Sensitive and portable point-of-care monitoring of the breast cancer biomarker CA15-3 remains constrained by the antibody dependence, multistep incubation procedures, and specialized instrumentation required for conventional immunoassays. Herein, a smartphone-integrated microfluidic biosensor was developed by integrating few-layer molybdenum boride MBene nanosheets (MBene NSs) with a triple-helix molecular switch (THMS)-mediated CHA-CRISPR/Cas12a cascade system for quantitative CA15-3 detection. Few-layer MBene NSs were synthesized via NaOH-assisted hydrothermal etching of MoAlB followed by ultrasonic exfoliation, establishing a fluorine-free route for boron-terminated two-dimensional quenching materials. First-principles calculations and ELF analyses predicted stronger adsorption interactions between MBene NSs and DNA nucleobases than those observed for Ti₃C₂ MXene, mainly mediated by π-π stacking interactions. This enhanced interaction facilitates efficient adsorption of fluorescent DNA probes, thereby suppressing background fluorescence and improving signal-to-noise performance. The calculated results agree with previously reported MXene-mediated ssDNA fluorescence quenching behavior and are further supported by our fluorescence quenching experiments. Aptamer recognition of CA15-3 released the K-strand from THMS, triggering enzyme-free CHA strand displacement and Cas12a-mediated trans-cleavage of the FAM-ssDNA probe, thereby restoring fluorescence at the MBene NSs interface. The sensing platform was integrated into a 3D-printed four-chamber microfluidic chip and analyzed using a smartphone-based green-to-blue (G/B) channel ratio. The biosensor exhibited a linear response over 10−2-102 U·mL−1, with a detection limit of 0.01 U·mL−1, a relative standard deviation of 1.75%, and high specificity against CA12–5, CEA, ER, and PR. Clinical validation using serum samples from preoperative patients, postoperative patients, and healthy individuals (n = 30) yielded an AUC of 0.99, in close agreement with ELISA results (AUC = 1.00). This study establishes MBene NSs surface engineering as a tunable strategy for CRISPR-based protein detection and provides a scalable framework for resource-adaptive point-of-care cancer diagnostics.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer correlates with high aggressiveness, an increased recurrence rate, and poor survival, holding significant diagnostic value. In this work, a HER2-specific aptamer (Apt) was engineered into a Apt nanoswitch (hApt) and revealed the interaction of HER2-aptamers through molecular docking and quantified the binding energy and dynamic behavior through molecular dynamics simulations. A lock-expose mechanism was designed by combining the target-induced conformational switch of hApt with template-prefabricated rolling circle amplification (rRCA), forming a T-hApt-rRCA sandwich structure. Coupled with CRISPR/Cas12a and fluorescent probes, this sensor enabled highly sensitive detection, with a linear range of 10 fg/mL to 10 ng/mL and a limit of detection of 1.42 fg/mL. Using HUVEC, A549, MCF-7, and SK-BR-3 cell lines to model HER2 heterogeneity in circulating tumor cells (CTCs) enabled in situ imaging, differentiation, and quantitative detection of membrane HER2 expression, thereby providing direct visualization of expression levels and highlighting the translational promise of this approach. The accurate detection and clear differentiation between the eight healthy samples and the twenty-eight breast cancer patient samples further underscore the practical applicability of this sensing strategy.

Redox dysregulation is a hallmark of numerous pathological conditions, including cancer, chronic inflammatory diseases, fibrosis, neurodegenerative disorders and cardiovascular diseases, where aberrant production of reactive oxygen, nitrogen, sulfur and carbonyl species disrupts cellular signalling, metabolism, immune responses and tissue homeostasis. Despite its central role in disease progression, clinical assessment and therapeutic modulation of redox imbalance remain largely indirect, non-specific, failing to adequately capture the spatially heterogeneous and highly dynamic nature of redox dysregulation. Redox-responsive nanosystems offers the potential to bridge this gap by incorporating redox-sensitive materials that can spatiotemporally sense and respond to disease-associated oxidative or reductive microenvironments. Through predictable chemical or structural transformations triggered by endogenous redox cues, these nanoplatforms enable site-specific imaging, on-demand drug release, catalytic therapy and signal amplification. These capabilities position redox-responsive nanomedicine as a powerful approach for enhancing therapeutic precision while minimising off-target toxicity. This review provides a comprehensive and mechanism-informed overview of redox-responsive nanosystems for advanced drug delivery and precision medicine. We systematically connect disease-associated redox mechanisms with nanoplatform design strategies and functional outcomes across diagnostic, therapeutic and combined theranostic applications. In addition, we critically discuss key translational challenges, including biosafety, pharmacokinetics, large-scale manufacturing and regulatory considerations. Finally, we outline future directions toward clinically translatable, redox-guided nanomedicine.

Replicating biological systems using non-living materials, from the foundational molecular level to complex tissue structures, is central to abiotic mimicry. Enzymes play a vital role in these systems; however, replicating their enzymatic power with minimal components remains a key challenge. Here we show that gallium in the liquid state exhibits nuclease-like activity with preferred cleaving sites. The mechanism involves nucleotide-biased adsorption and hydroxyl radical-assisted phosphodiester hydrolysis. Compared with previously reported artificial metallonucleases, the liquid gallium uniquely integrates its oxide layer for substrate adsorption and its metallic core with electrons as a cleavage active center, forming a ligand- and cofactor-free artificial nuclease platform. Moreover, their activity is tunable through synthesis parameters and external stimuli, enabling programmable control with spatial or temporal precision. This work presents a minimalistic yet functional approach to enzyme mimicry, expanding the design space for abiotic enzymatic systems and offering potential opportunities in therapeutic applications, synthetic biology, and biomaterials.

Circular DNA and RNA have found broad application in biosensing, gene therapy, diagnostics, and fundamental nucleic acid research due to their high structural stability. Their covalently closed topology provides strong resistance to exonuclease degradation, enabling applications such as rolling circle amplification or transcription (RCA/RCT), and highly sensitive CRISPR-based biosensing. Various strategies have been developed for the preparation of circular nucleic acids, primarily including chemical and enzymatic ligation, while circular RNA can additionally be generated via ribozyme or self-splicing intron mediated approaches. Each synthesis method has distinct features that influence its suitability for particular applications. In this review, we summarize recent advances in the synthesis of circular DNA, RNA, and RNA-DNA hybrids, discuss key factors affecting circularization, and how these methods can support the development of CRISPR biosensing and help broaden the future landscape of molecular diagnostics.

Creatinine is a critical biomarker for assessing renal function, and its accurate detection is essential for early diagnosis of kidney diseases. However, real-time and label-free specificity creatinine sensing remains challenging. Herein, we present an integrated microfluidic surface-enhanced Raman scattering (SERS) platform incorporating an in situ polymerized Au NPs@Cu MOF/hydrogel substrate for sensitive and selective creatinine detection. The Au NPs@Cu MOF composite serves as the core SERS active unit, in which Cu MOF provides specific Cu-N coordination sites for direct, label-free specificity creatinine capture. Meanwhile, Au NPs provide electromagnetic enhancement (EM) through localized surface plasmon resonance (LSPR), which synergistically couples with the chemical enhancement (CM) of the Cu MOF to amplify the Raman signal. The hydrogel matrix provides good morphological stability and improves signal reproducibility. In addition, a three-layer microfluidic architecture was designed to prevent hydrogel dehydration and sample cross contamination. This integrated sensor enables creatinine detection using only 10 μL of sample with a 10 min incubation, achieving a low detection limit of 2.54 × 10-4 mg/dL. Further, it exhibited excellent performance in both artificial urine and real human urine, highlighting its potential for practical bioanalytical applications.

Redox-responsive lipid nanoparticles (LNPs) are emerging as a powerful platform for precision nanomedicine by exploiting disease-associated redox imbalances, such as elevated glutathione and reactive oxygen species, to trigger controlled cargo release and structural activation. This strategy is of great importance for developing gene-based therapeutics, where efficient cytosolic delivery is essential. In addition to nucleic acids, redox-responsive LNPs have also been explored in delivering small molecules, proteins, and theranostic agents, broadening their potential in both cancer and non-cancer diseases. This review summarizes the biological basis of redox responsiveness, key design principles for responsive chemical structures, and major advances in payload delivery and targeting capability of LNP carriers. Moreover, some major bottlenecks, including redox heterogeneity, stability, responsiveness trade-offs, and translational complexity, are critically discussed. Future directions are also highlighted, particularly for organ-selective delivery, multifunctional theranostics, and clinically translatable LNP-based medicines.

Circulating tumor DNA (ctDNA) is a key biomarker that directly reflects tumor-specific gene mutations, but current biosensing strategies often suffer from inefficient molecular-to-electrocatalytic signal transduction and limited amplification efficiency. Herein, we reported for the first time a Cascade-Activated DNA Nano-Gating (CANDG) strategy integrated with a phosphorus-doped iron single-atom nanozyme (P/Fe-SAN) electrocatalyst to achieve dual-mode ctDNA sensing. The CANDG strategy was first employed to enhance detection specificity and sensitivity. In the presence of target ctDNA-PIK3CA, the initially inhibited DNA substrate was activated, initiating a strand-displacement reaction that converted the locked conformation into an open state, thereby autonomously driving subsequent strand-displacement events. This cascade process not only amplifies the nucleic-acid signaling but also triggers the precise release of TMB from an Exo III-assisted nano-switch (HP@TMB@MOF) probe. Meanwhile, phosphorus doping modulates the electronic structure of Fe-Na centers, thereby enhancing O2 activation and catalytic kinetics. Acting as an efficient electrocatalytic signal output unit for secondary amplification, it enables simultaneous electrochemical and colorimetric dual-mode detection. Benefiting from this cascade-catalysis synergy, the biosensor achieves ultralow detection limits of 63.8 zM (electrochemical) and 111.2 aM(colorimetric), along with excellent selectivity, reproducibility, and practical applicability. Furthermore, the single-atom catalytic configuration and reaction mechanism were elucidated by extended X-ray absorption fine structure (EXAFS) analysis and density functional theory (DFT) simulations. This work pioneers the CANDG-P/Fe-SAN coupled platform, effectively overcoming the long-standing bottleneck of weak recognition-catalysis coupling, and offers a generalizable strategy for ultrasensitive ctDNA detection toward early cancer diagnosis and therapeutic monitoring.

CA15-3 is a vital biomarker for monitoring and evaluating treatment efficacy in breast cancer patients, especially those with advanced-stage disease. Conventional immunoassays face limitations, including high cost, limited sensitivity, and dependence on potentially error-prone single-fluorescence signal outputs. To address these challenges, we designed a dual-signal-emitting sensor based on CdTe quantum dots@ZIF-8 nanomaterials, incorporating both the CRISPR/Cas12a system and catalytic hairpin assembly (CHA) technology. The binding of CA15-3 to its specific aptamer inhibits the CHA cascade reaction, thereby preventing the generation of doublestranded DNA activators necessary to initiate Cas12a-mediated trans-cleavage activity. As a result, FAM-ssDNA is extensively adsorbed onto the CdTe QDs@ZIF-8 surface, where efficient electron transfer effectively quenches its fluorescence. The incorporation of the aptamer facilitates dual-signal amplification through both the CHA and CRISPR/Cas12a systems, achieving a detection limit of 0.131 U/mL. This dual-signal output mechanism provides the system with an internal reference function, thereby significantly improving the reliability of the results. Overall, this work establishes a novel tumor biomarker detection platform that combines high sensitivity with excellent robustness.

Inspired by the highly specific recognition and signal amplification of pollen germination on a stigma, we propose a biomimetic electrochemical sensing strategy for ultrasensitive aflatoxin B1 (AFB1) detection. In this pollen-mimetic system, CuCo2O4 nanoflowers serve as "pollen-mimetic grains", with their bimetallic Cu/Co centers functioning as an internal "energy conversion system" for robust signal output. AFB1 acts as the "stigmatic secretion", triggering the release of CuCo2O4 nanoprobes from the aptamer complex via target-induced strand displacement. The released nanoprobes are subsequently captured by a hairpin probe on the electrode, which mimics the "stigmatic receptor", through specific DNA hybridization. Finally, the captured CuCo2O4 nanozyme catalyzes the oxidation of TMB, mimicking "pollen germination and tube growth" to amplify the recognition event into a measurable electrochemical signal. Under optimized conditions, this biomimetic sensor achieves a wide linear range (10-11 to 10-4 mg mL-1) and an ultralow detection limit of 2.59 fg mL-1, with excellent reproducibility, stability, and selectivity. Recovery rates in real food samples range from 97.61% to 101.53%. This pollen-stigma biomimetic framework not only provides a highly sensitive platform for AFB1 detection but also establishes a new paradigm for designing high signal-to-noise ratio biosensors for complex matrices.

Early and accurate detection of breast cancer is essential for improving patient prognosis. Carbohydrate antigen 15-3 (CA15-3) is a key serum biomarker used in clinical monitoring, yet its low abundance in early stages requires highly sensitive detection methods. Herein, we report an electrochemical biosensor that synergistically integrates a DNAzyme-driven DNA walker with bimetallic MnAu nanozymes for CA15-3 detection. Target recognition activates the DNAzyme walker, which progressively cleaves substrate strands and releases MnAuNP signal probes from the electrode surface. This process attenuates the nanozyme-catalyzed electrochemical signal for quantitative analysis. The core-shell MnAuNP provides robust oxidase-like activity, while the DNA walker enables efficient enzyme-free signal amplification. This synergistic amplification strategy achieves a wide linear range (0.05 U/mL-200 U/mL) and a low limit of detection (LOD) of 0.0017 U/mL with excellent selectivity. Satisfactory recovery rates (97.58-105.35%) in human serum confirm clinical potential. This work presents a novel strategy for breast cancer biomarker detection by combining DNA walker with nanozyme catalysis amplification, demonstrating promising potential for point-of-care breast cancer diagnostics.

DNA and RNA methylation are key epigenetic and epitranscriptomic modifications involved in gene regulation, genome stability, RNA metabolism, and disease progression. Aberrant methylation patterns in cell-free DNA and RNA have emerged as valuable biomarkers for cancer detection, disease monitoring, and therapeutic stratification. However, conventional methods such as bisulfite sequencing, methylation-specific PCR, MeRIP-seq, SCARLET, and LC-MS/MS often require harsh processing, high sample input, complex instrumentation, or lack site-specific resolution, limiting their clinical and point-of-care applications. CRISPR-based diagnostics provide a promising alternative by combining programmable nucleic acid recognition with collateral cleavage-mediated signal amplification. This review summarizes recent CRISPR strategies for detecting DNA and RNA methylation, including chemical conversion-assisted assays, restriction enzyme-mediated detection, direct amplification-free sensing based on methylation-modulated Cas activity, detection of oxidized cytosine derivatives, reverse transcription-mediated Cas12 detection of m6A, and structure-sensitive Cas13 sensing. We highlight how methylation-dependent sequence conversion, enzyme accessibility, polymerase behavior, and nucleic acid structure can be translated into CRISPR-readable signals. Finally, we discuss current translational challenges and emerging opportunities in point-of-care methylation diagnostics, integrated DNA-RNA profiling, engineered Cas effectors, AI-guided assay design, and CRISPR-compatible methylome analysis.

The continual evolution of pathogens such as SARS-CoV-2 exposes a fundamental limitation of static molecular diagnostics, where fixed primer probe designs are vulnerable to single-nucleotide variation and recombination. This review critically examines CRISPR-enabled point-of-care diagnostics as programmable analytical systems capable of maintaining diagnostic performance under viral evolutionary pressure. Rather than providing a comprehensive survey of platforms, we focus on design principles that govern variant-resilient detection, including guide RNA (gRNA) mismatch engineering, target redundancy, multiplexed architectures, and workflow-constrained assay chemistry. We analyse how amplification-free and autocatalytic CRISPR strategies reduce operational complexity while introducing new trade-offs in sensitivity, robustness, and deployability. Emerging and orthogonal CRISPR effectors are evaluated in terms of their practical value for point-of-care testing, distinguishing translationally viable approaches from proof-of-concept demonstrations. By aligning molecular detection strategies with SARS-CoV-2 evolutionary dynamics, this review provides a critical framework for designing adaptable diagnostics that can evolve alongside rapidly changing pathogens.

Achieving precise control of CRISPR/Cas trans-cleavage depends on understanding how nucleic acid activators engage Cas effectors, yet the fundamental principles of split-trigger activation of Cas12a remain unclear. Here, we uncover the mechanistic determinants that enable fragmented nucleic acids to collectively initiate Cas12a activity. We show that split triggers bearing external extensions fully support the R-loop formation, whereas internal extensions which disrupt the spacer complementarity abolish Csa12a activation. We further demonstrate that covalent linkage of split-trigger fragments prevents R-loop propagation, revealing that Cas12a's activation strictly requires two physically independent split fragments. Together, these findings establish a synergistic split-trigger activation mechanism in which cooperative hybridization of two individually fragments nucleates and extends the Cas12a R-loop with high efficiency. Conceptually, this mechanism enables a cascade architecture that transforms CRISPR diagnostics from a one-target one-Cas ribonucleoprotein (RNP) paradigm into a highly amplifying process in which a single target molecule activates numerous downstream Cas RNPs. Building on this principle, we show that the cleavage of a rationally designed linear DNA-RNA-DNA mediator by LbuCas13a generates optimally configured split triggers for Cas12a activation, thereby coupling RNA recognition to large-scale Cas12a activation without enzymatic preamplification. The resulting Split Trigger Activated Cas13-Cas12 Cascade System (STACS) achieves amplification-free detection down to 1 copy/uL within 15 minutes and maintains robust performance in complex biological (serum, saliva) and environmental (mud) matrices. This work establishes a generalizable strategy for engineering programmable CRISPR cascades with high Cas RNP activation multiplicity for ultrasensitive molecular diagnostics.

Cas13a-based diagnostic systems have been widely utilized for the detection of RNA targets. However, without preamplification such systems are difficult to realize ultrasensitive (RT-PCR level) single pot RNA detection. Here, we found that double strand RNA can effectively activate the trans-cleavage activity of Cas13a RNP, while the cleavage rates of dsRNA by activated Cas13a RNP are very low. In addition, specially designed RNA-Nanocircle has limited ability to activate Cas13a RNP, but this activation is restored once the circular structures are cleaved and become linear. Based on this original method to control trans-cleavage activity of Cas13a RNP, we developed a Cas13a autocatalytic biosensing system assisted by RNA-Nanocircles, which allows one target RNA to activate numerous Cas13a RNPs. With this approach we show ultrasensitive detection of 1aM of synthetic RNA targets without preamplification within 15 min. The clinical utility of this biosensor was validated by monitoring miRNA-21 levels in plasma samples from colorectal cancer patients. This innovative approach highlights the potential of Cas13a-based biosensors in precision oncology, offering a rapid, non-invasive, and ultrasensitive method for RNA biomarker detection in liquid biopsies.

CRISPR-Cas systems have emerged as powerful biosensing platforms capable of highly sensitive and specific nucleic acid detection. The performance of these assays critically depends on the design of the guide RNA (gRNA), which governs target recognition and activates cis- and trans-cleavage. However, native gRNAs often exhibit limited stability, weak mismatch discrimination, reduced activity in clinical matrices, and inconsistent collateral cleavage, thereby limiting the diagnostic potential of CRISPR sensors. To address these constraints, a wide spectrum of gRNA engineering strategies has been developed, including length change, structural reconfiguration, DNA modified strategies, chemical modifications, mismatch as well as multiple modifications. These modifications substantially enhance sensitivity, specificity, stability, and programmability across Cas12a, Cas13a, and emerging CRISPR effectors. This review systematically summarizes recent advances, compares mechanistic principles and functional outcomes, and highlights future opportunities for rational gRNA engineering to enable amplification-free detection, improve clinical robustness, and drive the development of next-generation CRISPR diagnostics.

Accurate detection of human epidermal growth factor receptor 2 (HER2) is critical for early breast cancer screening and personalized therapy. This study constructed a target-triggered, hairpin-enhanced CRISPR/Cas12a biosensor named "Tribos" for ultrasensitive HER2 detection. The system integrates an aptamer hairpin switch (HAS), HAS-allosterically triggered rolling circle amplification (RCA), and a hairpin-enhanced CRISPR/Cas12a fluorescence reporter module. Taking advantage of Cas12a's high affinity for stem-loop structures, we designed a double-stem-loop reporter probe (DS-FQ) and validated its trans-cleavage enhancement mechanism via molecular docking. Under optimal conditions, Tribos exhibited a linear range from 10 fg/mL to 10 ng/mL, with a limit of detection as low as 1.08 fg/mL. In clinical validation with 29 breast cancer patients and 13 healthy controls, the sensor achieved a sensitivity of 82.76% and a specificity of 100%, which were highly consistent with clinical diagnoses and ELISA results, and it effectively distinguished different HER2 expression levels. The modular design of Tribos offers a new strategy for high-performance CRISPR diagnostics and lays a foundation for next-generation molecular diagnostic technologies based on nucleic acid conformational regulation.

BACKGROUND:Accurate detection of low-frequency DNA mutations in body fluids is essential for cancer monitoring and treatment evaluation. However, the high abundance of wild-type DNA often masks rare mutant signals, making sensitive detection particularly challenging. RESULTS:We developed a screening strategy termed the CbAgo-enriched Cas12a mutation screening system (CECMS). By integrating the single-nucleotide resolution of CbAgo with the trans-cleavage activity of CRISPR-Cas12a, this system selectively eliminates wild-type DNA while enriching targeted mutant alleles. CECMS achieves up to 100-fold higher sensitivity at 37 °C compared with conventional Cas12a biosensors, enabling reliable detection of variant allele frequencies (VAFs) as low as 0.01%. In undiluted serum spiked samples for circulating tumor DNA (ctDNA) detection, the method successfully detected pancreatic cancer-associated KRAS G12D mutations at a VAF of 0.1%. SIGNIFICANCE:By leveraging CbAgo-mediated enrichment, the capability of exposing rare SNV for downstream detection is markedly improved. With its high efficiency and ease of use, CECMS holds strong potential as a convenient tool for clinical cancer diagnostics and monitoring.

Although Cas12f (Cas14) is among the smallest Class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effectors, it assembles into dimeric ribonucleoprotein (RNP) complexes with guide RNA, substantially increasing its functional size and limiting its suitability for gene editing and biosensing applications. To overcome this limitation, we systematically investigate the structural and functional roles of Cas12f dimerization using a combination of computational modeling and experimental validation. Structural analysis using Protein Data Bank data and AlphaFold-3 predictions revealed that the 5'-end sequence of tracrRNA is essential for dimer formation but dispensable for substrate cleavage. Based on this, we designed a truncated tracrRNA by removing 70 nucleotides from its 5'-end. This shortened tracrRNA successfully loaded into Cas12f to form a one guide RNA-one Cas12f monomer RNP. This functionally monomeric RNP demonstrated substantially enhanced trans-cleavage activity: 4.5-fold for ssDNA, 3.5-fold for dsDNA, and 2.5-fold for RNA, resulting in markedly improved detection sensitivity: 10-fold for ssDNA and dsDNA, and 4-fold for RNA. In addition, the functionally monomeric RNP exhibits cis-cleavage activity and gene editing efficiency comparable to that of the dimeric RNP, thereby restoring the advantage of Cas12f as a compact enzyme for in vivo gene editing. These results highlight that the functionally monomeric Cas12f RNP combines enhanced biosensing performance with retention of its uniquely compact size, benefiting gene editing applications.

Helicobacter pylori (H. pylori) infection poses a significant threat to public health. In this work, we combined Thermus thermophilus Argonaute (TtAgo) with loop-mediated isothermal amplification (LAMP) to establish a tri-color fluorescent biosensing for detection and genotyping of H. pylori from human salivary samples, termed as ALT-Hp. Leveraging the programmability, site specific cleaving-activity of TtAgo, ALT-Hp enabled the multiplexed detection of H. pylori-specific genes ureA, vacA, and cagA. Furthermore, a customized 3D-printed visualizer compatible with App-enabled smartphones was developed, highlighting its potential for on-site detection. ALT-Hp utilized the amplification capability of LAMP and the signal transduction and amplification "lighting-up" functions of TtAgo, for building a highly sensitive, specific and robust platform for on-site detection. It demonstrated robust performance in complex matrices, achieving a LOD of 1 CFU/mL within 30 min. Clinical validation using 72 samples showed 100% sensitivity and specificity. ALT-Hp was not only able to detect H. pylori, but also capable of genotyping by classifying H. pylori as type I and type II. The classification is primarily based on whether the strains carry virulence factors, and the two types differed significantly in pathogenicity and clinical outcomes, serving as a critical reference for clinical diagnosis and treatment decision-making. Thus, this ALT-Hp biosensing represented a powerful molecular diagnostic tool for monitoring and management of H. pylori infections.