Analytica Chimica Acta最新文献

{"title":"A ratiometric fluorescent probe consisting of Zn-MOF and calcium ion for sensitive and visual detection of doxycycline","authors":"Nan Yang ,&nbsp;Le Kang ,&nbsp;Wei Yao ,&nbsp;Baotong Xu ,&nbsp;Yunhui Feng ,&nbsp;Vladimir P. Fedin ,&nbsp;Enjun Gao","doi":"10.1016/j.aca.2026.345232","DOIUrl":"10.1016/j.aca.2026.345232","url":null,"abstract":"<div><h3>Background</h3><div>The global consumption of antibiotics is steadily rising, with doxycycline being extensively utilized in both animal husbandry and the treatment of COVID-19. Overuse of this antibiotic may result in residual accumulation and pose potential health risks. Conventional detection methods are often complex and labor-intensive, whereas fluorescence analysis offers advantages such as high sensitivity and rapid response. In this work, the development of a simple and efficient MOF-based fluorescent sensor for the detection of doxycycline holds considerable promise for practical applications.</div></div><div><h3>Results</h3><div>In this paper, a unique 3D metal-organic framework Zn[C₁₈H₁₆O₄N₄]·C₂H₆SO·2H₂O (Zn-MOF) for rapid detection of doxycycline was successfully synthesized and analyzed by SEM, PXRD, FTIR, TGA and UV-vis. The fluorescence of Zn-MOF at 430 nm is quenched by the static quenching and inner filter effect between Zn-MOF and doxycycline with detection limit of 2.01 μM. Concurrently, Ca²⁺ can combine with doxycycline and form the Zn-MOF that generates a new fluorescence signal at 550 nm. By mixing the Zn-MOF and Ca²⁺, a ratiometric fluorescent probe can be created. The ratio of fluorescence intensity (F₅₅₀/F₄₃₀) to doxycycline concentration revealed a good linear relationship and detection limit of 1.25 μM. The fluorescence detection efficiency of the ratiometric fluorescent probe is almost twice that of the single metal-organic framework.</div></div><div><h3>Significance</h3><div>The ratiometric fluorescent probe also exhibited excellent selectivity and stability in both ionic and antibiotic solution. As with the concentration of doxycycline increase, the color of the probe and Ca²⁺ shifted from blue to yellow, allowing for visual detection of doxycycline and facilitating its application in rapid detection in real life.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1395 ","pages":"Article 345232"},"PeriodicalIF":6.0,"publicationDate":"2026-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146187536","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ultrafast peptide preparation brings shotgun proteomics into the minute era","authors":"Haowei Ma ,&nbsp;Ming Bi ,&nbsp;Lang Zhao ,&nbsp;Zhixin Tian","doi":"10.1016/j.aca.2026.345223","DOIUrl":"10.1016/j.aca.2026.345223","url":null,"abstract":"<div><h3>Background</h3><div>Mass spectrometry-based shotgun proteomics relies on efficient sample preparation, where proteins are reduced, alkylated, and digested into peptides for LC-MS/MS analysis. While LC-MS/MS and bioinformatic identification have advanced to minute-scale workflows, sample preparation remains a major bottleneck, typically requiring hours to days. Although methods such as high-pressure and droplet-based reactions have reduced processing times, achieving minute-scale preparation has remained challenging. There is a clear need for an integrated, rapid sample preparation strategy to match the speed of modern LC-MS/MS and data analysis platforms.</div></div><div><h3>Results</h3><div>We developed OPPRAD (One-Pot Protein Reduction, Alkylation, and Digestion), an ultrafast sample preparation method conducted in water-in-oil nano/micro-droplets. This one-pot process completes all three key reactions within 5 min. When integrated with rapid LC-MS/MS on the Orbitrap Astral platform and MSFragger- or DIA–NN–based identification, the entire workflow achieved serum-to-peptide identification in 25.9 min and tissue-to-identification in 35.9 min—the fastest reported proteomic pipeline. The method showed high peptide recovery (∼68–71%), excellent cysteine alkylation (&gt;99%), and miss cleavage rates comparable to conventional bulk digestion. It enabled deep proteome coverage across a wide dynamic range and was successfully applied to identify differentially expressed proteins in paired liver cancer tissues.</div></div><div><h3>Significance</h3><div>OPPRAD drastically shortens proteomic sample preparation from hours to minutes, reducing steps, cost, and hands-on time. This innovation bridges a critical gap in high-throughput proteomics, enabling rapid translational applications and large-scale clinical studies. The method's speed and efficiency pave the way for real-time proteomic analysis and future integration with online desalting and database searching.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1395 ","pages":"Article 345223"},"PeriodicalIF":6.0,"publicationDate":"2026-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146161816","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optical-fiber sensors for dissociation constant measurement of aptamer and rapid detection of ampicillin in river water and milk","authors":"Guangxin Zhang ,&nbsp;Haixu Zhao ,&nbsp;Xiaoqi Li ,&nbsp;Yi Zhang,&nbsp;Haijing Jia,&nbsp;Qiaoqiao Zhang,&nbsp;Tianyu Dai,&nbsp;Xinhui Lou","doi":"10.1016/j.aca.2026.345221","DOIUrl":"10.1016/j.aca.2026.345221","url":null,"abstract":"<div><h3>Background</h3><div>The conflicted affinity evaluation results of small molecule-binding aptamers (SM-Apts) are increasing, raising the concerns on if a reported aptamer is a real aptamer. AMP17 is the first published controversial aptamer, which has been applied in at least 37 reported aptasensors for the detection of ampicillin, but was disproved by several homogeneous affinity methods including isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and mass spectrometry. Only the original paper reported its dissociation constant (K_D = 13.4 nM) by the affinity column-based fluorescence method. If is AMP17 a real aptamer? What are the possible reasons that result in the conflicted results?</div></div><div><h3>Results</h3><div>We carried out four most popularly used homogeneous assays (FRET-based strand displacement assays, circular dichroism, EvaGreen and thioflavin T–based label-free fluorescence assays, and DNase I assay). None of them proved the binding of AMP17 to ampicillin. We then successfully measured the K_D using a fiber optic evanescent wave sensor (FOEW), in which AMP17 was covalent immobilized on the SiO₂ fiber without (K_D = 2.6 ± 0.2 nM) or with BSA coating (K_D = 5.1 ± 0.5 nM). The high specificity of AMP17 was proved by testing other molecules and replacing AMP17 by a non-binding sequence. We finally constructed a FOEW for the detection of ampicillin via one-step competitive or two step-noncompetitive detection modes, respectively. The latter showed 25.9-fold lower limit of detection (LOD = 2.8 fM, S/N = 3) than the former. Both detection modes possessed the good anti-matrix interference capability and were capable for the detection of ampicillin spiked in river water and milk without the need of sample purification.</div></div><div><h3>Significance</h3><div>Our results support that AMP17 is a real aptamer with high affinity and specificity. The surface immobilization favors the binding of AMP17 with ampicillin. Our study highlights the limited application scope of homogeneous assays for affinity evaluation of small molecule-binding aptamers. FOEW is a facile platform for both K_D measurements and rapid detection.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1395 ","pages":"Article 345221"},"PeriodicalIF":6.0,"publicationDate":"2026-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146146204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nanozyme fabrication based on magnetic COFs and gold nanorods and its SERS detection of zearalenone","authors":"Xiaoyuan Ma ,&nbsp;Xi Ma ,&nbsp;Muxi Zhang ,&nbsp;Jinchi Han ,&nbsp;Zhouping Wang","doi":"10.1016/j.aca.2026.345229","DOIUrl":"10.1016/j.aca.2026.345229","url":null,"abstract":"<div><h3>Background</h3><div>As one of the most common mycotoxins, Zearalenone (ZEN) not only contaminates cereal crops but also exists in processed grain products for its chemical stability. After entering human body through the food chain, ZEN exhibits remarkable bioaccumulation properties, which poses varying degrees of harmful effects to human health. The traditional chromatography instrumental methods require expensive equipment and complex experimental procedures. The aim of this manuscript is to develop a facile and accurate method for ZEN detection in food samples.</div></div><div><h3>Results</h3><div>Here, a SERS platform was developed for ZEN detection using COF and noble metal nanomaterial with nitroreductase (NTR)-like activity. First, magnetic COF loaded with gold nanoparticles (MCOF-Au) were synthesized as SERS enhancement substrates with partial NTR activity. Simultaneously, palladium-loaded dumbbell-shaped gold nanorods (AuNR/Pd) were prepared and served as the primary NTR in the 4-NTP/NaBH₄ system, significantly enhancing both catalytic activity and SERS signal. After nucleic acid functionalization, the two components assembled. The addition of ZEN regulated the assemble of AuNR/Pd on MCOF-Au. Following magnetic separation, the catalytic and SERS activities changed in the precipitate. Using the Raman peak of the output product 4-ATP at 390 cm⁻¹ as the quantitative marker, the method showed a linear range in 0.001 μg/kg-1000 μg/kg. The LOD was calculated as 1.88 × 10⁻⁴ μg/kg, and spiked recoveries in wheat flour and corn ranged from 88.91% to 115.78%, demonstrating high sensitivity and reliability for real-sample detection.</div></div><div><h3>Significance</h3><div>This aptasensor integrates the high sensitivity of Raman signals with the selectivity of aptamers, providing a robust and efficient tool for accurate ZEN detection in food. COF based nanocomposites are proved to have remarkable NTR activity and the catalytic product 4-ATP exhibits prominent Raman signal, making it suitable for quantitative detection. This design offers a versatile platform for food safety monitoring and public health protection.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1395 ","pages":"Article 345229"},"PeriodicalIF":6.0,"publicationDate":"2026-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146153507","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An improved ion-exchange-to-silver-salt procedure for δ18O measurement of nitrate in natural samples","authors":"Bo Wang ,&nbsp;Chao Li ,&nbsp;Ying Wang ,&nbsp;Linbao Chen ,&nbsp;Yuting Wang ,&nbsp;Ran Ma ,&nbsp;Zhengyu Zhu ,&nbsp;Youping Zhou","doi":"10.1016/j.aca.2026.345228","DOIUrl":"10.1016/j.aca.2026.345228","url":null,"abstract":"<div><h3>Background</h3><div>The oxygen isotope composition (δ¹⁸O) of natural nitrate is a proven effective indicator of its sources and the (bio)geochemical processes that it has experienced. Removing interfering species (in particular oxyanions such as SO₄²⁻, PO₄³⁻, HPO₄²⁻ and H₂PO₄⁻) from natural samples with the currently established <strong>ion-exchange-to-silver-nitrate (IE2AgN)</strong> for reliable δ¹⁸O measurement with high temperature conversion-isotope ratio mass spectrometry (HTC-IRMS) involves a lengthy 5-step procedure. A less tedious and lower-cost <strong>IE2AgN</strong> procedure is highly desirable for high throughput analysis.</div></div><div><h3>Results</h3><div>We report an improved ion-exchange procedure based on the vast difference in solubilities in <em>n</em>-butyronitrile between AgNO₃ and interfering inorganic oxyanions. Nitrate was isolated from interfering inorganic oxyanions (and organic matter) as its silver salt following a 3-step procedure (anion-exchange, neutralization with Ag₂O and selective dissolution of AgNO₃ in <em>n</em>-butyronitrile). The δ¹⁸O of the purified nitrate was then analyzed by HTC-IRMS. A comparative δ¹⁸O analysis of the certified international nitrate O isotope standards before and after processing through our new 3-step and the current 5-step procedures showed them to yield comparably accurate and precise determinations with minimal isotope fractionation. The time required was more than halved (from 3-5 days to 1-2 days) as was the amount of expensive Ag₂O.</div></div><div><h3>Significance</h3><div>The simplified <strong>IE2AgN</strong> procedure will prove useful for high throughput and lower cost extraction of nitrate from natural samples for oxygen (and nitrogen) isotope analysis.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1395 ","pages":"Article 345228"},"PeriodicalIF":6.0,"publicationDate":"2026-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146160609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microscale native size exclusion chromatography high resolution mass spectrometry for the determination of the chelator-to-antibody ratio of immunoconjugates","authors":"Annika A.M. van der Zon ,&nbsp;Bram Weijers ,&nbsp;Danielle J. Vugts ,&nbsp;Andrea F.G. Gargano","doi":"10.1016/j.aca.2026.345214","DOIUrl":"10.1016/j.aca.2026.345214","url":null,"abstract":"<div><h3>Background</h3><div>Positron emission tomography (PET) using radiolabeled antibodies (radioimmunoconjugates) is a powerful imaging technique to track the distribution of therapeutic antibodies. Accurate determination of the average number of chelators per antibody (CAR) is essential for (radio)immunoconjugate characterization, which is important for maximizing tumor uptake and image quality. Determination of the CAR is considered a critical quality attribute. Radiometric titration, although widely used, is labor-intensive, material- and time-consuming, and requires approximately half a day per sample. To overcome this analytical bottleneck, we developed a microscale size exclusion chromatography – mass spectrometry (SEC-MS) analysis.</div></div><div><h3>Results</h3><div>By applying microscale flow, high concentrations of volatile salts, up to 1000 mM ammonium acetate, could be used to minimize secondary interactions. Moreover, high isCID energy of 140 eV was applied to reduce adduct formation and enhance the MS sensitivity. The method was optimized using model radioimmunoconjugates based on intact trastuzumab, with a particular focus on the impact of buffer concentration on both chromatographic effects and CAR determination. A final buffer concentration of 600 mM ammonium acetate was selected. The method was compared with radionuclide titration for analysis of the same immunoconjugate model and yielded similar results. Finally, we demonstrated the broad applicability of the method by testing diverse immunoconjugates (<em>e.g.</em>, ipilimumab, rituximab), including cysteine- and lysine-linked chelators (<em>e.g.</em>, DFO, DOTA, RESCA), as well as other smaller protein models, such as nanobodies.</div></div><div><h3>Significance</h3><div>The optimized microflow native SEC-MS method, with a rapid 20-min run time, provides comprehensive characterization, including determination of the average CAR, dispersity index, glycosylation profile, and mAb impurities. Crucially, this native approach is applicable to all tested immunoconjugates, including chemically sensitive species such as those with maleimide and thiourea linkages, which often challenge denaturing methods like RPLC-MS. This establishes SEC-MS as a broadly applicable alternative to radiometric titration for the comprehensive characterization of immunoconjugates.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1395 ","pages":"Article 345214"},"PeriodicalIF":6.0,"publicationDate":"2026-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146135286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Measuring absolute gas amounts with gas chromatography by using a novel setup for pressure based gas control","authors":"Andreas Hofmann,&nbsp;Ingo Reuter,&nbsp;Freya Müller,&nbsp;Anna Smith","doi":"10.1016/j.aca.2026.345217","DOIUrl":"10.1016/j.aca.2026.345217","url":null,"abstract":"<div><div>The characteristics of the gas itself, such as its volatility, compressibility, or weight, often complicate the process of feeding gas into a measuring system, such as a gas chromatograph. This is particularly relevant when quantifying individual gas components. While it is possible to inject gas with syringes, this method has several disadvantages, such as gas tightness and syringe and gas volume, especially for small gas quantities. Therefore, there is an urgent need for a simple, reliable, and improved method of gas injection into a measuring device. This study describes the development of a novel gas injection system that reliably and contaminant-free injects gas into gas chromatography (GC) instruments and other gas-related devices. The system consists of an injection unit, a vacuum system, and a loop assembly, and it connects directly to the corresponding device. Sample gas can then be introduced directly into the evacuated injection system and delivered to the measuring device. The system has been validated using a range of configurations and has undergone extensive testing. Additionally, two applications are presented to demonstrate the system's wide range of potential uses. Notably, the setup enables a contamination-free gas supply from battery pouch-bag cells. For this configuration, an adapter assembly was required for the pouch-bag cell and was integrated into the battery cell. Additionally, it is demonstrated how the setup can be used to determine the gas composition of a thermal decomposition reaction. This setup is a new, platform-independent option for introducing gas into a low-pressure environment and connecting it to devices without causing contamination. Reduced pressure enables multi-point calibration with a single reference or calibration gas mixture, which is pressure-dependent. Additionally, the integrated loop technology allows for the quantitative calculation of gas concentrations. Errors in calculating the amount of substance are less than 10–20%, even with flexible pouch cells.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1395 ","pages":"Article 345217"},"PeriodicalIF":6.0,"publicationDate":"2026-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146146661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preparation of ZIF-8 grafted magnetic solid-phase extraction sorbent for sensitive determination of four cannabinoids in urine and oral fluid","authors":"Hongyu Ning ,&nbsp;Yilei Fan ,&nbsp;Haodong Wang ,&nbsp;Huijun Liu ,&nbsp;Zhongping Huang ,&nbsp;Haixing Wang ,&nbsp;Xing Ke ,&nbsp;Yu Xu","doi":"10.1016/j.aca.2026.345194","DOIUrl":"10.1016/j.aca.2026.345194","url":null,"abstract":"<div><h3>Background</h3><div>The global increase in cannabis use has created a strong demand for reliable and efficient detection methods. Traditional urine-based assays are widely used and standardized, but suffer from complex hydrolysis requirements, long detection windows, and an inability to monitor recent use. In contrast, oral fluid testing enables non-invasive, real-time detection of parent cannabinoids, but is limited by low sample volume, matrix interference, and insufficient sensitivity in common rapid tests. It is clear that a sample preparation and detection approach is needed that is robust, highly sensitive, streamlined, and compatible with high-throughput confirmatory analysis such as LC-MS/MS.</div></div><div><h3>Results</h3><div>In this study, a magnetic solid-phase extraction (MSPE) method was developed using a novel magnetic metal–organic framework composite, Fe₃O₄@poly(GMA/DVB-ZIF-8), for the efficient extraction of four cannabinoids from urine and oral fluid. The sorbent uses multiple interactions—including hydrophobic, π–π, electrostatic, and Zn–O coordination—to achieve high analyte recovery (exceeding 85%). Coupled with UHPLC-MS/MS analysis, the method demonstrates wide linearity (0.05–25 ng/mL, r² &gt; 0.9945), low detection limits (as low as 0.017 ng/mL), and good repeatability (RSDs &lt;13.8%). Comparative evaluation with conventional liquid–liquid extraction showed minimal deviation (relative percent difference &lt;6.0%), confirming high trueness and reliability for cannabinoid monitoring in biological samples.</div></div><div><h3>Significance</h3><div>This MSPE approach provides a simple, efficient and high-throughput sample preparation method for reliable and rapid forensic analysis of cannabinoids in complex biological samples.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1394 ","pages":"Article 345194"},"PeriodicalIF":6.0,"publicationDate":"2026-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146153231","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative all-in-one high-performance thin-layer chromatography–planar start-zone enzyme assay applied for invertase activity","authors":"Isabel Müller,&nbsp;Viktoria H. Englert,&nbsp;Gertrud E. Morlock","doi":"10.1016/j.aca.2026.345213","DOIUrl":"10.1016/j.aca.2026.345213","url":null,"abstract":"<div><h3>Background</h3><div>Conventional methods for assessing enzyme activity often rely on <em>in vitro</em> assays with spectrophotometric detection; however, they lack accuracy for complex substrates. The recently introduced nanoGIT concept demonstrated the potential of on-surface enzyme assays performed directly in the start zone, followed by high-performance thin-layer chromatography (HPTLC) of the formed products on the same adsorbent surface. For the first time, the potential of this all-in-one HPTLC concept was investigated for the quantification of enzyme (invertase) activity using a complex substrate.</div></div><div><h3>Results</h3><div>Enzymatic hydrolysis of sucrose or egg biscuit extract was carried out in the start zone under identical conditions for all studied samples. On the same adsorbent surface, the hydrolysis products were separated, detected via densitometric fluorescence measurement, and quantified. Even with complex substrates, the integrated separation provided accurate, in-depth information on enzyme characteristics from the individual products formed and their respective intensities. The method was validated and proved to be accurate and reliable in the nanomolar range, outperforming current spectrophotometric <em>in vitro</em> assays that operate in the millimolar range. The assay in the start zone exhibited remarkable sensitivity, requiring a 12,500-fold lower substrate quantity while using equivalent enzyme amounts. Limitations and benefits were discussed.</div></div><div><h3>Significance</h3><div>For the first time, a validated quantitative all-in-one HPTLC–start-zone enzyme assay–FLD method was successfully demonstrated for the determination of the enzyme activity. It enabled the quantification of even minor amounts of individual enzyme reaction products, providing improved sensitivity and more accurate insights into enzyme characteristics. Low enzyme levels can be analysed, extending current analytical capabilities.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1394 ","pages":"Article 345213"},"PeriodicalIF":6.0,"publicationDate":"2026-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146138739","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Standardized digital PCR assay validation using PCR-ValiPal, demonstrated in cross-platform quantification of bovine papilloma virus","authors":"David Gleerup ,&nbsp;Matthijs Vynck ,&nbsp;Lien Gysens ,&nbsp;Cindy De Baere ,&nbsp;Wim Trypsteen ,&nbsp;Jo Vandesompele ,&nbsp;Olivier Thas ,&nbsp;Ann Martens ,&nbsp;Maarten Haspeslagh ,&nbsp;Ward De Spiegelaere","doi":"10.1016/j.aca.2026.345210","DOIUrl":"10.1016/j.aca.2026.345210","url":null,"abstract":"<div><h3>Background</h3><div>Digital PCR (dPCR) enables precise and absolute quantification of nucleic acids by partitioning samples into thousands of reactions, improving reproducibility and reducing reliance on standard curves compared to qPCR. However, rigorous assay validation remains essential to ensure reliability, particularly for parameters such as limit of detection, limit of quantification, trueness, and linearity. Existing guidelines (e.g., MIQE, dMIQE, ISO 20395:2019) highlight these requirements, but implementation is laborious and inconsistent across laboratories. To address this, we developed PCR-ValiPal, a user-friendly web application that standardizes and streamlines dPCR assay validation and reporting.</div></div><div><h3>Results</h3><div>PCR-ValiPal calculates the full range of analytical parameters required for ISO-compliant assay validation, including limit of blank, limit of detection, limit of quantification, precision, trueness, and linearity. While broadly applicable to any nucleic acid target, we demonstrate its use with a three-color PCR assay for bovine papillomavirus (BPV), a clinically relevant representative DNA assay, types 1 and 2, benchmarked across four platforms: Naica (droplet dPCR), QIAcuity (microwell dPCR), LOAA (real-time dPCR), and CFX96 (qPCR). Cross-platform comparisons revealed Naica and QIAcuity achieved low LOB and LOQ values with minimal bias, while LOAA exhibited stable but negative bias. qPCR performed best for BPV-2 sensitivity but was less reliable for BPV-1 at low concentrations. These results illustrate both the value of platform-specific optimization and the utility of PCR-ValiPal in providing transparent, standardized validation outputs.</div></div><div><h3>Significance</h3><div>PCR-ValiPal supports transparent, reproducible, and ISO-aligned validation of PCR-based assays, lowering barriers for both expert and non-expert users. By centralizing statistical analyses in a single tool, it enables reliable comparison across platforms and targets, facilitating adoption in research, diagnostics, and regulatory contexts. This work underscores the importance of standardized validation for ensuring confidence in nucleic acid quantification.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1394 ","pages":"Article 345210"},"PeriodicalIF":6.0,"publicationDate":"2026-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146135312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}