Urine-based PSMA detection for noninvasive prostate cancer diagnosis: recent advances, translational challenges, and future perspectives

Abstract

Prostate cancer (PCa) is one of the most prevalent malignancies in men worldwide and continues to impose a substantial public health burden. Although prostate-specific antigen (PSA) testing remains the most widely used tool for PCa screening, its suboptimal specificity and moderate sensitivity, especially within the diagnostic gray zone of 4–10 ng/mL—frequently leads to unnecessary prostate biopsies, overdiagnosis, and overtreatment. Therefore, the development of sensitive, specific, and noninvasive diagnostic strategies has become a major priority in PCa management. Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein, is markedly overexpressed in PCa tissues and is closely associated with tumor aggressiveness, pathological progression, metastatic potential, and castration resistance. Owing to its strong disease association and established clinical relevance in molecular imaging and targeted therapy, PSMA has emerged as an attractive candidate for noninvasive diagnostic development. At the same time, urine has become an appealing liquid-biopsy substrate because it can be collected noninvasively, repeatedly, and with direct anatomical relevance to the prostate. Increasing evidence suggests that urinary PSMA-related analytes, including soluble PSMA, PSMA-positive extracellular vesicles (EVs), and associated transcripts, may provide a biological basis for noninvasive PCa detection. In this review, we summarize the biological rationale for PSMA as a urinary biomarker and critically examine recent advances in urinary PSMA detection technologies. Particular attention is given to antibody-based immunoassays and integrated biosensing systems that combine aptamer-mediated molecular recognition, CRISPR/Cas12a-assisted signal amplification, magnetic enrichment, and lateral flow assay (LFA) readout. We further discuss the major challenges hindering clinical implementation, including pre-analytical variability, urinary analyte heterogeneity, insufficient large-scale validation, and the limitations of single-biomarker strategies. Finally, future perspectives are outlined with emphasis on assay standardization, multimarker integration, and the development of clinically deployable point-of-care testing platforms. Collectively, urinary PSMA detection represents a promising but still emerging route toward more specific and noninvasive PCa diagnostics.

1. Introduction

PCa is among the most frequently diagnosed malignancies of the male genitourinary system and represents a growing global health burden. With population aging, increasing public health awareness, and broader implementation of screening programs, the incidence of PCa has continued to rise. Because disease stage at diagnosis is strongly associated with prognosis, early and accurate detection remains a critical determinant of clinical outcome. Localized PCa can often be effectively managed with curative treatment, whereas advanced or metastatic disease is associated with significantly higher morbidity and mortality. Therefore, improving early diagnostic strategies remains a major objective in PCa care (1, 2).

At present, serum PSA testing remains the cornerstone of PCa screening and early detection (3). However, PSA is organ-specific rather than tumor-specific, and elevated PSA levels may also occur in benign prostatic hyperplasia, prostatitis, urinary retention, or following urological procedures (4). This limitation is especially problematic in the diagnostic gray zone of 4–10 ng/mL, where PSA testing has suboptimal specificity and may lead to unnecessary biopsies, overdiagnosis, and overtreatment (5). Consequently, there is an urgent need to identify more specific biomarkers and to develop noninvasive assays with improved diagnostic accuracy.

Liquid biopsy has emerged as a promising strategy for noninvasive cancer detection because it allows repeated sampling and may dynamically reflect disease progression and tumor heterogeneity (6, 7). Among the available biological fluids, urine is particularly attractive for PCa detection because it can be obtained noninvasively, repeatedly, and at low cost, while also having direct anatomical relevance to the prostate. Proteins, exfoliated cells, EVs, cell-free nucleic acids, and other prostate-derived components can enter the urinary tract, making urine a valuable substrate for biomarker discovery and diagnostic assay development (8, 9). Urinary EVs and urine proteomes have been reported to reflect prostate-derived molecular information and show utility for diagnosis and risk stratification in PCa (8, 10).

PSMA is one of the most widely studied and clinically applied molecular targets in PCa. PSMA is expressed at low levels in normal prostate tissue but is markedly upregulated in PCa cells, particularly in high-grade, advanced, and castration-resistant disease (11, 12). Because of these properties, PSMA has been extensively exploited in molecular imaging and targeted therapy (13). More recently, growing attention has focused on its potential as a urinary biomarker, raising the possibility that PSMA-based detection may be translated into noninvasive diagnostic applications (14, 15). Urinary PSMA-positive EVs and urine-derived exosomal PSMA have both been reported as promising noninvasive biomarkers for PCa detection (14).

In this review, we aim to provide a comprehensive and critical overview of urinary PSMA detection for noninvasive PCa diagnosis. We first summarize the biological basis of PSMA as a urinary biomarker and its relevance in PCa. We then discuss recent advances in detection technologies, with particular emphasis on integrated biosensing strategies. Finally, we highlight key translational challenges and future perspectives, focusing on clinical applicability and standardization.

2. Biological basis of PSMA as a urinary biomarker

2.1. PSMA biology and its relevance to PCa

PSMA, also known as folate hydrolase 1 (FOLH1) or glutamate carboxypeptidase II, is a type II transmembrane glycoprotein with established overexpression in PCa (11, 12). Its expression is generally low in normal prostate tissue but becomes markedly elevated in malignant transformation and may further increase in aggressive, metastatic, and castration-resistant disease (12, 16).

Importantly, the same biological specificity that underlies PSMA-based theranostics also supports its exploration as a diagnostic biomarker (12, 13). Compared with many exploratory biomarkers that have limited clinical context, PSMA benefits from a strong translational foundation. Nevertheless, PSMA is not uniformly expressed in all PCas, and intertumoral as well as intratumoral heterogeneity may influence its diagnostic performance (12, 17). Therefore, while PSMA is highly promising, it should be viewed as a biologically enriched and clinically relevant marker rather than a universally sufficient one (17).

2.2. Why urine is a plausible matrix for PSMA-based testing

Urine is uniquely suited for PCa biomarker development because of its noninvasive accessibility and direct anatomical relevance to the genitourinary tract (6, 8). Prostate-derived material may enter urine through direct secretion, cellular shedding, or extracellular vesicle release (9). This makes urine a biologically meaningful matrix for detecting tumor-associated molecules.

The potential sources of urinary PSMA-related signals are likely heterogeneous (14, 18, 19). These may include soluble PSMA shed from tumor-associated membranes, PSMA-positive EVs, tumor-derived cellular debris, and PSMA-related transcripts such as FOLH1 mRNA (19, 20). This diversity strengthens the biological rationale for urinary detection but simultaneously complicates assay development, because different analytical platforms may target fundamentally different molecular forms under the same broad label of “urinary PSMA.”

Different detection strategies are inherently biased toward specific molecular forms of PSMA—soluble protein, EV-associated PSMA, or PSMA-related nucleic acids. For example, ELISA-based assays primarily target the soluble form, whereas immunomagnetic or aptamer-based enrichment approaches preferentially capture PSMA-positive EVs. CRISPR-based systems, on the other hand, typically rely on nucleic acid signal conversion strategies.

Each approach presents distinct advantages and limitations. Soluble PSMA detection is relatively straightforward but may suffer from low specificity, whereas EV-associated PSMA may better reflect tumor biology but requires more complex isolation procedures. Understanding these differences is critical for interpreting results and for selecting appropriate analytical platforms in future clinical applications.

2.3. Urinary PSMA in the context of other urinary biomarkers

Several urinary biomarkers have been investigated for the noninvasive detection of PCa, including PCA3, the TMPRSS2: ERG fusion transcript, exosomal RNAs, and various urinary miRNAs (21). These biomarkers have shown varying degrees of utility in early detection, risk stratification, and biopsy decision-making (8, 22). Compared with these markers, PSMA is particularly attractive because it is not only associated with molecular alterations in PCa but also strongly linked to disease aggressiveness and clinical progression (11–14, 18).

In this broader biomarker landscape, urinary PSMA offers several advantages. First, it is closely connected to PCa biology and tumor behavior. Second, it already has established clinical value in imaging and therapeutic targeting (12, 13). Third, it can be detected through a variety of assay formats, including protein-based, vesicle-based, and nucleic acid-assisted strategies (14, 18–20). However, urinary PSMA must still demonstrate robust analytical performance and clinically meaningful diagnostic value beyond currently available urine-based tests (8). Representative urinary biomarkers for PCa detection and their characteristics are summarized in Table 1. As shown, although several urinary biomarkers have demonstrated clinical utility, each has inherent limitations related to sensitivity, specificity, standardization, or workflow complexity. In this context, urinary PSMA represents a biologically relevant and technically versatile target, but its clinical value will ultimately depend on robust analytical validation and its ability to provide incremental diagnostic benefit beyond existing biomarkers.

Table 1.

Representative urinary biomarkers for prostate cancer detection and their analytical characteristics.

Biomarker	Detection method	Clinical significance	Limitations	Representative references
PCA3	RT-PCR	Supports early detection and biopsy decision-making	Often requires prostate massage before urine collection	(21, 23)
TMPRSS2:ERG	qPCR	Improves diagnostic specificity, especially when combined with other markers	Limited sensitivity when used alone	(21, 23)
PSMA	ELISA/aptamer-based assay/integrated biosensor	Promising biomarker for noninvasive diagnosis and therapeutic stratification	Low abundance and incomplete standardization	(14, 18)
Exosomal RNA markers	RNA-based assays	May distinguish clinically significant disease from benign conditions	High cost and relatively complex workflow	(8, 22)
SelectMDx/multiplex gene panels	qPCR-based panels	Predicts the risk of clinically significant PCa	Interpretation may depend on algorithmic models	(21)
Urinary miRNAs	qPCR/sequencing	Potential biomarkers for diagnosis and prognosis	Poor inter-study standardization	(21)

3. Current landscape of urine-based PCa biomarkers

The field of urine-based PCa diagnostics has developed rapidly over the past decade (21). Commercial or late-stage tests based on transcripts, exosomes, and multiplex gene panels have demonstrated that urine can serve as a clinically practical source of tumor-associated information (24, 25). These tests are generally positioned as adjuncts to PSA and imaging, particularly in men with equivocal PSA levels or borderline clinical findings (24–26).

This context is important because urinary PSMA is not being developed in isolation (21). New PSMA-centered assays must ultimately demonstrate not only technical feasibility but also clinical value relative to established urinary biomarkers. In particular, they should ideally improve the detection of clinically significant PCa, reduce unnecessary biopsies, or enable more accessible point-of-care testing strategies. Thus, urinary PSMA should be viewed as part of a broader diagnostic ecosystem rather than as a standalone replacement for existing tests (24).

Importantly, the clinical utility of urinary PSMA detection should be evaluated in the context of established diagnostic tools. For example, PSA testing typically shows a sensitivity of approximately 70–80% but a specificity of only ~25–40% in the diagnostic gray zone (4–10 ng/mL), leading to substantial overdiagnosis (27). In contrast, urinary biomarkers such as PCA3 and SelectMDx have reported AUC values ranging from 0.68 to 0.85 in detecting clinically significant PCa (28).

Emerging studies on urinary PSMA detection, although still limited, have reported promising diagnostic performance. For instance, urinary exosomal PSMA assays have demonstrated AUC values approaching 0.80–0.90 in small cohorts, with improved specificity compared to PSA alone (18, 29). However, these findings remain preliminary and require validation in larger, well-characterized populations.

Therefore, the true translational value of urinary PSMA detection will depend on its ability to provide incremental diagnostic benefit beyond PSA and existing urinary biomarkers, particularly in clinically relevant populations such as patients within the PSA gray zone (30).

4. Recent advances in urinary PSMA detection technologies

4.1. Antibody-based immunoassays

Antibody-based immunoassays represent the most established and clinically accessible approach for protein detection and provide an important reference framework for evaluating emerging urinary PSMA detection technologies. These methods are widely used due to their standardized protocols, quantitative output, and compatibility with existing clinical laboratory infrastructure.

Representative studies have demonstrated that urinary PSMA can be detected using conventional immunoassay formats with moderate diagnostic performance. For example, Wang et al. reported that urinary exosomal PSMA achieved an AUC of approximately 0.82 for distinguishing PCa from benign conditions (18). Similarly, ELISA-based assays typically achieve limits of detection in the ng/mL range, although their performance in clinical settings remains constrained by matrix interference and low analyte abundance (31, 32).

In contrast, amplification-based biosensing platforms, particularly CRISPR/Cas12a-enabled systems, have demonstrated substantially improved analytical sensitivity under optimized experimental conditions. However, these systems remain largely at the proof-of-concept stage and lack sufficient clinical validation.

4.1.1. Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) remains one of the most widely used methods for protein quantification. In the context of urinary PSMA, ELISA is attractive because it relies on well-established antibody–antigen interactions, provides quantitative output, and can be readily implemented in conventional laboratory settings. It also serves as a useful benchmark method for validating newer detection platforms (18, 20, 33, 34).

However, urinary PSMA analysis by ELISA faces several limitations. Urine is a complex biological matrix containing salts, metabolites, endogenous proteins, and other components that may interfere with antigen–antibody binding or reduce assay reproducibility. In addition, PSMA may be present at relatively low abundance in urine, necessitating sample concentration, purification, or enrichment steps. These additional procedures increase workflow complexity and may introduce variability (9, 35, 36). As a result, while ELISA remains valuable for analytical validation and exploratory studies, its direct suitability for rapid or decentralized clinical testing remains limited (34, 36).

4.1.2. Chemiluminescence immunoassay and advanced affinity-based detection

Chemiluminescence immunoassay (CLIA) offers higher sensitivity, a broader dynamic range, and greater automation potential than conventional colorimetric ELISA. These features make CLIA theoretically attractive for detecting low-abundance urinary targets such as PSMA (37). In addition, advanced affinity-based systems incorporating nanoparticle amplification, fluorescence labels, or glycoform-specific recognition may further enhance analytical performance. A recent study describing an assay for aberrantly glycosylated urinary PSMA illustrates how analytically enriched PSMA subclasses may offer improved discrimination relative to total target measurement (20, 38).

Nevertheless, the utility of such methods in urinary PSMA detection remains limited by several factors, including instrumentation requirements, operational cost, and insufficient validation in real clinical samples (35, 38). Their translational value will therefore depend not only on increased sensitivity but also on whether they can detect clinically informative forms of urinary PSMA reproducibly and cost-effectively (38).

4.2. Integrated biosensing strategies for urinary PSMA detection

Recent advances in urinary PSMA detection have increasingly moved beyond single-technology platforms toward integrated biosensing systems that combine molecular recognition, signal conversion, target enrichment, and portable readout within a unified analytical workflow (39). This trend reflects the practical reality that no individual technology is sufficient to simultaneously address all major analytical challenges associated with urinary PSMA detection, including low target abundance, matrix complexity, limited assay sensitivity, and the need for clinically deployable formats (18, 20, 35, 36, 39–41). As a result, hybrid platforms that incorporate aptamer-based recognition, CRISPR/Cas12a-mediated amplification, magnetic enrichment, and LFA readout have emerged as a particularly promising direction for next-generation noninvasive PCa diagnostics (40–43). As illustrated in Figure 1, these integrated platforms typically follow a modular workflow consisting of molecular recognition, signal amplification, target enrichment, and portable readout.

Figure 1.

Integrated biosensing workflow for urine-based PSMA detection in noninvasive prostate cancer diagnosis. The platform combines aptamer-mediated molecular recognition, CRISPR/Cas12a-driven signal amplification, magnetic bead-based enrichment, and LFA readout, enabling sensitive, specific, and point-of-care-compatible detection of urinary PSMA.

Within these integrated systems, aptamers often serve as the primary molecular recognition element (44, 45). Compared with antibodies, PSMA-targeting aptamers offer several advantages, including chemical synthesis, facile functional modification, batch-to-batch consistency, and direct compatibility with nucleic acid-responsive detection circuits (46). More importantly, aptamer–target binding can be rationally engineered to induce conformational switching or strand displacement, thereby converting protein recognition into a nucleic acid signal. This property makes aptamers especially suitable for incorporation into signal amplification platforms, where the binding event itself can act as the trigger for downstream molecular processing (46–48).

CRISPR/Cas12a represents a highly effective amplification engine for integrated biosensing platforms, primarily because target recognition activates its collateral ssDNA cleavage activity and enables strong signal multiplication through trans-cleavage of labeled reporters (49, 50). For protein analysis, this enzymatic property can be rationally coupled to aptamer-based molecular transduction, in which target binding induces strand release, strand displacement, or conformational exposure of a predesigned DNA activator that subsequently switches on Cas12a cleavage. This strategy is particularly well suited to urinary PSMA analysis, where low analyte abundance and matrix complexity often limit the performance of direct detection schemes. By introducing an additional programmable amplification layer downstream of target recognition, Cas12a-based designs can substantially improve assay sensitivity without sacrificing modularity or compatibility with diverse signal-output modalities (49–52). Several CRISPR/Cas12a-based biosensing platforms have achieved detection limits down to the pM–fM range under optimized experimental conditions (53, 54). However, these results are largely derived from proof-of-concept studies using spiked samples rather than real clinical cohorts.

Magnetic enrichment provides a complementary upstream module that improves the analytical performance of urine-based assays by increasing target recovery, reducing nonspecific background, and simplifying post-capture washing and purification steps (14, 32). In practice, magnetic particles can be functionalized with antibodies, aptamers, or nucleic-acid probes to selectively isolate soluble biomarkers, EVs, or reporter-bearing complexes from urine (8, 14, 32). This is especially valuable in urinary diagnostics, where biomarker concentrations are low and pre-analytical variability is high. Notably, PSMA-positive EVs have already been isolated from urine by immunomagnetic approaches, supporting the feasibility of PSMA-directed vesicle enrichment as a PCa liquid-biopsy strategy (14). In parallel, recent urinary proteomic evidence indicates that extracellular-vesicle fractions can preserve prostate-derived molecular signatures and may provide biologically more informative material than unfractionated urine for downstream tumor-associated analysis (8, 9).

For practical deployment, LFA-based and other portable readout formats are particularly valuable because they enable rapid, low-cost, and user-friendly detection (55, 56). Traditional LFAs, however, often lack the sensitivity required for low-abundance tumor biomarkers, and this limitation can be partially overcome in integrated systems by upstream amplification and enrichment modules (56, 57). For example, aptamer recognition may first generate a nucleic acid trigger, CRISPR/Cas12a may then amplify the signal through collateral reporter cleavage, magnetic particles may concentrate the relevant analytes or signal complexes, and the final output may be visualized on an LFA strip (57–59). Such multistage designs preserve the operational simplicity of strip-based detection while markedly improving analytical performance (57–59). Similar strategies may also be adapted to fluorescence, electrochemical, or microfluidic readout formats, depending on the intended clinical setting (40, 60, 61).

Taken together, these integrated biosensing strategies represent a major evolution in urinary PSMA assay design. Rather than relying on a single recognition or readout mechanism, they exploit the complementary strengths of different technologies: aptamers provide selective and programmable recognition, CRISPR/Cas12a enables ultrasensitive signal amplification, magnetic particles improve analyte enrichment and matrix cleanup, and LFA supports portable point-of-care readout (32, 40, 56, 62). This modular architecture is highly attractive for translational development because each component can be independently optimized while remaining compatible with the overall workflow (40, 56, 62).

Nevertheless, several important challenges must be addressed before such integrated systems can be translated into routine clinical practice (63, 64). The combination of multiple functional modules may increase assay complexity, reagent instability, background leakage, and manufacturing variability (63, 65). Moreover, strong analytical performance in proof-of-concept studies does not necessarily guarantee reproducibility in real clinical urine samples. Therefore, future development should focus not only on maximizing sensitivity, but also on simplifying workflow design, improving matrix tolerance, enhancing batch consistency, and validating performance in large prospective patient cohorts (63–66).

Despite their conceptual advantages, integrated biosensing platforms remain at an early developmental stage and face several nontrivial challenges. First, the integration of multiple functional modules increases system complexity, which may compromise robustness and reproducibility (67).Second, batch-to-batch variability in reagents such as aptamers, enzymes, and nanoparticles can introduce significant analytical variability (68). Third, many reported studies rely on spiked samples or small clinical cohorts, and their performance in heterogeneous real-world urine samples remains insufficiently validated (69).

Importantly, there is a tendency in the literature to overstate analytical performance without adequately addressing practical constraints such as workflow complexity, assay time, and operator dependency (70). Therefore, distinguishing clearly between proof-of-concept demonstrations and clinically validated technologies is essential for accurately assessing the translational potential of these platforms (69).

Taken together, integrated biosensing strategies represent a significant advancement in urinary PSMA assay design by combining complementary technologies to overcome individual limitations. However, their translational potential should be interpreted with caution (71) Despite their conceptual advantages, these systems remain at an early developmental stage. The integration of multiple functional modules increases assay complexity and may compromise reproducibility (72). In addition, batch-to-batch variability in key components such as aptamers, enzymes, and nanomaterials introduces further uncertainty (72). Importantly, many reported studies emphasize analytical sensitivity without adequately addressing practical constraints, including workflow complexity, assay time, and operator dependence (73). Therefore, it is essential to distinguish clearly between proof-of-concept demonstrations and clinically validated technologies (71). Future studies should prioritize standardized evaluation, simplified workflows, and large-scale clinical validation to define the real-world applicability of these platforms.

5. Comparative advantages and limitations of current approaches

A comparison of the major technology classes for urinary PSMA detection is provided in Table 2. Importantly, these platforms differ not only in their underlying analytical principles but also in quantitative performance, clinical applicability, operational complexity, and translational readiness. As summarized, current analytical approaches can be broadly categorized into conventional immunoassays, advanced affinity-based detection systems, and integrated biosensing strategies (76). To further delineate these differences, Table 3 provides a quantitative and clinically oriented comparison, including key parameters such as limit of detection, dynamic range, time to result, and clinical role, thereby enabling a more comprehensive evaluation of their translational potential.

Table 2.

Comparison of major technology classes for urinary PSMA detection.

Platform	Core principle	Major strengths	Limitations	Clinical context	Translational potential	Representative references
ELISA/conventional immunoassay	Antibody–antigen binding	Familiar, quantitative, relatively standardized	Matrix interference, low analyte abundance, laboratory dependence	Validation/research	Useful for validation, limited for POCT	(18, 32)
CLIA/advanced affinity assay	Enhanced immunodetection with luminescent or nanoparticle-assisted readout	High sensitivity, broader dynamic range	Cost, instrumentation, limited urinary validation	Hospital lab	Promising in centralized settings	(8, 32, 74)
Integrated biosensing platform	Aptamer recognition + CRISPR amplification + magnetic enrichment + LFA/portable readout	High sensitivity, modularity, point-of-care potential	Multicomponent complexity, reproducibility challenges	POCT/screening	Highly promising but still early-stage	(8, 9, 32, 39, 75)

Table 3.

Comparative overview of urinary PSMA detection platforms.

Platform	Target analyte	Limit of detection	Dynamic range	Time to result	Major strengths	Key limitations	Clinical role	Translational stage
ELISA	Soluble PSMA protein	1–10pM	10pM–100nM	2–4h	Quantitative, standardized	Matrix interference, low sensitivity	Reference method	Clinically established
CLIA	Soluble PSMA/glycoforms	10–100fM	100fM–10nM	1–2h	High sensitivity, automated	Cost, instrumentation	Central lab diagnostics	Early clinical adoption
EV-based assays	PSMA+ EVs (protein/RNA)	100fM–10pM	pM–nM	4–24h	High biological relevance	Isolation complexity	Biomarker discovery	Emerging
Aptamer–CRISPR biosensor	Nucleic acid-converted signal	1–100fM	fM–nM	30–90min	Ultra-sensitive, modular	Limited clinical validation	POCT potential	Proof-of-concept
Integrated biosensing platforms	Multi-module hybrid signal	1–100fM	fM–nM	30–120min	Amplification + enrichment	Reproducibility issues	Next-gen POCT	Early translational

Data summarized from representative studies on urinary PSMA detection and related biosensing platforms (19, 21, 30–33, 37, 49, 54–58).

Conventional immunoassays, such as ELISA, are widely used due to their established protocols, quantitative output, and relatively high reproducibility. These methods are particularly valuable as reference techniques for analytical validation. However, their performance in urinary PSMA detection is often limited by matrix interference, low analyte abundance, and the need for laboratory-based instrumentation, which restricts their applicability in decentralized or point-of-care settings (32–34). As summarized in Table 3, ELISA typically exhibits detection limits in the ng/mL range and relatively long assay times, which further constrain its utility in rapid clinical decision-making. Therefore, ELISA is best positioned as a reference and benchmarking tool rather than a frontline diagnostic modality, particularly in the context of decentralized testing.

Advanced affinity-based assays, including chemiluminescence immunoassays and nanoparticle-enhanced detection systems, offer improved sensitivity and broader dynamic range. These approaches may enable the detection of low-abundance PSMA species or specific molecular subtypes, such as aberrantly glycosylated PSMA. Nevertheless, their clinical translation is constrained by high cost, complex instrumentation, and limited validation in large-scale urinary studies (33, 36). Compared with ELISA, these methods demonstrate improved analytical sensitivity (typically in the pg/mL range, as shown in Table 3), but their dependence on centralized laboratory infrastructure limits their applicability in decentralized clinical settings. As indicated in Table 3, although sensitivity is enhanced, operational complexity and cost remain significant barriers to widespread clinical adoption. Accordingly, these approaches are more suitable for high-throughput hospital-based diagnostics rather than point-of-care applications.

In contrast, integrated biosensing platforms that combine aptamer-mediated recognition, CRISPR/Cas12a-based signal amplification, magnetic enrichment, and portable readout formats (e.g., lateral flow assays) have emerged as a promising next-generation strategy. These systems are designed to address multiple analytical challenges simultaneously, including low target abundance, complex sample matrices, and the need for rapid and user-friendly detection. By leveraging the complementary strengths of each module, integrated platforms can achieve enhanced sensitivity, modularity, and point-of-care applicability (37, 49, 58). Importantly, these systems are uniquely positioned for future POCT deployment, provided that their robustness and reproducibility can be adequately addressed. Notably, as summarized in Table 3, these platforms can achieve detection limits down to the pM–fM range with significantly reduced assay time (typically within 30–120 min), highlighting their potential for rapid and highly sensitive detection. This performance advantage is clearly illustrated in Table 3, where integrated platforms outperform conventional immunoassays in analytical sensitivity and turnaround time. Importantly, these features make integrated biosensing systems particularly attractive for future point-of-care testing applications and decentralized diagnostic scenarios. However, it remains unclear whether such improvements in analytical sensitivity translate into meaningful clinical benefits, such as improved detection of clinically significant prostate cancer or reduction of unnecessary biopsies.

However, despite their conceptual advantages, integrated systems remain in an early stage of development. Their multicomponent nature may introduce challenges related to assay complexity, reagent stability, background signal leakage, and batch-to-batch variability. In addition, robust clinical validation in large and well-defined patient cohorts is still lacking. Therefore, while integrated biosensing strategies hold considerable promise, further optimization and standardization are required before they can be translated into routine clinical practice. Furthermore, the discrepancy between analytical performance under controlled experimental conditions and real-world clinical performance remains a critical unresolved issue. These limitations collectively represent major barriers to clinical translation and large-scale implementation.

Overall, as highlighted in Table 2, no single analytical platform currently satisfies all clinical and operational requirements. Conventional immunoassays provide robustness and standardization but lack sensitivity and portability; advanced affinity-based assays improve analytical performance but remain infrastructure-dependent; whereas integrated biosensing platforms offer superior sensitivity and point-of-care testing potential but require further validation and simplification. Future development should therefore focus not only on improving analytical sensitivity but also on enhancing robustness, reproducibility, workflow simplicity, and clinical relevance. Importantly, standardized head-to-head comparisons across different detection platforms under harmonized experimental conditions will be essential to define the clinical positioning of urinary PSMA assays and to determine whether they provide meaningful incremental value over existing diagnostic strategies. In particular, future studies should evaluate these platforms in clinically relevant populations, such as patients within the PSA gray zone, where improved specificity is most urgently needed.

6. Major challenges in urinary PSMA detection

6.1. Pre-analytical variability and lack of standardization

One of the major barriers to urinary PSMA assay development is the absence of standardized pre-analytical procedures. Variations in urine collection timing, first-void versus random sampling, prostate massage or digital rectal examination prior to collection, centrifugation protocols, storage conditions, and freeze–thaw cycles may all influence analyte recovery and assay performance (77–82). Without harmonized procedures, inter-study comparison remains difficult and clinical reproducibility is compromised (77–81).

To address these challenges, several standardization strategies should be considered (77). First, urine collection protocols should be harmonized, including recommendations on first-void versus random sampling, timing relative to digital rectal examination, and patient preparation (83). Second, sample processing procedures such as centrifugation speed, filtration, and storage conditions should be standardized to minimize variability in analyte recovery (84). Third, normalization strategies—such as creatinine normalization, total protein normalization, or extracellular vesicle quantification—should be incorporated to account for inter-sample variability (77).

In addition, the adoption of reporting guidelines, similar to MISEV standards for EVs, may help improve reproducibility and comparability across studies (69). Establishing such frameworks will be critical for advancing urinary PSMA detection toward clinical implementation (77).

6.2. Analyte heterogeneity

Urinary PSMA is not a single molecular entity. Different platforms may detect soluble PSMA protein, PSMA-associated EVs, or PSMA-related transcripts. These analytes differ in biological origin, abundance, stability, and potential clinical significance. This lack of analytical uniformity complicates cross-study comparison and hinders the establishment of clinically meaningful thresholds (80, 81).

6.3. Insufficient clinical validation

Most current studies on urinary PSMA detection remain exploratory or proof-of-concept in nature (18, 81, 85). Sample sizes are often limited, patient cohorts are heterogeneous, and external validation is rare. In particular, there is insufficient evidence regarding the performance of urinary PSMA assays in clinically important populations such as men with PSA values in the gray zone, patients with clinically significant disease, or those undergoing longitudinal monitoring. Large prospective multicenter studies are therefore needed (86, 87).

6.4. Limitations of single-biomarker strategies

PCa is a biologically heterogeneous disease, and no single biomarker is likely to capture its full complexity. Although PSMA is biologically compelling, its standalone diagnostic performance may be insufficient in certain settings. Multimarker approaches that combine PSMA with PCA3, TMPRSS2:ERG, urinary miRNAs, extracellular vesicle signatures, or imaging findings may improve diagnostic accuracy and clinical relevance (9, 86–90).

6.5. Clinical translation considerations

Beyond analytical performance, successful clinical translation of urinary PSMA assays requires careful consideration of regulatory approval, cost-effectiveness, scalability, and integration into existing clinical workflows.

From a regulatory perspective, diagnostic assays must demonstrate analytical validity, clinical validity, and clinical utility in accordance with guidelines from agencies such as the FDA or EMA. Currently, most urinary PSMA detection platforms remain at the proof-of-concept stage and have not yet undergone rigorous regulatory evaluation (71).

Cost and scalability are also critical factors (70). While integrated biosensing platforms offer high sensitivity, their multicomponent design may increase manufacturing complexity and cost, potentially limiting widespread adoption (67). Simplified assay formats with minimal instrumentation requirements will be more compatible with point-of-care deployment (73).

In terms of clinical workflow integration, urinary PSMA assays are most likely to function as adjunctive tools rather than standalone diagnostics. For example, they may be incorporated into decision-making pathways alongside PSA testing, MRI findings, and other urinary biomarkers to improve biopsy selection and risk stratification (91, 92).

Therefore, future development should prioritize not only analytical innovation but also real-world feasibility, regulatory readiness, and clinical usability.

7. Future perspectives

The future development of urinary PSMA detection should focus on both analytical optimization and clinical translation. First, standardized protocols for urine collection, pretreatment, storage, and normalization are urgently needed to improve reproducibility and comparability across studies (90). Second, assay development should emphasize not only analytical sensitivity but also matrix tolerance, ease of operation, batch consistency, and cost-effectiveness (93).Third, future studies should be designed around clinically relevant questions, especially whether urinary PSMA can help distinguish clinically significant PCa from indolent disease and reduce unnecessary biopsies in patients with equivocal PSA results. In this regard, multimarker integration may represent one of the most promising directions. PSMA-based detection could be combined with transcriptomic markers, exosomal biomarkers, urinary proteomics, or clinical risk models to generate more robust diagnostic frameworks (23, 94).

From a technological standpoint, hybrid systems that integrate aptamer recognition, CRISPR amplification, magnetic enrichment, and LFA or microfluidic readout may eventually yield clinically deployable point-of-care devices. However, for such systems to become truly viable, they must demonstrate not only excellent analytical performance under controlled conditions but also reproducibility, scalability, operational simplicity, and diagnostic value in real-world patient cohorts.

Ultimately, the field must move beyond elegant proof-of-concept biosensors toward rigorously validated assays aligned with genuine clinical decision-making (81, 87). This transition will require close collaboration among molecular biologists, analytical chemists, biomedical engineers, and clinicians.

8. Conclusion

Urinary PSMA detection represents a scientifically compelling and clinically relevant strategy for the noninvasive diagnosis of PCa. Its appeal arises from the convergence of three major factors: the strong biological relevance of PSMA in PCa, the practical advantages of urine as a liquid-biopsy substrate, and rapid advances in biosensing technologies capable of detecting low-abundance analytes with increasing sensitivity.

Among currently developing approaches, integrated biosensing systems that combine aptamer-mediated recognition, CRISPR/Cas12a-assisted amplification, magnetic enrichment, and portable readout have emerged as particularly promising. These hybrid strategies offer a pathway toward more sensitive, specific, and clinically deployable urinary PSMA assays.

Nevertheless, significant obstacles remain before urinary PSMA testing can be translated into routine clinical practice. These include pre-analytical variability, analyte heterogeneity, limited large-scale validation, and the need to demonstrate clear incremental value over existing biomarkers and diagnostic workflows. With continued technological refinement, rigorous clinical validation, and deeper integration into multimodal diagnostic frameworks, urinary PSMA-based assays may ultimately become valuable tools for PCa screening, biopsy triage, risk stratification, and disease monitoring.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by grants from the National Natural Science Foundation of China (Grant No. 82203217), the Top Talent Support Program for Young and Middle-Aged People of Wuxi Health Committee (No. BJ2023035), and the Scientific Technological Innovation and Venture Capital Fund in Wuxi (No. Y20232004).

Author contributions

ZJ: Writing – original draft, Writing – review & editing, Methodology, Supervision. CD: Writing – original draft, Methodology. JH: Formal Analysis, Writing – original draft. SZ: Conceptualization, Project administration, Formal Analysis, Validation, Visualization, Methodology, Data curation, Supervision, Writing – original draft, Software, Writing – review & editing, Funding acquisition, Resources, Investigation.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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