# The role of S100B protein as a diagnostic biomarker for brain injury

Nataliia Gnyliukh, James Wei, Winfried Neuhaus, Rabah Boukherroub, Sabine Szunerits

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Nataliia Gnyliukh, James Wei, Winfried Neuhaus, Rabah Boukherroub, Sabine Szunerits. The role of S100B protein as a diagnostic biomarker for brain injury. Sensing and Bio-Sensing Research, 2025, 50, 10.1016/j.sbsr.2025.100888. hal-05308229

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# The role of S100B protein as a diagnostic biomarker for brain injury

Nataliia Gnyliukh<sup>a,*</sup>, James Wei<sup>b</sup>, Winfried Neuhaus<sup>a,c</sup>, Rabah Boukherroub<sup>d</sup>, Sabine Szunerits<sup>a,d,*</sup>

## ARTICLE INFO

**Keywords:**

S100B protein

Biomarker

Diagnostics

Traumatic brain injury

Neurological diseases

## ABSTRACT

S100B is a brain protein, produced mainly by astrocytes, that indicates neurological injury by leaking into the bloodstream, cerebrospinal fluid (CSF), and urine. Elevated levels of S100B in blood and CSF serve as a marker for acute neural injury such as traumatic brain injury (TBI) and stroke. The extent of S100B elevation can help predict clinical outcomes after brain injury and monitor the effectiveness of treatment. Measuring S100B levels over time, or using a trajectory analysis, can provide more reliable information about injury progression and help predict secondary injuries. In order to predict clinical outcomes after brain injury, as well as to provide a basis for appropriate treatment and indicate treatment success, it is imperative to have appropriate analytical tools at hand. In this review, we focus on the research progress of S100B as an “alert” signalling molecule in the connection of brain injuries and critically assess current diagnostic assays for S100B, including Enzyme-Linked Immunosorbent Assay (ELISA) kits, biosensors, and point-of-care (PoC) devices.

## 1. Introduction

Neurological conditions are a leading cause of illness and disability globally, with over 3.4 billion people worldwide affected by a neurological condition, including Alzheimer's disease (AD) and other dementias, stroke and traumatic brain injury (TBI) [1]. These conditions have surpassed cardiovascular diseases as the leading cause of ill health and disability globally, according to a study published in The Lancet Neurology [2]. A global ageing population and increased exposure to environmental, metabolic, and lifestyle risk factors are contributing to the rising burden of these conditions. In the case of TBI, the number of global prevalent cases exceeded 38 million, caused primarily by falls, road injuries, and exposure to mechanical forces, such as falls or sports

injuries [3]. Falls are a particularly significant cause, accounting for many hospitalisations, while exposure to mechanical forces from explosions can also lead to TBI, especially in a military setting. In the case of stroke, around 12 million new cases are reported annually worldwide, with globally more than 94 million people having suffered from stroke at some point in their lives [4]. While stroke remains the leading cause of death and disability, scientists predict a 50 % increase in stroke-related deaths by 2050, with numbers reaching up to 9.7 million deaths annually.

It was in the 1990s, when S100B, known as a potential melanoma biomarker previously, was identified as a versatile screening, monitoring and prediction biomarker in both cerebrospinal fluid (CSF) and blood for the management of acute brain injury and neural distress

<footnote><sup>a</sup> Laboratory for Life Sciences and Technology (LiST), Faculty of Medicine and Dentistry, Danube Private University (DPU), Viktor-Kaplan-Straße 2, Geb. E, 2700 Wiener Neustadt, Austria</footnote>

<footnote><sup>b</sup> LynxSurfBio (LSB), Velocity - Innovation Arena, 280 Joseph Street, Suite 2000, Kitchener N2G 4Z5, Ontario, Canada</footnote>

<footnote><sup>c</sup> AIT - Austrian Institute of Technology GmbH, Center Health and Bioresources, Competence Unit Molecular Diagnostics, 1210 Vienna, Austria</footnote>

<footnote><sup>d</sup> University Lille, CNRS, Univ. Polytechnique, Hauts-de-France, UMR 8520 - IEMN, F 59000 Lille, France</footnote>

<footnote>Abbreviations: AD, Alzheimer's disease; AI, Artificial Intelligence; Au NPs, Gold nanoparticles; Au IDEs, Interdigitated gold electrodes; Aβ42, Amyloid-β 1-42; BBB, Blood-brain barrier; CI, Confidential interval; CSF, Cerebrospinal fluid; CT, Computed tomography; DAMP, Damage-associated molecular pattern; DPM-Cu(II), Dipyromethene complexed with Cu(II) ions; ELISA, Enzyme-linked Immunosorbent Assay; ER, Endoplasmic reticulum; FDA, Food and Drug Administration; GAD, Generalized anxiety disorder; GFAP, Fibrillary acidic protein; HAuNPs, Hollow gold nanoparticles; ISF, Interstitial fluid; ITO, Indium tin oxide; K<sub>D</sub>, Dissociation constant; LFA, Lateral flow assay; LFIA, Lateral flow immunoassay; LoD, Limit of Detection; MBT, Mercaptobutanol; mTBI, Mild Traumatic Brain Injury; MUC, Mercaptoundecanoic acid; MVB, Multivesicular body; NFL, National football league; NPV, Negative predictive values; NSE, Neuron-specific enolase; PLFS, Paper lateral flow assay; PoC, Point-of-care; RAGE, Receptor for advanced glycation end products; rGO, Reduced graphene oxide; SERS, Surface-enhanced Raman scattering; SPR, Surface plasmon resonance; TBI, Traumatic brain injury; TJ, Tight junctions.</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: nataliia.gnyliukh@dp-uni.ac.at (N. Gnyliukh), sabine.szunerits@univ-lille.fr (S. Szunerits).</footnote>
[5,6]. The strong correlation between elevated S100B levels and abnormal findings on computer tomography (CT) scans was a key reason for S100B's established prognostic value in TBI outcomes. Conversely, low S100B levels can predict normal CT findings, making it useful as a screening tool, potentially decreasing the number of unnecessary CT scans. While S100B has been shown to be a reliable indicator of damage, its role in both predicting outcome and detecting immediate structural abnormalities is still being refined [7].

For accurately measuring S100B in biological fluids, supporting its use in predicting patient outcomes and guiding treatment, Enzyme-Linked Immunosorbent Assay (ELISA) concepts have become an essential part. Such conventional diagnostic platforms lack point-of-care (PoC), simple, economical, prompt and personalized detection features [8]. The growing sensitivity and accuracy of alternative detection schemes, notably biosensors, are refining the temporal profile of S100B concentration fluctuations in blood and CSF after trauma for ultra-early diagnostics. While in recent years, numerous excellent reviews focused on the topic of S100B biology and use as a biomarker in the context of various diseases have been published [9–13], this review presents a comprehensive and up-to-date discussion of the technical capabilities and clinical translation of ELISA and biosensor-based detection methods for S100B in the context of neurological diagnostics. These diagnostic tests take advantage of the high stability of S100B, being resistant to

degradation during sample handling and storage, contributing to its reliability in clinical assays [14]. The required analytical sensitivities for S100B and the ability to differentiate between healthy and diseased patients are currently set at serum S100B levels between 20 and 150 pg mL⁻¹ [15,16], while the levels in CSF can vary between 75 and 810 pg mL⁻¹ [17–19]. However, it needs to be mentioned that some of the studies also reported concentrations of S100B in CSF in the control group as high as 2200 pg mL⁻¹, which is considerably high [20]. The wider concentration range indicates that S100B levels can fluctuate, depending on stress and other external factors. This also brings another level of complexity to draw conclusions based solely on the S100B levels in CSF. Therefore, any comparison results between control and diseased/treated groups of S100B CSF concentrations need to be carefully analysed. Before discussing the analytical S100B concepts, a better understanding of the physiological and pathological specifications of S100B proteins in a human organism is outlined.

## 2. Biological functions and cellular distribution of S100B

S100B [21,22] exists predominantly as a non-covalently associated homodimer S100B(ββ) that plays crucial roles in various cellular processes, including regulating calcium homeostasis, cell proliferation, and signal transduction [23]. This dimer consists of two identical subunits,

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Different levels of S100B: (A) Schematic representation of S100B homodimer interaction with a representative target peptide in the presence of Ca²⁺ (Image references from RCSB PDB: Kilby, P.M., Vaneldik, L.J., Roberts, G.C.K. (1997) https://doi.org/10.2210/pdb1CFP/pdb; Smith, S.P., Shaw, G.S. (1998) https://doi.org/10.2210/pdb1UWO/pdb [28,29]). (B) Flow of S100B (presented in yellow) through an intact and damaged blood-brain barrier (BBB). In the case of an intact brain, S100B is mostly present in astrocytes (violet). The endothelial cells (red) are interconnected with tight junctions (TJs) preventing the passage of S100B. In the case of a damaged BBB, disruption of the endothelial layer is observed and S100B can pass endothelial layer and released into the lumen. (C) Schematic transportation pathway of S100B. Illustration of the expression of monomeric in S100B, homodimerisation forming S100B(ββ), binding to target peptide in the presence of Ca²⁺ forming a S100B(ββ)/protein complex in cytoplasm. This complex is released into the extracellular space, diffuses across the BBB into the lumen due to TJ destabilisation that occurred by the effect of inflammatory cytokines, induced via the S100B-RAGE-NFkB pathway. Abbreviations: DNA - deoxyribonucleic acid, RNA - ribonucleic acid, ER - endoplasmic reticulum, RAGE - Receptor for Advanced Glycation Endproducts, NF-κB - nuclear factor kappa-light-chain-enhancer of activated B cells, TNFα - tumour necrosis factor-alpha, IL-1β - interleukin. Created in **https://BioRender.com**. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
each with a molecular weight of 10.7 kDa, resulting in a total dimeric mass of approximately 21.4 kDa [24]. The dimeric structure is remarkably stable, with a dissociation constant ($K_D$) in the picomolar range, ensuring that under physiological conditions, the monomeric form [23] is rarely observed [25]. The dimer's stability was pointed out to be crucial for its biological activity, as it enables efficient calcium binding and subsequent interaction with target proteins (Fig. 1A). S100B has, however, also been observed as a tetramer and higher-level oligomers. Different to the S100B dimer, tetrameric and higher-order oligomers may play a role in the activation of Receptor for Advanced Glycation End Products (RAGE) (Fig. 1C) [26], with a reported neuro-protective role in Alzheimer's disease (AD) [27]. It was demonstrated that supra-stoichiometric ratios of dimeric S100B-Ca²⁺ drastically decrease (95 %) the amyloid-β 1-42 (Aβ42) peptide [27] oligomerisation rate and Aβ42 oligomer levels.

S100B possesses many important intra- and extracellular functions that, in their mechanisms, often intertwine (Table 1). One of the key physiological roles of S100B is its involvement in calcium homeostasis. As a calcium-chelating protein, S100B undergoes conformational changes upon binding calcium ions, exposing hydrophobic surfaces that facilitate interactions with target proteins [30]. Upon binding to target proteins, S100B's calcium-binding affinity increases dramatically, by as much as 200- to 400-fold. This calcium-dependent regulation allows S100B to modulate cellular processes in response to fluctuations in intracellular calcium levels [31]. Additionally, S100B has been shown to bind zinc with high affinity, suggesting a potential role in zinc homeostasis as well.

S100B's biological significance extends, however, beyond its intra-cellular functions, as it can also be secreted into the extracellular space, where it acts as a damage-associated molecular pattern (DAMP) molecule interacting with the RAGE receptor (Fig. 1C). At nanoMolar (nM) concentrations (3 nM or 30,000 pg mL⁻¹), extracellular S100B exhibits neuroprotective and neurotrophic effects, promoting neuronal survival and stimulating neurite outgrowth in cerebral cortex neurons [32]. It has been found to enhance the survival of neurons during development and stimulate both neuronal maturation and glial cell proliferation [33]. Conversely, at micromolar (µM) (3.4 µM or 4,000,000 pg mL⁻¹) levels, it can trigger inflammatory responses due to its neurotoxicity and contribute to neuronal damage [34]. This dual intra- and extracellular functionality underscores the versatility of S100B in maintaining cellular homeostasis and responding to pathological conditions. Its specific cellular distribution, primarily but not exclusively, in astrocytes, and its ability to function both intra- and extracellularly make S100B a crucial player in various physiological and pathological processes, particularly within the nervous system. S100B is also expressed in such glial cells as oligodendrocytes, Schwann cells, ependymal cells, retinal Müller cells, and enteric glial cells [9]. Even more so, only mature astrocytes that enclose blood vessels and NG2-expressing cells can express S100B [35].

Furthermore, S100B can undergo post-translational modifications, such as phosphorylation and nitrosylation, potentially modulating its functional properties. S100B has indeed been shown to inhibit the phosphorylation of various target proteins, including the tumour suppressor p53. By interacting with and inhibiting the calcium-dependent phosphorylation of p53 by protein kinase C, S100B may influence cell cycle regulation and apoptosis. This interaction has implications in cancer biology, potentially contributing to uncontrolled tumour growth in certain contexts [43].

### 3. Secretion of S100B into blood and cerebral fluid

The secretion mechanism of S100B by brain cells is a complex process with specific characteristics. S100B is constitutively secreted by astrocytes into the extracellular space under normal physiological conditions, even in the absence of specific stimuli [34]. Increased concentrations of cytosolic Ca²⁺ ions, released from the endoplasmic reticulum

<table><caption>**Table 1**
S100B biological functions, distribution, and target interactors.</caption><thead><tr><th>Function</th><th>Description</th><th>Specifications</th><th>References</th></tr></thead><tbody><tr><td>Intracellular regulation</td><td>Regulates calcium homeostasis, enzyme activities, and protein phosphorylation</td><td>Cell Cycle Regulation: S100B acts as a trans-activator that negatively regulates cell division by controlling a set of genes required for this process.<br>Cytoskeletal Organization: The S100B dimer interacts with two molecules of CAPZA1, suggesting a role in cytoskeletal regulation.<br>Enzyme Modulation: S100B interacts with and activates aldolase A and aldolase C. It inhibits phosphoglucomutase activity. S100B binds to glycogen phosphorylase, though without affecting its activity.<br>Protein Degradation: S100B binds with calcyclin-binding protein/Siah-1- interacting protein (CacyBP/SIP), an enzyme component involved in protein ubiquitination.<br>Phosphatase Regulation: S100B interacts with PPP5C (Protein Phosphatase 5, Catalytic Subunit) via its TPR repeats.<br>Transcriptional Regulation: S100B interacts with p53, p63, and p73, affecting their biological functions.<br>Cell Signalling: Acts as a paracrine/autocrine trophic factor at low concentrations. S100B interacts with AGER (Advanced Glycation End-product Receptor), suggesting a role in receptor-mediated signalling.<br>Neurite Outgrowth: S100B promotes neurite outgrowth at nM concentrations through binding to RAGE. This activates pathways like NF-κB, which are involved in inflammation and immune responses. PKC is another target of S100B, playing a role in signal transduction pathways that affect cell growth and differentiation.<br>Neuronal Survival: Supports neuronal survival at low concentrations. The Ras/MEK/ERK1/2/NF-κB pathway plays a major role in S100B/RAGE-</td><td>[31,36-39]</td></tr><tr><td>Extracellular regulation</td><td>Involved in inflammation regulation, neuroprotection & neurotoxicity</td><td></td><td>[33,34,40-42]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Function</th><th>Description</th><th>Specifications</th><th>References</th></tr></thead><tbody><tr><td></td><td></td><td>induced neuronal survival at low concentrations of S100B.</td><td></td></tr></tbody></table>

(ER), have been reported to trigger S100B secretion. Studies showed that S100B follows the alternative to the traditional endoplasmic reticulum-Golgi pathway, like secretory autophagy and Multivesicular Body (MVB)-exosome pathway, which allows cells to secrete proteins that lack the typical signal peptide required for entry into the classical secretory pathway (ER-Golgi pathway) (Fig. 1C) [44]. While S100B is mainly expressed in the brain, the presence of S100B in blood samples is not restricted to the neuronal system and was found to be produced in adipocytes as well as in melanocytes and melanoma tissue, contributing to increased serum levels [45].

Under normal conditions, S100B plays a neuroprotective role through the production of low amounts of signalling oxygen radicals that lead to the activation of the anti-apoptotic factor Bcl-2, which protects neurons from programmed cell death [6]. At low physiological concentrations, S100B interacts with cell surface receptors to stimulate several beneficial processes, like neurite outgrowth, and enhances neuronal survival, calcium homeostasis, anti-inflammatory effects, and glial cell activation [46]. It also plays a role in cell proliferation, survival, and differentiation during brain development, affecting the maturation of astrocytes, oligodendrocytes, and neurons [33].

While low concentrations of secreted S100B have protective, beneficial effects on the organism, at high concentrations, S100B can have properties of a DAMP protein [34]. These include induction of neuronal apoptosis, stimulation of pro-inflammatory cytokine production by astrocytes and microglia, activation of inducible nitric oxide synthase leading to increased neurotoxic nitric oxide levels, and disruption of intracellular calcium homeostasis. High S100B levels may finally contribute to BBB disruption and promote excessive glial activation, potentially leading to glial scar formation. The mechanism underlying this concentration-dependent switch involves different receptor interactions and signalling pathways, like RAGE, triggering inflammatory cascades. Furthermore, excessive S100B can induce oxidative stress and impair mitochondrial function, contributing to cellular damage and energy deficits [42]. Interestingly, S100B levels have lately also been used for diagnostics of the clinical severity of SARS-CoV-2 infected patients, since S100B is associated with inflammation and organ damage markers [47]. This dual nature of S100B highlights its complex role in brain physiology and pathology, emphasising the importance of tightly regulated S100B levels in maintaining neural health.

Once S100B has been released into the extracellular space, the protein diffuses towards the BBB, facilitated by its rather low molecular mass of 21 kDa. S100B is generally contained within the brain under normal circumstances [48], as it is too large to passively diffuse (paracellular transport) through the tight junctions (TJ) of the BBB, a highly selective barrier preventing most substances from freely entering or exiting the brain (Fig. 1B). Vesicular transcytosis is a process of active transportation of molecules across the BBB endothelial cells [49]. Although its levels are relatively low at the BBB, and so far, this mechanism is not well-understood for S100B specifically. Transcellular transport of S100B across the BBB via receptor-mediated transcytosis has also been suggested [42]. Brain endothelial cells express RAGE, a receptor currently believed to be involved in the transcellular transport of S100B across the BBB [48,50]. It is in addition suggested that by interacting and activating the RAGE pathway, pro-inflammatory cytokines are expressed, which in turn destabilise the tight junction integrity (Fig. 1C) [48]. On the other hand, when the integrity of the BBB is compromised due to Traumatic Brain Injury (TBI), stroke, or various neurological diseases, the temporary rupture of the TJs occurs with open pores of 20 nm, allowing the passage of large proteins [48]. Under these

conditions, S100B can easily pass. This transport from the brain to the peripheral circulation is a critical process that allows for its detection in blood samples. Once S100B crosses the BBB, it enters the bloodstream, where it can be detected and measured as a biomarker of neural injury or stress [51]. It must be, however, considered that the efficiency of S100B transport from the brain to the blood is influenced by factors such as the severity of BBB disruption and the overall inflammatory state of the central nervous system. Understanding these mechanisms is crucial for interpreting S100B levels in blood samples and developing potential therapeutic strategies targeting S100B-mediated processes in neurological disorders.

It is thus clear that elevated levels of S100B are observed in patients under many different health conditions. The elevated levels of dimeric S100B are due to a potential disruption of the BBB integrity [52] linked to various neurological conditions. In the case of Alzheimer's disease (AD), its production is altered as part of the chronic neuroinflammatory process [53] with increased S100B levels being associated with neurotic plaques and neurofibrillary tangles. In mild Traumatic Brain Injury (mTBI), ischemic or haemorrhagic stroke, the S100B-producing astrocytes become activated, and subsequent release of S100B occurs into the extracellular space [54], being the CSF and the brain interstitial fluid (ISF), with the CSF being segregated from the blood by the blood-CSF barrier, while the ISF is segregated from the blood by the BBB (Fig. 1C).

## 4. Diagnostic and prognostic value of S100B

It is clear from the discussion above that the multifaceted nature of S100B in both physiological and pathological conditions underscores its importance in neurobiology and its potential as a diagnostic and prognostic tool in clinical settings (Table 1) [9,55,56]. Various diseases like stroke, neurodegenerative diseases including Alzheimer's and Parkinson's diseases, as well as some types of cancer, are characterised by increased levels of S100B in blood (Table 2).

In the case of TBI, the concentration of S100B in the CSF rises dramatically from 660 $pg mL^{-1}$ (healthy subject) to levels as high as 1,231,000 $pg mL^{-1}$ (1231 ng $mL^{-1}$) with clearly unfavourable outcomes [57]. In serum, the values of S100B are significantly lower than in CSF, with blood serum levels >100 $pg mL^{-1}$ within 3 h of injury being considered clinically significant and indicating the need for further neuroimaging [58]. Furthermore, it has been postulated that detectable elevations of S100B in the blood occur as early as 15 min post-injury, with peak serum S100B concentrations in patients with severe TBI being as high as 730 $pg mL^{-1}$ for a favourable outcome and 6030 $pg mL^{-1}$ for an unfavourable outcome. Indeed, serum S100B levels below 1200 $pg mL^{-1}$ have been associated with a low risk of significant neuroradiological changes, potentially reducing the need for computed tomography (CT) scans and associated radiation exposure [59]. S100B

<caption>**Table 2**</caption>

Concentration levels of S100B in serum and CSF determined for TBI, stroke and other neurodegenerative diseases.

<table><thead><tr><th>Disease</th><th>S100B / [pg mL<sup>-1</sup>]<sup>a</sup></th><th>References</th></tr></thead><tbody><tr><td>Traumatic Brain Injury</td><td>&gt; 660 (CSF, after 3 h injury)<br/>&gt; 100 (serum, adults, after 15–30 min)<br/>150–160 (serum, 2–18 years)<br/>230 (serum, children 10–24 months)<br/>350 (serum, &lt; 1 month)</td><td>[18,64–66]</td></tr><tr><td>Stroke</td><td>&gt;200 (serum, 48–72 h after injury)</td><td>[13,67,68]</td></tr><tr><td>Neurodegenerative Diseases</td><td>Alzheimer's disease: &lt;100 (serum)<br/>Parkinson's disease: 3100 (CSF)<br/>Multiple Sclerosis: 1510 (serum, acute phase)</td><td>[20,69–72]</td></tr><tr><td>Malignant Melanoma</td><td>stages I and II: &lt;100 (serum)<br/>stage III: ≈200 (serum, adult)<br/>stage IV: &gt;500 (serum, adult)</td><td>[73–75]</td></tr></tbody></table>

<footnote>a Healthy control: serum S100B = 20–150 $pg mL^{-1}$ [15,16]; CSF S100B = 75–810 $pg mL^{-1}$ [17–19], 2200 $pg mL^{-1}$ [20].</footnote>
concentrations are age-dependent, as underlined in a special study on neurologically healthy children. Reference levels of 150–160 pg mL⁻¹ are noted as a cut-off value for children above 2 years up to 18 years, while these levels are higher in children of younger age, notably 350 pg mL⁻¹ for children below one month and 230 pg mL⁻¹ for children between 10 and 24 months. The higher S100 value of newborns can be attributed to several factors, including brain injury during vaginal delivery, especially in cases of prolonged labour, the still immature or “leaky” BBB in neonates [60], as well as the elevated umbilical cord blood in babies [61]. The high values are also consistent with the protein's role in brain maturation, as S100B has neurotrophic effects, stimulating neurite outgrowth and regulating neuron survival at physiological concentrations.

Different to TBI, caused primarily by external damage to the brain, such as a blow or jolt to the head, strokes are caused by blood clots or internal haemorrhage in the brain. In the case of a stroke, data from 210 ischemic stroke patients revealed that the peak of S100B in serum was observed only 48–72 h after symptom onset with concentrations of 200–1500 pg mL⁻¹ [62,63]. This may go along with the observation that the BBB exhibits biphasic openings after stroke, immediately, once after 24 h and once later.

The use of S100B as a biomarker for TBI, stroke and neurodegenerative diseases is further complicated during comorbidities, as cancer might also result in fluctuation of S100B serum levels. Patients with early-stage melanoma (stages I and II) exhibit serum S100B concentrations below 100 pg mL⁻¹ with highly elevated levels in stage IV patients [73–75]. In the case of acutely ill patients suffering from cardiac arrest and cardiogenic shock, S100B could reflect concurrent hypoxic brain injury, which is a major driver of mortality in these patients. In one study, it has been shown that monitoring of S100B levels in blood could predict with high sensitivity (>70 %) and specificity (>93 %) a good prognosis with serum concentration = 0.23 pg mL⁻¹ and severe neurological defects or death when serum concentration = 1.64 pg mL⁻¹ [76]. This result needs to be, however, carefully interpreted since other studies show that in healthy control the levels of serum S100B is between 20 and 150 pg mL⁻¹ [15,16].

This also highlights the potential use of rapid assessment of S100B levels in patients suffering from severe cardiogenic shock or acute coronary syndrome, where S100B levels could significantly enhance multi-organ risk assessment [77,78]. Researchers also suggested the use of S100B as a biomarker for the complementary examination of patients with acute brain injury for the prediction of brain death, since it has been noticed in separate case studies that the increase of S100B occurs before brain death [12].

So, what makes S100B a valuable biomarker where specificity is one of the criteria next to being quantifiable, stable over time, easy to access and disease-specific?

S100B is consistently expressed in various neurological conditions. However, extra-cranial injuries (e.g. bone fractures, soft tissue damage) as well as cancer can influence its expression, which may lead to false positives if not carefully considered [79,80]. Disease specificity is thus moderate and S100B should be used in conjunction with clinical evaluation and possibly other diagnostic tools to minimise false-positive results. However, S100B is easy to access through minimally invasive methods based on blood drawing. Additionally, S100B protein is not affected by haemolysis in the sample, making it a robust sample to use in acute settings [81,82].

The specificity of S100B, defined by the possibility of S100B to accurately identify the disease, while minimising false positives and false negatives, varies depending on the context and the disease being assessed. In the framework of TBI, the specificity of S100B was found to be 47 % (95 % confidence interval (CI)) when using a cut-off value of ≥100 pg mL⁻¹, and it dropped to 42 % when applied to all patients regardless of the SNC (Scandinavian Neurotrauma Committee) classification [83]. This study analysed data from 530 patients who suffered mTBI, showing pre-hospital application feasibility. For spinal cord

injury, data from 56 acute injuries revealed a specificity of 55 % for a serum S100B concentration of 147 pg mL⁻¹ [79]. In detecting brain metastases, the specificity was 43 % at a serum S100B level of 58 pg mL⁻¹, which improved to 58.2 % when combined with anti-S100B IgG levels [84].

Moreover, S100B is relatively unaffected by storage, changes in temperature and freeze-thaw cycles, which greatly facilitate the handling of the protein and reliability of the analyses. The half-life of S100B in serum after TBI is difficult to access, as there is a continuous release of the protein from the damaged brain [52]. Typically, serum S100B concentrations peak within the first few hours after TBI and then begin to decline, with a reported half-life ranging from 1 to 6 h. As the serum half-life of S100B is rather short, an extended elevation of S100B indicates an ongoing influx. The diagnostic utility of S100B extends to its prognostic value.

## 4.1. Laboratory-based analysis of S100B in biological samples

While S100B is thus clearly a valuable biomarker, it needs to be quantifiable and measurable using reliable and reproducible analytical methods. Like most fluid-derived biomarkers, Enzyme-Linked Immunosorbent Assay (ELISA) is the gold standard for quantifying S100B in laboratory settings in serum and CSF [80,85]. Several commercial kits are available for S100B dosing from several manufacturers, giving a great variety of sensitivity options. Depending on the desired sensitivity, laboratory set-up, and sample number, the market offers a wide range of available, ready-to-use kits for the detection of S100B in serum, plasma, cell culture, supernatant, and CSF samples. The linear range lies between 2.7 up to 3.500,000 pg mL⁻¹ (with various LoD), depending on the kit component specifications, described in Table 3.

Several comparisons of different ELISA systems for detecting S100B have been reported. Smith et al. made a side-by-side comparison of four different ELISA assays for the detection of S100B protein in serum samples in 2005 [85]. The comparison was performed for two automated assays (LIAISON® Sangtec®100 and Elecsys®S100) and two manual ones (Sangtec®100 ELISA and CanAg S100 EI). Indeed, the two most frequently used quantitative automated systems in clinical settings are luminometric immunoassay LIAISON® Sangtec®100 (Sangtec; Italy) and the electrochemiluminescence immunoassay Elecsys®S100B® (Roche Diagnostics). LIAISON® Sangtec®100 assay is an automated chemiluminescence immunoassay for the quantitative determination of S100B protein in human serum and CSF with a Limit of Detection (LoD) of 20 pg mL⁻¹ and specificity of 97.7 % (95 % CI: 95.8–99.0) to 99.0 % (95 % CI: 94.6–100) depending on the sample set. The Elecsys S100B® assay and the new automated Cobas® system detect S100B in the range of 15–30,000 pg mL⁻¹ with a LoD < 15 pg mL⁻¹ and specificity of 93.8 % within 6 h of injury and 82.9 % within 24 h, with high negative predictive values (NPV) of 97.3 % and 94.3 %, respectively, for ruling out intracranial pathology [86].

A comparison of both methods revealed correlation coefficients from 0.76 to 0.95. The Elecsys S100B® assay displayed a superior ability to detect S100B in urine samples compared to the LIAISON® Sangtec®100 system [87]. Using a cut-off value of 100 pg mL⁻¹, the Elecsys S100B® demonstrated a sensitivity of 41 %, with positive and negative predictive values of 0.50 and 0.91, respectively, in melanoma patients. The Elecsys S100B® system takes only 18 min to run a serum sample, making it more suitable for TBI and possible for both emergency room and neuro-intensive care units use [88]. The system exhibited high sensitivity and negative predictive value for ruling out intracranial pathology, especially within 6 h of injury [86]. In the context of out-of-hospital cardiac arrest, the Elecsys S100B® system has shown that S100B levels at 48 h can independently predict unfavourable neurological outcomes with high specificity (100 %) and moderate sensitivity (39 %) when using a cut-off value of 370 pg mL⁻¹ [88]. This study was performed on 120 comatose patients post-cardiac arrest and gave promising cut-off values; however, it requires more in-depth studies to manage the prognostic
<table><caption>**Table 3**

Characteristics of selected commercially available S100B ELISA kits.</caption><thead><tr><th>Kit</th><th>Company</th><th>Linear range [pg mL<sup>-1</sup>]</th><th>LoD [pg mL<sup>-1</sup>]</th><th>Samples</th><th>Comments</th></tr></thead><tbody><tr><td>S100B ELISA Kit (ab234573)</td><td>Abcam</td><td>940–60,000</td><td>410</td><td>Serum<br/>Citrate plasma<br/>EDTA plasma<br/>Cell culture<br/>Supernatant</td><td>Single-wash<br/>90-min assay</td></tr><tr><td>Human Protein S100-B ELISA Kit<br/>(MBS762374)</td><td>MyBioSource</td><td>500–8000</td><td>100</td><td>Serum<br/>Plasma<br/>CSF<br/>Cell culture<br/>Supernatant</td><td>High specificity</td></tr><tr><td>S100B ELISA Kit</td><td>Antibodies online</td><td>78–5000</td><td>19.5</td><td>Serum<br/>Plasma<br/>CSF</td><td>Intra-assay Precision: &lt;8 %</td></tr><tr><td>Human S100B ELISA Kit</td><td>Invitrogen (Thermo<br/>Fisher)</td><td>31.25 –<br/>2000</td><td>18.75</td><td>Serum<br/>Blood</td><td></td></tr><tr><td>Sangtec®100 ELISA</td><td>DiaSorin</td><td>32–30,000</td><td>20</td><td>Serum</td><td>LIAISON® XL analyzer required</td></tr><tr><td>Elecsys S100B®</td><td>Roche</td><td>15–30,000</td><td>&gt;15</td><td>Serum<br/>CSF</td><td>Automated Cobas® system</td></tr><tr><td>CanAg S100 EIA</td><td>Diasourse-Diagnostics</td><td>50,000 –<br/>3,500,000</td><td>10</td><td>Serum</td><td>Uses biotin anti-S100 monoclonal antibody</td></tr><tr><td>Human S100B ELISA, EZHS100B-33 K</td><td>Millipore</td><td>2.7–2000</td><td>2.7</td><td>Serum<br/>Plasma<br/>CSF</td><td>Antibody pair used measures Human S100B<br/>no cross-reactivity with S100A1, S100A6 and<br/>S100A13</td></tr><tr><td>Human S100 Beta ELISA Kit</td><td>Proteintech</td><td>15.6–1000</td><td>1.0</td><td>Serum<br/>Plasma<br/>Cell culture<br/>Supernatant</td><td>Intra-assay Precision: &lt;5.3 %</td></tr></tbody></table>

level of main cardiac arrest markers, like neuron imaging. While the ELISA method remains the most used, its sensitivity and accuracy highly depend on the quality of the antibodies, and the reliability of the ELISA must be checked and maybe validated with other methods, e.g. the Roche platform.

## 4.2. Biosensors and point-of-care diagnostics for S100B

ELISA platforms offer high sensitivity and low-cost using enzyme-catalysed colour changes to detect S100B. While this approach is one of the most widely used analytical platforms and well accepted by clinicians and medical staff, ELISA platforms face clinical translation challenges, like slow turnaround times, cross-reactivity, single-target limitations, and batch-to-batch variability. The relatively long procedure times can be critical in emergency cases and in acute care settings, where rapid decision-making is crucial. Low signal intensity can occur leading to false positive or negative results, which reduces reliability in complex clinical samples. Biosensors provide real-time, on-site, and potentially multi-target detection by converting a biological interaction into a measurable signal. The possibility for miniaturisation and development of handheld devices makes them ideally suited as PoC devices, with several concepts having been proposed for swiftly detecting S100B [89] (Fig. 2A). Clinical translation challenges for biosensors include currently maintaining sensitivity and specificity in complex samples, ensuring environmental robustness (stability under varying conditions), achieving rapid response times, and developing user-friendly, cost-effective platforms for widespread adoption. For widespread clinical adoption, biosensors need to be developed into user-friendly and scalable devices, which requires significant innovation in materials, fabrication, and integration. For a biosensor to be clinically viable, critical optimisation of its design is required, focusing on the bioreceptor choice (e.g., antibody, aptamer), immobilisation technique on the transducer surface to ensure stability and function, and strategies to prevent non-specific binding (surface fouling) [90,91].

Table 4 summarises some of the main S100B biosensors developed in the last years. Most of the biosensors are based on the use of anti-S100B antibody or His-tagged RAGE protein with a wide linear range from 10

to 10,000 pg mL⁻¹ and LoD as low as 4.6 pg mL⁻¹. While this creates a foundation for the development of a PoC device suitable for clinical use, more work needs to be dedicated towards the diversification of the bioreceptor part of the sensor to improve the stability and shelf-life of PoC.

Electrochemical-based biosensors are one of the most widely used systems for detecting S100B, primarily through monitoring changes in the charge transfer resistance [97]. An electrochemical sensor based on His-tagged VC1 domains of RAGE as a bioreceptor for S100B, with a monolayer of a thiol derivative of dipyrromethene complexed with Cu (II) exhibited redox activity changes upon binding with S100B [100]. The RAGE-based bioreceptor proved to be rather specific for S100B, even though RAGE is known to have multiple binding partners. Testing the performance of the sensor in the presence of potential interfering factors, such as Aβ1–40 peptide (10 nM), did not significantly influence the S100B determination, further supporting the specificity of the biosensor. More recent electrochemical biosensors are comparable to ELISA in terms of sensitivity. The use of chitosan/reduced graphene oxide nanocomposites loaded on glassy carbon electrodes decreased the current signal of redox probes, due to the formation of antigen-antibody complexes, offering a wide dynamic range and a low LoD [97].

Optical methods such as Surface-Enhanced Raman Scattering (SERS)-based sensors are emerging as potent diagnostic tools for the clinical monitoring of S100B. A study that utilises a paper lateral flow assay (PLFS) with SERS signal transduction reported an impressive LoD of 5.0 pg mL⁻¹ for S100B [95]. Gold nanoparticle-based systems in the form of lateral flow immunoassays (LFIA) hold, in addition, significant promise for the rapid point-of-care detection of S100B protein (Fig. 2B) in real-life samples. The detection limit of this advanced LFIA system was as low as 50 pg mL⁻¹, and the integration of gold nanoparticles improves the colourimetric signal readout [94]. In clinical settings, these assays are valuable for quickly ruling out traumatic intracranial lesions due to their high negative predictive value, although they exhibited lower specificity. Therefore, S100B measurements obtained via LFIA should be interpreted in conjunction with clinical evaluation to minimise false positives and unnecessary CT scans. The combination of SERS with LFA resulted in a LoD = 0.14 pg mL⁻¹ in serum samples [99].
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** S100B assay kits: (A) Diagnostic concepts for S100B in CSF and blood using various techniques, like ELISA, biosensors and PoC devices. In the presented sandwich ELISA, S100B (symbolised by red circle) binds to a surface-bound capture antibody (orange) and a second detection antibody (blue). The formation of an immune complex is revealed via an enzymatic reaction between this immune complex and the interaction with an enzyme-linked antibody (violet). In this assay, the capture antibody and the detection antibody must bind to different, non-overlapping parts (epitopes) of the S100B protein. This ensures that both antibodies can bind simultaneously to the antigen, leading to higher specificity and accuracy compared to other ELISA formats. In the case of a biosensor, a capture-antibody is linked to the sensing surface and upon S100B binding, a change in the transducer signal is observed. In this scheme, a change in the position of a plasmonic signal is presented. PoC sensors can have different formats. Exemplified is a lateral flow assay (LFAs) with sensing and control line as well as a multiplexed field effect transistor. (B) Scheme of Later-Flow Immuno Assay (LFIA) for S100B based on poly(4-styrenesulfonic acid-co-maleic acid) (PSS-MA) coated GoldMag for the detection of S100B. The LFIA device comprises a nitrocellulose membrane, an absorbent pad, a sample pad, and a conjugate pad. Anti-S100 B antibody B (capture) and goat anti-mouse IgG (control, green) are pre-immobilised in a defined test line (T-line) and a control line (C-line) on the nitrocellulose membrane, respectively. PSS-MA-GoldMag is sprayed on the conjugate pad. During the test, S100B would combine with anti-S100B antibody, a modified PSS-MA-GoldMag. During migration, the anti-S100B antibody-modified PSS-MA-GoldMag conjugate would be captured by the other anti-S100B antibody B and goat anti-mouse IgG, resulting in a red band in the T-line and C-line, respectively. The magnetic response signals of the test and control lines would read in 30 min, and the signals of the T-line would be proportional to the concentration of S100B. Image of LFA strips after assays run with different standard solutions of S100B from 0 to 3 ng mL⁻¹, and linearity effect of the different standard solutions of S100B (adapted from Ref. [94]). Created in https://BioRender.com. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

Alternatively, immunosensors utilising hollow gold nanospheres (HAuNPs) with redox molecules like 4-mercaptobenzoic acid and Nile blue A have been developed, offering detection limits of 60 pg mL⁻¹ for S100B [101] in serum. These SERS-based sensors exhibit high sensitivity and specificity, with minimal interference from other substances. For instance, studies have demonstrated that the presence of bovine serum albumin, glucose, and carcinoembryonic antigen does not significantly affect the Raman signal of S100B [101].

More lately, several PoC sensors for S100B entered the market, such as the Tbit™ system, based on silicon nanotechnology that is integrated in a single-use disposable cartridge linked to a portable analyzer. The resistive diagnostic concept based on nanowires for the rapid detection of protein biomarkers, including S100B, in the context of TBI, was positioned as a predictive tool for a positive computed tomography (CT) scan and Food and Drug Administration (FDA) approved in 2017 [102,103]. The system is capable of providing test results in less than 2 min from a single drop of blood, with false negative results, although it had a specificity level of 41 %. This indicates that the system is highly effective in ruling out the presence of significant TBI, but may require further refinement to reduce false positives.

Table 5 compares the key analytical parameters of the discussed sensing concepts to [104].

## 5. Perspectives

The economic and healthcare impact of efficient and timely S100B-based diagnostics is high when considering the fact that S100B testing has the potential to reduce the number of unnecessary CT scans, particularly in cases of mild traumatic brain injury. This leads not only to decreased radiation exposure for patients to CT, but also results in a cost-effective analysis method. The price of S100B testing relative to CT scans is indeed crucial, with an estimation of the cost-effectiveness ensured when the S100B molecular test costs less than 33 % of a CT scan. Independent of cost considerations, S100B testing has the potential to improve patient flow in emergency departments, owing to shorter waiting times, faster results, and faster-adapted treatment. Another argument for PoC sensors for S100B can be seen in the context of sports notably American Football. In 2007, the National Football League (NFL) mandated neuropsychological baseline tests and retests for players before they could return to play after sustaining TBI. If TBI is too strong, they are not allowed to play anymore. For these cases, PoC giving fast results is of great value.

One of the hurdles that remains is that while S100B, as a biomarker, has high sensitivity (99 %), specificity is as low as 30 % for detecting head trauma-related lesions. This high sensitivity makes it valuable for ruling out the need for CT scans in certain cases of mTBI but makes it not a useful biomarker for a specific disease. Combining S100B with cytokines, such as IL-1β, IL-2 and IL-4, has shown promise in improving diagnostic accuracy. For example, a study on Generalized Anxiety Disorder (GAD) reported that the combination of S100B and cytokines had a high diagnostic accuracy of 94.47 % [105]. This approach could be extended to other conditions, such as TBI or brain metastases, by identifying specific cytokine profiles that correlate with the presence of these conditions. Other brain-derived proteins, like neuron-specific enolase (NSE) or glial fibrillary acidic protein (GFAP), could enhance the specificity of the assay. These proteins are also released into the bloodstream following brain injury and could provide a more comprehensive biomarker profile. Multiplexed assays are thus the future in the field with S100B being one of the essential elements.

Another important aspect of all diagnostics platforms is sample preparation, which is particularly essential when considering blood as an analytical medium. With regard to sample preparation, the fact that S100B can experience polymerization under low antioxidant conditions should be studied more deeply. Lee et al. showed that preventing the formation of the disulfide-bridged S100B dimers when copper and ascorbic acid are present, results in S100B of different reactivity and
<table><caption>**Table 4**
Selected literature on S100B biosensors developed in the last 7 years.</caption><thead><tr><th>Detection Method</th><th>Sensing element</th><th>Linear Range [pg mL<sup>-1</sup>]</th><th>LoD [pg mL<sup>-1</sup>]</th><th>Comments<sup>a</sup></th><th>References</th></tr></thead><tbody><tr><td>Electrochemical</td><td>His-tagged RAGE</td><td>1000–7400</td><td>1000 (plasma)</td><td>Au/MBT/DPM-Cu(II)</td><td>[92]</td></tr><tr><td>SPR</td><td>anti-S100</td><td>250–10,000</td><td>136</td><td>Au/MUC</td><td>[93]</td></tr><tr><td>LFA</td><td>anti-S100</td><td>10–2000</td><td>50</td><td>PSS-MA-GoldMag nanoparticles</td><td>[94]</td></tr><tr><td>Electrochemical</td><td>anti-S100</td><td>10–1000</td><td>6</td><td>AuIDEs</td><td>[92]</td></tr><tr><td>Paper lateral flow assay with SERS signal</td><td>anti-S100</td><td>5 pg mL<sup>-1</sup> up to low ng mL<sup>-1</sup></td><td>5</td><td>Hierarchical three-dimensional nanostructure</td><td>[95]</td></tr><tr><td>LFA</td><td>anti-S100</td><td>10–2000</td><td>4.6</td><td>Au NPs</td><td>[96]</td></tr><tr><td>Electrochemical</td><td>anti-S100</td><td>10–1000</td><td>1.9</td><td>chitosan/rGO</td><td>[97]</td></tr><tr><td>Photoelectrochemical</td><td>anti-S100</td><td>0.25–1000</td><td>0.15</td><td>rGO-Au/ITO</td><td>[98]</td></tr><tr><td>SERS - LFA</td><td>anti-S100</td><td>200–22,000</td><td>0.14</td><td>Au NPs</td><td>[99]</td></tr><tr><td>Nanophotonic biosensor</td><td>Capillary-assembled heterochain arrays</td><td>1–100,000</td><td>1</td><td>500-nm polystyrene and 250-nm selenium nanoparticles</td><td>[8]</td></tr></tbody></table>

<sup>a</sup> Au NPs: gold nanoparticles; AuIDEs: interdigitated gold electrodes. ITO: indium tin oxide. LFA: lateral flow assay. SPR: surface plasmon resonance. SERS: Surface-Enhanced Raman Scattering. RAGE: Receptors for Advanced Glycation End. rGO: reduced graphene oxide. MUC: mercaptoundecanoic acid. MBT: mercaptobutanol. DPM-Cu(II): dipyromethene complexed with Cu(II) ions.

<table><caption>**Table 5**
Comparison of analytical performance of ELISA vs Biosensor for S100B.</caption><thead><tr><th>Parameter</th><th>ELISA</th><th>Biosensors</th></tr></thead><tbody><tr><td>Selectivity</td><td>Depends on quality of antibodies used correlation coefficients: 0.76–0.95<br>Negative predictive value up to 97.3 % for TBI</td><td>Depends on used bioreceptor<br>Normally high selectivity, with Some PoC biosensors achieving near 100 % specificity</td></tr><tr><td>Sensitivity</td><td>Most sensitive ELISA has a LoD = 1 pg mL<sup>-1</sup></td><td>depending on technology LoD as low as 0.14–1.9 pg mL<sup>-1</sup> reported</td></tr><tr><td>Linearity</td><td>Linear ranges vary by kit 15–30,000 pg mL<sup>-1</sup> (Elecsys), 31.25–2000 pg mL<sup>-1</sup> (Invitrogen)</td><td>Linear ranges vary by assay technology chitosan/rGO systems: 10–1000 pg mL<sup>-1</sup> SERS LFAs: 0.14–22,000 pg mL<sup>-1</sup></td></tr><tr><td>Precision</td><td>S100B ELISA intra-assay precision can reach <8 %</td><td>Comparable to ELISA in recent studies</td></tr><tr><td>Speed</td><td>Typically, between 90 min and several hours</td><td>Results in 5–10 min</td></tr><tr><td>Robustness</td><td>Automated ELISAs are robust when standardized kit and sample variations may affect results</td><td>Biosensor robustness is an ongoing concern, especially regarding interference</td></tr><tr><td>Stability</td><td>ELISA-targeted S100B is stable in serum (unaffected by freeze-thaw or temperature changes)</td><td>Biosensor stability is highly dependent on the type of molecule used in the device</td></tr></tbody></table>

properties [106]. In some of our own in vitro experiments, we indeed observed a loss in S100B detectability with commercial ELISA assays when having fewer antioxidants (ascorbic acid, glutathione) in the medium. This points towards a change in binding affinity to the antibody-based bioreceptors of the ELISA kits. Testing of engineered antibodies, as well as antibody alternatives, would largely add to the understanding of this question.

Indeed, while antibodies feature high specificity and low toxicity, development costs are high and the stability of these bioreceptors at room temperature is limited. Due to their small size, simple structure, high antigen binding affinity, and remarkable stability under extreme conditions, engineered antibodies (e.g. nanobodies, nanoCLAMPs) possess the potential to overcome several of the limitations of conventional monoclonal antibodies and might be ideal bioreceptors to be considered also for S100B [107–111]. The same is valid for aptamers, artificial single-stranded oligonucleotides that bind a specific target molecule or a family of target molecules, including proteins. These “chemical antibodies” with three-dimensional structures exhibit excellent thermal stability, high affinity and specificity for the homologous protein targets proposed for therapeutic means, but not widely used for diagnostic purposes [112].

Saliva is also considered for evaluating neurological disorders. The

compelling reasons to use saliva over blood are related to its non-invasive nature, can be sampled without the help of a medical professional, and provides *improved safety*. There is currently no translation of S100B blood-based sensing platforms to S100B saliva-based PoC devices in the context of neurological disorders [48,113]. It was shown that salivary S100B levels are as effective in differentiating TBI patients as using serum samples. In some of the studies, it could be confirmed that saliva S100B is higher than blood concentrations of the same persons. Using model cell culture experiments, it could be seen that S100B permeation takes place from the blood to saliva across salivary gland epithelial cells, which seem to be tightness-dependent and suggest a paracellular route. In the case of inflamed cells, where maybe RAGE receptors are involved in the transport, increased permeability was seen. Sensing in saliva requires obtaining a better understanding of where saliva S100B comes from, with testing the role of the blood-saliva barrier in this context becoming of importance. Another point in this context is that sex differences were found in a set of blood and saliva samples. This might have important implications when setting the thresholds for healthy assessments.

The analysis of endogenous metabolites, rather than S100B, which has not been widely considered until now, might offer an interesting alternative [114]. The question of whether the detection of metabolites by liquid chromatography-mass spectrometry analysis in post-mortem CSF is suitable to identify TBI and to distinguish it from acute cardiovascular control fatalities has been recently addressed. Non-invasive diagnosis of brain injuries using breathomics, through analysing volatile organic compounds (VOCs) in exhaled breath, is indeed an emerging and promising area of research. Studies are underway to identify specific “breath biomarkers” associated with TBIs, which could allow for rapid, early diagnosis and routine assessment, particularly in high-risk settings like sports and the military [115]. While promising, this is still in the early stages, with challenges in developing standard analytical and statistical frameworks for analysing the samples [116]. One limitation with breathomics of VOC using mass spectrometry when adapting to PoC is the need for remote (not in the hospital) or emergency screening of the sample. This would be only possible if scientists and engineers develop small, light and cheap enough mass spectrometry machines.

While from the discussion above it is clear that S100B shows promise as a biomarker for neurological conditions and has potential as a therapeutic target, ongoing challenges include establishing consistent correlation with disease severity, determining its precise role in pathogenesis, and developing reliable methods for real-time monitoring.

## 6. Conclusion

In conclusion, the dimeric form of S100B, with its unique structural
stability and functional properties, has established itself as a crucial
biomarker in neurological diagnostics, by reflecting active cellular
damage and injury in biological fluids like blood and cerebrospinal fluid.
Its ability to accurately reflect neuropathological conditions, guide
clinical decision-making, and potentially monitor treatment efficacy
underscores its significance in modern neurocritical care and neuro-
logical practice. Clinical applications of S100B, however, still struggle
due to its lack of specificity for disease states, its rapid and complex
concentration fluctuations after injury, and the need for standardized
protocols and extensive validation across diverse patient populations
and clinical settings. While S100B has been included in clinical guide-
lines for mTBI in Europe, one of the unresolved issues is linked to the
robustness of the biomarker. Large-scale studies and national registries
might provide the basis for such evidence. Moreover, because other
markers compete with S100B, the use of artificial intelligence might be
helpful to build a marker hierarchy or perhaps to combine different
markers to reach a higher sensitivity and specificity in prognosticating
clinical outcomes. Converging evidence indicates a possible involve-
ment of the interplay between S100B and inflammation markers, such as
C-reactive protein [105]. Since both markers can interact with RAGE,
multiplexed sensing of S100B with inflammation biomarkers might be
a way to consider in the future. The same might be true for the other
astrocytic proteins, GFAP [48]. There is, in addition, a pending need for
validated cut-off levels and further multi-centre studies to establish
specificity for conditions such as acute brain injury, traumatic brain
injury, stroke, brain death and others like Alzheimer's disease.

On the sensor side, despite advances in biosensor technologies shown
to be promising to bring higher sensitivity, speed, and portability to
S100B detection, they remain challenging to introduce into clinical
laboratories. While often they need to be tested in clinical settings, many
biosensors still struggle with accurate detection of low S100B concen-
trations in complex samples, like CSF, blood or saliva [117]. Other ob-
stacles on the way of biosensor-based PoC devices becoming a regular
practice in medicine include adjusting the technical infrastructure in
clinical laboratories and reducing the costs of the PoC device
manufacturing. PoCs often use expensive materials or have a complex
fabrication process, limiting scalable production and accessibility.

Several further steps should thus be taken in order to advance the acceptance of PoC devices in clinical settings. First and foremost, standardisation of the manufacturing procedure and calibration protocols can significantly improve performance consistency and result trustworthiness. Another important issue that needs to be considered, when obtaining, processing, and storing patients' personal data and test results, is integrating robust cybersecurity frameworks when designing biosensor-connected workflows. This will protect sensitive information, increasing trust from clinicians and patients. Finally, with the rapid progress of Artificial Intelligence (AI), scientists could use the possibility of combining gathered data with other health records to create a complex picture of patient's history and deliver context-aware diagnosis. These important aspects need to be taken into consideration, while promoting novel biosensors for clinical applications.

CRediT authorship contribution statement

**Nataliai Gnyliukh:** Writing – review & editing, Writing – original draft. **James Wei:** Writing – review & editing, Writing – original draft. **Winfried Neuhaus:** Writing – original draft, Conceptualization. **Rabah Boukherroub:** Writing – review & editing, Writing – original draft. **Sabine Szunerits:** Writing – review & editing, Writing – original draft, Funding acquisition, Conceptualization.

Declaration of competing interest

JW has a commercial interest in S100B for diagnostics means. The
other authors declare that they have no known competing financial in-
terests or personal relationships that could have appeared to influence

the work reported in this paper.

Acknowledgements

The project is funded by the Horizon 2020 Framework Program of the European Union under grant agreement No. 101129095 (LungCare). Support of the government of Lower Austria and Tyrol under the COMET Project « PI-SENS » (Personalized Medicine enabled by intelligent sensing systems) FFG grant no. FO 999915477 is acknowledged.

Data availability statement

No data was used for the research described in the article.

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