/NHS coupling to link cTnT and cTnI antibodies to carbon paste electrodes (CPEs).⁴³ After functionalization, this biosensor exhibited a linear range of 0.01 to 50 ng mL⁻¹ and 100 to 500 ng mL⁻¹ and a LOD of 0.6 pg mL⁻¹ and 2.5 pg mL⁻¹ for cTnT and cTnI respectively. This was achieved in conjunction with reverse iontophoresis (RI), looking to improve the non-invasive capabilities of electrochemical sensors. The variety of different techniques, nanostructures, metals, and polymers tested are continuing to push the capabilities of electrochemical detection of cTnI and cTnT for CVDs.

Fig. 4 (A) Electrochemical immunosensor reported for the detection simultaneous detection of cTnI and cTnT⁴² (reprinted with permission from Elsevier @ copyright 2016); (B) detection of myoglobin using AuPtAg porous hollow nanorods in human serum⁴⁴ (reprinted with permission from Springer Nature).

Myoglobin

Myoglobin, a cytoplasmic hemoprotein found exclusively in cardiac myocytes and oxidative skeletal muscle fibers, is used for the reversible binding of oxygen. The structure of myoglobin is described as a conjugated protein with about 153 amino acid residues that interact with the central heme group, an iron–porphyrin complex.⁴⁵ This interaction between the heme group and the polypeptide chain takes place at the fifth coordination position of the iron link as well as at additional binding sites between the porphyrin and the polypeptide chains. The myoglobin structure differs from the hemoglobin structure because the affinity for oxygen is higher in myoglobin and there is no cooperative binding with oxygen as in hemoglobin. This specific structure of myoglobin allows oxygen binding in a reversible reaction as the heme group controls the environment of the molecule.⁴⁶ Myoglobin plays an important role in the regulation of hypoxic vasodilation by controlling the binding of nitric oxide (NO), catalyzing NO deoxygenation, and reducing nitrite, thus contributing to the control of blood pressure.⁴⁷ Thus, myoglobin plays a central role in oxygen storage, intracellular oxygen transport, and the management of homeostasis.

Cardiac myoglobin is not found outside of muscle tissue but can enter the bloodstream in the event of muscle injury.²⁸ Myoglobin is rapidly released from the myocardium, the muscle layer of the heart, and increases within the first 30 minutes after an acute event or injury. Due to its small size and rapid kinetic properties, myoglobin is quickly released into the bloodstream, indicating its importance in the early detection of heart disease and the exclusion of heart damage.⁴⁸ The physiological range of myoglobin is below 91 ng mL⁻¹ in men and below 63 ng mL⁻¹ in women. A serum myoglobin level of more than 110 ng mL⁻¹ is considered pathological and may indicate an AMI.⁴⁹ Although myoglobin is useful as an early marker for detection, it is not specific and requires a troponin test to confirm myocardial injury to rule out possible false positive myoglobin values.⁵⁰

Often being released due to cardiac muscle injury, myoglobin compliments the cardiac troponins in its potential to aid early diagnosis of AMI. This has pushed the development of electrochemical immunosensors for the determination of cardiac myoglobin. One such sensor adopted a glassy carbon electrode (GCE) with AuPtAg porous hollow nanorods (PHNR)'s for myoglobin detection in human serum.⁴⁴ Once the AuPtAg PHNRs are immobilized on the electrode, antibodies are deposited, and remaining active sites are blocked with BSA to prevent nonspecific interactions (Fig. 4B). In human serum, the biosensor demonstrated a high sensitivity, detecting within a linear range of 0.0001 to 1000 ng mL⁻¹ and a LOD of 0.46 pg mL⁻¹. Its rapid and sensitive response could find potential application in the clinical monitoring of myoglobin.

An ITO-coated glass plate with Au nanoparticles (NPs) expressed a wide range in both PBS and human serum.⁵¹ The AuNPs are bound by a 3-aminopropyltriethoxy silane (APTES) monolayer, and then the antibody is applied, incubated for 2 hours, and washed with PBS–EDTA. After full preparation, the sensor displayed a linear range of 0.02 to 1 μg mL⁻¹ and a LOD of 5.5 ng mL⁻¹. Another ITO/AuNP immunosensor utilizing the same functionalization process but with BSA reported a linear range of 0.01 to 1 μg mL⁻¹ and a LOD of 2.7 ng mL⁻¹.⁵² The unique functionalization of these two sensors provides a desirable biocompatibility due to their AuNP-based bioelectrode structure.

Multiwalled carbon nanotubes (MWCNTs) modified screen-printed electrodes (SPEs) were used to immobilize antibodies for myoglobin determination in serum.⁵³ To do this monoclonal myoglobin antibodies were used, with BSA being used to maintain specificity. This platform was able to detect myoglobin within a range from 0.1 to 90 ng mL⁻¹ and with a small LOD of 0.08 ng mL⁻¹.

A myoglobin immunosensor was fabricated utilizing a myoglobin-specific peptide for detection in serum.⁵⁴ To do this, a gold electrode was activated using dithiobis(succinimidyl propionate) (DSP) which immobilized the myoglobin-specific peptide. Once immobilized, BSA was added to prevent nonspecific interactions from occurring. This produced a linear range of 17.8 to 1780 ng mL⁻¹ and a limit of detection of 9.8 ng mL⁻¹. This unique adoption of a myoglobin-specific peptide offers a rapid and sensitive method of myoglobin detection.

C-reactive protein

Chronic inflammation also plays an important role in the stages of the atherosclerotic process. Acute phase reactions are non-specific responses that occur in response to most forms of infection, inflammation, tissue damage, and neoplasia.⁵⁵ As a result, proteins in the form of inflammatory biomarkers are rapidly released and upregulated under the control of a cytokine cascade originating at the site of injury.^56,57

C-reactive protein (CRP) is a primitive biomarker of acute inflammation that is released in inflammatory conditions and is primarily synthesized in liver hepatocytes as a homo-pentameric protein, known as native CRP, and then breaks down irreversibly into the respective monomers at the corresponding sites of infection or inflammation.

CRP is of major interest for global cardiovascular risk and helps clinicians predict the risk of recurrent cardiac ischemia and death in patients with stable and unstable angina, in patients admitted to the hospital with acute coronary syndromes, and in patients undergoing percutaneous angioplasty.⁵⁸ Physiologically, CRP is thought to stimulate the production of tissue factors, which in turn stimulate coagulation and complement activation, both of which play a crucial role in cardiovascular disease.⁵⁹ Because CRP is an acute-phase reactant, it acts as an inflammatory marker with peak levels occurring within 50 hours of cardiac injury and then declining once the inflammatory stimulus has disappeared, with a half-life of approximately 19 hours.⁵⁶ The physiological range of CRP in the human population, without signs of acute illness, is 2 mg L⁻¹ and can increase to 300 mg L⁻¹ in various inflammatory diseases.⁵⁹ Increases in CRP levels of up to 500 times can be observed during inflammatory episodes.⁶⁰ Regular monitoring of CRP levels may be useful for the early detection of intermediate complications after a myocardial infarction. Being largely expressed in inflammatory responses, reliable and rapid CRP monitoring could reshape how CVDs and diseases are clinically treated. This has resulted in electrochemical CRP immunosensors being a large focus in biosensor research.

A CeO₂ nanorod-based biosensor was developed for the rapid detection of CRP.⁶¹ To functionalize the CeO₂ nanorods, they were made hydrophilic with an acetic acid solution followed by cystamine (Cys) and GA to allow the CRP antibodies to immobilize on the surface (Fig. 5A). This immunosensor demonstrated detection of CRP within a range of 0.3 to 7.0 μg mL⁻¹ and an LOD of 0.18 μg mL⁻¹. The electrodes used were low-cost, portable, and flexible allowing for increased accessibility as a point-of-care device.

Fig. 5 (A) Detection of CRP utilizing CeO₂ nanorods on screen-printed electrodes using differential pulse voltammetry technique⁶¹ (reprinted with permission from Elsevier); (B) H-FABP detection on modified glassy carbon electrode using labeled antibodies⁶² (reprinted with permission from Elsevier); (C) detection of CK-MB using anti-CK-MB antibody⁶³ (reprinted with permission from Elsevier).

A CRP immunosensor utilizing polypyrrole (PPy)–Au nanocomposite exhibited a wide range of detection from 0.0005 to 60 μg mL⁻¹ and a LOD of 0.17 ng mL⁻¹ in serum.⁶⁴ The PPy–Au was prepared as a black suspension from a reaction between 80% ethanol-dissolved Py and 0.1% chloroauric acid (HAuCl₄). This suspension was then deposited onto GCEs, covered in CRP-antibody, and blocked off with BSA to create a fully functional sensor.

Using an indole probe to link antibodies to AuNPs, a recently reported CRP immunosensor displayed a wide physiological range of 0.0001 to 100 μg mL⁻¹ and LOD of 0.03 ng mL⁻¹.⁶⁵ Functionalized through four general steps, AuNPs were deposited onto screen-printed carbon electrodes (SPCEs), followed by indoles, CRP antibody, and BSA. Further pushing the variety of distinct immunosensors, an Au wire-based sensor was developed for rapid detection of CRP.⁶⁶ The gold wire array was first functionalized with 3-mercaptopropionic acid (MPA) and then EDC/NHS linkage, allowing for CRP antibody to be immobilized and BSA applied. Once prepared, the sensor reported an incredibly low 5.0 to 220 fg mL⁻¹ range and an LOD of 2.25 fg mL⁻¹ in saliva and serum. Additionally, this sensor was specifically developed to be used for heart failure diagnostics.

H-FABP

Fatty acid binding proteins (FABPs) are a group of low molecular weight cytoplasmic proteins found in various organ systems and play a functional role in lipid metabolism as well as in energy homeostasis. FABPs are involved in cellular fatty acid metabolism as they reversibly bind to long-chain polyunsaturated fatty acids and transport them from the cell membranes to the mitochondria.⁶⁷ Heart-specific fatty acid binding protein (H-FABP) is an early and highly sensitive biomarker of myocardial ischemia and is usually measured together with troponin. H-FABP is one of the most abundant cytoplasmic proteins in cardiac muscle and plays a role in the uptake, transport, and metabolism of fatty acids.⁶⁸ Its main function is to transport intracellular long-chain fatty acids and protect cardiac muscle cells from these fatty acids, which accumulate in high concentrations during ischemic injury. FABP is expressed in various cells, including adipose tissue, macrophages, liver, intestine, central and peripheral nervous tissue, and skeletal and cardiac muscle tissue.⁶⁹ Concentrations peak during ischemic stroke and are released early into the cytosol during myocardial infarction and necrosis of cardiac muscle cells.⁷⁰ FABPs are released from injured cells into the plasma quickly, with a half-life of approximately 20 minutes after release into circulation. Physiological levels of H-FABP are in the single digits 0.0 to 5.5 ng mL⁻¹ range, which can be attributed to the physiological role itself, but can peak as early as 1 hour after onset of AMI symptoms, including chest pain, and return to normal limits after 12–24 hours. Although values of H-FABP are lower than those of other markers, such as myoglobin, it is very useful in AMI diagnosis as it is a direct insight into metabolism, which is controlled by the kidneys. While the distinction between healthy and infected individuals is not as drastic with H-FABP levels as it may be with myoglobin or troponin, its direct insight into metabolic processes can be vital in supplementing AMI diagnosis. While not as deeply explored as many of the other cardiac markers, H-FABP has still made some progress in the development of electrochemical immunosensors.

Recently, a polycrystalline gold electrode immunosensor was developed for H-FABP detection in serum.⁷¹ Upon the gold electrodes were CNTs enhanced with methylene blue (MB) assembled on a polymer film of polythionine (PTh). This initial functionalization continued with the addition of EDC/NHS, H-FABP antibody, as well as Gly to prevent nonspecific bindings. Its sensitive detection, enabled by the combination of MB and PTh, ranges from 3.0 to 25.0 ng mL⁻¹ with an LOD of 0.47 ng mL⁻¹. Feng et al. reported the utilization of Au nano dendrites/chitosan-grafted-ferrocene and thionine-absorbed AuPt nanocrystals/polydopamine/open-pored hollow carbon spheres as substrate and label material to generate multi-signal output biosensor for successful capturing of H-FABP as shown in Fig. 5B.⁶²

One H-FABP sensor utilized gold-interdigitated electrodes (GIDs), immobilizing antibodies with EDC/NHS linkers.⁷² Being able to detect H-FABP from 0.098 to 100 ng mL⁻¹ with an LOD of 0.836 ng mL⁻¹ in human serum, this sensor has the sensitivity and width for the physiological range. Another gold-electrode-based sensor was developed by treating the gold with 1-mercaptoundecanoic acid (11-MUA), and then immobilizing the antibody with EDC/NHS and blocking it with BSA. This reported a slightly slimmer range of 0.098 to 25 ng mL⁻¹ with a lower LOD of 0.236 pg mL⁻¹ in serum. A third sensor was fabricated in the same method as instead citing a wider range of 0.098 to 100 ng mL⁻¹ and a larger LOD of 117 pg mL⁻¹.⁷³

CK-MB

CK-MB, the cardiac-specific isoenzyme of creatine kinase (CK), is also used for the detection of myocardial infarction. CK is an enzyme that catalyzes the transfer of a phosphate group from creatine phosphate to adenosine diphosphate to generate adenosine triphosphate. Disruption of cell membranes due to hypoxia or cell injury results in the release of CK from the cytosol of the cell into the systemic circulation. CK has been used as a sensitive but non-specific test for myocardial infarction because CK is present in a variety of tissues other than the myocardium. CK is a dimeric molecule composed of monomeric subtypes M and B, resulting in different isoenzymes.⁷⁴ The three dimeric isoenzymes of CK, including CK-MB, CK-MM and CK-BB, are named after the tissue in which they were identified. The MB isoenzyme of creatine kinase is the biochemical marker currently used clinically in the evaluation of patients with suspected myocardial infarction.⁷⁵

CK-MB was the biomarker of choice for the diagnosis of AMI prior to the introduction of cardiac troponin determination. CK-MB first appears 3–5 hours after the onset of clinical symptoms, peaks at around 24 hours and returns to physiological levels within 48–72 hours. Physiological levels of CK-MB are below 7.8 ng mL⁻¹ in men and below 4.4 ng mL⁻¹ in women. The value of CK-MB in the late diagnosis of an AMI is limited, but due to its early release characteristics it is useful for the diagnosis of reinfarction when levels rise again after a decline following an AMI.⁷⁶ CK-MB levels normalize within 24–36 hours. Because troponin remains in the circulation longer than CK-MB, CK-MB is useful for detecting reinfarction because it has a shorter duration of rise at detectable levels in plasma. Although CK-MB has a high sensitivity for AMI diagnosis, its specificity is rather low due to release kinetics. CK is not a sufficient marker for the detection of myocardial damage that occurs in non-ST elevation MI.²⁷ Damage to the myocardial tissue leads to the release of CK-MB into the systemic circulation, which allows clinicians to conclude the extent of damage and possible reinfarction⁷⁵ and has drawn substantial interest as a target for electrochemical immunosensing. Glassy carbon electrodes (GCE) were configured with SU-8 nanofibers embedded with MWCNTs (MWCNTs-SU-8) through electrospinning.⁷⁷ The CK-MB monoclonal antibodies were then immobilized through EDC/NHS coupling and tested within an extensive range of 10 ng mL⁻¹ to 10 mg mL⁻¹ with its LOD at 1 ng mL⁻¹ in human serum. Another GCE-based serum immunosensor was developed with AuPdCu nanowire networks (NWNs).⁷⁸ CK-MB antibody was then deposited followed by BSA to maintain specificity. This platform demonstrated a range of 0.001 to 2000 ng mL⁻¹ and a detection limit of 0.88 pg mL⁻¹.

Gold SPEs were used in a serum CK-MB biosensor using immobilized creatine phosphate (Pcrea) in place of CK-MB antibodies.⁷⁹ To link the Pcrea to the gold surface, the Au-SPEs were first aminated with cysteamine (Cys), followed by EDAC/NHS coupling to bind Pcrea to the sensor. This unique technique showed a linear range of 0.19 to 28.8 μg mL⁻¹ and LOD of 0.11 μg mL⁻¹.

Notably, two platforms were fabricated for performance in urine and saliva instead of human serum. The first utilized graphite paper electrodes with gold nanoparticles deposited on their surface.⁶³ The CK-MB antibody was then linked to the gold nanostructure through the addition of 6-mercapto-1-hexanol (6-MH) and then (3-glycidyloxypropyl)-trimethoxysilane (3-GOPE) (Fig. 5C). This provided a linear range of 0.1 to 50 pg mL⁻¹ and LOD of 0.045 pg mL⁻¹. The second employed a gold electrode functionalized first with 11-MUA carboxyl groups and then poly(ethyleneimine) (PEI) and poly(vinyl sulfonic acid) (PVS).⁸⁰ This film was then further augmented with gold nanoparticles stabilized with cysteamine, and then linked to Pcrea through EDC/NHS coupling. This immunosensor presented a higher linear range of 5.0 to 100.0 ng mL⁻¹ and LOD of 0.209 ng mL⁻¹.

D-dimer

In the hemostatic process, there is a balance between the formation of fibrin clots and the breakdown of clots by the fibrinolytic system.⁸¹ D-dimer is formed when plasmin, an enzyme activated as part of the fibrinolytic pathway, breaks down fibrin to further dissolve blood clots. D-dimer is a soluble product of fibrin degradation that directly results from thrombin degradation by the fibrinolytic system. This fibrin fragment forms and circulates in the bloodstream for several days after a thrombotic event. The coagulation cascade triggers the production of thrombin, which then breaks down fibrinogen into fibrin, leading to the aggregation of fibrin monomers. Factor XIIIa causes cross-linking of the gamma chains at the C-terminal appendages by covalent binding and this dimerization of the D-domains of two fibrin monomer units. This cross-linked fibrin leads to the term dimeric D-domain or D-dimer.⁸²

The measurement of D-dimer in the bloodstream plays an important role in hypercoagulable states. Physiologic levels of D-dimer are less than 0.50 mg L⁻¹, with levels that rise above 0.5 mg considered a positive D-dimer test.⁸³ D-dimer has a half-life of approximately 4 to 6 hours and can remain elevated for approximately 7 days. Thrombotic events are common and should be followed closely as they are a major cause of morbidity and mortality. In venous pathologies such as deep vein thrombosis, the gold standard for invasive testing is costly, so non-invasive diagnostic strategies must be used.⁸² In recent decades, D-dimer has proven to be a highly sensitive marker in the initial evaluation of patients with suspected venous thromboembolism. D-dimer testing is potentially useful in the diagnosis and management of various other thrombosis-related clinical conditions, including disseminated intravascular coagulation, ischemic cardiopathy, stroke, myocardial infarction, and thrombolytic therapy.⁸⁴ D-dimer is a reliable and sensitive biomarker that can be used to assess fibrin deposition and stabilization, which is a critical factor in cardiovascular injury. Despite its limited specificity due to the occurrence of D-dimer in a variety of cardiovascular pathologies, D-dimer can be used as a valuable laboratory tool for the diagnosis and treatment of many thrombosis-related cardiovascular syndromes.

As a valuable biomarker for the assessment of patients with an assortment of diseases including thrombosis-related cardiovascular syndromes, many electrochemical immunosensors for D-dimer have been reported. Multiplex sensor was reported to detect D-dimer in human plasma samples along with other diagnostic and prognostic biomarkers of COVID-19.⁸⁵ Anti-D dimer antibody was immobilized onto ZnO deposited gold electrode using a DSP crosslinker, and Tween-20 was used to block potential sites of non-specific binding. Once functionalized, this sensor demonstrated a linear range of 0.05 to 10 ng mL⁻¹ and a LOD of down to 0.6 pg mL⁻¹. Another plasma-based platform of MWCNTs on an SPE was modified first with chitosan nanoparticles (CSNPs), then protein A (PrA), and finally D-dimer antibody and BSA.⁸⁶ With a rapid response time of 5 seconds, this achieved a functional range of 2.0–500.0 ng mL⁻¹ and LOD of 0.6 ng mL⁻¹.

A low-cost test was fabricated for D-dimer detection utilizing interdigitated microelectrodes (IDMEs) with a price estimated at $1 per test.⁸⁷ The sensor was configured first by depositing a layer of PPy followed by APTES and GA for antibody immobilization. Once immobilized, superblock was utilized to block the remaining binding sites. The linear range of the sensor was reported from 1 pg mL⁻¹ to 10 ng mL⁻¹ with the LOD being 1.0 pg mL⁻¹ in PBS.

A serum biosensor comprising of a bi-functional self-assembled monolayer (SAM) was developed constituted from two different thiols 16-mercaptohexadecanoic acid (16-MHDA) and 11-(ferrocenyl)-undecanethiol (11-F-UDT).⁸⁸ With a gold electrode foundation, the two thiol solutions allowed for both antibody attachment and signal transduction. Once the thiol monolayer is assembled, the antibody is immobilized utilizing EDC/NHS coupling and blocked with BSA. Demonstrating a shared linear range of 1–1000 ng mL⁻¹ in both PBS and serum and LODs of 0.01 and 1.4 ng mL⁻¹ respectively. Table 1 shows the list of electrochemical biosensors reported for the detection of cardiac biomarkers.

Table 1 List of electrochemical biosensors reported for the detection of cardiac biomarkers

Biomarker	Functionalization	Detection range	LOD	Buffer/biofluid	Reference
cTnT	CPE/NHS–EDC/cTnT–Ab	0.01–50 ng mL^−1	0.6 pg mL^−1	—	43
	GCE/COOH–Py/NHS–EDC/GA/cTnT–Ab/Gly	2.5–125 pg mL^−1	0.70 pg mL^−1	PBS	37
		1.0–10 pg mL^−1	0.35 pg mL^−1	Serum
	Au/ZnO/DSP/cTnT–Ab/SB	1 pg mL^−1 to 100 ng mL^−1	1 pg mL^−1	Serum	89
cTnI	CPE/NHS–EDC/cTnI–Ab	100–500 ng mL^−1	2.5 pg mL^−1	—	43
	GCE/AuPtPd–FND/cTnI–Ab/BSA	0.01–100.0 ng mL^−1	3 pg mL^−1	Serum	78
	ITO-GE/PDATT/NHS–EDC/cTnI–Ab/BSA	0.01–100.0 ng mL^−1	0.033 pg mL^−1	Blood plasma	39
	Au/Nf–DILHCNT/cTnI–Ab/BSA	0.05–30 ng mL^−1	0.02 ng mL^−1	—	90
	SPCE/GO/NHS–EDC/cTnI/BSA	0.001–250 ng mL	0.16 pg mL^−1	Serum	38
	SPCE/Cu3(BTC)2–PANI/NHS–EDC/cTnI–Ab/T-20	1–400 ng mL^−1	0.8 ng mL^−1	Mouse serum	91
	GO-SPE/ATA/cTnI/T-20	0.1–100 ng mL^−1	0.08 ng mL^−1	Serum	92
	ITO-GE/rGO/nMo3Se4/APTES/NHS–EDC/cTnI–Ab/BSA	1 fg mL^−1 to 100 ng mL^−1	1 fg mL^−1	Serum	93
Cardiac myoglobin	GCE/AuPtAg–PHNR/Mb–Ab/BSA	0.0001–1000 ng mL^−1	0.46 pg mL^−1	Serum	94
	ITO-GE/APTES/Au-NPs/Mb–Ab	0.02–1 μg mL^−1	5.5 ng mL^−1	PBS	51
	ITO-GE/APTES/Au-NPs/Mb–Ab	0.01–1 μg mL^−1	2.7 ng mL^−1	PBS	52
	SPE/MWCNT/Mb–Ab–IgG/BSA	0.1–90 ng mL^−1	0.08 ng mL^−1	Serum	53
	SPE/rGO/Au-NPs/Mb-Ab/BSA	1–1400 ng mL^−1	0.67 ng mL^−1	Serum	28
	GQD-SPE/NHS–EDC/Mb–Ab/BSA	0.01–100 ng mL^−1	0.01 ng mL^−1	Serum	28
	Au/DSP/Mb–Pep/BSA	17.8–1780 ng mL^−1	9.8 ng mL^−1	Serum	95
	ITO-GE/APTES/Pt(MPA)-NPs/Mb–Ab/BSA	0.01–1 μg mL^−1	1.70 ng mL^−1	PBS	96
	Br–Py–GCE/AuNP–PEI/glyoxal/Mb–Ab/Gly	9.96–72.8 ng mL^−1	6.29/ng mL	PBS	97
	ITO-GE/APTES/NHS–EDC/ZnS(MPA)/Mb–Ab/BSA	10–1000 ng mL^−1	—	PBS	98
CRP	CeO2–NRs/Cys–GA/CRP–Ab	0.3–7.0 μg mL^−1	0.18 μg mL^−1	PBS	61
	GCE/PPy–AuNPs/CRP-Ab/BSA	0.0005–60 μg mL^−1	0.17 ng mL^−1	Serum	64
	SPCE/AuNPs/indole/CRP–Ab/BSA	0.0001–100 μg mL^−1	0.03 ng mL^−1	Serum	65
	SPCE/PS/rGO–MNP–PDA–ENF/CRP–Ab/BSA	0.5–60 ng mL^−1	0.33 ng mL^−1	PBS	99
	SPCE/ErGO/NH2–VMSF/GA/CRP–Ab/BSA	10 pg mL^−1 to 100 ng mL^−1	8 pg mL^−1	Serum	100
	SPCE/GO/NHS–EDC/CRP–Ab/BSA	0.001–100 μg mL^−1	0.38 ng mL^−1	Serum	38
	Au/MPA/CRP–Ab/BSA	5.0–220 fg mL^−1	2.25 fg mL^−1	Serum/saliva	66
	SPCE/AuNPs/SAM/CRP–Ab/BSA	0.047–23.6 μg mL^−1	17.0 ng mL^−1	Serum	101
H-FABP	Au/PTh/MB–CNT/H-FABP–Ab/Gly	3.0–25.0 ng mL^−1	0.47 ng mL^−1	Serum	71
	Au/NHS–EDC/H-FABP–Ab	0.098–100 ng mL	0.836 ng mL^−1	Serum	72
	Au/NHS–EDC/11-MUA/H-FABP–Ab/BSA	0.098–25 ng mL^−1	0.236 pg mL^−1	Serum	102
CK-MB	GPE/AuNPs/6-MH/3-GOPE/CK-MB–Ab/BSA	0.1–50 pg mL^−1	0.045 pg mL^−1	Saliva/urine	63
	GCE/AuPdCu–NWNs/CK-MB–Ab/BSA	0.001–2000 ng mL^−1	0.88 pg mL^−1	Serum	78
	Au/PEI–PVS/Au-NPs–Cys/CP	5.0–100.0 ng mL^−1	0.209 ng mL^−1	Saliva/urine	80
	GCE/CK-MB/CP/GA–Cys	0.1–2000 ng mL^−1	0.04 ng mL^−1	Serum	21
	GCE–MWCNTs-SU-8/NHS–EDC/CK-MB–Ab	10–10 mg mL^−1	1 ng mL^−1	—	77
	Au-SPE/Cys/NHS–EDAC–CP	0.19–28.8 μg mL^−1	0.11 μg mL^−1	Serum	79
	PANI-NW/NHS–EDC/CK-MB–Ab/BSA	50 fg mL^−1 to 5 pg mL^−1	150 fg mL^−1	PBS	103
	Au/NHS–EDC/ferrocene–CK-MB–Ab/BSA	0.001–50 ng mL	0.5 pg mL^−1	Krebs–Henseleit solution	104
	Au-SPR/ThA–SAMs/NHS–EDC/CK-MB–Ab	0.00–300 ng mL^−1	13 ng mL^−1	Serum	105
D-dimer	Au/ZnO/DSP/D-dimer–Ab/T-20	0.05–10 ng mL^−1	0.6 pg mL^−1	Plasma	85
	SPE/MWCNTs/CSNPs/PA/D-dimer–Ab/BSA	2.0–500.0 ng mL^−1	0.6 ng mL^−1	Plasma	86
	IDME/PPy/APTES/GA/D-dimer–Ab/T-20	1–10 000 pg mL^−1	1.0 pg mL^−1	PBS	106
	Pt/AuNPChi/D-dimer–Ab	—	0.9 ng mL^−1	—	107
	Au/16-MHDA–11-F-UDT/NHS-EDC/D-dimer–Ab/BSA	1–1000 ng mL^−1	0.01 ng mL^−1	PBS	88
		1–1000 ng mL^−1	1.4 ng mL^−1	Serum
	Cr–Au–GE/ZnO-NR/lipid-film/D-dimer–Ab	100 pg mL^−1 to 1 mg mL^−1	0.1 ng mL^−1	—	108
	Cu–GE/lipid-layer/D-dimer–Ab	1–1000 pg mL^−1	0.3 pg mL^−1	Serum	109
	Au/MWCNTs/H2SO4–HNO3/NHS–EDC/NTA/Cu^2+/D-dimer–Ab	100 pg mL^−1 to 10 mg mL^−1	100 pg mL	Plasma	110

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Point of care devices

Usage of these electrochemical sensors described above with the capability to detect various proteins has been shown to find certain biomarkers associated with cardiovascular diseases (CVD), such as troponin I, procalcitonin, and C-reactive protein.³⁸ These devices have a multitude of advantages, including both cost efficiency and time efficiency. From a point of care perspective, these sensors are useful due to their “easy portability, simple operation, and reliable performance”. In vitro diagnosis methods like electrochemical sensors are increasing in prominence because of “their convenience and rapidity”.¹¹¹ They offer “point-of-care testing, continuous monitoring, miniaturization, high specificity and sensitivity, rapid response time, ease of use, and low costs. These kinds of devices have already been used to detect tumor markers such as circulating tumor cells, alpha-fetoprotein, and prostate-specific antigens. They specialize in detecting prostate cancer and hepatocellular carcinoma. Continuous monitoring is possible using these devices and has proven their ability to track and monitor metabolic biomarkers such as glucose.¹¹² These sensors can have become increasingly sensitive and detect very low concentrations of CVD biomarkers like cardiac troponin I,¹¹³ as well as cardiac myoglobin.²⁸ They also have the potential to detect biomarkers for heart failure such as BNP and NT-proBNP.¹¹⁴

Electrochemical sensor usage has been further expanded through the usage of attachable sensors that give it bedside monitoring capabilities. Using comfortable and flexible materials with the sensors allows them to be worn for long stretches of time while still being effective. There is a certain level of comfort and high fit that are needed to reduce patient discomfort and risk the accuracy of detecting the disease biomarkers. These flexible devices have proven useful in detecting biomarkers for diseases like Zika, AIDs, Ebola, and COVID-19.¹¹⁵ Devices with these capabilities, especially when in a smaller form, are ideal for biomarker detection at the bedside of a patient in part due to their cost effectiveness, ease of production, and ability to utilize small volumes.¹¹⁶ These point-of-care electrochemical sensors, such as impedance-based sensors, are sensitive enough to match common immunoassays but also pragmatic for bedside monitoring.¹¹⁴ These types of rapid analysis sensors have been reported for bedside monitoring of COVID-19 biomarkers.¹¹⁷

The point-of-care biosensor offers a significant advantage in cardiac biomarker measurement, reducing the turnaround time from approximately 2 hours for centralized laboratory tests to just 10–14 minutes for point-of-care tests.¹¹⁸ The quick diagnosis allows for more timely and structured decision-making. This benefits patients by reducing their length of stay and increasing the likelihood of successful treatment and discharge to their homes. However, these sensors have limitations. They often have narrow linear analyte ranges, inadequate detection limits, and limited selectivity for detection purposes. One more challenge is the size of the equipment, as benchtop tools are not easily used as portable. Despite these challenges, point-of-care biosensors, especially electrochemical biosensors, offer numerous advantages, including simple sample preparation, affordability, miniaturization of complex equipment, and a rapid response time.¹¹⁹

The table (Table 2) above compares cardiac biomarker detection devices from different companies, including the type of device and the biomarkers tested. This information is essential for timely interventions and improved access to care.¹⁴⁵ Many of these are multiplex devices, that allow simultaneous detection of multiple biomarkers at once. The combination of multiple biomarkers provides an understanding of cardiovascular pathology related to different cardiovascular disorders based on the levels of multiple markers.¹⁴⁶ Despite this, point-of-care (POC) devices often lag behind benchtop or lab-system counterparts in terms of sensitivity and the variety of markers they can detect. This is evident in the use of high-sensitivity (hs) testing for specific biomarkers, which is crucial for detecting and monitoring cardiovascular diseases.¹⁴⁷ Utilized by non-POC devices, hs detection allows for enhanced detection range, particularly of lower concentrations of certain markers compared to standard assays. This increased sensitivity can detect cardiac disease earlier, leading to the prospect of earlier intervention.¹⁴⁸ Advancements in the sensitivity of electrochemical immunosensors are crucial for improving point-of-care (POC) treatment of cardiovascular diseases (CVDs). In addition to high-sensitivity detection of markers of interest, a wider variety of biomarkers enhances the specificity of CVD diagnosis, addressing the issue of poor or delayed diagnosis rates.¹⁴⁹ The simultaneous detection of underutilized markers such as H-FABP, non-specific markers like CRP, and the current standard cardiovascular markers such as cTnI and CK-MB will enhance diagnostic capabilities and differentiation of CVDs with similar manifestations.¹⁵⁰

Table 2 List of cardiac biomarker detection devices available and the type and number of biomarkers that could be tested using them

Product name	Company	Type	No. of biomarkers	POC	Biomarkers										Ref.
					hscTnT	cTnT	hscTnI	cTnI	Cardiac myoglobin	hsCRP	CRP	H-FABP	CK-MB	D-dimer
Cobas h 232	Roche	Handheld	4	✓		✓			✓				✓	✓	120
i-STAT	Abbott	Handheld	2	✓				✓					✓		121
Minicare I-20	Phillips	Handheld	1	✓				✓							122
PATHFAST	Polymedco	Benchtop	6	✓			✓	✓	✓	✓			✓	✓	123
AFIAS	Boditech	Benchtop	6			✓		✓	✓	✓			✓	✓	124
Ichroma II	Boditech	Benchtop	6	✓		✓		✓	✓	✓			✓	✓	125
Mini VIDAS	BioMerieux	Benchtop	4				✓		✓				✓	✓	126
DZ-Lite c270	Diazyme	Benchtop	3						✓	✓				✓	127
RAMP	Response Biomedical	Benchtop	4					✓	✓				✓	✓	128
AQT90 Flex	Radiometer	Benchtop	5	✓				✓	✓		✓		✓	✓	130
QuickSens Ω100	8sens.biognostic	Benchtop	4					✓		✓	✓	✓			129
Triage MeterPro	Quidel	Benchtop	4	✓				✓	✓				✓	✓	131
SMART/CUBE	EuroLyser	Benchtop	4					✓		✓	✓			✓	132
Fluoro-Checker TRF	Nano-Ditech	Benchtop	3					✓			✓			✓	133
Dimension Vista	Siemens	Lab-system	6				✓	✓	✓	✓	✓		✓		134
ARCHITECT	Abbott	Lab-system	5				✓	✓	✓		✓		✓		135
Stratus CS Acute Care	Siemens	Lab-system	5	✓				✓	✓	✓			✓	✓	136
ADVIA Centaur	Siemens	Lab-system	3				✓		✓				✓		137
RX Series	Randox	Lab-system	3							✓	✓		✓		138
Access 2	Beckman Coulter	Lab-system	3				✓		✓				✓		139
Cobas e analyzer	Roche	Lab-system	4		✓			✓	✓				✓		140
RapiCard InstaTest	Cortez Diagnostics	Readerless	5	✓				✓	✓		✓		✓	✓	141
Nano-Check	Nano-Ditech	Readerless	5	✓				✓	✓		✓		✓	✓	142
LifeSign	Princeton BioMeditech	Readerless	3	✓				✓	✓				✓		143
Roche CARDIAC	Roche	Readerless	1	✓		✓									144

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Key challenges and future perspectives

The field of cardiovascular disease detection and monitoring has been revolutionized by the rapid advancement of technology, particularly through electrochemical immunosensors. These devices offer fast, reliable, and cost-effective detection of cardiac biomarkers, such as H-FABP, which previously lacked sufficient detection methods. It is crucial to detect these biomarkers at lower concentrations with high reliability for early diagnosis and intervention. Multiplex systems further enhance diagnostic capabilities by allowing simultaneous tracking of multiple biomarkers, which is essential for accurately assessing disease status and progression. In emergency settings, this capability facilitates prompt initiation of appropriate treatments based on a comprehensive understanding of cardiovascular pathology. Continuous monitoring of cardiac and metabolic biomarkers post-surgery or therapy is vital for evaluating patient well-being and recovery progress. While diagnostic biomarkers are valuable for initial disease detection, monitoring biomarkers play a pivotal role in ongoing patient management, providing clinicians with real-time data to assess treatment efficacy and adjust therapeutic strategies as necessary. The convergence of medicine and technology in electrochemical immunosensors represents a critical advancement, promising significant improvements in cardiovascular disease management. As both fields continue to evolve synergistically, the potential to comprehend, detect, and monitor heart disease will continue to expand, ultimately leading to improved patient outcomes and saving more lives without a doubt.

Addressing these challenges will be crucial for realizing the full potential of electrochemical immunosensors in transforming cardiovascular disease management, enhancing patient care, and saving lives. The field of electrochemical immunosensors for cardiac biomarkers is advancing rapidly, yet significant challenges remain. Standardization and validation are crucial to ensure consistency and reliability across different sensor platforms and assay techniques in clinical settings. Achieving high sensitivity to detect biomarkers at pico-level concentrations without compromising specificity is a major technical hurdle, as is effectively integrating multiple biomarkers (multiplexing) into a single platform while managing cross-reactivity and interpreting complex data outputs. Ensuring biocompatibility, stability over time, and suitability for diverse clinical environments are essential for practical implementation. Cost-effective development and global accessibility of these sensors, particularly in resource-limited settings, present barriers to widespread adoption. Obtaining regulatory approval through rigorous testing and validation processes adds complexity and time to the pathway to clinical use. Additionally, seamless integration of sensor data into existing healthcare systems and workflows is necessary for effective clinical implementation and utilization. Addressing these challenges through collaborative efforts will significantly enhance diagnostic and monitoring capabilities for cardiovascular diseases, ultimately improving patient outcomes and healthcare delivery worldwide.

Data availability

No primary research results have been included and no new data were generated or analyzed as part of this review.

Conflicts of interest

There are no conflicts to declare.

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