A Review of Detection Probes for Vanin‐1: Development and Biological Applications in Various Inflammatory Diseases

ABSTRACT

Vascular non‐inflammatory molecule‐1 (Vanin‐1), also known as pantetheinase, is a key enzyme that regulates oxidative stress, inflammatory responses, and metabolic processes by catalyzing the hydrolysis of pantetheine into cysteamine and pantothenic acid. Recent studies have progressively elucidated its pivotal role in various pathological processes, including acute kidney injury (AKI), inflammatory bowel disease (IBD), liver injury, and rheumatoid arthritis (RA). As an important biomarker for early diagnosis and assessment of treatment efficacy, it has attracted widespread attention and driven the rapid development of highly sensitive and specific detection tools. This article provides a systematic review of Vanin‐1 probes developed in recent years, covering fluorescent, chemiluminescent, bioluminescent, and electrochemical sensors, with a focus on their molecular design principles, detection mechanisms, performance characteristics, and biological applications. In addition, this article outlines future trends in Vanin‐1 probe technologies, providing a comprehensive theoretical and methodological reference to support further basic research and clinical translation applications related to Vanin‐1.

Vanin has emerged as a diagnostic biomarker for inflammation‐related diseases. Vanin‐1 probes have seen continuous improvements in sensitivity, selectivity, and practicality, evolving from fluorescent probes to bioluminescent probes, chemiluminescent probes, and electrochemical probes. These probe tools have not only deepened understanding of Vanin‐1 in related diseases but also provided vital support for early diagnosis, treatment monitoring, and drug discovery.

1. Introduction

Vascular non‐inflammatory molecule‐1 (Vanin‐1) was initially characterized from a crude extract of horse kidney, and its enzymatic activity was subsequently confirmed through purification from horse kidney [1, 2]. Vanin‐1 has been identified as a pantetheinase that hydrolyzes pantetheine into pantothenic acid and the amino‐thiol cysteamine [3], and these products play essential roles in diverse biological processes (Figure 1) [4, 5, 6]. This enzyme is extensively involved in regulating oxidative stress, glutathione (GSH) synthesis, energy metabolism, and cellular homeostasis. It is highly expressed in metabolically active tissues, including the liver, kidneys, and intestine, and acts as a key mediator in inflammatory responses through its involvement in immune cells such as macrophages [7, 8, 9]. Studies have shown that abnormal Vanin‐1 expression is closely associated with various pathological processes, such as inflammatory diseases like inflammatory bowel disease (IBD) [10, 11], acute kidney injury (AKI) [12, 13, 14], as well as metabolic disorders like diabetes [15, 16]. Consequently, Vanin‐1 has emerged as a promising early diagnostic biomarker and a potential therapeutic target. Accurate and real‐time monitoring of its expression is crucial for early disease diagnosis, pathological mechanism investigation, and therapeutic evaluation. However, traditional detection methods (e.g., Western blot, enzyme‐linked immunosorbent assay) are often constrained by high background signals, limited sensitivity, and laborious sample processing. These shortcomings impede their utility for real‐time, dynamic, and noninvasive in vivo detection, thereby restricting both mechanistic studies and clinical diagnostic applications. Consequently, the development of novel chemical probes capable of monitoring Vanin‐1 activity with high spatiotemporal resolution is therefore essential for elucidating its biological functions and facilitating clinical translation.

FIGURE 1.

Vanin‐1 hydrolysis and downstream pathways. Vanin‐1 has been identified as pantetheinase that hydrolyzes pantetheine into pantothenic acid and the amino‐thiol cysteamine. These products are extensively involved in regulating oxidative stress, glutathione (GSH) synthesis, energy metabolism, and cellular homeostasis.

The detection of Vanin‐1 primarily relies on measuring its amide hydrolysis activity, with the majority of existing probes following an enzyme‐activated design strategy. The core design incorporates pantetheine (a natural substrate of Vanin‐1) or its analogues as the recognition unit, which is linked to a signal reporter group (such as fluorophore, chemiluminescent moiety, electrochemical reporter) via a cleavable amide bond. Upon enzymatic cleavage of the amide bond by Vanin‐1, the reporter group is released, resulting in a measurable signal output, such as fluorescence turn‐on, chemiluminescence emission, or an electrochemical current shift. In this review, we systematically summarize the design strategies, detection mechanisms, performance metrics, and practical applications of various Vanin‐1 probes, and further explore future directions for probe development. Our aim is to offer a comprehensive reference to support both fundamental studies and translational efforts targeting Vanin‐1.

2. Development of Vanin‐1 Probes

In recent years, various types of probes have been developed for detecting Vanin‐1 activity, encompassing a range of detection modalities, including fluorescence, chemiluminescence, bioluminescence, and electrochemistry. These advances have driven research from qualitative analysis in vitro toward real‐time quantitative imaging in vivo. Table 1 summarizes the limits of detection, advantages, and representative applications of each probe type.

TABLE 1.

Advantages and representative applications of Vanin‐1 probes.

Probes	Types	LD a ng/mL	Advantages	Representative applications
Fluorescent probes
Pantothenate‐AMC [17]	Fluorescent probe λ ex/λ em c  = 340/460 nm	/ b	First Vanin‐1 fluorescent probe	Inhibitor screening, Vanin‐1 quantification in cells and tissues
PA‐AFC [18]	Fluorescent probe λ ex/λ em = 420/510 nm	/	Excellent sensitivity and specificity	In vitro evaluation of inhibitors
CV‐PA [19]	Ratiometric fluorescent probe λ ex = 525 nm, λ em = I628 nm/ I582 nm	4.7	First ratiometric design	Evaluation of inhibitors, detection of Vanin‐1 in sera, fluorescence imaging of Vanin‐1 in living cells
CYLP [20]	NIR fluorescent probe λ ex/λ em = 590/710 nm	0.02	Extremely low detection limit, deep tissue penetration, minimal background interference	Imaging of Vanin‐1 in cells and in mice
Cy‐Pa [21]	NIR fluorescent probe λ ex/λ em = 730/780 nm	/	High selectivity and sensitivity for in situ real‐time monitoring of endogenous Vanin‐1	Imaging and evaluating the role of Vanin‐1 in chemotherapy
DDAV [22]	NIR fluorescent probe λ ex/λ em = 600/670 nm	/	Low cytotoxicity with ultra‐sensitivity/specificity for in vitro and in vivo in situ imaging	In situ imaging Vanin‐1 activity in biosamples, early kidney injury diagnosis, visual screening gut bacteria, and high‐throughput screening Vanin‐1 inhibitors
TMN‐PA [23]	Near‐infrared emission ratiometric fluorescent probe λ ex = 470 nm, λ em = I645 nm/I568 nm	0.37	Self‐calibrating, accurate quantification, rapid detection within 15 min	Fluorescence imaging in cells and mice
DCM‐PA [24]	Red emission ratio fluorescent probe λ ex = 440 nm, λ em = I640 nm/I564 nm	0.69	Has the maximum stokes shift (∼ 200 nm), short response time (30 min), low sensitivity	In vivo imaging and activity monitoring of Vanin‐1
MB‐Van1 [25]	Dual‐modal fluorescent and colorimetric probe λ ex/λ em = 665/710 nm	0.031	Dual‐mode detection, low background, rapid imaging	Detection of Vanin‐1 activity in various biological tissues, monitoring the dynamic changes of Vanin‐1 during inflammation
PHPO [26]	Fluorescent probe λ ex/λ em = 530/590 nm	0.054	High reactivity, excellent biocompatibility, and low cytotoxicity	High‐throughput screening for Vanin‐1 regulator, diagnosis of Vanin‐1‐related diseases
Bioluminescent probes
PA‐AL [27]	Bioluminescent probe	1.14	Exceptional sensitivity suitable for deep tissue imaging	Imaging and evaluating the role of Vanin‐1 in cells and in vivo
Chemiluminescent probes
CL‐Pa [28]	Chemiluminescent probe	0.008	Ultra‐high signal‐to‐noise ratio with no excitation light interference	Early diagnosis of AKI
BPan‐CL [29]	Dual‐locked chemiluminescent probe	/	Dual‐locked, higher signal‐to‐noise ratio, higher accuracy, noninvasive diagnosis	Identify IBD and healthy mice by in vivo imaging, early prediction of IBD
Electrochemical probes
VaninLP [30]	Electrochemical probe	2.47	No sample pretreatment required; direct detection of turbid samples	Monitor and quantify the activity of Vanin‐1 of HepG2 tumor cells, blood, and urine samples

^a Limits of detection.

^b Not detected in the reference article.

^c λ _ex: excitation wavelengths, λ _em: emission wavelengths, λ _ex/λ _em: excitation/emission wavelengths, I₆₂₈ nm/I₅₈₂ nm: the ratio of fluorescence intensity at 628 and 582 nm.

All Vanin‐1 probes reported in the literature to date have adopted a uniform three‐component architecture consisting of a substrate–linker–reporter design, and their general detection principle is illustrated in Figure 2. These probes employ pantetheine as a specific recognition substrate for Vanin‐1. Pantetheine is conjugated via an amide bond to a self‐immolative linker, which is further connected to a reporter group, such as a fluorophore, chemiluminescent moiety, or electrochemical group. In the intact probe, the reporter group remains in a quenched or silent state, resulting in an extremely low background signal. Upon exposure to Vanin‐1, the enzyme catalyzes the hydrolysis of the amide bond between pantetheine and the linker, releasing pantothenic acid. This step exhibits high substrate specificity, other proteases, including trypsin and elastase, cannot efficiently recognize the pantetheine structure, thereby ensuring the specificity of the assay. At the same time, the cleavage of the amide bond triggers a 1,6‐elimination or an electron cascade reaction within the linker, leading to its spontaneous decomposition and the subsequent release of the free reporter group. The rational design of the self‐immolative linker is critical for probe performance. The linker maintains an optimal distance between the cleavage site and the reporter group to avoid steric hindrance, while ensuring that the reporter group is completely released upon enzymatic cleavage. Once released, the reporter group recovers its intrinsic optical or electrochemical properties, generating a detectable signal, such as enhanced fluorescence, chemiluminescence, or a redox current, proportional to the Vanin‐1 activity.

FIGURE 2.

Schematic diagram of the detection principle of Vanin‐1 probes. The detection of Vanin‐1 primarily relies on measuring its amide hydrolysis activity, with the majority of existing probes following an enzyme‐activated design strategy. The core design incorporates pantetheine (a natural substrate of Vanin‐1) or its analogues as the recognition unit, which is linked to a signal reporter group (such as fluorophore, chemiluminescent moiety, or electrochemical reporter) via a cleavable amide bond. Upon enzymatic cleavage of the amide bond by Vanin‐1, the reporter group is released, resulting in a measurable signal output, such as fluorescence turn‐on, chemiluminescence emission, or an electrochemical current shift.

2.1. Fluorescent Probes

Fluorescent probes are the most extensively studied category of probes for Vanin‐1 detection, due to their operational simplicity, strong visualization capability, and suitability for real‐time imaging [31, 32]. Their evolution has advanced from visible‐light to near‐infrared (NIR) emission, and from simple turn‐on systems to ratiometric sensing mechanisms. Figure 3 illustrates the general structure and reaction scheme of the Vanin‐1 fluorescent probe.

FIGURE 3.

Structure and reaction scheme of fluorescent probes with Vanin‐1. (A) pantothenate–AMC, (B) PA‐AFC, (C) CV‐PA, and (D) CYLP, (E) Cy‐Pa, (F) DDAV, (G) TMN‐PA, (H) DCM‐PA, (I) MB‐Van1, and (J) PHPO.

2.1.1. Conventional Fluorescent Probes

Pantetheine is a natural substrate for Vanin‐1. Ruan et al. demonstrated that the pantothenate moiety is essential for Vanin‐1 recognition, whereas the cysteamine portion is dispensable. By replacing the cysteamine moiety of pantetheine with a fluorescent group, the researchers developed a novel Vanin‐1 substrate, pantothenate‐AMC (Figure 3A) [17]. This probe could be recognized and hydrolyzed efficiently by both the native Vanin‐1 and its soluble recombinant form, generating a strong turn‐on fluorescence signal (λ _ex/λ _em = 340 /460 nm). The pantothenate‐AMC probe exhibits high sensitivity and is suitable for multiple applications, including the characterization of human recombinant Vanin‐1, high‐throughput screening (HTS) for inhibitors, and quantitative analysis of Vanin‐1 activity in cells and tissues. In a 384‐well plate assay, this probe demonstrated robust performance with a signal‐to‐background ratio (S/B) of 7 and a Z′‐factor of 0.75. Using this probe, a library of 1280 pharmacologically active compounds was successfully screened, leading to the identification of inhibitors with novel chemical scaffolds. Thus, pantothenate‐AMC provides a valuable tool for target validation and early‐stage drug discovery.

2.1.2. Ratiometric Fluorescent Probes

Ratiometric fluorescent probes enable self‐calibration by measuring the ratio of fluorescence intensities at two distinct wavelengths, which significantly reduce interference from environmental factors and improve detection accuracy. Hu et al. developed a CV‐PA probe by conjugating PA with a cresyl violet fluorophore (Figure 3C) [19]. Upon enzymatic cleavage, the probe releases the fluorophore, generating a ratiometric signal (I_{628 n m}/I_{582 n m}) that allows quantitative imaging of Vanin‐1 activity in live cells. The probe exhibits a linear response in the range of 5–400 ng/mL with a detection limit of 4.7 ng/mL. CV‐PA probe has been applied to evaluate the efficiency of different inhibitors and to quantitatively determine Vanin‐1 levels in serum samples. The results show that enzyme activity is significantly higher in fetal bovine serum and newborn calf serum compared to normal human serum. Notably, this probe enabled the first ratiometric comparison and in situ quantification of Vanin‐1 activity in different live cell lines—LO2 and HK‐2 cells—showing higher Vanin‐1 levels in LO2 cells, a result subsequently confirmed by Western blot analysis. Although this probe has not yet been employed for in vivo imaging, CV‐PA provides a valuable tool for investigating the role of Vanin‐1 in pathophysiological contexts.

2.1.3. Near‐Infrared (NIR) Fluorescent Probes

NIR fluorescent probes provide significant advantages for in vivo imaging due to their deep tissue penetration and minimal interference from biological autofluorescence. Yang et al. developed a novel NIR probe, CYLP for Vanin‐1 bioimaging, by conjugating PA as the recognition unit with amino hemicyanine (CyOH) as the fluorochrome group via a carbamate‐based self‐immolative linker (Figure 3D) [20]. This probe operates at excitation and emission wavelengths of 590 and 710 nm and exhibits high selectivity and sensitivity, with a minimum detection limit of 0.02 ng/mL. CYLP probe has been successfully used for imaging Vanin‐1 in both cells and mice. Moreover, it revealed significantly elevated Vanin‐1 levels in the tissues from inflammatory mouse as well as in the intestines of mice with IBD, thereby establishing CYLP as a valuable tool for studying the biological functions of Vanin‐1 and related diseases.

Another NIR fluorescent probe, Cy‐Pa, was designed to detect the level of Vanin‐1 in live cells and in vivo (λ _ex/λ _em = 730/780 nm) (Figure 3E) [21]. This probe also demonstrates high selectivity and sensitivity, enabling real‐time, in situ monitoring of endogenous Vanin‐1. It enables qualitative and quantitative tracking of Vanin‐1 fluctuations in HepG2 and HepG2/DDP cells, as well as in tumor tissues of xenograft mice. Through its application, Cy‐Pa has helped reveal that Vanin‐1 promotes tumor resistance to cisplatin by inhibiting GSH synthesis, thereby providing a new perspective for optimizing therapeutic strategies in cancer.

DDAV, reported by Qian et al., represents a further advance in the field of NIR fluorescent probe design (Figure 3F) [22]. This probe links PA to the NIR fluorophore DDAN, with excitation and emission wavelengths of 600 and 670 nm, respectively. DDAV allows real‐time, sensitive, and specific detection of Vanin‐1 activity both in vitro and in vivo, with low cytotoxicity. It has been applied for early diagnosis of renal injury via urinary Vanin‐1 detection and supports noninvasive in vivo imaging. Using DDAV‐based imaging, the intestinal bacterium Enterococcus faecalis strain 20247 was identified as a microorganism that highly expresses Vanin‐1. Furthermore, a DDAV‐assisted high‐throughput screen led to the discovery of a novel potent inhibitor, oleuropein, from natural herbal sources.

In summary, NIR probes such as CYLP, Cy‐Pa, and DDAV have significantly expanded the capacity to visualize and quantitatively measure Vanin‐1 activity in complex biological environments. These tools not only deepen our understanding of Vanin‐1 in inflammation, cancer, and host–microbial interactions but also hold promise for advancing the development of early disease diagnosis and targeted therapy.

2.1.4. Near‐Infrared Emission Ratiometric Fluorescent Probe

Building on previous research, Qian et al. conjugated pantothenic acid as the recognition unit with a NIR dicyanoisophorone derivative fluorophore TMN‐NH₂ as the fluorophore (Figure 3G) [23]. This yielded a novel NIR ratio‐based probe, TMN‐PA (I_{645 n m}/I_{568 n m}), capable of rapidly detecting Vanin‐1 within 15 min, with a detection limit of 0.37 ng/mL. This probe offers a sensitive means for in situ, real‐time monitoring of Vanin‐1 activity in vivo.

Subsequently, the same research team reported another novel green‐to‐red emission ratiometric fluorescent probe, DCM‐PA (I_{640 n m}/I_{564 n m}), by conjugating PA with the NIR fluorophore DCM‐NH₂ (Figure 3H) [24]. DCM‐PA allows rapid (30‐min) detection of Vanin‐1, with a lower minimum detection limit of 0.69 ng/mL. This probe has been successfully applied for detecting endogenous Vanin‐1 and has undergone ratiometric fluorescence imaging in HeLa cells and mouse models, highlighting its utility for in situ monitoring of enzyme activity in living systems.

Overall, TMN‐PA and DCM‐PA exemplify the advancement of ratiometric NIR probes for Vanin‐1 detection. By enabling more accurate quantitative imaging in complex biological environments, these tools provide a robust platform for investigating the role of Vanin‐1 in physiological and pathological processes.

2.1.5. Dual‐Modal Fluorescent Probe

The fluorescence mode offers high sensitivity and can be used for the precise quantification of trace amounts of Vanin‐1 as well as in vivo imaging of deep tissues. Although the colorimetric mode has lower sensitivity, it requires no specialized equipment and allows for rapid semi‐quantitative screening through visual observation of color changes, making it suitable for rapid screening in resource‐limited settings. The dual‐mode design strikes a balance between sensitivity and portability, expanding the probe's range of applications. A dual‐mode (fluorescent/colorimetric) probe MB‐Van1, reported by Dai et al., was synthesized by linking the fluorophore methylene blue to the recognition group pantothenic acid using 4‐aminobenzyl alcohol as a self‐eliminating linker (Figure 3I) [25]. By modifying the nitrogen atom on the phenothiazine ring of methylene blue, this modification disrupts the molecule's original conjugated structure, conferring the probe with the distinctive advantage of extremely low background fluorescence, which significantly enhances the detection sensitivity. MB‐Van1 probe appears colorless and negligible fluorescence in its initial state, and the pantothenic acid group in the probe is selectively cleaved, releasing free methylene blue molecules in the presence of Vanin‐1. This cleavage causes MB‐Van1 to change color from colorless to blue, while significantly enhancing the fluorescence intensity, thereby enabling dual‐mode fluorescence and colorimetric detection of Vanin‐1. With a remarkably low detection limit of 0.031 ng/mL, this probe enables rapid (within 5 min) monitoring Vanin‐1 activity in multiple inflammatory mouse models. It has successfully detected elevated Vanin‐1 levels in RA, drug‐induced liver injury (DILI), and nonsteroidal anti‐inflammatory drugs (NSAIDs)‐induced colitis, offering a powerful tool for dynamic inflammation monitoring.

Meanwhile, Tian et al. developed the enzyme‐activated fluorescent probe PHPO, which demonstrates high specificity and sensitivity for Vanin‐1 (Figure 3J) [26]. Its selectivity has been validated using VNN1‐knockout mice. PHPO allows imaging of the distribution of endogenous Vanin‐1 in intestinal tissue and can monitor expression changes during organoid differentiation. Moreover, this probe has been used to visualize Vanin‐1 activation in a mouse model of IBD, providing new insights into disease pathogenesis. Additionally, PHPO supports HTS for Vanin‐1 modulators and has facilitated the identification of a potent novel inhibitor, octyl gallate (IC₅₀  =  29.9 nM). Together, these probes expand the toolkit for studying Vanin‐1 in inflammation and facilitate inhibitor discovery for related disorders.

2.2. Bioluminescent Probes

Bioluminescent probes generate signals through enzyme‐catalyzed bioluminescent reactions, offering distinct advantages, including high sensitivity, low background noise, and favorable biocompatibility. Among these, the firefly luciferin–luciferase system is widely employed in pharmaceutical and bioanalytical applications. The reaction relies on luciferin or aminoluciferin as the substrate and proceeds in the presence of ATP, Mg^{2 +}, and O₂, with the 6′‐amino (or 6′‐hydroxy) group serving as the key site for luciferase interaction. Since the bioluminescence of luciferin can penetrate tissues of living animals, this platform has been adapted to develop numerous probes for in vivo imaging of various analytes.

In 2018, Lin et al. reported the first bioluminescent probe targeting Vanin‐1, designated PA‐AL (Figure 4) [27]. The probe was constructed by conjugating PA as the recognition unit to aminoluciferin, achieving a minimum detection limit of 1.14 ng/mL. PA‐AL enabled noninvasive detection of endogenous Vanin‐1 activity in living animals, thereby filling a gap in the field of bioluminescent imaging of this enzyme. Its subsequent use in studying starvation‐induced upregulation of Vanin‐1 provided a new tool for exploring the biological roles of Vanin‐1 in metabolism and related disorders.

FIGURE 4.

Structure and reaction scheme of bioluminescent probe PA‐AL with Vanin‐1.

In parallel, Wang et al. developed the fluorescent probe PA‐AFC for in vitro inhibitor evaluation [18] (Figure 3B). This probe, which links PA to the fluorophore AFC, displays excellent sensitivity and specificity for Vanin‐1. Together, PA‐AFC (for in vitro screening) and PA‐AL (for cellular and in vivo validation) form a complementary assay system that supports a complete workflow spanning in vitro screening, cellular verification, and in vivo evaluation. When applied to validate candidate inhibitors in a mouse colitis model, this integrated platform helped confirm Vanin‐1 as a promising therapeutic target for IBD.

2.3. Chemiluminescent Probes

Chemiluminescent probes operate without requiring an external excitation source, thereby eliminating autofluorescence interference entirely and providing an exceptionally high signal‐to‐noise ratio. These characteristics confer distinct advantages for applications in biological fluid analysis and in vivo imaging.

Feng et al. developed the first chemiluminescent probe CL‐Pa for urinary Vanin‐1 detection. The probe was designed by conjugating PA to a phenoxy‐dioxetane scaffold via a self‐immolative linker (Figure 5A) [28]. CL‐Pa demonstrates outstanding sensitivity with a detection limit of 0.008 ng/mL, prominent and fast response (146‐fold signal increase within 30 min), and great selectivity toward Vanin‐1 under physiological conditions. Leveraging these features, CL‐Pa successfully detected overexpressed Vanin‐1 in HepG2 cells as well as upregulated Vanin‐1 in HK‐2 cells following ethylene glycol stimulation. Notably, the probe enabled detection of cisplatin‐induced AKI as early as 12 h after cisplatin administration—significantly earlier than conventional clinical biomarkers (serum creatinine (sCr) and blood urea nitrogen (BUN)) and preceding observable renal histopathological changes (48 h). Furthermore, CL‐Pa allowed for the monitoring of urinary Vanin‐1 fluctuations in mice with varying degrees of AKI severity, offering a novel approach for early AKI diagnosis and therapeutic evaluation.

FIGURE 5.

Structure and reaction scheme of chemiluminescent probes with Vanin‐1. (A) CL‐Pa, (B) BPan‐CL.

To address the complexity of the inflammatory microenvironment, Jin et al. designed a dual‐locked chemiluminescent probe BPan‐CL (Figure 5B) [29]. This probe operates via a cascade activation mechanism involving the IBD‐related biomarkers chymotrypsin (CHT) and Vanin‐1, producing a specific chemiluminescent signal only when both enzymes are sequentially activated. Compared to fluorescent probes with similar dual‐lock designs, BPan‐CL exhibits a superior signal‐to‐noise ratio, enabling precise imaging and early diagnosis of IBD with enhanced specificity. In both live‐cell assays and IBD mouse models, BPan‐CL demonstrated significantly higher sensitivity than its single‐locked counterpart, Pan‐CL. This probe accurately distinguished IBD mice from healthy controls via in vivo imaging and enabled early IBD prediction through noninvasive fecal detection. These results validate that the combination of CHT and Vanin‐1 serves as an effective biomarker for precise, early IBD diagnosis, while also highlighting the application potential of logic‐gated probes in complex pathological scenarios.

2.4. Electrochemical Switching Probes

Electrochemical probes provide notable advantages, including simple operation, minimal instrumentation requirements, and the ability to perform direct detection in biological fluids. These characteristics position them as promising tools for point‐of‐care diagnostics and liquid biopsy applications.

Kumaragurubaran et al. developed VaninLP, the first activity‐based ratiometric electrochemical switch probe for Vanin‐1 (Figure 6) [30]. The probe operates through Vanin‐1‐catalyzed cleavage of an amide bond, which triggers a self‐immolative cascade that releases an amino ferrocene reporter, thereby generating a measurable electrochemical signal specific to the enzyme. A key strength of VaninLP is its ability to directly quantitatively measure Vanin‐1 activity in turbid biofluids—such as blood and urine—without requiring extensive sample pretreatment. The probe exhibits a wide linear range of 8–300 ng/mL, a low detection limit of 2.47 ng/mL, high selectivity, and strong enzyme affinity, with no observable interference from other electroactive substances present in biological matrices.

FIGURE 6.

Structure and reaction scheme of electrochemical probe VaninLP with Vanin‐1.

VaninLP has been successfully applied to detect Vanin‐1 activity on the surface of HepG2 cells, as well as in blood and urine samples. These results underscore its potential as a point‐of‐care diagnostic tool for early‐stage kidney injury diagnosis and for monitoring disease progression in conditions such as malaria.

3. Biological Applications of Vanin‐1 Probes

The design strategies for the probes described above are not isolated technical demonstrations, but rather precise responses to specific biological and clinical challenges. Traditional detection methods, such as Western blotting and ELISA, often face significant limitations when applied to Vanin‐1 activity measurement. These limitations include labor‐intensive and time‐consuming procedures that hinder rapid detection, insufficient sensitivity preventing reliable quantitative detection of Vanin‐1 in high background or turbid biological samples, and an inherent inability to support real‐time monitoring in living systems. The development of diverse probes has been specifically driven by the need to overcome these obstacles.

The design strategies outlined above are not merely technical demonstrations but are driven by specific biological and clinical demands. Fluorescent probes offer operational simplicity and robust visualization capabilities, making them suitable for cellular and in vivo imaging. NIR probes further enable deep tissue penetration, rendering them favorable for imaging visceral organs. Ratiometric probes improve quantitative accuracy through self‐calibration based on dual‐wavelength signal measurement. Chemiluminescent probes require no external excitation source and eliminate autofluorescence interference entirely, offering significant advantages in the analysis of biofluids such as urine and in deep tissue imaging. Electrochemical probes can directly detect turbid biological samples without complex pretreatment, positioning them as promising tools for point‐of‐care testing. Consequently, clinical needs are the primary determinant in probe selection. In the following sections, we will detail the applications of each probe type in early disease diagnosis and stratification, pathological mechanism investigation, as well as drug development and therapeutic efficacy evaluation (Figure 7).

FIGURE 7.

Applications of Vanin‐1 probes. Real‐time imaging using Vanin‐1 probes in living cells, organs, and small animals can be used for early disease diagnosis and stratification, pathological mechanism investigation, as well as drug development and therapeutic efficacy evaluation. The images in this figure are sourced from the references [19, 21, 22, 23, 24, 25, 26, 29].

3.1. Early Diagnosis and Disease Stratification

Vanin‐1 probes have demonstrated significant practical value in the early diagnosis and classification of various diseases. In AKI, the chemiluminescent probe CL‐Pa advances the diagnostic window to 12 h post‐administration, which is more than 36 h earlier than conventional biomarkers, such as sCr and BUN, and is capable of distinguishing between different grades of AKI [28]. The electrochemical probe VaninLP also serves as a practical tool for early AKI detection [30]. The NIR probe DDAV not only enables imaging of early‐stage renal injury but also allows for simultaneous monitoring of disease progression [22]. For IBD, the dual‐locked chemiluminescent probe BPan‐CL permits early IBD prediction via noninvasive fecal testing [29]. Probes such as PHPO and CYLP can visualize the activation and distribution of Vanin‐1 in inflamed intestinal regions, aiding in the precise pathological subtyping of IBD [20, 26]. Furthermore, the dual‐mode probe MB‐Van1 can rapidly detect Vanin‐1 upregulation in inflammatory models such as RA and DILI, while VaninLP demonstrates potential for monitoring the progression of malaria [25, 30].

3.2. Pathological Mechanism Research

Vanin‐1 probes provide critical tools for elucidating its physiological and pathological functions. The ratiometric probe CV‐PA first enabled quantitative in situ comparisons of Vanin‐1 across different live cells [19]; studies using CYLP and DCM‐PA probes further confirmed the association between Vanin‐1 and inflammatory responses as well as metabolic disorders such as diabetes [20, 24]. The bioluminescent probe PA‐AL revealed that starvation induces Vanin‐1 upregulation, whereas the DDAV probe identified gut‐microbial strains with high Vanin‐1 expression, providing new insights into the interactions between the microbiota and the host [22, 27]. Using the NIR probe Cy‐Pa, researchers demonstrated that Vanin‐1 modulates the sensitivity of tumor cells to cisplatin by regulating the synthesis of GSH. Combination therapy with the Vanin‐1 inhibitor RR6 enhanced cellular resistance to cisplatin, providing insight into the mechanisms of chemotherapy resistance and a basis for the rational design of combination therapies [21]. Additionally, the PHPO probe successfully visualized the dynamic changes in Vanin‐1 expression during organoid differentiation, opening new avenues for research in developmental and disease‐modeling [26].

3.3. Drug Development and Efficacy Evaluation

As a promising therapeutic target, significant progress has been made in the development of platforms for the discovery and evaluation of Vanin‐1 inhibitors. Early studies employed the fluorescent substrate pantothenate‐AMC for HTS, leading to the identification of several novel inhibitor scaffolds [17]. More recently, probes such as PHPO, DDAV, and PA‐AFC/PA‐AL have enabled multidimensional screening across in vitro, cellular, and in vivo levels [18, 22, 26, 27]. These studies have identified several potent natural inhibitors, including octyl gallate and oleuropein, which show promise in the treatment of diseases such as IBD and cancer [22]. In terms of efficacy assessment, CL‐Pa and BPan‐CL allow real‐time, noninvasive monitoring of the drugs responses to AKI or IBD, while Cy‐Pa provides critical data for evaluating the combination of Vanin‐1 inhibitors with chemotherapeutic agents such as cisplatin [21, 28, 29]. Moreover, analysis of gut microbiota with high Vanin‐1 expression based on DDAV provides guidance for optimizing the use of NSAIDs and mitigating associated intestinal risks [22].

4. Summary and Outlook

Vanin‐1 has emerged as an important pathological biomarker, and significant progress has been made in related probe technologies in recent years. From conventional fluorescent probes to chemiluminescent systems with high signal‐to‐noise ratios, pretreatment‐free electrochemical sensors, and logic‐gated probes with enhanced diagnostic specificity, continuous improvements in sensitivity, selectivity, and practicality have been achieved. These tools have not only deepened our understanding of Vanin‐1 in diseases such as AKI and IBD but also have provided vital support for early diagnosis, therapy monitoring, and drug discovery. In the future, multi‐lock probes capable of responding simultaneously to multiple pathological microenvironmental signals, such as pH, reactive oxygen species (ROS), and specific enzymes, could be designed to enable more precise discrimination of disease subtypes through Boolean logic operations (AND, OR, NOT). For example, a pH/enzyme dual‐responsive probe could be developed to simultaneously detect an acidic tumor microenvironment and Vanin‐1 activity. Furthermore, current probes are largely limited to detection functions and cannot simultaneously achieve targeted intervention. A promising strategy is to construct bifunctional conjugated molecules consisting of a probe‐Vanin‐1 inhibitor complex. Using a pantetheine recognition fragment as a linking scaffold, a signal reporter group could be attached to one terminus and the pharmacophore of a Vanin‐1 inhibitor to the other. Upon entering the target lesion, Vanin‐1 cleaves the amide bond, simultaneously releasing a reporter signal and the free inhibitor. This approach integrates in situ imaging diagnosis with concurrent targeted inhibitor therapy, providing a novel synthetic strategy for the development of therapeutic and diagnostic probes for inflammation and cancer. Although significant progress has been made with Vanin‐1 probes, future research may focus on the following areas: Enhancing clinical translation capabilities. Currently, most probes remain in the preclinical stages, future efforts should validate their stability, safety, and diagnostic performance using disease models that more closely resemble human conditions and clinical samples. Expanding detection capabilities. Integration of multiple imaging modalities (e.g., photoacoustic imaging) or sensing principles could yield multifunctional probes capable of simultaneously detecting enzyme activity, subcellular localization, and tissue‐microenvironment signals. Broadening the scope of biological and clinical applications. High‐sensitivity probes should be employed to investigate the role of Vanin‐1 in neurological and metabolic diseases, as well as other conditions, and to explore its potential as a therapeutic target across a wider spectrum of pathologies. Improving probe performance. Next‐generation probes should combine ultra‐high sensitivity, favorable pharmacokinetic properties (rapid clearance, tissue‐specific accumulation), and smart activation mechanisms (dual‐lock or multi‐lock designs) to minimize background signals and improve reliability in complex biological environments. Fostering technological integration. Convergence of probe chemistry with microfluidics, nanomaterials, and portable sensing platforms will accelerate the development of compact, integrated diagnostic devices suitable for point‐of‐care testing and home monitoring. Enabling precision medicine. Dynamic, non‐invasive monitoring of Vanin‐1 levels could support patient stratification, the development of personalized treatment plans, and real‐time assessment of therapeutic efficacy, thereby contributing to precision‐medicine frameworks.

Moving forward, continued innovation in molecular design, deeper interdisciplinary integration, and rigorous clinical validation will further establish Vanin‐1 probes as indispensable instruments in disease diagnosis, mechanistic investigation, and therapeutic development, ultimately strengthening the foundation for precise prevention, diagnosis, and treatment of Vanin‐1‐associated diseases.

Author Contributions

Shumei Wang: conceptualization, methodology, data curation, visualization, writing – original draft preparation, and writing – review and editing. Xing Li: conceptualization, supervision, and writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Biographies

Shumei Wang obtained her PhD in Medicinal Chemistry from the School of Pharmaceutical Sciences, Shandong University, China, and currently serves as a researcher at Shandong University of Commerce and Technology, National Engineering Research Center for Agricultural Products Logistics. Her research focuses on the design, synthesis, and bioactivity evaluation of compounds and probes exhibiting anti‐inflammatory, anticancer, and antibacterial properties.

Xing Li holds a Master's degree from Shandong University, China, and currently serves as a Senior Lecturer at Shandong Labor Vocational and Technical College. His research primarily focuses on the application of artificial intelligence within drug discovery and development.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.