Molecular Testing in Early Diagnosis and Clinical Assessment of Alzheimer’s Disease: A Narrative Review

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and one of the leading causes of dementia worldwide. With the increasing prevalence driven by population aging, there is a growing demand for early, accurate, and biologically grounded diagnostic approaches. Advances in molecular diagnostics have created new opportunities for early disease detection, staging, and monitoring of therapeutic responses, reshaping contemporary diagnostic workflows. Validated cerebrospinal fluid biomarkers—amyloid-β, total tau, and phosphorylated tau—form the core of current biologically based diagnostic criteria, while blood-based biomarkers such as plasma p-tau and neurofilament light chain are gaining prominence due to their minimally invasive nature and scalability. Advanced imaging techniques, including amyloid and tau positron emission tomography, further enhance diagnostic accuracy and support differentiation of AD from other neurodegenerative disorders. Despite these advances, the clinical implementation of molecular diagnostics remains limited by methodological heterogeneity, biological variability, and the lack of standardized analytical and clinical frameworks. Addressing these translational challenges is essential for integrating molecular biomarkers into routine clinical practice and for enabling reliable, large-scale screening and early diagnosis of AD.

1. Introduction

Alzheimer’s disease (AD) is a neurodegenerative proteinopathy that constitutes an increasingly significant global health challenge. This progressive and debilitating disorder is clinically characterized by a gradual decline in cognitive function, most notably severe memory impairment, accompanied by progressive deterioration of executive function, language abilities, and visuospatial skills [1,2]. The disease course is typically divided into three stages: prodromal or preclinical AD, mildly symptomatic AD, and clinically manifest AD (Figure 1).

Two major clinical variants are recognized, early-onset AD (EOAD) and late-onset AD (LOAD) [3], the key differences between which are summarized in

Table 1. Comparative neuropsychological, neuroimaging, and biomarker profiles of early-onset and late-onset Alzheimer’s disease.

Feature	EOAD	LOAD	References
Age at symptom onset	Early (<65 years)	Late (≥65 years)	[5]
Cause of the disease	Often associated with genetic factors; more aggressive disease course	Predominantly sporadic; multifactorial etiology;	[3,6]
Memory impairment	Mild to moderate in early stages	Severe; prominent early feature	[6,7,8]
Cognitive decline	Greater involvement of non-memory domains (language, visuospatial function, executive function)	Slower, memory-dominant; cognitive decline	[2,6,8]
Neuroimaging characteristics	Predominant posterior cortical atrophy with relative hippocampal sparing	Prominent medial temporal lobe atrophy (hippocampus)	[6]
Biomarker profile (CSF/plasma)	Earlier and more pronounced changes in CSF Aβ42 reduction and tau elevation; higher frequency of abnormal biomarker profiles at a younger age	Gradual biomarker changes with stronger age-related variability; plasma biomarkers influenced by comorbidities	[9,10,11]

Aβ—amyloid-β; CSF—cerebrospinal fluid; EOAD—early-onset Alzheimer’s disease; LOAD—late-onset Alzheimer’s disease.

. The global burden of AD has increased substantially over recent decades, primarily as a consequence of population aging [4].

An estimated 44 million people worldwide are currently affected by AD, and this number is projected to double approximately every 20 years. The risk of developing AD increases markedly with age, reaching nearly 30% by 85 years of age. AD represents the most common cause of dementia, accounting for approximately 60–80% of cases globally. At present, more than 55 million individuals live with dementia worldwide, with nearly 10 million new diagnoses reported annually [2]. According to a World Health Organization report, the global number of dementia cases is expected to triple by 2050 compared with the estimated 35.6 million cases reported in 2010. The prevalence of AD is strongly age-dependent, affecting approximately 5–8% of individuals over 65 years of age and increasing sharply to 25–50% among those over 85 [1,2].

At the biological level, AD is characterized by the accumulation of extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein (p-tau). These pathological alterations lead to synaptic dysfunction, chronic neuroinflammation, and progressive neuronal loss, particularly within the hippocampus and association cortices. The dynamic interplay between amyloid deposition, tau pathology, and neurodegeneration constitutes the core pathogenic cascade of AD and provides a fundamental biological framework for the development of molecular diagnostic strategies [1,4].

The current standard for diagnosing AD in many countries is based on the diagnostic criteria outlined in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) [4,12]. This approach is predominantly symptom-based and relies largely on clinical history and cognitive assessment rather than on objectively measurable biological markers. Although a DSM-5-based evaluation often allows for a reasonably accurate clinical diagnosis, it has important limitations. In most cases, significant cognitive impairment reflects an advanced stage of the disease, and patients with substantial cognitive decline may experience difficulties in undergoing comprehensive diagnostic procedures or therapeutic interventions [4]. In response to these limitations, contemporary diagnostic frameworks have shifted toward a biologically grounded definition of AD. The National Institute on Aging–Alzheimer’s Association introduced the AT(N) classification system, which categorizes biomarkers into three core domains: amyloid pathology, tau pathology, and neurodegeneration. This framework more accurately reflects the underlying molecular and pathological processes of AD and provides a structured basis for the early identification and biological staging of disease pathology [4,8]. Early and precise detection of AD-related symptoms and underlying biological changes is essential for effective screening, diagnosis, and disease management [2]. Timely identification enables patients and caregivers to plan and implement appropriate medical and lifestyle interventions that may help preserve functional independence and quality of life. Nevertheless, the detection of early-stage AD in routine clinical practice remains challenging due to limited consultation time, difficulties in reliably identifying underlying pathology, and the frequent misattribution of early cognitive symptoms to normal aging [2,4,13,14]. As the prevalence of AD continues to rise, the current diagnostic and therapeutic paradigm increasingly requires strategies that enable earlier and more accurate detection.

Molecular testing has therefore emerged as a key strategy to address these diagnostic challenges. Cerebrospinal fluid (CSF) biomarkers and positron emission tomography (PET) imaging allow for the direct in vivo detection of amyloid and tau pathology, while recently developed blood-based biomarkers provide a less invasive and more accessible alternative. These approaches create the opportunity to identify AD at preclinical or mildly symptomatic stages, long before extensive neuronal damage has occurred [6,9]. Despite rapid technological progress, however, molecular diagnostics are not yet routinely implemented in everyday clinical practice due to limitations related to availability, cost, and variability in testing protocols. Bridging this translational gap requires standardized methodologies and extensive clinical validation.

Accordingly, this review highlights the importance of early diagnosis of AD and provides a comprehensive overview of molecular diagnostic approaches that may support the detection of AD at its earliest stages and facilitate future integration into clinical practice.

2. Alzheimer’s Disease Pathogenesis

2.1. The Amyloid Cascade Hypothesis and Amyloid-β in Alzheimer’s Disease Pathogenesis

AD is a complex, multifactorial neurodegenerative disorder characterized by progressive deterioration of cognitive functions accompanied by marked cerebral atrophy [1,2,4]. Its pathogenesis is driven by an intricate interplay of molecular and cellular mechanisms, primarily involving two hallmark proteinopathies: the extracellular accumulation of Aβ peptides forming amyloid plaques and the intracellular aggregation of hyperphosphorylated p-tau into NFTs. These pathological alterations disrupt neuronal connectivity, impair synaptic integrity, and ultimately lead to neuronal dysfunction and neuronal loss [15]. Beyond these core proteinopathies, the pathogenesis of AD encompasses a broader network of interconnected biological processes. As schematically illustrated in Figure 2, the neurobiological environment affected by AD pathology exhibits a multifactorial etiology that contrasts sharply with the healthy brain. This framework highlights several key pathogenic mechanisms that interact with and exacerbate protein aggregation, including increased oxidative stress, chronic neuroinflammation, and metabolic dysfunction, all of which contribute to disease progression [16,17].

The amyloid cascade hypothesis has served as the dominant conceptual framework for understanding the pathogenesis of AD for more than three decades [18,19]. According to this model, the aberrant accumulation of Aβ peptides within cerebral tissue represents the initiating pathological event that triggers a cascade of downstream molecular and cellular processes characteristic of AD. Progressive Aβ accumulation leads to the formation of extracellular amyloid plaques, one of the principal histopathological hallmarks of the disease [18].

Aβ peptides—most notably β-amyloid 40 (Aβ40) and the more aggregation-prone β-amyloid 42 (Aβ42)—are central to AD pathophysiology and form the molecular basis of the amyloid cascade hypothesis. These peptides are generated through sequential proteolytic cleavage of amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase [9,18]. As illustrated in Figure 3, this proteolytic pathway results in the production of Aβ42, which exhibits a strong propensity to self-aggregate into soluble oligomers, extracellular fibrils, and ultimately amyloid plaques [20,21].

The accumulation of amyloid plaques contributes to AD progression through a network of interrelated mechanisms [22]. Soluble oligomeric forms of Aβ, particularly Aβ42 oligomers, are considered highly neurotoxic species that directly impair synaptic transmission and synaptic plasticity. These oligomers disrupt glutamatergic signaling essential for learning and memory and perturb intracellular calcium homeostasis [22]. Sustained exposure to Aβ oligomers activates intrinsic apoptotic pathways, ultimately leading to neuronal loss and progressive cognitive decline [20].

Beyond their direct neurotoxic effects, amyloid plaques also promote chronic microglial activation and sustained neuroinflammatory responses [20]. While early microglial activation may initially exert a protective role by facilitating Aβ clearance, persistent activation—often exacerbated by impaired clearance mechanisms—becomes deleterious. This prolonged inflammatory state significantly contributes to synaptic dysfunction, neuronal injury, and the progressive neurodegenerative trajectory characteristic of AD [20,21].

2.2. Molecular Mechanism of Tauopathy in Alzheimer’s Disease Pathogenesis

Tau, encoded by the MAPT gene located on chromosome 17q21, is a neuron-specific microtubule-associated protein (MAP) predominantly localized within axonal compartments. Tau plays a critical role in the assembly and stabilization of microtubules, which are essential components of the neuronal cytoskeleton responsible for maintaining intracellular transport and structural integrity [10,23]. The interaction of tau with the microtubule lattice is tightly regulated by a range of post-translational modifications (PTMs), including phosphorylation, acetylation, and ubiquitination [24]. These modifications modulate tau binding affinity and functional dynamics, thereby supporting cytoskeletal homeostasis and normal neuronal function. Dysregulation of tau PTMs is a key feature of several neurodegenerative disorders, most notably AD, where aberrant phosphorylation promotes tau detachment, aggregation, and the formation of NFTs [24,25].

Under physiological conditions, tau is indispensable for maintaining neuronal structural and functional integrity through its stabilizing effects on microtubules. In AD, however, tau undergoes aberrant hyperphosphorylation, a pathological modification driven largely by dysregulated kinase activity, particularly that of glycogen synthase kinase-3β and cyclin-dependent kinase 5 [9,23]. As illustrated in Figure 4, hyperphosphorylation markedly reduces the affinity of tau for microtubules, converting the protein from a microtubule assembly-promoting factor into a disruptive species. This pathological transition leads to tau dissociation from the axonal cytoskeleton and its subsequent aggregation into paired helical filaments, which represent the principal structural components of NFTs—one of the defining histopathological hallmarks of AD [23,26].

The formation of pathological tau is fundamentally driven by an imbalance between kinase and phosphatase activities. Under normal conditions, tau phosphorylation is tightly controlled by a dynamic equilibrium; in AD, however, the activity of key phosphatases, particularly protein phosphatase 2A, is significantly reduced [24]. This shift promotes the accumulation of hyperphosphorylated tau, with levels reported to be up to threefold higher in AD brains compared with healthy controls [23,24]. This enzymatic disequilibrium not only facilitates tau misfolding and aggregation but also enables hyperphosphorylated tau to sequester normal tau, as well as other microtubule-associated proteins such as MAP1 and MAP2, thereby promoting their co-aggregation and functional impairment. As a result, tau transitions from a physiological MAP into a neurotoxic aggregate, emerging as a central mediator of cytoskeletal destabilization, synaptic dysfunction, and progressive neurodegeneration in AD [23].

2.3. Oxidative Stress in Alzheimer’s Disease Pathogenesis

Reactive oxygen species (ROS), as inevitable by-products of aerobic metabolism, exert dual roles in cellular physiology. Under homeostatic conditions, they participate in essential signaling processes; however, excessive ROS accumulation results in oxidative stress, a deleterious state that compromises mitochondrial integrity and disrupts cellular homeostasis [16]. The central nervous system (CNS), owing to its high oxygen consumption, limited antioxidant capacity, and lipid-rich neuronal membranes, is particularly vulnerable to oxidative damage, rendering it a primary target in neurodegenerative disorders such as AD [27].

In the context of AD, oxidative stress is not merely a secondary consequence of neurodegeneration but constitutes a significant contributor to disease pathogenesis [16,27]. Aging is associated with a progressive imbalance between pro-oxidant and antioxidant mechanisms, which exacerbates neuronal dysfunction and accelerates neurodegenerative processes. This vulnerability is further amplified by the brain’s high metabolic demands and structural composition, promoting lipid peroxidation, protein oxidation, and damage to nucleic acids. Collectively, these oxidative alterations impair neuronal viability and contribute to the progressive cognitive decline observed in AD [27,28].

2.4. Chronic Inflammation in Alzheimer’s Disease Pathogenesis

Accumulating evidence from neurobiological research increasingly identifies neuroinflammation as a mechanistically central component of AD pathogenesis, with a contributory role that may rival or exceed that of classical neuropathological hallmarks such as Aβ plaques and NFTs [27]. Understanding and modulating the complex interactions between the immune system and the CNS are therefore considered critical determinants in delaying or preventing the onset of late-onset neurodegenerative disorders [28,29].

In AD, the core proteinopathies—Aβ and pathological tau species—act as potent activators of the innate immune response through the engagement of glial cells, particularly microglia and astrocytes. This activation initiates a state of chronic neuroinflammation within the brain parenchyma [16]. While early glial activation may exert protective effects by promoting Aβ clearance, sustained immune activation becomes maladaptive and significantly exacerbates disease progression [29].

Persistent neuroinflammation contributes to increased production and impaired clearance of both Aβ and tau, thereby amplifying synaptic dysfunction, neuronal injury, and widespread neurodegeneration [14]. Consequently, modulation of neuroinflammatory pathways has emerged as a major focus of therapeutic research, driving extensive investigation into anti-inflammatory strategies for AD. In addition, peripheral inflammatory conditions—including systemic infections, cerebrovascular events, and metabolic disorders such as obesity—are recognized as important extrinsic factors that can exacerbate central neuroinflammatory processes and accelerate AD pathology [28,29].

2.5. Metabolic Dysfunction in Alzheimer’s Disease Pathogenesis

AD is increasingly recognized as a disorder closely linked to metabolic dysfunction. Growing evidence indicates that mitochondrial impairment, altered glucose metabolism, and systemic metabolic disturbances play critical roles in neuronal energy homeostasis and represent key features of AD pathogenesis [14,30]. Studies have demonstrated that mitochondrial deoxyribonucleic acid (DNA) damage, impaired oxidative phosphorylation, and defective mitochondrial quality control occur early in the disease process and may precede microglial activation and neuroinflammation in the AD brain [14,30].

These alterations compromise adenosine-5′-triphosphate production and promote excessive generation of ROS, thereby contributing to oxidative stress and neuronal injury. Consistent with these findings, cerebral glucose hypometabolism is a well-established and reliable characteristic of AD, detectable even at preclinical stages [30,31].

Dysregulation of insulin signaling, frequently associated with type 2 diabetes mellitus, has also been implicated in hippocampal metabolic abnormalities and cognitive decline. This mechanistic link between insulin resistance and AD has led to the conceptualization of AD as “type 3 diabetes,” underscoring the contribution of neuroendocrine dysfunction to disease pathogenesis [32].

3. Diagnostic Approaches in Alzheimer’s Disease

3.1. Cerebrospinal Fluid Biomarker Profiling

Uncertainty associated with the clinical diagnosis of AD has driven intensive efforts to identify diagnostic approaches that enable earlier and more accurate detection. At present, a definitive diagnosis of AD still requires histopathological confirmation [33].

The initial assessment of patients with suspected early-stage AD represents a critical task in primary care and specialist settings and is primarily aimed at excluding reversible causes of cognitive impairment, including depression, vitamin deficiencies, hormonal imbalances, and electrolyte disturbances. This evaluation typically involves a detailed medical history to identify established risk factors, such as family history of AD, age, sex, apolipoprotein E genotype, physical inactivity, low educational attainment, diabetes, and obesity [13,34].

Comprehensive clinical assessment also requires careful review of comorbid conditions and medications that may affect cognitive performance, as well as structured interviews with patients and caregivers to evaluate changes in cognitive function and their impact on daily living [33,34].

Physical and neurological examinations, routine laboratory testing, and standardized assessments of cognitive and functional abilities form the basis of the initial differential diagnosis [3].

Building upon the original diagnostic criteria established in 1984, the National Institute on Aging–Alzheimer’s Association revised the clinical criteria for mild cognitive impairment and dementia due to AD in 2011, incorporating biomarker evidence derived from imaging, serum, and CSF analyses to improve diagnostic specificity. In parallel, the DSM-5 reclassified dementia and related cognitive syndromes under the broader category of neurocognitive disorders, facilitating improved differentiation among neurodegenerative diseases, including AD [12].

Biomarkers capable of detecting early neurodegenerative changes have therefore assumed an increasingly central role in the diagnostic framework of AD [21]. Consequently, CSF analysis and PET imaging are currently regarded as key diagnostic modalities, particularly for the assessment of amyloid pathology, which represents one of the earliest quantifiable biological indicators of AD-related neurodegenerative processes [35].

CSF analysis has long constituted the cornerstone of biochemical assessment in AD, providing direct insight into the molecular mechanisms underlying disease pathogenesis [36].

CSF is a specialized biological matrix that surrounds the CNS and reflects the neurochemical environment of the brain and spinal cord. Collection of CSF requires lumbar puncture (LP), an invasive diagnostic procedure that constitutes the principal limitation to its widespread use in routine clinical practice. The procedure involves percutaneous insertion of a needle into the subarachnoid space, typically at the L3/L4 or L4/L5 intervertebral level, enabling the acquisition of CSF for molecular profiling and quantitative biomarker analysis [21,36]. The most clinically relevant CSF biomarkers are those associated with amyloid and tau pathology [11,17,21,36].

A reduction in CSF amyloid-β 42 (Aβ42) concentration is widely recognized as a sensitive and reliable indicator of early AD-related pathology. This decrease reflects the sequestration of Aβ42 into insoluble amyloid plaques within the brain parenchyma, a process that begins many years, and often decades, before the onset of overt clinical symptoms [9,17,19]. Consequently, CSF Aβ biomarkers play a critical role in identifying individuals in the preclinical phase of AD.

Tau-related biomarkers are similarly central to the diagnosis and staging of AD [11,37]. Elevated concentrations of total tau (t-tau) and p-tau, particularly the p-tau181 isoform, are well-established indicators of neuronal injury and tau hyperphosphorylation, respectively. These biomarkers demonstrate robust diagnostic performance in differentiating AD from other neurodegenerative disorders [11,26,37,38].

Advances in analytical methodologies have expanded the spectrum of measurable tau isoforms to include p-tau217, p-tau231, and p-tau205, each providing distinct temporal and pathological resolution of disease progression. Notably, elevations in p-tau217 closely coincide with the earliest detectable amyloid plaque deposition, as confirmed by amyloid-PET imaging [34], whereas increased p-tau205 levels correlate more strongly with early neuronal dysfunction detectable by [¹⁸F]fluorodeoxyglucose positron emission tomography (FDG-PET) [10,39].

In addition to amyloid and tau markers, neurofilament light chain (NfL) has emerged as an important indicator of axonal injury [40]. NfL is a cytoskeletal protein predominantly expressed in large-caliber, myelinated axons, where it contributes to structural stability [40,41,42]. Elevated CSF NfL concentrations reflect the extent of neuroaxonal degeneration and increase proportionally with disease severity across a wide range of neurological conditions, including inflammatory, traumatic, and neurodegenerative disorders [42,43]. Due to its limited disease specificity and sensitivity to age-related physiological changes, CSF NfL alone is insufficient for differential diagnosis of AD; however, its inclusion in multimodal biomarker panels enhances prognostic stratification and provides a more comprehensive assessment of neurodegenerative progression within the AD continuum [41,43].

Despite its high diagnostic validity, the clinical utility of CSF biomarker profiling is inherently limited by the invasiveness of lumbar puncture, particularly in elderly populations [41].

Age-related reductions in CSF turnover may affect sample reliability, while common geriatric conditions such as frailty, spinal degeneration, and comorbidities increase the risk of procedural complications. In addition, anticoagulant therapy may further elevate the risk of spinal hematoma, often contraindicating LP [21]. These limitations highlight the critical need for minimally invasive yet analytically robust diagnostic alternatives better suited to large-scale clinical application in aging populations [21,44,45].

3.2. The Role of PET Imaging in Amyloid and Tau Plaque Visualization and Monitoring

PET imaging complements CSF-based diagnostic approaches by enabling non-invasive, in vivo visualization and longitudinal monitoring of AD pathology. Owing to its high spatial resolution and reproducibility, PET is particularly valuable in clinical trials and observational studies focused on assessing disease progression, therapeutic efficacy, and temporal changes in neuropathological burden [21,46].

Amyloid PET imaging, employing Aβ-specific radioligands, has emerged as a robust modality for the detection of cerebral amyloid plaque deposition. In clinical practice, radiolabeled tracers such as florbetapir or flutemetamol are administered intravenously and selectively bind to aggregated Aβ within the brain parenchyma, enabling direct visualization of amyloid pathology in vivo [21,46,47,48]. Amyloid PET provides critical diagnostic information, particularly in the early and preclinical stages of AD, and supports differential diagnosis by distinguishing AD from other neurodegenerative conditions [46,47].

Tau positron emission tomography (tau-PET), which utilizes radiotracers targeting insoluble tau aggregates, has advanced substantially following regulatory approval of the first tracer, [¹⁸F]flortaucipir [10]. In agreement with neuropathological observations, recent tau-PET studies have identified the perirhinal cortex (Brodmann area 35) as one of the earliest cortical regions affected by tau pathology, even in cognitively unimpaired individuals [10,49,50]. Early tau accumulation in this region, located near the rhinal sulcus, has been detected independently of concurrent amyloid burden. Importantly, tau load quantified within the perirhinal cortex correlates with reduced functional connectivity to the medial prefrontal cortex and with subtle memory deficits, indicating early neurodegenerative compromise preceding overt clinical symptoms [21,46,47,48,49,51].

Overall, PET imaging enables real-time, quantitative assessment of both amyloid and tau proteinopathies in vivo, representing a significant advancement over conventional post-mortem or in vitro techniques. By facilitating longitudinal evaluation of disease trajectory, PET serves as an essential complement to CSF-based biomarkers [21,47]. Moreover, its reproducibility and high spatial precision render PET indispensable in clinical trials and longitudinal studies aimed at monitoring therapeutic response and quantifying neuropathological changes over extended time periods [21,47,48]. Together, these capabilities enhance early diagnostic accuracy, improve risk stratification, and support the development of personalized approaches to AD management [47,48].

3.3. Blood-Based Biomarkers in Alzheimer’s Disease

Blood-based biomarkers have become a central focus in efforts to improve diagnostic workflows and support the development and longitudinal monitoring of disease-modifying therapies in AD. Increasing emphasis has been placed on the implementation of accessible and cost-effective plasma biomarkers that may complement or partially replace more invasive diagnostic modalities [3,49,51].

In this context, blood screening—particularly assessment of the plasma amyloid-β 42/amyloid-β 40 (Aβ42/Aβ40) ratio—offers a minimally invasive alternative to CSF analysis [52,53].

Compared with CSF, blood represents a more complex analytical matrix due to lower concentrations of target proteins and the influence of peripheral synthesis, metabolism, and clearance mechanisms. Nevertheless, major technological advances, including immunoprecipitation–mass spectrometry and single-molecule array (Simoa) platforms, have markedly improved the analytical performance of plasma biomarker assays [52,54]. As a result, blood-based biomarkers such as Aβ, NfL, and tau species are increasingly recognized as valuable adjuncts to established diagnostic approaches, including CSF analysis and PET imaging, by enabling minimally invasive, scalable screening and longitudinal disease monitoring [54]. However, the integration of these ultra-sensitive analytical technologies into routine clinical practice remains constrained by significant technical and economic limitations. Although the Simoa platform enables relatively high-throughput automated analysis, it requires costly instrumentation and proprietary reagent kits, which restrict its accessibility primarily to specialized centers [9,52].

In contrast, immunoprecipitation–mass spectrometry, often regarded as the analytical gold standard due to its high specificity and quantitative precision, is characterized by lower throughput and complex, multi-step sample preparation requiring highly trained personnel. Furthermore, the substantial capital investment associated with mass spectrometry platforms and the challenges related to inter-laboratory standardization limit their widespread clinical adoption. Consequently, IP-MS currently serves predominantly as a research and validation tool, whereas more conventional immunoassay-based techniques remain more feasible for large-scale population screening [9,52,54]. A comparative overview of these diagnostic strategies is provided in

Table 2. Comparative analysis of diagnostic modalities for Alzheimer’s disease pathology.

Feature	CSF Biomarkers	PET Imaging (Aβ-PET/Tau-PET)	Blood Biomarkers	References
Method Type	Biochemical (fluid analysis)	In vivo molecular imaging	Biochemical (fluid analysis)	[3,9,21,22,49,51]
Invasiveness	High invasiveness (lumbar puncture); limited tolerability in the elderly	Minimally invasive (intravenous tracer injection); high cost and limited availability	Minimal invasiveness (blood draw); high accessibility	[3,12,47,49,51,52,53]
Key analytes measured	Aβ42 (reduction), Aβ42/Aβ40 ratio, t-tau, p-tau181, p-tau217, p-tau231	Aβ radioligands (e.g., florbetapir); tau tracers (e.g., [18F]Flortaucipir)	Aβ42/Aβ40 ratio, p-tau181, p-tau217, p-tau231, NfL	[11,26,47,52,53]
Pathology detected	Amyloidopathy, tauopathy, neuronal injury	Direct visualization of amyloid and tau aggregates	Peripheral surrogates of central pathology	[9,26,47,52,53,55,56]
Diagnostic value	Gold standard biochemistry; high sensitivity and specificity	Established imaging; gold standard; high spatial resolution and longitudinal assessment	High screening accuracy (AUC > 0.88)	[9,21,22,55,56,57]
Represented change	Reflection of Aβ sequestration, and tau hyperphosphorylation	Direct quantification of pathological burden in vivo	Peripheral reflection of cerebral pathology	[3,9,21,22,49,51]
Clinical readiness	Routine use in specialized centers	Clinical use in specialized centers; widely applied in research and trials	Early clinical implementation; limited FDA-cleared assays available	[9,15,21,46,55,58,59,60]

Aβ—amyloid-β; AUC—area under the curve; CSF—cerebrospinal fluid; NfL—neurofilament light chain; PET—positron emission tomography; p-tau—phosphorylated tau; t-tau—total tau.

.

The plasma Aβ42/Aβ40 ratio shows a strong and reproducible correlation with cerebral amyloid burden as assessed by amyloid PET imaging. This association has been consistently demonstrated in large, multicenter cohorts, including ADNI, BioFINDER, and AIBL, which reported high diagnostic accuracy with area under the receiver operating characteristic curve values exceeding 0.88 [9,55,56]. Importantly, these findings indicate that plasma amyloid biomarkers can predict brain amyloid pathology up to several years—often as early as eight years—before the onset of clinically detectable AD [9,55].

NfL has also emerged as a robust and versatile blood-based biomarker of axonal injury across a broad range of neurological conditions [9].

Following neuronal damage, NfL is released into the circulation, and its plasma concentration correlates closely with the severity and progression of neurodegeneration. Although NfL lacks disease specificity—being elevated in disorders such as AD nontemporal dementia, multiple sclerosis, and traumatic brain injury—its prognostic value in plasma is substantial [61,62,63]. Consequently, current research increasingly focuses on combining NfL with disease-specific markers, such as Aβ and p-tau, to enhance diagnostic precision and support multimodal biomarker panels for personalized disease monitoring [9,61].

Recent advances in blood-based p-tau biomarkers, particularly p-tau181, p-tau217, and p-tau231, represent a major breakthrough in the diagnostic and prognostic assessment of AD [9,10]. These markers, together with emerging indicators of neurodegeneration and glial activation, provide a minimally invasive means of capturing core AD pathology and may substantially influence future diagnostic algorithms [10,54,64].

Elevated plasma p-tau concentrations show strong concordance with cerebral amyloid plaque burden and tau aggregation, as quantified by PET imaging and confirmed by post-mortem analyses [54]. Longitudinal studies further demonstrate progressive increases in plasma p-tau levels in cognitively unimpaired but amyloid-positive individuals, in contrast to relative stability observed in amyloid-negative subjects. This dynamic trajectory underscores the utility of plasma p-tau as a sensitive indicator of disease progression along the AD continuum [10,54].

Despite these advances, widespread clinical implementation of blood-based biomarkers remains contingent upon further analytical harmonization, establishment of standardized clinical cut-off values, and mitigation of confounding biological variability. Addressing these challenges is essential to enhance the robustness, reproducibility, and clinical reliability of plasma biomarkers and to facilitate their integration into routine diagnostic pathways for AD [55,56]. A schematic overview of current molecular diagnostic modalities in AD is presented in Figure 5.

Importantly, recent regulatory milestones have substantially advanced the clinical translation of blood-based biomarkers in AD. In May 2025, the U.S. Food and Drug Administration (FDA) cleared the Lumipulse^® G pTau217/β-Amyloid 1–42 Plasma Ratio test—the first blood-based in vitro diagnostic assay authorized to aid in the diagnosis of AD in adults aged 55 years and older presenting with cognitive impairment. This decision represents a pivotal step toward integrating minimally invasive plasma biomarker testing into routine diagnostic workflows as an alternative to cerebrospinal fluid analysis and PET imaging [58,59].

Subsequently, the Elecsys^® pTau181 plasma assay also received FDA clearance for use as an aid in the evaluation of patients with cognitive decline in primary care settings, further expanding access to blood-based molecular diagnostics [60].

It is essential to distinguish these regulatory-approved diagnostic tools from the numerous plasma biomarkers that remain designated for research use only. While many assays targeting Aβ ratios, phosphorylated tau isoforms, and neurofilament light chain demonstrate strong diagnostic performance in research and clinical trial settings, only a limited number have undergone the rigorous analytical validation and regulatory review required for clinical authorization. This distinction is critical when assessing the current readiness and real-world applicability of molecular testing in AD.

Beyond regulatory approval, large-scale cohort studies further support the practical implementation of plasma biomarkers within structured diagnostic pathways. In particular, Therriault et al. (2024) [65] demonstrated in a multicenter clinical study that plasma phosphorylated tau assays can be incorporated into stepwise diagnostic algorithms to improve diagnostic accuracy while reducing reliance on more invasive and costly procedures, such as cerebrospinal fluid analysis and PET imaging. Their findings highlight the value of risk-adjusted, biomarker-guided strategies, in which blood-based testing serves as an initial screening tool to stratify patients according to the likelihood of underlying amyloid pathology, thereby optimizing referral for confirmatory imaging or CSF testing [65].

Similarly, Ferreira et al. (2023) [66] reported that combined profiling of plasma biomarkers—including p-tau181, glial fibrillary acidic protein (GFAP), NfL, and the Aβ42/Aβ40 ratio—improves the identification of individuals along the AD continuum in population-based settings. In this study, reduced plasma Aβ42/Aβ40 ratios were associated with cerebral amyloid deposition and were detectable prior to overt clinical decline, whereas elevations in NfL and GFAP in the absence of amyloid abnormalities were more frequently observed in non-AD neurodegenerative conditions. These findings support the use of multimodal plasma biomarker panels to enhance diagnostic specificity and enable cost-effective, minimally invasive screening strategies compared with lumbar puncture or PET imaging [66].

Such structured implementation frameworks underscore the emerging role of plasma biomarkers not only as investigational tools but as increasingly actionable components of real-world clinical decision-making, particularly in memory clinics and primary care settings.

Together, regulatory progress and robust cohort-based validation support the transition of plasma biomarkers from research-oriented assays toward scalable elements of precision-oriented diagnostic pathways in AD.

4. New Opportunities in Molecular Testing and Its Potential in the Clinical Assessment of Alzheimer’s Disease

4.1. Non-Coding RNA Perspectives in Alzheimer’s Disease Diagnosis

Despite extensive research efforts, current therapeutic strategies for AD remain largely limited to symptomatic management and are unable to halt or reverse disease progression. Consequently, increasing emphasis has been placed on early-stage intervention, as treatment initiated during the initial phases of the disease is more likely to preserve neuronal function and delay neurodegenerative processes [15,67]. This paradigm shift has intensified research aimed at identifying reliable biomarkers for early diagnosis and at developing diagnostic tools capable of detecting pathological changes before the onset of overt clinical symptoms [67].

While Aβ and p-tau represent the core pathological hallmarks of AD, growing evidence indicates that additional molecular biomarkers and cellular processes may contribute valuable diagnostic information. These include nucleic acid-based markers such as DNA and ribonucleic acid (RNA) species, as well as molecular signatures associated with oxidative stress, regulated cell death pathways, and inflammatory mechanisms, including PANoptosis [41,67]. Accurate quantification of such biomarkers in CSF or peripheral blood is essential for enabling timely diagnosis and for guiding emerging disease-modifying therapeutic strategies.

The development of highly sensitive and specific molecular assays has made it increasingly feasible to identify individuals in the asymptomatic or preclinical stages of AD. Early biomarker-based detection not only enhances diagnostic precision but also provides a critical window of opportunity for therapeutic intervention at a stage when neurodegenerative changes may still be limited and potentially modifiable [67].

Recent advances in transcriptomic profiling have enabled the comprehensive characterization of multiple non-coding RNAs (ncRNA) classes implicated in AD pathogenesis. These include micro RNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), each of which contributes to disease-related molecular networks through distinct regulatory mechanisms. Importantly, dysregulated ncRNA expression has been detected in CSF, blood, and brain tissue of individuals across different stages of AD, highlighting their potential utility as minimally invasive biomarkers [68].

In this section, we synthesize current evidence on the diagnostic relevance of ncRNAs and discuss their applicability as preclinical biomarkers for the early detection and biological stratification of AD. Particular emphasis is placed on their translational potential, analytical feasibility, and limitations that must be addressed prior to routine clinical implementation.

4.1.1. Potential of microRNA in Early Diagnosis of Alzheimer’s Disease

miRNAs are short, single-stranded ncRNA molecules, typically consisting of 21–23 nucleotides, that play a central role in post-transcriptional regulation of gene expression [4,69]. Incorporated into the RNA-induced silencing complex, miRNAs bind to complementary sequences within target messenger RNAs (mRNAs), leading to translational repression or mRNA degradation. Through these mechanisms, miRNAs regulate a wide range of cellular processes, including neuronal development, differentiation, synaptic plasticity, oxidative stress responses, and apoptosis [68,69]. Numerous miRNAs are abundantly expressed in the central nervous system and are critically involved in neuronal maturation and maintenance, highlighting their relevance to neurodegenerative disorders such as AD [70,71].

Accumulating evidence indicates that dysregulated miRNA expression contributes to AD pathogenesis and may reflect early molecular alterations preceding overt neurodegeneration. Consequently, miRNAs represent the most extensively investigated class of ncRNAs with respect to their diagnostic applicability in AD [70,71].

Altered miRNA expression profiles have been consistently reported in brain tissue, CSF, blood, and extracellular vesicles of individuals with AD compared with cognitively healthy controls [4,67,70,72,73]. These observations underscore the potential of circulating miRNAs as minimally invasive biomarkers for early and preclinical diagnosis. However, their clinical translation necessitates the development of sensitive, specific, and standardized detection methodologies [70,71].

Recent studies have demonstrated the diagnostic promise of miRNAs in the early detection of AD [70,73]. Xie et al. (2024) [73] proposed a dual-signal amplification strategy combining rolling circle amplification with endonuclease-assisted signal enhancement for miRNA analysis. Using this approach, the authors demonstrated that serum miR-206 levels could reliably distinguish AD patients from healthy individuals, highlighting the applicability of advanced amplification techniques for sensitive miRNA-based diagnostics [73].

In parallel, Duan et al. (2024) [74] investigated the diagnostic value of serum-derived exosomal miRNAs using second-generation high-throughput sequencing and quantitative real-time polymerase chain reaction. Their findings showed that altered exosomal miRNA expression profiles effectively discriminated AD patients from controls, supporting the concept of circulating exosomal miRNAs as reliable, noninvasive liquid biopsy biomarkers for early AD screening [74].

Further evidence was provided by Devara et al. (2025) [75], who analyzed the expression of miR-501-3p and miR-502-3p in CSF exosomes, serum exosomes, fibroblasts, and B lymphocytes obtained from AD patients. The consistent upregulation of these miRNAs across multiple biological matrices, their strong correlation with pathological features such as Aβ burden, and their origin from key affected brain cell types (neurons and astrocytes) identify this miRNA pair as a particularly promising diagnostic panel. Importantly, these findings suggest the feasibility of a blood-based assay that reflects the severity of AD-related neuropathology [75].

Similarly, Sbriscia et al. (2025) [76] evaluated a panel of five miRNAs—miR-23a-3p, miR-223a-3p, miR-100-5p, miR-132-3p, and miR-212—and identified miR-132-3p as a particularly strong candidate biomarker. The successful isolation of neuron-derived extracellular vesicles and the detection of reduced miR-132-3p levels, even in mild AD, emphasize its relevance to the earliest stages of neurodegeneration and its potential utility as a preclinical biomarker [76].

Despite these findings, further large-scale, multicenter studies are required to validate the diagnostic accuracy, reproducibility, and clinical applicability of miRNA-based biomarkers. Standardization of analytical platforms, normalization strategies, and clinically meaningful cut-off values will be essential before miRNA profiling can be integrated into routine diagnostic workflows for AD.

Among the investigated candidates, miR-132-3p and the miR-501-3p/miR-502-3p pair appear particularly promising due to their reproducible alterations across multiple biological matrices and early-stage disease contexts. Nevertheless, their clinical implementation remains at the validation stage, and additional prospective studies are necessary to confirm their robustness and generalizability.

4.1.2. Potential of Long Non-Coding RNA in the Early Diagnosis of Alzheimer’s Disease

lncRNAs represent a heterogeneous class of non-coding transcripts exceeding 200 nucleotides in length that exert diverse regulatory functions across multiple levels of gene expression [77,78].

lncRNAs modulate epigenetic regulation, transcription, post-transcriptional processing, and translation through interactions with DNA, RNA, and proteins. In particular, they influence chromatin remodeling, transcriptional activation or repression, mRNA stability, and the activity of translational machinery [77,79].

Given their extensive involvement in regulatory networks within the CNS, lncRNAs are increasingly recognized as important contributors to AD pathophysiology. Recent evidence suggests that dysregulated lncRNA expression may serve as a source of clinically relevant biomarkers for AD [79,80].

Several studies have demonstrated that plasma levels of the lncRNA BACE1 antisense transcript (BACE1-AS) are significantly elevated in individuals with AD compared with cognitively healthy controls. Importantly, increased BACE1-AS expression correlates positively with the severity of cognitive impairment and exhibits high diagnostic specificity, reported to reach approximately 88%, supporting its potential utility as a blood-based biomarker for AD [79,81,82].

Further support for the diagnostic relevance of circulating lncRNAs was provided by Khodayi et al. (2022) [83], who investigated plasma lncRNA expression profiles in AD patients using a multi-step screening and validation approach. Among 90 candidate lncRNAs initially screened, five were validated, with lncRNA NEAT1 and BC200 showing significantly increased expression in the plasma of AD patients compared with controls [83].

NEAT1 has been implicated in neuroinflammatory signaling and innate immune activation, particularly through modulation of the NF-κB pathway and inflammasome-related responses, which are well-established contributors to AD pathogenesis. Increased NEAT1 expression has also been associated with microglial activation and oxidative stress, further linking this lncRNA to recognized inflammatory mechanisms underlying disease progression [83,84].

BC200, a neuron-specific lncRNA, is involved in the regulation of local synaptic protein translation and synaptic plasticity. Dysregulation of BC200 has been associated with synaptic dysfunction, an early and central event in AD pathophysiology, and may contribute to impaired neuronal communication and cognitive decline [85,86].

Complementary in silico RNA sequencing analyses revealed widespread dysregulation of lncRNAs in AD, with 33 transcripts upregulated and 13 downregulated relative to healthy individuals, underscoring the complexity of lncRNA-mediated regulatory alterations in the disease [83].

In addition, Kazemi et al. (2024) [87] performed an in-depth in silico analysis combined with experimental validation in peripheral blood mononuclear cells using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Their findings identified the lncRNA RN7SK and a network of closely associated genes, including TNF, TNFAIP3, CCL3, and FLT3, as key contributors to AD pathogenesis. Notably, these genes are strongly linked to inflammatory pathways, suggesting that RN7SK may represent a molecular interface between neuroinflammation and transcriptional dysregulation in AD. Given its established roles in brain development, neuronal function, and immune regulation, RN7SK emerges as a promising candidate for inclusion in blood-based diagnostic panels [87].

Collectively, these studies support the concept that lncRNA–mRNA co-expression networks may provide a novel and accessible strategy for the noninvasive diagnosis of AD. Such approaches have the potential to overcome limitations associated with conventional blood biomarkers and to improve the accuracy of early and preclinical disease detection. However, large-scale validation and methodological standardization are still essential prerequisites for their translation into routine clinical practice.

Among circulating lncRNAs, BACE1-AS, NEAT1, and RN7SK emerge as the most clinically relevant candidates, primarily due to their association with core AD pathological pathways and detectable alterations in peripheral blood. At present, these biomarkers remain in the exploratory and early validation phase, and additional longitudinal studies are required to confirm their diagnostic robustness and clinical utility.

4.1.3. Potential of Circular RNA in Early Diagnosis of Alzheimer’s Disease

circRNAs are a class of single-stranded ncRNAs characterized by a covalently closed loop structure, most commonly generated through a back-splicing mechanism during pre-mRNA processing [67,88]. As a result of their circular conformation, circRNAs exhibit enhanced resistance to exonuclease-mediated degradation, conferring high molecular stability. Their expression is dynamically regulated and displays distinct spatiotemporal patterns across cell types, tissues, and developmental stages. circRNAs participate in diverse physiological and pathological processes by acting as miRNA sponges, modulating transcriptional and post-transcriptional regulation, and interacting with RNA-binding proteins [67,78,89].

circRNAs are particularly abundant in the central nervous system, including the cerebral cortex, hippocampus, white matter, and photoreceptor neurons, and accumulate with aging, suggesting a potential role in neuroaging. Notably, altered circRNA expression profiles have been detected in both peripheral blood and synaptic compartments of individuals with AD, where they are associated with disease onset and progression [67,78,89]. These characteristics highlight circRNAs as attractive candidates for biomarker development in neurodegenerative disorders.

Chi et al. (2024) [90] demonstrated the development of a compact yet highly informative circRNA-based biomarker panel by integrating circRNA expression profiles with three-dimensional (3D) genome conformation data. Their approach revealed that circRNAs exhibiting similar expression patterns tend to share spatial proximity within the 3D genomic architecture, enabling the selection of the most diagnostically relevant circRNAs across distinct chromosomal regions. Integration of 3D genomic information significantly enhanced the prognostic performance of the circRNA signature for AD [90].

Complementary evidence was provided by Li et al. (2020) [91], who investigated circRNA expression in CSF samples to identify novel biomarkers for AD. In an initial screening phase, 163 circRNAs exhibiting differential expression were associated with key AD-related pathways. Among these, circ-AXL, circ-GPHN, and circ-PCCA emerged as candidates with potential clinical relevance for predicting disease risk and progression. These circRNAs demonstrated associations with cognitive performance and structural brain changes, supporting their utility in clinical risk stratification and disease monitoring [91].

Further studies have confirmed the remarkable stability and CNS enrichment of circRNAs, reinforcing their suitability as molecular biomarkers for AD [92]. Importantly, several circRNAs correlate with established clinical parameters, including cognitive assessment scores such as the Mini-Mental State Examination (MMSE) and hippocampal atrophy measured by neuroimaging. Mechanistically, circRNAs implicated in AD appear to participate in key pathological processes, including regulation of apoptosis, neuroinflammation, and synaptic function, underscoring their biological relevance and potential value for early diagnosis and differential assessment [92].

Among these candidates, circ-AXL, circ-GPHN, and circ-PCCA demonstrate particularly promising diagnostic potential, especially in relation to cognitive decline and structural brain alterations. Although these molecules exhibit favorable biological characteristics, their clinical implementation remains at the research and early validation stage and requires confirmation in larger prospective and multicenter cohorts.

ncRNAs constitute a diverse and functionally complex class of RNA transcripts that do not encode proteins but exert critical regulatory roles in cellular homeostasis [68]. Given their regulatory capacity and dynamic expression patterns, ncRNAs have emerged as promising molecular indicators of pathological alterations associated with neurodegenerative diseases, including AD [67,68]. A comparison of the ncRNAs discussed above as potential diagnostic biomarkers for AD is presented in

Table 3. Comparison of non-coding RNAs in the diagnosis of Alzheimer’s disease.

Feature	miRNA	lncRNA	circRNA	References
Structure	Single-stranded, 21–23 nucleotides	>200 nucleotides; linear transcripts lacking an open reading frame	Covalently closed loop structure	[67,79]
Mechanism of action	Regulate post-transcriptional gene expression by binding to target mRNAs	Regulate gene expression as molecular scaffolds, miRNA sponges, and chromatin modifiers	Act as miRNA sponges, regulators of gene expression, and splicing modulators	[67,72,79]
Impact on Aβ	Regulate Aβ levels mainly via modulation of BACE1 activity and APP processing	Major modulators of amyloid plaque formation, primarily through regulation of BACE1 expression	Function as molecular sponges sequestering deleterious miRNAs, thereby indirectly protecting neurons	[4,67,70,79]
Impact on p-tau	Modulate formation of pathological neurofibrillary tangles	Control tau stability and translation, preventing or promoting toxic aggregation	Indirect regulation of tau pathology via miRNA sequestration	[67,79]
Selected potential biomarkers	miR-501-3p, miR-502-3p, miR-132-3p	RN7SK, NEAT1, BC200	circ-AXL, circ-GPHN, circ-PCCA	[75,76,83,87,91]
Sample source	Plasma, serum whole blood, CSF	Plasma, whole blood	CSF	[73,74,75,76,83,87,91]

Aβ—amyloid-β, APP—amyloid precursor protein, BACE1—β-site amyloid precursor protein cleaving enzyme 1, circRNA—circular RNA, CSF—cerebrospinal fluid, lncRNA—long non-coding RNA, miRNA—microRNA.

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4.1.4. Limitations and Standardization of ncRNA-Based Diagnostics

Despite the growing body of evidence supporting the diagnostic relevance of ncRNAs in AD, several limitations currently restrict their translation into routine clinical practice. One of the primary challenges is the substantial heterogeneity of ncRNA expression profiles reported across studies, arising from differences in patient cohorts, disease stage, biological matrices, and analytical methodologies [67,68,80]. Variability in sample collection, processing, and storage further contributes to inconsistent results and limits cross-study comparability [67,83].

Analytical standardization represents an additional critical obstacle. ncRNA detection relies on diverse platforms, including qRT-PCR, microarrays, and next-generation sequencing, each characterized by distinct sensitivities, normalization strategies, and susceptibility to technical bias [68,77,78]. The lack of universally accepted reference controls, normalization procedures, and validated clinical cut-off values complicates the interpretation of ncRNA expression levels and hampers their integration into standardized diagnostic workflows [67,80,83].

Biological complexity also poses significant challenges. ncRNA expression is influenced by age, sex, genetic background, comorbidities, medication use, and systemic inflammatory states, all of which may confound disease-specific molecular signatures [28,29,67]. In peripheral biofluids, additional variability arises from the contribution of non-neuronal tissues and from differences in extracellular vesicle release, stability, and clearance mechanisms [74,76]. These factors necessitate careful biomarker selection and support the development of multi-marker panels rather than reliance on single ncRNA species [70,74,76].

Finally, most ncRNA-based diagnostic candidates have been evaluated in relatively small, cross-sectional cohorts, with limited longitudinal validation [67,80,83]. Large-scale, multicenter studies employing standardized protocols and diverse populations are essential to establish clinical robustness, reproducibility, and predictive value across the AD continuum [21,55,56]. Addressing these limitations through coordinated efforts in assay harmonization, reference standard development, and regulatory validation will be pivotal for advancing ncRNA-based diagnostics from exploratory research tools to clinically actionable biomarkers.

4.2. The Potential of Nanotechnology in the Early Diagnosis of Alzheimer Disease

Nanoscience and nanotechnology have driven transformative advances across biomedicine. This rapidly developing field focuses on the design of structures, devices, and systems that exhibit unique physicochemical and functional properties arising from nanoscale dimensions [93]. In the context of AD, nanotechnology has introduced novel opportunities for early diagnosis through the development of highly sensitive and selective molecular detection platforms [14].

The diagnostic potential of nanotechnology is largely attributed to the distinctive characteristics of nanomaterials, including their small size and high surface-to-volume ratio. These features facilitate efficient interactions with biological systems and enable the construction of advanced biosensors capable of detecting low-abundance biomarkers in complex biological matrices [14]. Nanoparticles, nanoprobes, and surface-functionalized nanomaterials have therefore been extensively explored as components of diagnostic systems targeting key AD-associated biomarkers, including Aβ, tau species, inflammatory mediators, and ncRNAs [94,95,96,97].

The integration of nanotechnology with molecular biology has enabled the development of multifunctional diagnostic platforms that combine high analytical sensitivity with multiplexing capabilities. For example, Park et al. (2019) [98] developed a diagnostic approach employing fluorescent nanoparticles (quantum dots, QD565 with red fluorescence) as molecular imaging sensors for the simultaneous detection of selected miRNAs and inflammatory markers associated with AD. This strategy allows concurrent visualization of multiple molecular targets, thereby enhancing diagnostic accuracy and supporting early disease stratification [98].

Further translational progress was demonstrated by Lee et al. (2023) [99], who reported the clinical validation of an advanced diagnostic platform termed surface-functionalized nanomaterial-based immunofluorescence assay (SNAFIA). In this study, antibody-immobilized nanoparticles were employed, including iron(II,III) oxide core nanoparticles coated with a silica (SiO₂) shell and polyethylene glycol (PEG), as well as antibody-functionalized nanosensors composed of a poly(D,L-lactide) core and an outer PEG shell. Signal generation in the SNAFIA system is based on Förster resonance energy transfer (FRET). Fluorescence is produced by a dye pair encapsulated within a hydrophobic polymer membrane, consisting of DiO (3,3′-dioctadecyloxycarbocyanine perchlorate) and DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate). This nanoparticle-based platform enables non-invasive detection of adenylyl cyclase-associated protein 1 (CAP1) in tear fluid, providing a novel liquid biopsy approach for AD and mild cognitive impairment. Importantly, SNAFIA demonstrated high analytical sensitivity, strong diagnostic performance, and a significant negative correlation with MMSE scores, supporting its potential utility for early screening and longitudinal monitoring of cognitive decline [99]. A schematic representation of the nanotechnology-based diagnostic platforms discussed above is presented in Figure 6.

Collectively, these studies illustrate the capacity of nanotechnology-based diagnostic tools to achieve ultra-sensitive, minimally invasive detection of AD-related molecular signatures. Nevertheless, widespread clinical implementation will depend on further validation in large-scale cohorts, standardization of nanomaterial synthesis and assay protocols, and rigorous assessment of reproducibility and cost-effectiveness. Addressing these challenges will be essential for translating nanotechnology-enabled diagnostics from experimental platforms into clinically viable tools for early AD detection.

4.3. Other Promising Techniques for Molecular Diagnosis of Alzheimer’s Disease

4.3.1. Induced Pluripotent Stem Cell-Based Models

In addition to ncRNA-based diagnostics and nanotechnology-enabled platforms, several other emerging approaches show promise for improving the early molecular diagnosis of AD. These strategies aim to capture early pathological changes through patient-specific cellular models, peripheral molecular signatures, and regulated cell death pathways, thereby complementing established biomarker frameworks [100,101,102,103,104,105].

Induced pluripotent stem cell (iPSC) technology represents a powerful tool for modeling neurodegenerative diseases at the cellular level using patient-derived cells [100]. A key advantage of iPSCs lies in their ability to retain the complete genetic background of the donor, enabling accurate in vitro recapitulation of disease-specific molecular and cellular phenotypes. This approach is particularly valuable in neurodegenerative disorders, where access to primary neuronal tissue is highly limited and invasive diagnostic procedures remain a major constraint [104,105].

iPSC-derived neural progenitor cells and neurons have been increasingly applied to investigate disease mechanisms, identify molecular biomarkers, and assess responses to experimental therapies. Cavalli et al. (2020) [106] demonstrated the utility of iPSC-based neuronal models as a less invasive platform for biomarker discovery in AD. By analyzing whole-transcriptome datasets derived from patient-specific iPSC-derived neurons, the authors identified gene expression signatures capable of distinguishing AD samples from healthy controls. Importantly, these signatures enabled improved segregation of samples originating from the entorhinal cortex, a region affected early in AD, indicating that iPSC-based models can faithfully reflect early transcriptional alterations underlying disease progression. Although these findings are promising, further validation in larger cohorts, inclusion of additional cell types such as astrocytes and microglia, and integration of epigenetic analyses are required to confirm their diagnostic applicability [106].

4.3.2. Oxidative Stress-Related Biomarkers

Compelling evidence indicates that molecular alterations associated with oxidative stress precede the onset of clinical dementia by several decades [2]. Although the precise temporal sequence of these early changes remains incompletely understood, oxidative imbalance is recognized as a core contributor to AD pathophysiology. Importantly, several studies suggest that oxidative stress-related alterations in the brain are mirrored in peripheral tissues, particularly in circulating blood cells, making them attractive targets for minimally invasive biomarker development [101,102].

Polak-Szabela et al. (2021) [107] demonstrated that the combined assessment of oxidative stress markers and neuropsychological testing improves diagnostic accuracy in AD. Notably, the integration of superoxide dismutase activity with performance on the Rey Auditory Verbal Learning Test, particularly delayed recall, yielded high diagnostic value in differentiating AD patients from controls [107].

In addition to enzymatic antioxidant markers, products of oxidative damage have also been investigated. Elevated levels of lipid peroxidation markers, such as malondialdehyde and F2-isoprostanes, have been consistently reported in AD and are considered reliable indicators of systemic oxidative injury, further supporting the role of oxidative stress as an early and measurable component of disease progression [16,102].

Chiricosta et al. (2022) [108] applied machine learning algorithms to high-throughput transcriptomic data derived from peripheral blood samples and identified a molecular signature involving mitochondrial ribosomal proteins and electron transport chain subunits. These findings underscore mitochondrial dysfunction and oxidative stress as central drivers of AD pathology and suggest that redox-related transcriptomic patterns may serve as early peripheral biomarkers [108].

Furthermore, Peña-Bautista et al. (2019) [109] used ultra-high-performance liquid chromatography coupled with tandem mass spectrometry to evaluate oxidative stress biomarkers in urine samples from individuals with MCI due to AD. They reported significantly elevated levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and an increased 8-OHdG/2-dG ratio, reflecting DNA oxidative damage. Importantly, these markers correlated with established CSF biomarkers such as Aβ and p-tau, highlighting the potential utility of urine-based oxidative profiling as a non-invasive tool for early diagnosis and disease monitoring [109].

4.3.3. Blood Transcriptomics and Multi-Omics Approaches

Peripheral blood transcriptome analysis has emerged as another promising strategy for identifying molecular signatures associated with AD. Given that gene expression changes in blood cells occur early and persist throughout disease progression, transcriptomic profiling may provide insight into both pathophysiological mechanisms and diagnostic biomarkers [110,111,112]. Advances in high-throughput sequencing and genomic technologies now enable large-scale analysis of gene expression patterns and genetic variation across thousands of individuals, substantially improving the resolution of AD risk assessment and disease stratification [110,113].

Zhong et al. (2024) [111] demonstrated that blood-based transcriptomic signatures can effectively distinguish individuals with AD from cognitively healthy controls. Due to the relative ease and minimal invasiveness of blood sampling, transcriptomics holds considerable potential as a diagnostic and monitoring tool, supporting early intervention and the development of precision medicine approaches for AD [111].

4.3.4. Regulated Cell Death Pathways and PANoptosis Signatures

Neuronal loss in AD involves multiple regulated cell death mechanisms, including apoptosis, pyroptosis, and necroptosis [103]. Recent studies have revealed extensive crosstalk among these pathways, leading to the concept of PANoptosis, a coordinated and inflammatory form of programmed cell death mediated by the assembly of a multiprotein complex termed the PANoptosome [103,114,115].

PANoptosis represents an integrated inflammatory cell death program that synchronizes molecular components of apoptosis, pyroptosis, and necroptosis into a unified regulatory framework. Unlike classical cell death pathways, which operate through relatively distinct signaling cascades, PANoptosis is characterized by simultaneous activation and functional interaction of multiple effector mechanisms. Central to this process is the PANoptosome, a multiprotein signaling platform that integrates upstream sensors, adaptor proteins, and executioner molecules derived from the three canonical pathways [103,114,115].

Within this framework, apoptosis-related mediators include caspase-8 and caspase-3; pyroptotic signaling involves inflammasome components such as NLRP3 and gasdermin D (GSDMD), whereas necroptosis is primarily mediated by receptor-interacting protein kinases RIPK1 and RIPK3 together with mixed lineage kinase domain-like protein (MLKL) [103,115]. Increasing evidence suggests that coordinated activation of these pathways contributes to Aβ accumulation, tau pathology, neuroinflammation, and progressive neuronal degeneration in AD [114,116,117].

Wang et al. (2024) further proposed PANoptosis as a key pathological mechanism in AD and identified a gene signature including SLC17A7, SNAP25, GAD1, and GRIN2A, associated with early disease stages [117]. Importantly, these genes do not represent structural components of the PANoptosome itself. Rather, they encode proteins involved in synaptic transmission and neuronal excitability SLC17A7 as a vesicular glutamate transporter, SNAP25 in synaptic vesicle fusion, GAD1 in GABA synthesis, and GRIN2A as a subunit of the NMDA receptor. Their dysregulation likely reflects neuronal vulnerability and synaptic dysfunction occurring in the context of PANoptosis-associated neuroinflammation, rather than direct execution of the cell death machinery [114,117,118].

Given that PANoptosis remains a relatively recent conceptual framework, further mechanistic and translational studies are required to clarify its precise contribution to AD pathogenesis and to determine its potential utility as a diagnostic or therapeutic target [117].

4.4. Barriers to Clinical Implementation of Molecular Diagnostics in Alzheimer’s Disease

Despite the rapid development of molecular diagnostic tools for AD, their translation from research settings into routine clinical practice remains limited. This gap results from a convergence of analytical, biological, clinical, and regulatory challenges that collectively hinder the widespread implementation of molecular testing in real-world diagnostic workflows [10,15,21].

One of the principal obstacles is the lack of analytical and methodological standardization across laboratories. This limitation is particularly evident for CSF and blood-based biomarkers, including p-tau isoforms (p-tau181, p-tau217, p-tau231), Aβ ratios, and emerging ncRNA signatures. Variability in pre-analytical factors—such as sample collection procedures, storage conditions, assay platforms, and calibration standards—leads to substantial inter-laboratory differences in biomarker quantification [11,44]. Consequently, result reproducibility and data comparability across studies and clinical centers remain suboptimal.

Biological heterogeneity further complicates the clinical interpretation of molecular biomarkers. Biomarker concentrations are influenced by age, sex, genetic background (including APOE genotype), and the presence of comorbid conditions such as metabolic disorders, vascular disease, or systemic inflammation [10,44]. For blood-based biomarkers, additional confounding factors arise from peripheral production, altered clearance mechanisms, and variability in blood–brain barrier integrity, which may obscure the relationship between peripheral biomarker levels and central nervous system pathology [9,44]. These sources of biological variability highlight the need for population-adjusted reference ranges and disease stage-specific thresholds.

Another critical limitation is the absence of universally accepted clinical cut-off values that clearly distinguish pathological from non-pathological biomarker profiles. While many molecular biomarkers demonstrate high diagnostic accuracy at the group level, their application at the individual patient level—particularly during preclinical or mildly symptomatic stages of AD—remains challenging [21,37]. Overlap between biomarker changes associated with normal aging, early AD pathology, and other neurodegenerative disorders increases the risk of diagnostic uncertainty, including false-positive and false-negative classifications [10,15].

Economic and infrastructural constraints also restrict clinical adoption. Advanced imaging modalities such as amyloid- and tau-PET, although highly informative, are costly, resource-intensive, and largely confined to specialized centers [15,46]. Similarly, ultra-sensitive analytical platforms required for reliable plasma biomarker detection, including single-molecule array (Simoa) and mass spectrometry-based techniques, are not yet routinely available in standard clinical laboratories [9,52]. These limitations underscore the need for simplified, cost-effective assays that preserve diagnostic reliability while enabling large-scale screening and longitudinal monitoring.

Regulatory and clinical validation barriers represent an additional challenge. Many emerging molecular approaches—particularly those based on ncRNAs, transcriptomic signatures, and nanotechnology-enabled biosensors—lack large-scale prospective validation in diverse patient populations [41,67]. Moreover, regulatory approval requires robust evidence demonstrating clinical utility, reproducibility, and a measurable impact on patient management. Until such evidence is established, most novel molecular diagnostic tools are likely to remain confined to research and clinical trial settings [15,21].

In particular, ncRNA-based diagnostic approaches currently lack clearly defined regulatory pathways and standardized validation frameworks. The absence of harmonized regulatory guidelines for RNA-based assays, together with insufficient multicenter prospective validation, represents a significant barrier to their clinical translation. Establishing consensus-driven validation criteria and fostering international collaborative studies will be essential to ensure analytical reliability, clinical utility, and regulatory approval.

Addressing these limitations will require coordinated international efforts focused on assay harmonization, development of certified reference materials, longitudinal multicenter validation studies, and systematic integration of molecular biomarkers into standardized diagnostic algorithms. Only through such translational and collaborative strategies can molecular diagnostics evolve from advanced research tools into reliable components of routine clinical assessment and monitoring of AD. Together, these challenges underscore the need for integrative, multimodal diagnostic strategies that balance analytical rigor with clinical feasibility, thereby providing a foundation for future precision medicine approaches in AD.

5. Conclusions

AD remains one of the most prevalent and challenging neurodegenerative disorders worldwide, associated with profound medical, social, and economic consequences. Despite decades of intensive research, current therapeutic strategies remain largely symptomatic and do not halt or reverse disease progression. In this context, early and biologically grounded diagnosis represents one of the most promising avenues for improving patient outcomes by enabling timely intervention before irreversible neurodegeneration occurs.

Recent advances in molecular diagnostics have substantially reshaped the contemporary framework of AD assessment. CSF biomarkers and PET imaging currently represent the most validated approaches for in vivo detection of amyloid and tau pathology, providing a biologically defined framework for diagnosis. However, their invasiveness, high cost, and limited accessibility restrict widespread implementation in routine clinical practice. Consequently, increasing emphasis has been placed on blood-based biomarkers, which offer minimally invasive, scalable, and cost-effective alternatives for screening and longitudinal disease monitoring.

Beyond established protein biomarkers, emerging molecular strategies, including ncRNAs, transcriptomic signatures, nanotechnology-enabled biosensors, and patient-specific iPSC-derived cellular models, further expand the diagnostic horizon of AD. These approaches provide complementary insights into disease biology and hold promise for detecting pathological changes at the earliest, preclinical stages. Nevertheless, their translation into clinical practice remains constrained by methodological heterogeneity, limited large-scale validation, and the absence of standardized analytical and clinical frameworks.

As highlighted throughout this review, the primary challenge facing molecular diagnostics in AD is no longer the identification of novel candidate biomarkers, but rather their reliable and reproducible implementation in real-world clinical settings. Addressing barriers related to assay standardization, clinical cut-off definition, cost, infrastructure, and regulatory validation is essential for transforming advanced molecular tools into clinically actionable diagnostics.

Future progress will depend on coordinated international efforts aimed at harmonizing analytical methodologies, establishing robust reference standards, and integrating molecular biomarkers into standardized diagnostic algorithms. Such translational strategies are critical for bridging the gap between research innovation and clinical practice.

Ultimately, the successful implementation of molecular diagnostics may enable the detection of AD at asymptomatic or minimally symptomatic stages, fundamentally altering disease management and opening new opportunities for effective, personalized intervention.