Recent approaches in the sensing strategy for the detection of cardiovascular disease biomarkers

Abstract

Cardiovascular diseases (CVDs) remain among the leading non-communicable diseases worldwide, accounting for nearly half of the mortalities annually in both the developing and developed countries. Early diagnosis and timely intervention are therefore critical for effective disease management. In clinical practice, the diagnosis of most cardiac conditions relies on the evaluation of circulating cardiac biomarkers, which offer direct insight into myocardial injury and cardiac function. Beyond traditional gold-standard biomarkers such as cardiac troponins (cTn), B-type natriuretic peptide (BNP), and myoglobin, ongoing research aims to identify novel biomarkers with diagnostic and prognostic value. This review overviews cardiac troponin (cTn), the gold-standard CVD biomarker, and discusses emerging candidates including interleukine-6 (IL-6), the soluble isoform of suppression of tumorigenicity 2 (sST2) and microRNAs. Particular attention is given to their potential integration into point-of-care (PoC) biosensor platforms for real-time monitoring of cardiovascular conditions. The development of rapid, sensitive, specific, precise, and cost-efficient biosensing platforms for cardiac biomarker detection is essential to address the alarmingly high CVD burden. Here, we summarize recent biosensing approaches for the detection of several biomarkers reported in the literature, including the biosensors construction, the materials used for the sensor surface, the detection method, such as electrochemical, chemiluminescent, colorimetric, or fluorescent, and signal amplification techniques. Finally, we review recently reported and commercially available multiplexed PoC systems for cardiac biomarker analysis, highlighting current trends and future directions in CVD diagnostics. The graphical abstract shows the main CVD-biomarker biosensing approaches.

A detailed review on sensing strategies for the recognition of emerging and standard biomarkers for the early diagnosis and prognosis of cardiovascular diseases, including their potential integration into point-of-care biosensor platforms.

Introduction

Cardiovascular diseases (CVDs) are the leading cause of illness and death in developed and underdeveloped countries. The number of deaths attributed to CVDs globally was 20.5 million in 2021.¹ In 2026, according to the American Heart Association update, CVDs affect 48.9% of US adults, with a prevalence of 5.2% for coronary heart disease, 3.9% for Stroke, 4.48% for atrial fibrillation, while Heart failure (HF) has risen to 7.7 million.² CVDs are among the most prevalent and costly health problems worldwide. Between 2025 and 2050, the prevalence of cardiovascular disease and crude mortality is expected to increase by 90% and 73.4%, respectively.³

Among different CVDs types are heart failure, atrial fibrillation, cardiomyopathy, coronary artery disease, stroke and arrhythmia (Fig. 1).

Fig. 1. Cardiovascular disease types.

Although statins are the primary drugs for preventing cardiac events, patient responses vary, with some not reaching therapeutic targets and others experiencing side effects. Regular monitoring of risk factors like hypertension, dyslipidemia, and diabetes is recommended by the Ministry of Health, especially for women over 40 and those in peri- and post-menopausal periods. The National Heart, Lung, and Blood Institute has launched the SABER CVD initiative, a proteomics platform aiming to identify biomarker signatures for atherosclerotic CVD and its risk factors. This analysis links circulating protein biomarkers to various cardiovascular conditions, reflecting biological pathways such as inflammation, oxidative stress, adiposity, metabolism, fibrosis, and tissue remodeling.⁴ A biomarker can be defined as a “molecular characteristic that is quantifiable and measurable to serve as an indicator of either the normal body function, pathogenic reaction or a pharmacological response to a therapeutic intervention”.⁵ Over the last decade, numerous studies have focused on identifying biomarkers relevant to cardiovascular disease (CVD) risk management. An ideal CVD biomarker (CVD-B) should provide predictive, diagnostic, and prognostic value. Biomarkers with high sensitivity and specificity to cardiac conditions can lead to earlier diagnosis and more effective therapeutic outcomes. In addition, it should be biologically pertinent to cardiovascular pathology and be objectively measurable and clinically validated in various populations. Importantly, it should be easily applicable in routine clinical practice to enhance heart disease prevention and management.^6,7 Advances in Molecular Biology and lab technologies have improved CVD-B screening through techniques like immunoassays (ELISA, CLIA), electrophoresis, chromatography (HPLC), genetic tests, and molecular imaging (PET, SPECT, MRI).

A recently published paper by Teertam and coworkers,⁸ reported an exhaustive review on cardiovascular biomarkers critical for the diagnosis, prognosis, and pathophysiology of cardiovascular diseases.

Soluble ST2 (sST2), lipoprotein-associated phospholipase A2 (Lp-PLA2), myeloperoxidase (MPO), and procalcitonin (PCT) have recently emerged as promising markers in the field of both acute and chronic cardiovascular diseases as reported by Huang and coworkers.⁹

Among the biomarkers commonly used in differential diagnosis and treatment monitoring are Creatine Kinase MB (CK-MB), an enzyme indicative of the extent and progression of myocardial damage,¹⁰ and B-type natriuretic peptide (BNP), which is widely used to diagnose heart failure, evaluate disease severity, and track treatment response and clinical progression.¹¹ Cardiac troponins (cTn) remain central to clinical practice, serving as the primary biomarkers for confirming or excluding acute myocardial infarction and for monitoring recovery following the ischemic event.¹² Beyond the well-established and standardized cardiovascular biomarkers, this review will describe novel candidates with strong potential for integration into point-of-care testing platforms for the management of cardiovascular diseases. The biomarkers analyzed are as follows: cardiac troponin, interleukin-6, soluble isoform of suppression of tumorigenicity-2, and microRNA.

Cardiac troponin (cTn)

Cardiac troponins (cTn) are firmly established as the gold standard biomarkers for the diagnosis of acute myocardial infarction (AMI). Troponins are regulatory proteins that form a complex regulating the calcium-dependent interaction between myosin and actin, thereby facilitating muscle contraction and relaxation. This complex consists of three different subunits: Troponin C (TnC), Troponin T (TnT), and Troponin I (TnI), all of which are expressed in both cardiac and skeletal muscle cells.¹³ However, because cardiac troponins are encoded by different genes, TnT and TnI exhibit higher myocardial specificity. Under normal health conditions, circulating troponin levels are negligible. During myocardial ischemia, a combination of oxidative stress, inflammatory responses, catecholamine release, and cardiomyocyte necrosis leads to the release of cTnI and cTnT into the bloodstream. Consequently, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) are routinely used for AMI diagnosis, whereas cTnC lacks sufficient cardiac specificity and is not clinically employed for this purpose.¹⁴

Nevertheless, elevated cTnT levels are not exclusively associated with AMI, as increases may also occur in the context of skeletal muscle injury, potentially leading to diagnostic uncertainty and delays.^15,16 In contrast, cardiac cTnI is considered the most cardiac-specific troponin, and its elevation is strongly indicative of myocardial infarction (MI).¹⁷ According to the Universal Definition of MI, diagnosis of myocardial infarction (MI) is defined by an increase in cTn levels, with at least one value above the 99th percentile upper reference limit (URL) of a healthy population.¹³ However, elevated cTn levels alone cannot reliably distinguish MI from other causes of myocardial injury,¹³ and may not be very effective for rapid diagnosis in the very first hours after symptom onset.¹⁸ Importantly, biological variables such as age, sex, and ethnicity significantly influence circulating cTn levels. Women generally exhibit lower baseline cTn concentrations than men, a factor that may contribute to the underdiagnosis of AMI in female patients. For this reason, the adoption of sex-specific cTn reference thresholds is essential to improve diagnostic accuracy in women and to reduce persistent gender disparities in cardiovascular care.¹⁹

Interleukin 6 (IL-6)

CVDs development is closely driven by a strong inflammatory response involving the recruitment of many types of immune cells, such as macrophages, neutrophils, and lymphocytes, which secrete pro- and anti-inflammatory cytokines. Among these mediators, interleukin-6 (IL-6) plays a central and multifaceted role.²⁰ IL-6 is a key factor involved in the development of plaque destabilization, contributing to the progression of atherosclerotic cardiovascular disease (ASCVD). Chronic elevated IL-6 levels are strongly associated with the high risk of myocardial infarction, stroke, peripheral artery disease, and heart failure.^21–23 In a clinical trial, elevated plasma IL-6 measured 2 days after an ST-elevation myocardial infarction (STEMI) has been associated with larger infarct size, more severe reperfusion injury, and adverse ventricular remodelling, suggesting a role in early risk stratification beyond infarct sizing based on troponin alone.²¹ In ischemic stroke, elevated IL-6 has been implicated as a marker of disease severity and functional outcome.²² It has been demonstrated that elevated serum IL-6 levels following acute ischemic stroke have been associated with greater functional impairment and poorer neurological outcomes.²⁴ Furthermore, systematic evidence in peripheral artery disease similarly identifies IL-6 among the interleukins with the strongest support as a diagnostic and prognostic biomarker.²³

Recent studies have suggested that monitoring IL-6 levels can help predict the risk of future CVDs, even in individuals without previously diagnosed CVD.²⁵ In the Multi-Ethnic Study of Atherosclerosis (MESA), higher circulating IL-6 in initially healthy adults was independently associated with incident cardiovascular events over long-term follow-up, underscoring its potential value for primary-prevention risk assessment.²⁵ Due to its prominent inflammatory activity, IL-6 serves as an indicator of many pathological conditions.²⁶ In the cardiovascular system, IL-6 influences disease progression by regulating cardiomyocyte hypertrophy and apoptosis.²⁷

Studies have demonstrated that cardiac IL-6 levels are markedly increased in patients with end-stage heart failure, supporting its prognostic value in severe cardiac dysfunction.²⁸ Notably, increased circulating IL-6 levels have also been observed in individuals with asymptomatic left ventricular systolic dysfunction, indicating that IL-6 elevation may occur before the clinical onset of heart failure.²⁸ An increase in IL-6 levels over prolonged periods is also associated with atherosclerosis, further exacerbating cardiac conditions.²⁹ Beyond IL-6 itself, its receptor system has also been reported to be dysregulated in coronary artery disease and acute myocardial infarction, further supporting a mechanistic link between IL-6 signalling and ischemic injury.³⁰ Collectively, these findings indicate that IL-6 testing may provide valuable support for the timely evaluation and clinical management of heart failure and other cardiovascular conditions. However, IL-6 has not yet been incorporated in clinical practice guidelines for cardiovascular disease, and no universally validated cut-off values currently exist for cardiovascular risk stratification.³¹

IL-6 should therefore be regarded as a promising but still investigational biomarker whose diagnostic and prognostic utility remains context-dependent and requires further validation in large prospective cohorts before its routine clinical implementation.³²

Resolvins

Resolvins belong to the family of specialized pro-resolving mediators (SPMs), known as “guardians” of tissue homeostasis, and actively promote the resolution of inflammation and the restoration of tissue homeostasis. Interest in their role as cardiovascular biomarkers is comparatively recent, and the available clinical evidence is still limited to a small number of observational studies.^33,34 Resolvins are lipid mediators derived from ω-3 fatty acids. SPMs derived from EPA include the E-series resolvins, such as resolvins E1 (RvE1) and RvE2, whilst DHA-derived resolvins include the D-series resolvins (RvD1, RvD2, RvD3, RvD4 and RvD5, as well as protectins and maresins).³³ Decreased levels of RvD1 as compared to healthy controls are shown to persist in patients with aneurysmal subarachnoid hemorrhage (aSAH). In this prospective cohort, the reduction in RvD1 was also independently associated with greater bleeding severity and a worse neurological outcome, supporting its potential prognostic value in the acute cerebrovascular setting.³⁵ Another study reports that RvD1 levels were lower in ST-segment myocardial infarction patients as compared to the control group. This reduction has also been associated with greater myocardial damage and a less positive hospital outcome, suggesting a possible role for RvD1 in both the diagnostic and short-term prognostic assessment of ST-segment elevation myocardial infarction.³⁶ Similarly, another study has reported that lower and higher levels of RvD2 are associated with a reduced risk of atherosclerotic cardiovascular disease (ASCVD), implying a U-shaped relationship. However, the results are not concrete, and future research is recommended on this subject.³⁷ Overall, the evidence supporting resolvins as cardiovascular biomarkers remains considerably more limited in the number of available studies and in cohort size. Resolvins should therefore currently be regarded as an early-stage, investigational biomarker family requiring confirmation in larger, adequately powered prospective studies before any clinical utility can be established.

Soluble isoform of suppression of tumorigenicity 2 (sST2)

ST2 is a molecule that belongs to the interleukin-1 receptor family and exists in two isoforms: the transmembrane form, known as ST2 ligand (ST2L), and the circulating soluble form, referred to as soluble isoform of suppression of tumorigenicity 2 (sST2).³⁸ Under physiological conditions, the receptor ST2L binds the cytokine interleukin-33 (IL-33), triggering intracellular signaling pathways that regulate inflammation, fibrosis, and ventricular remodeling.

The binding between ST2L and IL-33 has been shown to exert a cardioprotective effect on cardiomyocytes by blocking progressive hypertrophy and fibrosis. In vivo studies have demonstrated that genetic deletion of ST2 leads to increased cardiac fibrosis in animal models, highlighting its protective role in cardiac homeostasis.³⁹ On the contrary, when ST2 is released into the circulation as sST2, it prevents the beneficial IL-33/ST2L interaction, working as a decoy receptor. Elevated sST2 levels, therefore, reflect reduced IL-33-mediated cardioprotective signaling. Clinically, increased circulating sST2 concentrations have been associated with adverse cardiovascular outcomes. Elevated sST2 levels have been linked to a higher risk of severe events, including recurrent myocardial infarction and stroke, in patients with acute MI.⁴⁰ In particular, it offers additional prognostic value compared with high-sensitivity troponin T and is a strong predictor of outcome in heart failure (HF).⁴¹ Moreover, increased sST2 has also been reported in individuals with type 2 diabetes, suggesting its potential role as an early indicator of coronary artery disease and future cardiovascular complications.⁴² Collectively, these findings support sST2 as a promising biomarker for risk stratification and disease progression in cardiovascular disorders. Unlike cardiac troponins, however, sST2 has not yet been validated as a diagnostic marker, and its role remains confined to adjunctive risk stratification and prognostic assessment in chronic cardiovascular and metabolic conditions. sST2 should therefore be considered a promising but still investigational prognostic biomarker, whose broader clinical adoption will depend on further standardization of assays and on validation of disease-specific cut-off values in large, prospective cohorts.^43,44

Micro RNA

MicroRNAs (miRNAs) are small non-coding RNAs (19–25 nucleotides) that regulate gene expression post-transcriptionally and play a critical role in both physiological and pathological processes. Changes in miRNA expression are also linked to the development of cardiovascular conditions, including coronary artery disease (CAD), atherosclerosis, acute myocardial infarction (AMI), heart failure (HF), and cardiac remodeling. In recent years, numerous studies have identified specific miRNAs associated with different stages and phenotypes of cardiovascular disease, highlighting their potential for diagnosis and prognosis. For example, an increase in cardiac miRNAs, such as miR-208a, miR-499, miR-1 and miR-133a, has been reported in patients with acute myocardial infarction, suggesting their potential as candidate biomarkers of myocardial injury.⁴⁵ Beyond acute myocardial infarction, altered expression of specific miRNAs has also been described in heart failure and in the progression of atherosclerotic and cardiometabolic disease, reflecting their broader involvement in processes such as cardiac remodeling, endothelial dysfunction, and lipid metabolism. Over the past decade, this has prompted growing interest in circulating miRNA signatures as next-generation, minimally invasive diagnostic tools for a range of cardiometabolic disorders. The high stability of miRNAs in biological fluids, their disease-specific expression and their rapid response to pathological stress have made them promising biomarkers.^45,46

However, a key feature of their mechanism of action is that a single miRNA can simultaneously regulate multiple target genes.⁴⁷ This complex capacity for pleiotropic regulation highlights the need for further research to fully elucidate miRNA-mediated regulation of cardiovascular phenotypes and associated pathological conditions. Despite this promise, several challenges currently limit the clinical translation of miRNAs as cardiovascular biomarkers, including the lack of standardized pre-analytical and analytical protocols, variability in normalization strategies across studies, and the absence of validated, disease-specific cut-off values.⁴⁸ Nevertheless, a recent meta-analysis suggests that circulating miR-21, when combined with high-sensitivity cardiac troponin, may offer incremental diagnostic accuracy for acute coronary syndrome compared with either marker alone, although the authors themselves note that this evidence remains limited by substantial heterogeneity across studies and requires confirmation before clinical application.⁴⁹ Unlike cardiac troponins, therefore, miRNAs are not yet part of clinical practice and remain an investigational class of biomarkers whose diagnostic and prognostic value will need to be confirmed through large, standardized, prospective studies before routine clinical implementation can be considered. Tables 1 and 2 provide an overview of the classification of biomarkers; the first table is sectioned into proteins, enzymes, and peptides and the second relates only to miRNAs.

Table 1. Basic summary of CVD biomarkers and their concentration range divided into proteins, enzymes and peptides. Legend: CHD: coronary heart disease, AMI: acute myocardial infraction, HF: heart failure, AVD: atherosclerotic vascular disease, CVD: cardiac valvular dysfunction, 1 pmol L⁻¹ = 0.15 ng ml⁻¹.

Biomarker	Name	Disease	Mean concentration range	Unit	References
Proteins	sST2	HF and acute coronary syndrome	10–33	ng ml−1	50
	cTn I	CHD	1.5–2.3	ng L−1	51
	hs-cTn	AMI	3.0–14.0	ng L−1	52
	H-FABP	Acute myocardial ischemia	0.1–25	ng ml−1	53
	HsCRP	AVD	1.0–3.0	mg L−1	54
	GDF-15	Coronary heart disease and HF	1.2–1.8	µg L−1	55
	Fibrinogen	Coronary artery disease	1.3–7.0	g L−1	56
	PAPP-A	AVD and acute coronary sindrome	10.0–64.26	ng mL−1	57
	CD40/CD40L	AVD	5.0–15.0	ng mL−1	58
	Copeptin	AMI, HF and stroke	51.2–122.0	pmol L−1	59
	IL-1/IL-6	Acute and chronic HF	10.0–50.0/3.0–10.0	pg mL−1	60
	Gal-3	HF and stroke	10.2–16.0	ng mL−1	61
	TNF-α	Chronical HF	7.8–500.0	pg mL−1	62
	PTX-3	Angina pectoris, AMI, HF, AVD	2.0–10.73	ng mL−1	63
	Procalcitonin	Chronical HF	0.85–2.0	ng mL−1	64
	Myoglobin	AMI	72.0–100.0	ng mL−1	65
	IMA	Myocardial ischemia	40.0–85.0	U mL−1	66
	NGAL	Myocardial ischemia and chronic HF	100.0–300.0	ng mL−1	67
	LDLC	AVD	130.0–190.0	mg dL−1	68
Enzymes	MPO	CHD (plaque instability), HF and AMI	15.0–101.0	ng mL−1	69
	MMP-9/MMP-2	AMI (plaque rupture) and CHD	90.0–200.0/80.0–100.0	ng mL−1	70 and 71
	Lp-PLA2/sPLA2	AVD, CHD and stroke	200.0–235.0/1.5–2.5	ng mL−1	72 and 73
	GPBB	Earlier myocardial ischemia and AMI	7.0–10.0	ng mL−1	74
	LDH	AMI	200.0–400.0	U L−1	75
	CK-MB	AMI	5.0–10.0	ng mL−1	76
Peptides	UA (purine)	HF	6.0–7.0	mg dL−1	77
	MR-proANP	Acute and chronic HF	121.5–165.0	ng mL−1	78
	BNP	CVD, hypertension, CHD, AMI, arrhythmias	0.12–0.4	ng mL−1	79
	ET-1	CHD, AMI, HF	1.5–3.5	pg mL−1	80
	NT-proBNP	HF	0.3–1.8	ng mL−1	81
	MR-proADM	Acute HF, hypertension, AMI, acute stroke	75.0–150.0	ng mL−1	82
	Resolvin D	Atherosclerosis and AMI	2.19–9.43	ng ml−1	83

Table 2. Summary of miRNA associated with CVD divided into 3 groups: downregulated, upregulated and independent of concentration but present in specific disease.

	Name		References
Down regulated	miR-1	HF, arrhythmia	84
	miR-30	HF, cardiac fibrosis	85
	miR-133, miR-150	HF, cardiac hypertrophy	86 and 87
Up regulated	miR-23, miR-195	Cardiac hypertrophy	88 and 89
	miR-21, miR-199	HF, cardiac fibrosis	90 and 91
	miR-214, miR-125	HF	92 and 93
Specific disease	miR-17-5p, miR-28, miR-106a	Dilated cardiomyopathy	94–96
	let-7e, miR-10a, miR-23b, miR-26b, miR-27b, miR-93, miR-126, miR-145, miR-181a, miR-374, miR-451, miR-499	Aortic stenosis	97

Strategy for using a biomarker pattern

Despite cardiac troponin remains the reference standard for the acute diagnosis of myocardial injury, reflecting cardiomyocyte necrosis with high specificity and well-validated diagnostic thresholds, combining these biomarkers discussed in this review leverages their complementary roles in inflammation, myocardial injury, resolution of inflammation, cardiac remodeling, and gene regulation.⁹⁸ In particular, IL-6 and sST2 reflect the inflammatory and fibrotic/remodelling processes that underlie disease progression, providing insights into prognosis and risk stratification rather than acute diagnosis; resolvins provide additional information on the resolution phase of inflammation; and miRNAs offer a further level of post-transcriptional regulatory information relevant to cardiac remodelling and gene expression. This multi-marker approach may offer enhanced sensitivity and specificity compared to single biomarkers alone, enabling earlier and more accurate detection of cardiovascular conditions.⁹⁹ However, this combined biomarker panel has not yet been clinically validated and remains investigational. Compared to the traditional methods, this pattern-based strategy could provide richer biological insights, improve risk stratification, prognosis, and treatment monitoring, heralding a promising shift toward personalized and precision cardiovascular care.¹⁰⁰

Recent advancements in the biosensing approaches for cardiac troponins (cTn)

Cardiac troponins are effective biomarkers for monitoring cardiovascular disease. Many factors such as high specificity and sensitivity, early detection and widespread clinical acceptance, make it the most reliable cardiac biomarker.¹⁰¹ As recommended by guidelines from the European Society of Cardiology (ESC), American College of Cardiology Foundation (ACCF), American Heart Association (AHA), and World Heart Federation (WHF), high-sensitivity tests enable precise measurement of troponin even at very low concentrations, allowing earlier detection of myocardial injury and infarction, aiding timely diagnosis and treatment.¹³ The current detection techniques employed for the detection of cardiac troponins are ELISA and chemiluminescent immunoassays (CLIA).¹⁰² These techniques are based on colorimetric or fluorometric detection of cTnI after its binding to the specific antibodies. These techniques are widely accepted thanks to their high sensitivity, specificity, and reliability, which are due to consistent and accurate results in clinical settings.¹⁰³

Researchers are currently working on developing point-of-care biosensing platforms for cTnI and cTnT to improve time to results. Also, it is essential to maintain the sensitivity, specificity and limit of detection offered by the traditional techniques. The recent major advancements in biosensor development of cardiac troponins are summarized here.

Optical biosensors

Optical biosensors are based on the binding interactions between cTn and the detector molecule which is immobilized on biosensor surface.¹⁰⁴ Target molecules should have a high specificity and binding affinity for cTn. These molecules such as aptamers, antibodies or labelled polymers are chosen precisely. Once cTn binds to the immobilized detector molecule, it forms a cTn-detector complex, thus altering the optical properties of the cTn biosensor such as refractive index, absorbance, fluorescence and surface-plasmon resonance.¹⁰⁵

A study reported the development of an SPR-based optical biosensor based on the interaction of cTnT with anti-cTnT functionalized gold triangular nano prisms (Au TNPs). The binding results in a shift of localized SPR wavelengths and thus, the reported LOD was about 0.0009 ng ml⁻¹. The linear range around the 47 nm edge length of Au TNPs was 0.0035–35 ng ml⁻¹.¹⁰⁶ In another study, researchers built a biosensor based on fluorescent properties pre and post interaction with the detector molecule. The common problem of fluorescent biosensors is the low sensitivity due to the fluorescent noise created by non-specific molecules in the body fluids. The surface acoustic wave separation technique (SAWST) was utilized to purify the target to increase the purity of the substrate. Additionally, the surface radiation force (SRF) exposed microfluidic beads on the sensor surface on which the immobilized probe is present to bind the cTn1. The LOD thus obtained was 44 pg ml⁻¹ in PBS and 0.34 ng ml⁻¹ in human serum.¹⁰⁷

In another approach, a platform to reduce fluorescent interference was developed by employing a horseradish peroxidase as a label (HRP). The fundamental principle behind the biosensor was the in situ fluorogenic interaction between two different polymers i.e. polyethyleneimine (PEI) and p-phenylenediamine (PPD) to create fluorescent copolymer nanoparticles (FCNPs). This modified ELISA exhibited a dynamic range of 5–180 ng ml⁻¹ and LOD 0.19 ng ml⁻¹ for cTn1.¹⁰⁸

Colorimetric biosensors are also an interesting approach to detect cardiac troponins. Colorimetric biosensors relay the information about the presence of a target analyte through colour changes that are induced. The colour changes can either be recorded through naked eye or simple spectrophotometers. Transducers are used in these biosensors which convert the biological interaction of the target analyte and the immobilized detection molecules (such as enzymes, antibodies, aptamers etc.) into colour signals. These transducers may include gold nanoparticles and other chromogenic substances. One such reported biosensor employed poly-dimethyl siloxane (PDMS)–gold nanoparticles (AuNPs) as the sensor surface. Additionally, silver enhancement for colorimetric detection of cardiac troponin 1 was done. This biosensor was able to detect cTn1 in less than 20 minutes and the LOD of 0.01 ng ml⁻¹ was obtained.¹⁰⁹

Surface plasmon resonance (SPR) based biosensors

SPR sensors are fast, label free and easy to use sensors. Most of the SPR sensors include long ranged plasmon surface polaritons, gold nanoparticle SPR, fluorescent spectroscopy for SPR, surface plasmon enhanced scattering and ultrasensitive plasmonic biosensors. One such reported biosensor is a fiber based SPR biosensor that employed aptamers with complimentary sequences to the cTn1. The researchers performed surface concentration optimizations to eventually achieve the LOD of 57.5 ng ml⁻¹.¹¹⁰

Electrochemical biosensors

Electrochemical biosensors may be built by using aptamers and antibodies as recognition elements. There are several different categories of electrochemical biosensors based on the type of output they measure for signal detection. Thus, electrochemical biosensors may be potentiometric, voltametric, electrochemical impedance spectroscopy, field effect transition and electrochemical luminescence.^107,111

A study reported a potentiometer that had screen printed graphite electrodes (SPGE) functionalized with gold nanoparticles (AuNPs). The cTn1 biomolecules were then immobilized on these electrodes. Afterwards, anti-cTn1 polyclonal rabbit antibodies were covalently bound to di-dimethylphenol dendrimers by carbodiimide coupling method. The increase in cTn1 targets results in reduction of the dendrimers acquired by the sensor and hence a change in electric potential can be observed. The biosensor exhibited a good range of detection of 1–10⁵ pg ml⁻¹ and a limit of detection of 7.3 pg ml⁻¹.¹¹²

Researchers have also developed a voltametric biosensor where they used a printed circuit board and spiked it with gold nanoparticles. The developed biosensor detected the cTn1 at the nucleic acid level by formation of a three-way DNA junction. Each fragment was compressed into three parts a cTn1 recognizer, methylene blue containing transduction part and an immobilizer with thiol group. The limit of detection observed was 0.001 pg ml⁻¹ in both human serum and HEPES buffer.¹¹³ Another interesting and promising approach is the use of electrochemical impedance spectroscopy (EIS). The technique relies on the quantity and electric conduction potential of the materials adsorbed on the sensor surface.¹⁰² Researchers have developed an immunosensor by functionalizing the sensor surface with metal organic framework (MOF) and nickel foam (NF). In this way, the modification resulted in the development of sensors functionalized with amine groups. The limit of detection obtained was 0.013 pg ml⁻¹ along with high specificity for cardiac troponins.¹¹⁴ A summary of biosensing approaches mentioned in this review for cTn is provided in (Table 3).

Table 3. Summary of the biosensing approaches for cTn mentioned in literature.

Sr. no.	Method	Materials of sensor	Range	LOD	References
1	SPR	Au TNPs rabbit anti-cTn Abs	0.0035–35 ng ml−1	0.0009 ng ml−1	106
2	Optical fluorescence, SAWST SRF	AuNPs ssDNA aptamer PS beads	Not mentioned	0.34 ng ml−1	107
3	Optical fluorescence, ELISA based	PPD, PEI, HRP, FCNPs	5–180 ng ml−1	0.19 ng ml−1	108
4	Colorimetric	PDMS, AuNPs	0.01–10 ng ml−1	0.01 ng ml−1	109
5	SPR	ssDNA aptamers, AuNPs	Not mentioned	57.5 ng ml−1	110
6	Potentiometer immunosensor	SPGE electrodes, AuNPs, anti-cTn rabbit polyclonal Abs	10–10 000 pg ml−1	7.3 pg ml−1	112
7	Voltametric immunosensor	AuNPs, aptamers	Not mentioned	0.001 pg ml−1	113
8	EIS	MOF, NF	Not mentioned	0.013 pg ml−1	114

Recent advancements in the biosensing approaches for interleukin-6 (IL-6)

IL-6 may serve as an emerging biomarker for CVD. Although IL-6 may not be used as a direct alternative to cardiac troponin testing, the gold standard for myocardial infarction, it can still be used for POC testing to obtain a rapid and timely assessment to reduce risk.

The following section illustrates recent approaches to measuring IL-6 levels in point-of-care systems reported in the literature:

Direct electrochemical immunoassay (dECIA)

Direct electrochemical immunoassay (dECIA) is a category of electrochemical sensors in which the sample is detected against an immobilized antibody affixed to the sensor.¹¹⁵

In one approach, the researchers developed an electrochemical biosensor based on enzyme antibody linkages. The sensor was developed using gold nanoparticles based on modified platinum carbon (PC). 4-Mercaptobenzoic acid (4-MBA) was immobilized on this sensor along with the linker IL-6 antibody.¹¹⁶ The sensor functionality was then analyzed by using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) analyses (Fig. 2A).

Fig. 2. Direct electrochemical immunoassay approach: (A) schematic diagram of the electrochemical sensor based on gold nanoparticles¹¹⁶ and (B) polypyrrole-based direct electrochemical immunoassay sensor.¹¹⁶ Copyrights belong to the authors and pictures taken with permission.

The surface of platinum carbon (PC) was modified by spherical gold nanoparticles. This process facilitated the signal molecule 4-MBA connection. The carboxyl group of 4-MBA was activated by using (1 (3-dimethylamino-propyl)-3-ethylcarbon diimide hydrochloride/N-hydroxysuccinimide) (EDC/NHS). Detection was done both in standard solution and animal samples. The results with the sensor were comparable with that of the sensor. The linear range of detection observed was 100–700 pg ml⁻¹ while a limit of detection LOD of 3 pg ml⁻¹ was calculated.¹¹⁶

In another study with direct electrochemical immunoassay, the antibody against IL-6 was immobilized on polypyrrole nanoparticles (PPy) decked with gold nanoparticles. This sensor exhibited a wide linear range of detection from 1 pg ml⁻¹ to 15 µg ml⁻¹ and the limit of detection observed was 0.33 pg ml⁻¹ (ref. 117) (Fig. 2B).

Another approach reported uses the technology of liquid-gated field effect transistor (FET) based on horizontally aligned single walled carbon nanotubes (SWCNT). The quantification through the sensor is achieved by recording its drain current responses post the attachment of the IL-6 to its corresponding immobilized antibody. Good LOD values were observed (1.37 pg ml⁻¹) and the dynamic range was 1–100 pg ml⁻¹.¹¹⁸

Indirect competitive electrochemical immunoassay (icECIA)

In this approach, a specific competitor is used that hampers the attachment of the targeted molecule to its receptor. The signal is generated and amplified by the interactions of the nonlabelled competitor and the receptor. This approach is because the signal has an inverse relation to the quantity of the molecule in the concerned sample to be tested.¹¹⁵

In one such study, the biosensor was built using electrochemically reduced graphene oxide (ERGO) and gold–palladium bimetallic nanoparticles (AuPdNPs). The base sensor was combined with silver nanoparticles functionalized polystyrene bionanolabel as a competitor. Additionally, electrically heated carbon electrode was utilized to further improve the sensitivity of the sensor. The sensor exhibited a wide linear range of 0.1–100 000 pg ml⁻¹ and a very good LOD of 0.059 pg ml⁻¹.¹¹⁹

Sandwiched nanoparticles labelled electrochemical immunoassay (sECIA-NP)

In this approach, a secondary antibody is employed to achieve the purposes of very high sensitivity and lowering the LOD. These types of sensors are very common with cancer detection. Upon the attachment of the molecule of interest to the antibody on the biosensor surface produced an electrochemical signal which is enhanced in turn by the attachment of the secondary antibody. The secondary antibody is bound by an electroactive label to achieve this goal.¹¹⁵ One example of this sensor is exhibited by the preparation of electrochemically sensitive immunosensors by coupling silver nanoparticle (AgNPs) and hollow titanium-phosphate spheres of 430 nm diameter and the average thickness of 40 nm. Then a secondary antibody (Ab2) was attached to this preparation. The amplified signal is then coupled with magnetic array sensing. The sensor showed excellent results in both the range (0.0005–10 ng ml⁻¹) and LOD (0.1 pg ml⁻¹).¹²⁰

Sandwich enzyme linked electrochemical immunoassay (sELECIA)

In this approach, an enzyme is used as a label with the secondary antibody. The concentration of the substrate catalyzed is then quantified to measure the quantity of the molecules concerned in the sample. Horseradish peroxidase (HRP), a very common ELISA enzyme is used frequently for the label.¹¹⁵

In one study, the researchers used gold nanoparticles and poly-dopamine (PDOP) as the base of the sensor along with carbon nanotubes (CNT). The enzyme used to label the secondary antibody was HRP. The sensor exhibited linear range of 4–800 pg ml⁻¹ and LOD of 1.0 pg ml⁻¹.¹²¹ The researchers further improved the LOD to 0.05 pg ml⁻¹ by using the sandwiched multienzyme-DNA labels.¹²²

Surface plasmon resonance based optical biosensors

Being cost-effective and robust while ensuring excellent sensitivity and specificity, surface plasmon resonance based optical biosensors are the most common. It can be classified into localized surface plasmon resonance (LSPR) when the phenomenon occurs on the sub-wavelength level while in SPR the same occurs on thin metallic strips.¹²³

The researchers in one study using this approach developed two different biosensors on surface plasmon resonance imaging (SPRi) assays. One of these biosensors were built by using mouse monoclonal antibodies for IL-6 immobilized using cysteamine linkers. The second biosensor incorporates galiellalactone as the receptor instead of the antibody which is an IL-6 specific inhibitor. It was immobilized using octadecanethiol (ODM) as the linker. The sensors depicted the ranges between 3–20 pg ml⁻¹ and 1.1–20 pg ml⁻¹ respectively. The LODs observed were 3 and 1.1 pg ml⁻¹ respectively.¹²⁴

Colorimetric based biosensors (CBB)

Colorimetric biosensors hold great potential for the detection of many biomarkers. These biomarkers are cost-efficient, and offer good sensitivity and selectivity, and considerably reduce the detection time which is the basic goal behind any point of care system.^123,125 Ever since the development of nanoparticles, colorimetric biosensors have become increasingly desirable for biomarker detection. Gold nanoparticles have been a usual preference in these biosensors for their rapid changes in color.

Researchers in one study have developed an optical aptasensor in which AuNPs were conjugated with two complimentary aptamers in a “sandwich model”. Both aptamers were of different regions of the IL-6 protein. As the IL-6 molecules were introduced, they attached to their complimentary regions on the aptamers thus causing the aggregation of the functionalized AuNPs. This caused a shift in the absorption wavelength (520–540 nm) and color (red to pink). The results can be observed visually or by a spectrophotometer. The researchers reported this aptasesnor to be a proof of concept with intentions of developing a similar sensor for clinical settings. This sensor exhibited a range of 3.3–125 µg ml⁻¹ and an LOD of 1.95 µg ml⁻¹.¹²⁶

Paper-based colorimetric optical biosensors

Smartphone based biosensors, due to their portability and ease of use have garnered much attention in point of care detection research. Therefore, smartphones particularly encompassing colorimetric optical biosensors offer huge potential. The color signal can be easily converted to quantitative and readable data that can be accessed through an app on the cell phone. Therefore, paper-based sensors are of much importance for future developments.¹²⁷

To check IL-6 as a biomarker for whole blood sepsis, researchers have developed a paper-based biosensor with a smartphone connectivity. The biosensor was developed by immobilizing the captured antibodies on a filter paper by dropping. This step was followed by the addition of IL-6 and their binding to the captured antibody. Lastly, AuNP functionalized anti-rabbit IgGs were introduced that produced colored changes to paper substrates. The colorimetric results were analyzed through the mobile densitometry app. The biosensor showed the linear range of 0.001 pg ml⁻¹ to 10 pg ml⁻¹ and LOD of 0.1 pg ml⁻¹. The biosensor was extremely time efficient with readable results available in only 17 min.¹²⁸

Lateral flow immunoassay

Among paper-based analytical techniques, lateral flow immunoassay (LFIA), is a very superlative detection technique due to its boundless advantages. LFIA is a cheap, rapid and easy to use analytical technique.¹²⁹ The LFIA strip has 4 main parts, a pad for placing the liquid sample, a conjugation pad that has the bioreceptor having nanoparticles labelling, a nitrocellulose membrane on which test, and control strips are sprayed, and an absorbent pad that collects the excessive liquid.¹³⁰ LFIA strips function in both the sandwich and competitive detection methods for the biomarkers wherein heavy molecular weight biomarkers are better detected with sandwich based LFIA strips.¹³¹

AuNPs are the most common labels used in LFIA strips, but they offer less sensitivity which can be improved by enhancers like using silver. In one such study, the researchers developed an AuNP-LFIA biosensor for the detection of IL-6. The biosensor was built by using AuNPs conjugated with the antibody for IL-6. The T line had biotinylated polyclonal human IL-6 antibody along with streptavidin while the C line had anti-mouse IgG directly dispensed at it. A 1 : 1 solution of silver nitrate and hydroquinone was applied to the test pads for enhancing the signal after the sandwich test. The sensor reported LOD of 1 pg ml⁻¹ in buffer medium and 5 pg ml⁻¹ in serum sample.¹³²

Another much-explored topic is the use of other nanoparticles like Pt having good optical properties and nanozymes that retain catalytic activity even in extreme conditions as well as resistance to proteases.¹³³ Nanomaterials with peroxidase activity have also been much explored due to their similarity to natural peroxidases and catalytic activity. One such magnetic colorimetric based immunoassay has also been reported. The oxidase activity of ceria nanospheres were used in this study. Fe₃O₄ nanospheres were employed to immobilize the IL-6 specific antibody while the secondary antibodies were labelled with CeO_2. First an immunocomplex is formed between the iron oxide labelled captured antibody and IL-6. This is followed by magnetic separation after which the secondary labelled antibody with copper oxide is introduced to form the sandwich complex. Post the magnetic separation, o-phenylenediamine (OPD) is added which is catalyzed by the ceria nanospheres to a yellow product, 2,3-diaminophenazine (oxOPD). Absorbance of oxOPD is measured at 480 nm which is dependent on the presence of IL-6. This immunoassay showed a LOD of 0.04 pg ml⁻¹ and a range of 0.0001–10 pg ml⁻¹.¹²⁰

A very interesting LFIA based immunosensor with dual mode of detection has been reported by Wen et al.¹³⁴ Au-nanoshells coupled with IL-6 detection antibody were utilized as LFIA reporters. These modified probes first detected IL6 via colorimetric effect by giving two different colored lines on the strip achieving a LOD of 5 ng ml⁻¹. Then the second colorimetric signal named as the test line by the authors was subjected to irradiation laser at 808 nm and the temperature changes were measured with a portable infrared camera. The method achieved greater efficiency and sensitivity having a range of detection of 1–1000 ng ml⁻¹ and LOD of 0.3 ng ml⁻¹.

Fluorescence based optical biosensors (FOB)

Fluorescence based biosensors due to their high precision are also one of the most desirable forms of biosensors to be developed. These biosensors mostly use organic dyes as fluorescent tags. These organic tags had several disadvantages such as light bleaching, pH sensitivity and insolubility in water which have been solved by the development of nanomaterials such as carbon nanotubes (CNT) and quantum dots.¹³⁵

Nitrogen coupled carbon quantum dots (N-CQD) were used to develop an aptasensor for IL-6 based on Förster resonance energy transfer (FRET). The aptasensor was made with N-CQD modified DNA aptamer at one end and AuNPs at the other end to form a pairing of a donor and quencher. In absence of IL-6, AuNPs acting as quenchers hamper the emission intensity from N-CQD being near the donor. Once IL-6 introduced, its binding to the aptamer causes conformational changes that changes that releases N-CQDs from aptamer and the consequential fluorescence is recorded. It showed a range of 1.5–5.9 pg ml⁻¹ and the LOD of 0.82 pg ml⁻¹.¹³⁶

Organic electrochemical transistor (OECT)

A study has reported that a biosensor functionalized with oligonucleotide aptamers specifically designed to bind to IL-6. The sensor is based on gold nanoparticles attached to a poly(3,4-ethylenedioxythiophene) further enhanced with polystyrene sulfonate (PEDOT: PSS) electrode to ensure the accumulation of thiolated aptamers electrically. LOD of 24 pg mol⁻¹ were observed by Diacci et al.¹³⁷

A summary of the above-mentioned biosensors is provided in (Table 4).

Table 4. Summary of reported biosensors for IL-6 detection.

Method	Materials of sensor	Range	LOD	Reference
dECIA	AuNPs, Pt-C 4-MBA	100–700 pg ml−1	3 pg ml−1	116
dECIA	AuNPs–PPyNPs	1–15 000 000 pg ml−1	0.33 pg ml−1	117
dECIA FET	SWCNT	1–100 pg ml−1	1.37 pg ml−1	118
icECIA	ERGO-AuPdNPs	0.1–100000 pg ml−1	0.059 pg ml−1	119
sECIA-NP	AgNPs-Ti-Pspheres-Ab2	0.0005–10 ng ml−1	0.1 pg ml−1	120
sELECIA	PDOP-CNT-HRP	4–800 pg ml−1	1 pg ml−1	122
SRPi	Mouse monoclonal Abs for IL-6	3–20 pg ml−1	3 pg ml−1	124
SRPi	Galiellalactone–octadecanethiol	1.1–20 pg ml−1	1.1 pg ml−1	124
OECT	AuNPs–PEDOT: PSS	pg ml−1 to ng ml−1	24 pg ml−1	137
CBB	AuNPs-IL-6-aptamers	3.3–125 µg ml−1	1.95 µg ml−1	126
CBB	AuNPs, IL-6 Abs-IgGs	0.001–10 pg ml−1	0.1 pg ml−1	128
LFIA	AuNPs-IL-6 Abs	Not reported	5 pg ml−1	132
LFIA	Ceria nanospheres-IL-Ab-Ab2	0.0001–10 pg ml−1	0.04 pg ml−1	120
LFIA	AuNPs nanoshell-IL-6 Abs	1–1000 ng ml−1	0.3 ng ml−1	134
FOB	N-CQD-DNA aptamer-AuNPs	1.5–5.9 pg ml−1	0.82 pg ml−1	136

Recent advancements in the biosensing approaches for soluble suppression of tumorigenicity 2 (sST2)

Soluble suppression of tumorigenicity 2 (sST2) is an important biomarker of CVD particularly being an indicator of heart failure. Precise, specific and rapid measurement of sST2 levels may result in crucial and timely assessment of heart health. Some of the recent approaches for the detection of sST2 reported in the literature are briefed as follows:

Electrochemical immunosensor

One study has explained the design and development of an electrochemical sensor for the detection of sST2 in human serum as a cardiac biomarker. Graphite paper (GP) electrodes were functionalized with fullerene – C₆₀. Anti sST2 antibodies were then immobilized on the surface of the electrode. The biomarker was detected using EIS and cyclic voltametric (CV) analyses. It showed wide range of 0.1–100 fg ml⁻¹ and LOD of 0.124 fg ml⁻¹.¹³⁸

Another study has reported the development of a field effect transistor (FET) based biosensor for sST2 detection. The researchers employed the technique of electro polymerization to immobilize several peptide epitopes specific to sST2 on the polymer surface. The polymer surface was made with poly(aniline-co-3-aminobenzenesulfonic acid), poly(AN-co-MSAN). Furthermore, three different Molybdenum based carbides were integrated onto the conductive polymer surface to enhance its performance. The biosensor had a range of 0.001–100 pg ml⁻¹ and LOD of 0.00005 pg ml⁻¹.¹³⁹

An amperometry immunosensor with magnetic beads has been reported as well. In this sensor, carboxylic acid micro sized magnetic beads (COOH-MB) were used to immobilize immunoconjugates for the selective recognition and binding of sST2. Moreover, a labelled secondary antibody modified with streptavidin peroxidase (Strep-HRP) was utilized to capture the target. The LOD reported was 39.6 pg ml⁻¹.¹⁴⁰

In another example of electrochemical immunosensor, Huang et al. used differential pulse voltammetry to measure the changes in current with the binding of sST₂ to the sensor surface. For this purpose, glassy carbon electrodes were first coated with nanocomposite polyaniline (PANI). The sensor is then further modified by the dispersion of MXene-Nafion and finally anti-sST₂ antibodies were immobilized. The study has reported a linear range of 1 pg ml⁻¹ to 1 µg ml⁻¹ and a LoD of 0.149 pg ml⁻¹. Additionally, the sensor depicted effective performance with the serum samples of both the diseased and healthy subjects.¹⁴¹

Optical biosensor

Huang et al. have recently reported an immunosensor for the detection of sST2. The sensor is based on the D-shaped extensively tilted fiber grating (ExTFG). The sensor's refractive index (RI) potential was enhanced by axis specific polishing of the surface in order to create a D-shape. The sensor was subsequently biomodified with Molybdenum disulphide (MoS₂) and anti-ST2 monoclonal antibodies. The sensor exhibited good sensitivity to ST₂ in standard as well as healthy controls and patient serums. The sensor exhibits localized changes of refractive index due the antigen–antibody reaction between ST₂ and ST₂ Abs, thus increasing red shift in the transmission spectra's resonance peaks with increasing concentration of antigen. The sensitivity of the shift was calculated to be 0.335 nm/log(mg mL⁻¹). Thus, a label free promising biosensor with good linear range (1–1000 ng ml⁻¹) and limit of detection (LoD 0.472 ng ml⁻¹) was obtained.¹⁴²

In a striking contrast to the label-free biosensor reported above, a study has reported an optical biosensor based on FRET mechanism. The sensor is designed by careful selection of a ssDNA aptamer based on optimal affinity for the target. The aptamer was then immobilized on the graphene oxide (GO) base of the sensor. It exhibited a wide range of 0.1–100 µg ml⁻¹ and LOD of 3.7 ng ml⁻¹.¹⁴³

Qiu et al. in another aptamer-based biosensor have reported a fluorescence biosensor. The sensor in based again on the binding affinity of the target ST₂ with the aptamer with specific analyte recognition. The sensor is accompanied by a reporter DNA sequence and Crispr/Cas12a system. Upon binding, trans-cleavage activation amplifies the signal of the fluorescent probe that increases with the increasing concentration of ST_2. The sensor has exhibited a linear range of 5–120 ng ml⁻¹ and a limit of detection (LoD) of 0.816 ng ml⁻¹. Additionally, the sensor had a good specificity in serum samples with a correlation (R² = 0.9794) with ELISA results.¹⁴⁴

Thermal biosensor

In a different approach of developing label-free biosensors, the researchers of a novel study have reported the development of molecularly imprinted nanoparticle polymers (nanoMIPs) for the sensing of h-FABP and sST₂ biomarkers. Synthetic antibodies are manufactured for both biomarkers via solid phase synthesis. SPR analyses exhibited comparable binding affinities for the structures to the commercially available options. The particles are then functionalized on thermocouples and arranged in 3d-printed flow cells. Upon binding of the target sST₂ on the surface, the change in thermal resistance was measured. The sensor arrangement with three parallel arranged thermocouples has exhibited good specificity in serum samples with a linear range of 3.15–315 ng ml⁻¹ and limit of detection of 8.79 ± 0.79 ng ml⁻¹.¹⁴⁵

A summary of the abovementioned biosensors for sST2 is shown in (Table 5).

Table 5. Summary of reported biosensors for sST2 detection.

Method	Materials of sensor	Range	LOD	Reference
EIS, CV	GP. Fullerene-C60, anti-sST2 Abs	0.1–100 fg ml−1	0.124 fg ml−1	138
FET	Peptide epitopes, MXenes, (AN-co-MSAN)	0.001–100 pg ml−1	0.00005 pg ml−1	139
Amperometry	COOH-MB, immunoconjugates for sST2, Strep-HRP	Not mentioned	39.6 pg ml−1	140
DPV	GCE, PANI-MXene Naffion, anti-sST2 Abs	1 pg ml−1–1 µg ml−1	0.149 pg ml−1	141
Refractive index biosensing	D-shaped ExTFG, MoS2, anti ST2 monoclonal Abs	1–1000 ng ml−1	0.472 ng ml−1	142
FRET	ssDNA, GO	0.1–100 µg ml−1	3.7 ng ml−1	143
Fluorescence aptasensor	cDNA-FQ, APT-1, Crispr/Cas12a system	5–120 ng ml−1	0.816 ng ml−1	144
Thermal resistance	nanoMIPs, thermocouples, 3d-cells	3.15–315 ng ml−1	8.79 ± 0.79 ng ml−1	145

Recent advancements in the biosensing approaches for miRNA

Conventionally, miRNA can be detected through various molecular biology techniques such as qRT-PCR, northern blotting, in situ hybridization and microarray analysis. However, just like other biomarkers, there has been much research in the development of rapid, sensitive and specific biosensors for individual miRNA sequences concerned with a specific pathology.¹⁴⁶ Some of the recent advancements in the biosensing approaches for miRNA identification are elucidated as follows:

FRET based detection of miRNA of interest has been particularly studied for the development of an optical biomarker for miRNA detection. A study has reported the development of a FRET based biosensor for miRNA of interest using semiconductor quantum dots. A microarray platform was developed to detect three different miRNAs at the same time. Terbium (Tb) complex was used as a donor in this setup. The resulting LOD was 1 nM.¹⁴⁷

In another study, graphene quantum dots were used as semiconductor platforms while the molecular beacon probes were functionalized with pyrene. The biosensor exhibited a wide range of 0.1–200 nM and LOD of 0.1 nM.¹⁴⁸

There have also been great developments in the design of label free optical approaches to detect miRNA, most prominently SPR approaches have been studied. Label free SPR approaches have been more effective compared to the fluorescent approaches.¹⁴⁹ Additionally, a label free SPR biosensor enhanced with streptavidin has been reported. The biosensor had a 5-fold improvement in its signal while the LOD was reduced to 1.7 fM for miRNA-122.¹⁵⁰

Additionally, in another approach, researchers developed a genosensor for miRNA-21 detection. They modified the sensor surface with graphene oxide (GO) and poly(diallyldimethylammonium chloride) (PDDA). These were formed in the form of a bilayer on a gold disk, previously enhanced with mercapto propane sulfonate (MPS). The sensor exhibited a wide linear range between 1 fM and 10 nM and a LOD of 0.3 fM.¹⁵¹

Researchers have also constructed a biosensor with GO and AuNPs both serving as the sensor surface and signal amplification units. The sensor was developed to detect the miRNA-141 and exhibited good range (0.1 pM–2 nM) and LOD (0.1 pM).¹⁵²

In another more recent approach, the researchers have developed a biosensor of molybdenum disulfide (MoS₂) nanosheets along with CRISPR/CAS12a enzymes. The system was developed for miRNA-499 which is involved in acute myocardial infraction. The CRISPR/CAS12a enzymes recognize, bind and cut which release the signal to the MoS₂ nanosheets to enhance fluorescence. Thus, the greater the miRNA, the greater the fluorescence is. This biosensor exhibited a linear range of 0.1–13.3 nM and LOD of 381.78 pM.¹⁵³ An advanced peptide nucleic acids-microarray system for the recognition of miRNA, composed by a multilayered Si/Al/agarose component, was developed by Petralia et al. The PNA-microarray was investigated for the detection of miRNA-122, resulting in a sensitivity of about 1.75 µM⁻¹ and LoD of 0.043 nM. The contribution, in terms of H-bonds amounts to 298 and 333 K of the agarose coating to the dsPNA-RNA interactions was demonstrated by Molecular dynamic simulations.¹⁵⁴

Samadishadlou et al. have reported a paper-based fluorescence biosensor for the detection of miRNA-499. The paper-based biosensor was developed by gold nanoparticles approximately 12 nm in size having functionalized with molecular beacons achieving an approximation of 18 probes per particle. Thus, prepared nanoprobes were then coated on the paper where in miRNA-499 binding activates the previously quenched fluorophore. The signals were quantified using a benchtop reader and standard benchtop camera followed by image analysis. The sensor has demonstrated a linear range of 10 fM–100 pM and LoD of 10 fM.¹⁵⁵

In another example of paper-based biosensor, Nylon discs were used for the paper-based assay by the immobilization of the DNA probe. Increasing concentrations of miRNA-21 were added to the analytical device for hybridization followed by the functionalization with cysteine modified gold nanoparticles which attach to the negatively charged phosphate backbone of the target nucleotide due to its positive charge. Finally, a solution H₂O₂ and 3,3′,5,5′ tetramethylbenzidine (TMB) was added. The developed nanozyme catalyzes this colorimetric reaction resulting in a blue color on the disc captured via smartphone camera. The sensor reported a linear range and LoD of 1 pM–1 nM and 0.5 pM respectively as well as a recovery rate of 97.6% in the human serum.¹⁵⁶

In another example of using TMB for the detection, an electrochemical biosensor for the detection of miRNA-155 using graphene quantum dots has also been reported recently. In this study, Hu et al. developed the sensor by attaching a catcher DNA to the electrode. Upon capturing the target, an amino modified DNA probe is linked forming a sandwich. Graphene quantum dots and horseradish peroxidase assemble is introduced that acts as the catalyzer upon the capturing of the target. Finally, H₂O₂/TMB is added which upon catalysis changes the color from colorless to blue as well as an increase in electrochemical current signal. The sensor exhibited a linear range of 1 fM–100 pM and LoD of 0.14 fM.¹⁵⁷

A summary of the recent biosensing approaches for miRNA mentioned above are provided in (Table 6).

Table 6. Summary of the biosensing approaches for miRNA.

Method	Materials of sensor	Range	LOD (miRNA)	References
FRET	Tb, semiconductor QDs	2–10 nM	1 nM (miRNA-20a, miRNA-20b, miRNA-21)	147
FRET	Pyrene, graphene QDs	0.1–200 nM	0.1 nM (miRNA-155, miRNA-210, miRNA-21, miRNA-196a)	148
SPR	Streptavidin	Not mentioned	1.7 fM (miRNA-122)	150
SPR genosensor	GO, PDDA, MPS	1 fM–10 nM	0.3 fM (miRNA-21)	151
SPR	GO, AuNPs	0.1 pM–2 nM	0.1 pM (miRNA-141)	152
Fluorescence, CRISPR/Cas12a	MoS2 nanosheets, CRISPR/Cas12a enzymes	0.1–13.3 nM	381.78 pM (miRNA-499)	153
Hybridization fluorescence	Peptide nuclei acids microarray	0.1–10 nM	0.043 nM (miRNA-122)	154
Paper-based nanoprobe fluorescence	Au-NPs, molecular beacons	10 fM–100 pM	10 fM (miRNA-499)	155
Paper-based colorimetric	Nylon disc, Cys/AuNPs, H2O2/TMB	1 pM–1 nM	0.5 pM (miRNA-21)	156
Paper-based electrochemical	GQDs, HRP, TMB, capture ssDNA	1 fM–100 pM	0.14 fM (miRNA-155)	157

Photoluminescent nanomaterials for CVD biomarkers sensing

Metal nanomaterials are widely investigated in bioanalytical sensing thanks to their distinctive optical features as good surface plasmon absorption, long fluorescence lifetime, large Stoke's shift and good emission quantum yield.¹⁵⁸ Since they demonstrate elevated signal-to-noise ratios and customizable surface functionalization, gold-nanostructures appear to be the best choice for sensing and targeting applications, especially in the biomedical field. In fact, these nanomaterials can detect ions, metabolites, proteins, and nucleic acids through various techniques, including fluorescence, phosphorescence, or electrochemiluminescence. Recently, gold nanoparticles formation has been used as a transducer system for the enzymatic recognition of amino acids in biological fluids.¹⁵⁹ Similarly, gold nanoclusters have been used for the ultrasensitive detection of nucleic acids by quenching photoluminescence upon hybridization reaction.¹⁶⁰ Metal nanoclusters, known for their dimensions below 2 nm and energy levels like those of molecules, represent a highly interesting category of nanomaterials;¹⁶¹ in fact, they possess both fluorescence and electrochemiluminescence (ECL) properties¹⁶² that can be tuned by varying their size or their core or surface atomic composition by inserting various types of molecular ligands for bioconjugation. The latest research works specifically illustrate how NCs enable the connection of organic emitters and plasmonic NPs, while remaining compatible with microfluidic and point-of-care (POC) formats.¹⁶³ Their easy surface chemical functionalization allows the integration of this nanomaterial on miniaturized systems for the development of point-of-care system,¹⁶⁴ paper sensors¹⁶⁵ or wearables,¹⁶⁶ allowing for the development of miniaturized smart devices for real-time monitoring. Recent advancements in the fabrication of quantum dots, carbon dots, perovskite nanocrystals, and metal nanoclusters have unveiled methodologies for meticulously regulating the wavelength, duration, and intensity of emission through composition, size, and surface ligands.^167,168

Recently nanomaterials have also been used for the quantification of CVD biomarkers, such as IL-6, cardiac troponins (cTnI/T), miRNAs, and resolvins and have become essential for smart diagnosis of all those markers considered crucial for cardiac disease.

Since 2020, several optical and electrochemical sensors have been developed for the sensing of CVD biomarkers mainly based on electric, electrochemiluminescence and optical readouts.¹⁶⁹ Diacci et al.¹³⁷ published a work based on an OECT aptasensor made on PEDOT: PSS, herein the binding of IL-6 to the aptamer that was immobilized on the surface caused the ionic transport using the polymer channel. This showed a real-time current modulation with a LOD of 0.48 pg ml⁻¹ in spiked serum, demonstrating both ultra-high sensitivity and operation in physiologically relevant matrices. In the subsequent year, the same group employed an antifouling strategy to enhance the performance of the OECT; the surface was engineered by modifying PEDOT: PSS with antifouling and aptamer-stabilizing chemistries, which improved effective binding efficiency and diminished non-specific adsorption. Their platform attained a limit of detection of 1.8 pg ml⁻¹ for IL-6 under label-free measurement conditions, validating the appropriateness of organic electrochemical transistors for portable, low-sample-volume detection of inflammatory biomarkers.¹⁷⁰ In 2024 Liu and his group¹⁷¹ have developed a Ratiometric Electrochemical aptasensor for Interleukin-6 by combining a methylene blue polymer as an internal reference with carbon nanotubes with a LOD of 0.54 pg ml⁻¹ and a linear range of six orders for IL6 sensing. Furthermore, although many studies still use NPs or small fluorophores for interleukin sensing, nanoclusters are becoming increasingly common as fluorescent labels for sensing related protein targets, and the conjugation and energy transfer strategies (i.e., how they bind to the antibody and how they emit light) can be directly repurposed for IL-6 as well. It is also important to note that immunosensing platforms using electrochemiluminescence have already achieved detection limits of less than one femtogram per milliliter for IL-6 in biofluids, such as plasma or serum, implying that performance targets for future nanocluster-based sensors will need to match or exceed these sensitivity levels to be competitive.¹⁷²

Recognition of cardiac troponins allows the early diagnosis of acute myocardial infarction. Their sensing is challenging due to low amount of these markers in the femtogram or picogram per milliliter (fg–pg mL⁻¹) range and to the biologic matrix interferences, where proteins, salts, and other molecules can easily interfere with the sensing. In 2024, Anju et al.¹⁷³ developed an immunosensor for troponin I (cTnI) based on gold nanoclusters (AuNCs) functionalized with specific antibodies reaching a LOD of 0.51 ng ml⁻¹. The sensing strategy is based on enhancement of the Au-nanocluster photoluminescence signal upon troponin-antibody complex formation at the Au-nanoparticles surface. In detail, once troponin binds to the antibody the antigen–antibody complex causes a separation between the NCs and the AuNPs, resulting in fluorescence engulfment. Therefore, the presence of troponin causes a measurable increase in the light signal, proportional to the troponin concentration. The same research group have developed a similar system for troponin T (cTnT) sensing, using AuNCs functionalized with folic acid, and then coupled to manganese dioxide nanosheets as fluorescence quencher. The troponin-T-folic acid interaction causes the release of quencher with the increase in the fluorescence signal. The LOD reported was about 0.037 ng Ml⁻¹ for cTnT antigen.¹⁷⁴ Field-Effect Transistor (FET)-based devices have also been developed by Hung & Lee,¹⁷⁵ this system uses aptamers to reduce the “Debye screening” effect, for the sensing of troponin in biological media with a LOD of 10 pg ml⁻¹. Electrochemiluminescence-based sensors could have a huge impact on the sensitivity of a system, as shown by Zhu et al.;¹⁷⁶ in fact, they have designed a highly sensitive immunosensor for cardiac troponin I (cTnI) based on ECL. The coupled gold nanoclusters (AuNCs), which served both as ECL emitters and bioconjugation platforms, with antibodies thanks to a hybridisation chain reaction (HCR). In this case the HCR process generated long DNA assemblies, increasing the number of AuNC labels per recognition event and thus strongly boosting the ECL output. The system achieved an ultralow limit of detection of 1.01 fg ml⁻¹. Furthermore, they have demonstrated that this system does not work well just under controlled laboratory conditions but also in fully validated patient serum, demonstrating that it is possible to use nanoclusters for specific troponin sensing.¹⁷⁶ DNA-templated metal nanoclusters system has been largely used for the recognition of cardiac-related microRNAs, such as miR-1, miR-133, miR-208 and miR-499. In this scenario, metal nanoclusters like gold and silver have been widely investigated due to their excellent luminescent properties and easy DNA-functionalization. The fluorescence of metal nanoclusters has quenched the hybridization of miRNA target with the specific complementary strand DNA probes anchored at nanocluster surface, as recently proposed by Yourston et al., who reported a miRNA sensing strategy based on the hybridization of miRNA target with specific DNA-templated AgNCs with a limit of detection of 1.6 × 10⁻⁶ M for miR-21sensing.¹⁷⁷ Hence, Cu-NC-based on ECL methodologies have demonstrated to be useful for miRNA sensing, Wang's group,¹⁷⁸ has developed a method based on aggregation-induced electrochemiluminescence for the detection of miR-208/499 with a limit of detection in the femtomolar region. They reported that copper nanoclusters (CuNCs) self-assembling within reverse microemulsions induce a quantum yield enhancement of system. This technology attained significant signal amplification and minimized non-radiative losses, providing a framework for ultrasensitive quantification of miRNA relevant to cardiac applications. Similarly, Cui et al.,¹⁷⁹ have used a Cu-NC electrochemiluminescence (ECL) platforms based on synthetic vesicles as a confinement tool to detect miRNA145 with a limit of detection of 30 fM over a linear range of 100 fM–10 nM with an analysis time <30 min. In contrast, Qi's group¹⁸⁰ has reported the use of the enzyme TdT (terminal deoxynucleotidyl transferase) for the femtomolar sensing of miRNA21 by the in situ formation of CuNCs. In fact, their design amplifies labels per binding event: the template-independent extension at the recognition site creates T/C-rich tails that nucleate numerous CuNCs in situ, pushing sensitivity into the attomolar–femtomolar regime without external fluorophores.

Resolvins are small amphipathic lipids, making their sensing challenging. RvD1 interacts with the GPCRs ALX/FPR2 and GPR32 with high stereoselectivity; Au or Ag nanostructures can be modified with receptor-derived peptides or antibodies to confer affinity and selectivity and find application in SERS/ECL platforms. This approach is supported by the well-characterized biology of RvD1–ALX/GPR32 binding, which provides a rational template for receptor-mimetic capture layers.¹⁸¹ Nanocarrier-inspired capture layers can also be used; Delivery studies indicate that nanoscale scaffolds can stabilize and present RvD1 in biological media (e.g., ROS-sensing vesicles and polymeric carriers). This implies that similar amphiphilic monolayers or vesicle coatings on plasmonic substrates could enhance the pre-concentration of resolvins at the interface, thus improving on-chip recognition and readout.¹⁸² A pictorial summary of the aforementioned discussion is provided in (Fig. 3).

Fig. 3. Photoluminescent nanomaterials for CVD biomarkers sensing: (A) illustration of (a) the synthesis of the controllable Cu NCA and (b) the AIECL sensing system based on Cu NCA and SnS2 nanosheets.¹⁷⁸ (B) The graphical representation of detection of cardiac troponin T antigen (cTnT) using cTnT specific antibody conjugated gold nanoclusters–MnO₂ nanosheet (Ab@FAAuNC/MNS) nano couple.¹⁷⁴ (C) Schematic of PLP-RvD1 fabrication and its targeting treatment for myocardial ischemia–reperfusion injury.¹⁸².

Point-of-care platform for cardiac disease biomarkers recognition

Point-of-Care (PoC) testing refers to the development of simplified means of detection of biological elements involved in the prognosis of specific diseases, simply biomarkers. In simpler terms, PoC refers to all forms of testing that can be done close to the patient without the requirement of sample preparation in equipped facilities. It is a simple sample-in-answer-out mechanism.¹⁸³

Even though the pioneers of PoC systems were developed as early as 1972, it was not until early 1980s when the term was first coined.¹⁸⁴ Additionally, after monumental feat in the form of the Human Genome Project in 2000 in molecular biology, the research has progressed exponentially to find rapid and accurate means of molecular diagnosis. The idea is to create chips or systems that can provide molecular diagnosis without having to go through the hassle of time taking procedures like PCR and ELISA.¹⁸⁵ The most prominent development in this sector is through the development of DNA microarrays.^186,187

Moreover, PoC systems offer several other benefits over traditional approaches such as operative simplicity, no need for trained personnel, cheap, mass level manufacturing and rapid results to name a few.¹⁸⁸ Any PoC system comprises of several key components which can be divided into an analyte separation system that picks out the target from the raw biological sample, a detection unit which using biochemical assays detects the target biomarker, a data processor to detect and translate signals for a reader module that converts the input signal into readable values. The reader module can now be as simple as a smartphone app.¹⁸⁹

A comparative overview of traditional testing and PoC systems is provided in (Fig. 4A) while (Fig. 4B) gives insight into the basic framework of a biosensor.

Fig. 4. (A) Comparative overview between traditional testing and PoC testing system, (B) schematic of a biosensor: an analytical device consisting of a biological system, capable of selectively interacting with the analyte of interest, in intimate contact with a transductor that converts the interaction process into a measurable signal.

Recent advancements in commercially available PoC platforms for cardiovascular diseases

There has been great development in the research for PoC platforms for CVD biomarker detection. The PoC platforms have been developed both commercially and in research. The recent and advanced reported commercially available PoC devices for CVD biomarkers are focused on multiplex screening. The devices target a minimum of two and a maximum of four biomarkers for detection with cTn being present in almost all of these. Other important, targeted biomarkers for detection include CK-MB and Myo. The biochip cardiac array developed by Randox, based on chemiluminescence read-out is the best performer in term of LoD for all the biomarkers recognized: cTn1, CK-MB, Myo and H-FABP.

These platform devices provide insight into the early detection of acute myocardial infractions. Majority of the commercially reported devices use chemiluminescence, colorimetric or fluorescent techniques for signal amplification and detection. Most of these mentioned devices have either been approved by the food and drug administration (FDA) in the US or have gotten the Conformité Européenne (CE) in Europe. There has been huge advocacy for the development of devices that can provide rapid and accurate results from the bedside or in the ambulance.^189–191 However, there is still a shortage of inexpensive, multiplexed devices that can be used at home. Ouyang et al.,¹⁹⁰ in their review have provided details of the commercially available PoC devices. Table 7 summarizes the commercially available PoC multiplex devices as reported by Ouyang et al.¹⁹⁰ and Mani et al.¹⁹² and in their papers respectively.

Table 7. Summary of the commercially available PoC multiplexed platform.

System name (company)	Detection technology	Biomarkers: dynamic range and LoD (ng mL−1)	Required sample and volume	Analysis time (min)	Regulatory
Fluoro-Check; Nano-Ditech	Chromatographic fluorescence	Myo: 20–1000 ng ml−1, 10 ng ml−1	Serum, whole blood, plasma (80 µL)	15	In vitro diagnostic use only
		cTn1: 0.1–30 ng ml−1, 0.03 ng ml−1
		CK-MB: 2–200 ng ml−1, 2 ng ml−1
Nano-Check AMI 2 in 1 CD 201S; AMI 3 in 1 CD 301S; Nano-Ditech	AuNPs based chromatographic assay	cTn1: 0.1–30 ng ml−1, 0.1 ng ml−1	Plasma, serum 200–250 µL	15	IVD use only, FDA clearex
		CK-MB: 2–200 ng ml−1, 2 ng ml−1
		Myo: 20–1000 ng ml−1, 20 ng ml−1
Randox biochip cardiac array; Randox	Chemiluminescence	cTn1: 0.18–50 ng ml−1, 0.18 ng ml−1	Serum	—	IVD use only
		CK-MB: 0.4–100 ng ml−1, 0.4 ng ml−1
		Myo: 1.8–700 ng ml−1, 1.8 ng ml−1
		H-FABP: 0.15–100 ng ml−1, 0.15 ng ml−1
Triage Cardio 3 Panel; Quidel	Microcapillary fluorescence	cTn1: 0.01–10 ng ml−1, 0.02 ng m−1)	Whole blood, plasma	20	CE marked IVD use only
		BNP: 0.005–5 ng ml−1, 0.005 ng ml−1
		CK-MB: 1–8 ng ml−1, 1 ng ml−1
Cobas h 232; Roche	AuNP based chromatography	cTnT: 0.04–2 ng ml−1, 0.04 ng ml−1	Whole blood 150 µL	8–12 min	IVD use only
		NT-proBNP: 0.06–9 ng ml−1
		Myo: 30–700 ng ml−1
		CK-MB: 1–40 ng ml−1
i-STAT; Abbott	Microchannel electrochemical ELISA	cTn1: 0.02–50 ng ml−1	Whole blood, plasma17–95 µL	5–10	IVD use only, CE marked, FDA cleared
		0.0016 ng ml−1 (blood)
		0.00105 ng ml−1 (plasma)
		BNP: 0.015–5 ng ml−1
		CK-MB: 0.6–150 ng ml−1
PATHFAST; Mitsubishi Chemical	Magnetic enzymatic chemiluminescence	cTn1: 0.019–50 ng ml−1, 0.0023, ng mL−1	Whole blood, plasma 100 µL	15	IVD use only FDA cleared
		NT-proBNP: 0.015–30 ng ml−1
		hs-CRP: 50–30 000 ng ml−1
		Myo: 5–1000 ng ml−1, 0.898 ng ml−1
		CK-MB: 1–250 ng ml−1, 0.106 ng ml−1
AFIAS; Boditech	Fluorescence immunoassay	cTn1: 0.015–15 ng ml−1, 0.01 ng ml−1	Whole blood, plasma, serum 10–50 µL	3–15	CE marked, IVD use only
		NT-proBNP: 0.013–30 ng ml−1, 0.00803 ng ml−1
		hs-CRP: 5 × 103–2 × 105 ng ml−1, 138 ng ml−1
		Myo: 2–500 ng ml−1
		CK-MB: 3–100 ng ml−1, 1.17 ng ml−1
Ichroma II; Boditech	Fluorescent chromatography	cTn1: 0.1–50 ng ml−1, 0.11 ng ml−1	Whole blood, plasma, serum	3–12	CE marked, IVD use only
		NT-proBNP: 0.01–30 ng ml−1
		hs-CRP: 2.5 × 103–3 × 105 ng mL−1
		Myo: 5–500 ng ml−1, 1.86 ng ml−1
		CK-MB: 3–100 ng ml−1, 1.34 ng ml−1
Mini VIDAS; BioMerieux	Enzymatic fluorescence	cTn1: 0.01–30 ng ml−1, 0.0008 ng ml−1	Plasma, serum 150–250 µL	17 – 30	CE marked, IVD use only
		NT-proBNP: 0.02–25 ng ml−1, 0.02 ng ml−1
		Myo: 5–1000 ng ml−1
		CK-MB: 0.8–300 ng ml−1
		Gal-3: 3.3–100 ng ml−1
DZ-Lite c270; Diazyme	Immunoturbidimetry enhanced with latex	cTn1: 0.4–10 ng ml−1	Plasma, serum 4–25 µL	10	CE marked, IVD use only, FDA clearance for hs-CRP and myoglobin
		hs-CRP: 200–2 × 104 ng ml−1
		Myo: 13.2–615.9 ng ml−1
		h-FABP: 0.74–120 ng ml−1
		MPO: 12–1000 ng ml−1
RAMP; Response Biomedical	Fluorescent chromatography	cTn1: 0.03–32 ng ml−1, 0.03 ng ml−1	Whole blood 75 µL	15–19	CE marked, IVD use only, FDA cleared
		NT-proBNP: 0.027–22 ng ml−1, 0.018 ng ml−1
		Myo: 2.36–400 ng ml−1, 2.36 ng ml−1
		CK-MB; 0.32–80 ng ml−1, 0.32 ng ml−1
AQT90 Flex; Radiometry	Time resolved fluorimetry	cTn1: 0.01–25 ng ml−1, <0.01 ng ml−1	Whole blood, plasma	11–19	IVD use only, CE marked, FDA clearance for cTn
		cTnT: 0.01–25 ng ml−1, <0.01 ng ml−1
		NT-proBNP: 0.012–35 ng ml−1, <0.02 ng ml−1
		hs-CRP: 5 × 103–5 × 105 ng mL−1
		Myo: 20–900 ng ml−1, 2 ng ml−1
		CK-MB: 1.5–300 ng ml−1, 1.5 ng ml−1
QuickSens Ω 100; 8sens.biognostic	AuNP based chromatography	cTn1: 1 ng ml−1	Whole blood, plasma, serum 120 µL	15	IVD use only, CE marked
		hs-CRP: 500 ng ml−1
		h-FABP: 0.6–75 ng ml−1
SMART/CUBE; EuroLyser	Immunoturbidimetry enhanced with latex	cTn1: 1–10 ng ml−1, 1 ng ml−1	Serum 20–50 µL	3–10	IVD use only, CE marked
		hs-CRP: 250–2 × 104 ng ml−1
Aspect-Plus; Critical diagnostics	Fluorescent chromatography	sST2: 12.5–25 ng ml−1, 12.5 ng ml−1	Plasma 35 µL	20	IVD use only, CE marked

Recent advancements in PoC platforms for cardiovascular diseases reported in literature

Like the commercially available multiplexed PoC systems for CVD biomarkers, several researchers have also reported such systems. Many efforts are being made to develop PoC systems that can offer rapid and accurate biomarker detection for the monitoring of heart health. Majority of the researchers have also been focused on developing household PoC systems integrated with machine learning and AI.^193,194 Also, there are many reports of the developed PoC systems where the users can get the results via smartphone apps in minutes.¹⁹⁵

An example of one such reported multiplexed platform, researchers utilized the lateral flow assay (LFA) technique for qualitative detection of acute myocardial infarction biomarkers cTnT and h-FABP. The LFA was based on a Zirconium metal organic framework (ZrMOF) and Cadmium Tellurium nanoparticles (CdTeNPs). This PoC system had an analysis time of 8 minutes. It was able to detect the h-FABP in concentrations as low as 1 ng ml⁻¹ (ref. 196) (Fig. 5A).

Fig. 5. Multiplexed PoC platforms for CVD biomarkers: (A) structural and functional framework of LFA PoC Zou et al.,¹⁹⁶ (B) structural and functional framework of multiplexed fluorescent Ab microarray PoC Colom et al.¹⁹⁷ (C) Structural and functional framework of electrochemical ePAD Immunosensor PoC Boonkaew et al.¹⁹⁸ and (D) structural and functional framework of Fluorescent lateral flow strip (UC-LFC) PoC You et al.¹⁹⁹ Copyrights belong to the authors and pictures taken with permission.

In another study, the researchers developed a fluorescent antibody microarray PoC for five different biomarkers, CRP, CysC, h-FABP, cTn1 and NT-proBNP. The microarray panel contained streptavidin tetramethyl rhodamine conjugate (Streptavidin TRITC) and antirabbit IgG tetramethyl rhodamine conjugate (AntiIgG-TRITC) for capturing and detecting the targets. The platform had an analysis time of 90 minutes and the LODs reported were 15 ± 5, 3 ± 1, 24 ± 3, 25 ± 3, and 3 ± 1 ng ml⁻¹ for CRP, CysC, h-FABP, cTn1 and NT-proBNP respectively. It was a proof-of-concept model and even though the reported detection limit is higher than ideal, the system holds much promise for future improvements and development into a functional and reliable PoC platform for a diverse range of cardiac biomarkers¹⁹⁷ (Fig. 5B). An example of an immunosensor for the detection of three different CVD biomarkers CRP, cTn1 and procalcitonin (PCT) is provided by Boonkaew et al.¹⁹⁸ (Fig. 5C). The immunosensor was a paper based electrochemical analytical device (ePAD). The ePAD was made from the stacking of a wax patterned paper and a film of transparency using laser cuts and dual sided adhesive. The antibodies against the specific biomarkers were immobilized on the carbon electrodes modified with graphene oxide (GO) which were then stencil printed on the ePAD. Square wave voltammetry (SWV) was used to determine the concentrations of the concerned biomarkers. LODs obtained were 0.38 ng ml⁻¹, 0.16 pg ml⁻¹ and 0.27 pg ml⁻¹ for CRP, cTn1 and PCT respectively. You et al.¹⁹⁹ have developed and reported a household PoC platform for the detection of brain natriuretic peptide (BNP) and suppression of tumorigenicity 2 (ST2) biomarkers, both of which are associated with heart failure (Fig. 5D). The PoC is a lateral flow strip based on the reading of fluorescence of target analytes. Upconversion nanoparticles (UCNPs) were employed for the system. The results can be read using a smartphone app. LOD obtained was 5 pg ml⁻¹ for BNP and 1 ng ml⁻¹ for ST2 in spiked samples. In testing with clinical samples, the detection limits were 29.92 ng ml⁻¹ and 17.46 pg ml⁻¹ for ST2 and BNP respectively. The results in both cases show good correlation to already approved established systems for detection of these biomarkers by FDA (Table 8).

Table 8. Summary of the literature reported PoC multiplexed platform.

Detection technology	Biomarkers	LoD ng ml−1	Analysis time (min)	Reference
Zr. metal organic framework, lateral flow assay	h-FABP	1	8	196
	cTnT	250
Fluorescent Ab microarray	CRP	15 ± 5	90	197
	CysC	3 ± 1
	h-FABP	24 ± 3
	cTn1	25 ± 3
	NT-proBNP	3 ± 1
Square wave voltametry, paper based electrochemical sensor	CRP	0.38	30	198
	cTn1	0.00016
	PCT	0.00027
Lateral flow assay, nanoparticles	sST2	29.92	20	199
	NT-proBNP	0.01746

Future perspective for CVD biomarkers biosensing

The CVD monitoring has been revolutionized by the rapid advancement of innovative technologies, particular through the emerging of new biomarkers, innovative nanostructured materials for enhanced optical and electrical reading, low-cost multiplexing technology and recently the enhancement of user-friendly features through artificial intelligence. These technologies integrated into CVD platforms will offer fast, reliable, and cost-effective monitoring of CVD's patient conditions. Multiplex systems further enhance diagnostic capabilities these assays are used for the simultaneously quantification of complementary biomarkers, which are employed to identify various conditions as inflammation and fibrotic/remodeling processes (IL6, sST2), myocardial injury (sST2), the resolution phase of inflammation (resolvins), and gene regulation associated with cardiac remodeling (miRNA). The multiplexing approaches offer enhanced diagnostic performance compared to single biomarkers, enabling more accurate detection of cardiovascular conditions. The integration of multiple biomarkers into a single CVD platform, managing cross-reactivity, and assuring high sensitivity without compromising selectivity are the major technical hurdles. Moreover, stability, cost-effectiveness, and the suitability for diverse clinical environments are essential for practical application. Meanwhile global accessibility will be assured by regulatory approval obtained through rigorous testing and validation processes. Addressing these challenges through multidisciplinary efforts will enhance CVD monitoring capabilities for cardiovascular diseases, improving healthcare. From a clinical and biochemical perspective, the analytical and clinical validation of this pattern of biomarkers will be useful for translating these technological advances into routine practice. Nevertheless, unlike cardiac troponin, IL-6, sST2, resolvins and miRNAs do not currently have standardized tests or universally validated cut-off values, which are necessary for routine clinical use. Nowadays, Il-6 emerged as the strongest individual predictor of the inflammatory response, but this value still needs to be integrated into a multi-marker inflammatory profile.³¹ Similar pre-analytical and normalization difficulties affect the quantification of circulating miRNAs,⁴⁸ whilst specific threshold values for sST2 have yet to be definitively established.⁴³ Another consideration is the biological context in which these biomarkers are interpreted. In general, troponin is released as a specific consequence of damage to cardiomyocytes. Still, the release of most of these new biomarkers, such as IL-6 and sST2, is not disease-specific and is influenced by comorbidities such as chronic inflammatory diseases and obesity, as well as by physiological factors such as age, sex, and circadian variation; all of these factors can confound their interpretation if they are not adequately taken into account.^44,200,201 Ultimately, demonstrating that these emerging biomarkers, individually or in combination, offer additional diagnostic or prognostic value compared with the standard troponin-based treatment approach will be required for their inclusion in clinical practice guidelines.

Conclusion

This review highlights the advancements in biosensing strategy and the read-out technologies for the sensitive and specific monitoring of cardiovascular biomarkers. Detection strategies relating to the reference biomarker, cardiac troponins, and to emerging biomarkers such as interleukin-6, the soluble isoform of suppression of tumorigenicity 2 (sST2) and various microRNAs were selected and analyzed, as they are considered promising tools for improving the clinical utility. A broad range of emerging biosensing technologies and signal amplification approaches has been discussed, including optical biosensors based on photoluminescence, colorimetry, and surface plasmon resonance (SPR), as well as electrochemical and organic electrochemical transistor (OECT) platforms. In addition, hybrid immunoassay-enzyme systems such as direct electrochemical immunoassay, sandwiched nanoparticle-labelled electrochemical immunoassay, sandwich enzyme-linked electrochemical immunoassay, and indirect competitive electrochemical immunoassay have been examined in detail.

An overview of the latest developments in photoluminescent nanomaterials for CVD biosensing, with a particular focus on recent advances in luminescent metal-nanoclusters, has been reported. Furthermore, an exhaustive analysis of seventy commercially available multiplexed point-of-care (PoC) systems for cardiac biomarkers is presented, together with a concise survey of recent literature-reported PoC platforms for CVD biomarkers. This synthesis highlights current technological trends and outlines future directions in cardiovascular diagnostics. By leveraging innovative nanotechnologies and integrating multiplexed biomarkers into personalized medicine, future advancements are poised to redefine the prevention, diagnosis, and management of CVDs, ultimately reducing the global disease burden.

Author contributions

SA, GF, EB, VP, MDL, CI and CV: conceptualisation, data curation, formal analysis, investigation, methodology, validation, visualisation, writing – original draft, writing – review & editing. AC, SP: resources, supervision, project administration, writing – original draft, review & editing.

Conflicts of interest

There are no conflicts to declare.

Biographies

Biography

Albino Carrizzo.

Prof. Albino Carrizzo received the PhD in Translational and Clinical Medicine from the University of Salerno, Italy, in 2015. He is Associate Professor at the University of Salerno, where he chairs the Master's Degree Program in Dentistry and Dental Prosthetics and heads the Vascular Pathophysiology Laboratory at IRCCS Neuromed. He has coauthored more than 100 scientific publications. His research focuses on endothelial dysfunction, hypertension, vascular aging, and cardiometabolic diseases, with particular emphasis on nitric oxide signaling and novel therapeutic approaches.

Biography

Salvatore Petralia.

Prof. Salvatore Petralia: received the PhD degree in chemistry from the University of Catania Italy, in 2003. He is associate Professor of Chemistry at University of Catania. He is coordinator of the operative Unit of Consorzio Interuniversitario per le Biotecnologie (CIB unit of Catania) and co-founder of AIDA srl (Advanced and Innovative Diagnostic Academy). He has coauthored 160 peer-reviewed scientific publications, chapters of book and is a coinventor of 15 applications patents. His research activities cover multidisciplinary aspects of chemistry, nanostructured materials, biosensing and photochemical processes.

Data availability

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