Analytica Chimica Acta最新文献

{"title":"A fully automated, high-throughput electro-extraction and analysis workflow for acylcarnitines in human plasma and mouse muscle tissues","authors":"Yupeng He ,&nbsp;Paul Miggiels ,&nbsp;Amy Harms ,&nbsp;Yvonne Rijksen ,&nbsp;Renata M.C. Brandt ,&nbsp;Wilbert P. Vermeij ,&nbsp;Bert Wouters ,&nbsp;Thomas Hankemeier","doi":"10.1016/j.aca.2025.344224","DOIUrl":"10.1016/j.aca.2025.344224","url":null,"abstract":"<div><h3>Background</h3><div>The labor-intensive and time-consuming nature of sample preparation poses significant challenges for bioanalysis, especially for large-scale samples characterized by limited volumes/mass, and low analyte abundance. Additionally, manual sample processing can compromise reproducibility. To overcome these limitations, automation and high-throughput methodologies are essential, highlighting the need for an automated, high-throughput sample preparation and analysis workflow.</div></div><div><h3>Results</h3><div>This study presents a fully automated, high-throughput electro-extraction (EE) platform integrated with a CTC PAL3 autosampler and liquid chromatography–mass spectrometry analyzer. The integrated platform underwent qualification, followed by optimization of EE parameters using a Design of Experiment approach. Ten acylcarnitines were selected as model analytes. The optimization models exhibited strong fits (p &lt; 0.006, R² &gt; 0.91). The optimized platform achieved an enrichment factor of up to 400 (an extraction recovery of up to 99 %) in designed academic samples, and was effectively implemented and evaluated using 20 μL of spiked human plasma samples. To test clinically relevant materials, the platform was utilized to study the effects of muscle tissue isolation speed on acylcarnitine stability, and to examine acylcarnitine abundance across muscle types in progeria (sarcopenia) mouse muscle. We found that the speed of muscle isolation does not affect measured levels of acylcarnitines, and detected higher acylcarnitine abundances are consistent with literature.</div></div><div><h3>Significance</h3><div>This study provides an automated, high-throughput, and cost-effective workflow enabling extraction and analysis of 120 samples per day, with a cost of &lt;0.1 Euro per sample. It presents a significant stride towards the creation of fully-automated, high-throughput bioanalysis workflows for large-scale studies involving biomass limited samples in the foreseeable future.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1364 ","pages":"Article 344224"},"PeriodicalIF":5.7,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144097266","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":"Analytical quality control in targeted lipidomics: Evaluating the performance of commercial plasma as a surrogate for pooled study samples","authors":"Alanah Grant-St James ,&nbsp;Aude-Claire Lee ,&nbsp;Alex J. Lee ,&nbsp;Julien Wist ,&nbsp;Ferdous Sohel ,&nbsp;Kok Wai Wong ,&nbsp;Bu B. Yeap ,&nbsp;Ruey Leng Loo ,&nbsp;Amanda Henry ,&nbsp;Daniella Susic ,&nbsp;Emad El-Omar ,&nbsp;Jeremy K. Nicholson ,&nbsp;Elaine Holmes ,&nbsp;Luke Whiley ,&nbsp;Nicola Gray","doi":"10.1016/j.aca.2025.344225","DOIUrl":"10.1016/j.aca.2025.344225","url":null,"abstract":"<div><h3>Background</h3><div>Pooled quality control (PQC) samples are the gold standard for data quality monitoring in metabolic phenotyping studies. Typically composed of equal parts from all study samples, PQCs can be challenging to generate in large cohorts or when sample volumes are low. As an alternative, externally sourced matrix-matched surrogate QCs (sQC) have been proposed. This study evaluates the performance of sQCs against PQCs for assessing analytical variation, data pre-processing, and downstream data analysis in a targeted lipidomics workflow.</div></div><div><h3>Results</h3><div>Plasma samples (n = 701) from the Microbiome Understanding in Maternity Study, along with PQC (n = 80) and sQC (n = 80) samples, were analyzed using a lipidomics assay targeting 1162 lipids. QC samples were injected throughout acquisition, and data pre-processing was performed using each strategy. For simplicity, a subset (n = 381) of the study samples was used to assess differences in downstream statistical analyses.</div><div>Both QC approaches demonstrated high analytical repeatability. While PQC and sQC compositions differed, use of PQCs retained less than 4 % more lipid species during pre-processing. Univariate analysis identified more statistically significant lipids with PQC-based pre-processing, but multivariate model performance was similar between datasets.</div></div><div><h3>Significance</h3><div>This study provides a comprehensive comparison of QC strategies and emphasizes the importance of careful QC workflow selection. While PQCs offer advantages, sQCs serve as a suitable alternative for quality assessment and pre-processing. Their commercial availability also supports use as intra- and inter-laboratory long-term references, aiding data harmonization across studies and laboratories.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1365 ","pages":"Article 344225"},"PeriodicalIF":5.7,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144104252","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":"Utilizing the total phosphorus content of lipid extracts as a normalization strategy in quantitative lipidomics studies","authors":"Johannes Scholz ,&nbsp;Anna Gremme ,&nbsp;Julia Hillebrand ,&nbsp;Jasper Baldauf ,&nbsp;Lennart J. Bendheuer ,&nbsp;Jonas Berreiter ,&nbsp;Anne C. Sehnal ,&nbsp;Julia Bornhorst ,&nbsp;Heiko Hayen","doi":"10.1016/j.aca.2025.344228","DOIUrl":"10.1016/j.aca.2025.344228","url":null,"abstract":"<div><h3>Background</h3><div>Normalization is a crucial element of quantitative lipidomics studies, for which there is still no uniform recommended strategy. There are various approaches for normalizing samples, including referencing to the total volume or weight, to the DNA content or the protein content. In addition, samples can be normalized to the phosphorus content, but either not all phospholipid classes are included or a less sensitive colorimetric assay is used. <strong>It is therefore necessary to develop new ways of normalization that are both specific and sensitive to compensate for the biological sample variance and the variance from pre-analytical and analytical steps.</strong></div></div><div><h3>Results</h3><div>We present a tailored normalization method for lipid quantification which is based on the total phosphorus content (TPC) of lipid extracts. The TPC reflects the amount of phospholipids present in the sample, because polar phosphorus-containing compounds like DNA and phosphorylated sugars are separated due to the extraction procedure. In order to test the normalization strategy, the (phospho)-lipids in the model organism <em>C. elegans</em> were quantified using SFC-TIMS-MS/MS. Subsequently, a digestion method was developed to degrade the lipids present in the organic extract. This was used to determine the TPC via ICP-OES and ICP-MS/MS analysis. In particular, very low detection limits (LOQ = 0.3 μg L⁻¹) can be achieved with the ICP-MS/MS method. The elemental and molecular information were then combined, and the samples were normalized to TPC. The normalization showed a significant improvement in the standard deviations compared to the initial concentrations as well as an improvement compared to the normalization based on the total protein content, which is very frequently used.</div></div><div><h3>Significance</h3><div>Our method shows a sensitive way to determine the TPC in total lipid extracts of various samples, exemplified for human plasma and nematode samples. The normalization minimizes the standard deviations in biological samples even in comparison with other normalization approaches, i.e. protein content, and can contribute to increasing the conclusiveness of quantitative lipidomics studies.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1364 ","pages":"Article 344228"},"PeriodicalIF":5.7,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144097269","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":"Controllable synthesis of chiral hierarchical porous Co(OH)2 and its application in rapid chiral recognition and separation","authors":"Jinhua Xu ,&nbsp;Jinyu Zhang ,&nbsp;Wenmin Zhang ,&nbsp;Shiye Xie ,&nbsp;Lan Zhang","doi":"10.1016/j.aca.2025.344212","DOIUrl":"10.1016/j.aca.2025.344212","url":null,"abstract":"<div><h3>Background</h3><div>Chiral pollutants present significant environmental and health concerns, with neurotoxic amino acid analogs like β-N-methylamino-L-alanine (BMAA) demonstrating enantiomer-specific bioaccumulation in aquatic ecosystems. Current analytical approaches for chiral environmental contaminants rely predominantly on chromatographic techniques that require extensive sample preparation (typically 24 h), limiting field deployment and high-throughput screening capabilities. Detection thresholds for these compounds (0.1–10 μg/L) necessitate sensitive methodologies that maintain stereochemical integrity. This study addresses these analytical challenges through the rational design of chiral cobalt hydroxide (CF–Co(OH)₂) nanomaterials engineered for dual-mode detection and separation functionality.</div></div><div><h3>Results</h3><div>A hierarchical porous α-Co(OH)₂ with tunable chirality (R/S/RS configurations) and morphologies (square, rough surface, snowflake, flower-like) via micelle-templated growth strategies. The materials demonstrated dual functionality: (1) Rapid colorimetric amino acid discrimination within 5 min with precision (RSD = 4.9 %, n = 6), validated through urinary <span>l</span>-tryptophan quantification (17.1 ± 0.4 μg/mL); (2) Ultrasensitive BMAA separation via SPE-HPLC-MS/MS with significantly improved detection limits (LOD = 0.02 μg/kg) and processing speed (144 × faster than conventional methods). The colorimetric detection mechanism exploits BMAA's ability to form cobalt-amine complexes that produce concentration-dependent yellow coloration, enabling visual detection at 50 μg/kg in freshwater samples. Field testing successfully detected BMAA in crucian carp (0.39 ± 0.03 μg/kg), confirming food-chain biomagnification with excellent recovery (90.1–102.7 %) across diverse matrices. The system's chiral specificity exhibited distinct affinity patterns (BMAA: R–Co(OH)₂; DAB: S–Co(OH)₂) with 91.4 % enantiomeric excess in just 10 min through configuration-specific “three-point binding” mechanisms (intramolecular binding energy: 18.7 kcal/mol).</div></div><div><h3>Significance</h3><div>This research establishes morphology-programmable chiral materials as a versatile analytical platform for rapid on-site environmental monitoring and high-throughput toxin analysis. The developed methodology directly addresses World Health Organization guidelines for algal toxin detection in drinking water while providing a generalizable approach for chiral pollutant discrimination in complex environmental and biological samples.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1364 ","pages":"Article 344212"},"PeriodicalIF":5.7,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144097270","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":"A versatile two-dimensional liquid chromatography coupled to high-resolution mass spectrometry platform for unraveling chromatographic profiles of large synthetic oligonucleotides","authors":"Bifan Chen,&nbsp;Bingchuan Wei,&nbsp;Lulu Dai,&nbsp;Jenny Wang,&nbsp;Dawen Kou,&nbsp;Kelly Zhang","doi":"10.1016/j.aca.2025.344218","DOIUrl":"10.1016/j.aca.2025.344218","url":null,"abstract":"<div><h3>Background</h3><div>Many gene expression-modulating therapeutics, as well as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing technology have greatly transformed the field of biomedical research. A number of trials already in clinic well exemplify the potential of oligonucleotide-based therapeutics to address many disease areas. The quality and the integrity of the oligonucleotides are therefore crucial to ensure efficacy and ultimately patient safety, which often require thorough characterization. In general, these oligonucleotides are synthetically made by solid phase phosphoramidite chemistry, during which many possible undesired byproducts from incomplete or side reactions can be generated. However, the examination of the overall chromatographic profile on the large synthetic oligonucleotides from the intact level has been limited and challenging by direct coupling to mass spectrometry (MS). This is due not only to the low abundance and heterogeneity of impurities, but also to difficulties in separation. Furthermore, many oligonucleotide separation methods coupled online to MS greatly suffer from suboptimal sensitivity of the MS detection.</div></div><div><h3>Results</h3><div>Here, to address these challenges, we develop a versatile two-dimensional liquid chromatography-mass spectrometry (2DLC-MS) platform involving multiple heart-cut fractionation, adduct removal, and acquisition through wide window quadrupole isolation. Using a 100-mer long single guide ribonucleic acid (sgRNA) as the model compound, we showcased the in-depth analysis of the sgRNA IP-RPLC profile as well as HILIC profile at lower temperature.</div></div><div><h3>Significance</h3><div>Overall, by utilizing the 2DLC-MS platform, we were able to gain a major sensitivity boost to examine the chromatographic profiles of large synthetic oligonucleotides, enabling lot to lot comparison and understanding of impurity profiles.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1364 ","pages":"Article 344218"},"PeriodicalIF":5.7,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144104251","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":"A homogeneous mass-encoded strategy for mass spectrometric biosensing of multiplex proteins","authors":"Junjie Hu ,&nbsp;Fei Liu ,&nbsp;Yunlong Chen ,&nbsp;Xinxin Xie ,&nbsp;Huangxian Ju","doi":"10.1016/j.aca.2025.344219","DOIUrl":"10.1016/j.aca.2025.344219","url":null,"abstract":"<div><h3>Background</h3><div>Protein kinases play important roles in fundamental biological processes. Aberrant activities may result in many diseases, thus the detection of multiplex kinase activities is important in clinical diagnosis. Recent progress has focused on the mass spectrometric biosensing technique to enable highly sensitive detection of multiplex targets. However, the application of the developed methods in multiplex enzyme analysis is greatly challenged due to the substrate immobilization on solid interfaces, which affect the contact between substrates and enzymes to reduce enzyme reaction efficiency.</div></div><div><h3>Results</h3><div>This work developed a homogeneous mass-encoded method for biosensing of multiplex proteins, which was performed using designed peptides containing the coding sequences and substrate regions. With the assistance of titanium dioxide coated magnetic beads (TiO₂-MBs) to capture the phosphopeptide products, followed by trypsin to cleave the products for releasing the coding sequences, the kinase assays were achieved by submitting the supernatant for ultrahigh performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) analysis. Using protein kinase A (PKA) and human epidermal growth factor receptor 2 (HER2) as model targets, the peak area ratios of the coding sequences to the internal standards showed linear relations of 1.0–100 ng mL⁻¹ and 0.2–20 U mL⁻¹, with the detection limits of 0.46 ng mL⁻¹ and 0.033 U mL⁻¹ for HER2 and PKA, respectively. The proposed strategy also demonstrated great practicability in inhibition analysis and kinase activity assays in cell lysates.</div></div><div><h3>Significance</h3><div>A homogeneous mass-encoded method for multiplex detection of kinase activities could simplify the assay procedure and reveal the enzyme activities with free substrates. The strategy enabled multiplex kinase activity assays with convenience, high sensitivity, and high specificity, demonstrating promising applications in clinical fields.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1364 ","pages":"Article 344219"},"PeriodicalIF":5.7,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144097271","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":"Method development and validation for analysis of 74 per- and polyfluoroalkyl substances (PFAS) in food of animal origin using QuEChERSER method and LC-MS/MS","authors":"Kevin M. Stroski,&nbsp;Yelena Sapozhnikova","doi":"10.1016/j.aca.2025.344216","DOIUrl":"10.1016/j.aca.2025.344216","url":null,"abstract":"<div><h3>Background</h3><div>The detection of per- and polyfluoroalkyl substances (PFAS) in food is extremely important for ensuring safe food consumption however most analytical methods focus on only a small group of known analytes. <strong>The goal of this study was to develop and validate a new method for analysis of 74 PFAS ranging across 15 different groups including legacy PFAS, short-chain alternatives, precursors and breakdown products in beef, chicken, pork, catfish and eggs.</strong></div></div><div><h3>Results</h3><div>The method was based on “quick, easy, cheap, effective, rugged, safe, efficient, and robust” (QuEChERSER) mega-method and LC-MS/MS analysis. Validation was performed at, above and below maximum levels set by the European Food Safety Authority (EFSA) in beef, chicken, pork, catfish, liquid egg, and powdered egg samples. Acceptable recoveries were obtained for 72 %–93 % of analytes using reagent only calibration curves across all matrices and concentrations tested. This increased to 84 %–97 % when using matrix matched calibration curves. Method performance was comparable (or better than) US Food and Drug Administration (FDA) and USDA Food Safety and Inspection Service (FSIS) methods. Further validation of the method with NIST standard reference materials (SRMs) 1946 and 1947 resulted in accuracies of 71 %–112 %. The validated method was applied to test catfish samples, and low levels of PFAS (0.020–2.24 ng/g wet weight) were measured in both farm raised and wild caught fish.</div></div><div><h3>Significance</h3><div>These results demonstrate the ability of this method to accurately measure a broad range of PFAS analytes in complex food matrices providing broad applicability and versatility to end users. To our knowledge, this is the largest current targeted method for analysis of PFAS in foods.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1364 ","pages":"Article 344216"},"PeriodicalIF":5.7,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144097272","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":"Gold nanoparticle-assisted, label-free SELEX coupled with high-throughput NGS for generating highly sensitive and specific DNA aptamers targeting Aflatoxin B1","authors":"Shazia Shareef,&nbsp;Hariprasad P.","doi":"10.1016/j.aca.2025.344201","DOIUrl":"10.1016/j.aca.2025.344201","url":null,"abstract":"<div><h3>Background</h3><div>Aflatoxin B₁ (AFB₁) is a highly toxic fungal secondary metabolite that poses serious health risks to humans and animals, prompting the development of sensitive and specific detection technologies. Aptamer-based detection strategies offer a potential alternative to traditional detection methods. This study aimed to develop AFB₁-specific aptamers using a gold nanoparticles-assisted SELEX (AuNP-SELEX) approach, eliminating the need for target immobilization. In this method, aptamers bound to AFB₁ detach from AuNPs while unbound or weakly bound sequences remain associated. The process was further strengthened using High-Throughput SELEX (HT-SELEX), enabling the efficient screening of large libraries for aptamers with minimal off-target binding.</div></div><div><h3>Results</h3><div>Successive rounds of SELEX led to the enrichment of high-affinity aptamers, which was monitored using a colorimetric AuNP-based assay. The top four aptamers- L2, CPMA1, L1, and L3, showed strong binding affinities with dissociation constants (<em>K</em>_d) of 1.24, 1.6, 2.55, and 5.26 nM, respectively. Circular dichroism spectroscopy confirmed conformational changes in the aptamers upon AFB₁ binding. A fluorescence assay using a FAM-labelled L2 aptamer and graphene oxide also achieved a detection limit of 0.53 nM.</div></div><div><h3>Significance</h3><div>This study demonstrates the effectiveness of the AuNP-assisted HT-SELEX platform as a rapid, visual, and refined method for selecting aptamers against small molecules like AFB₁. The approach enhances detection sensitivity and specificity, offering a valuable tool for food safety monitoring and mycotoxin detection.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1364 ","pages":"Article 344201"},"PeriodicalIF":5.7,"publicationDate":"2025-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144067321","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":"Specific detection of DNA and RNA by the CRISPR-Cas12a system containing spacer split crRNA","authors":"Wenjing Ren, Mingzhi Li, Xinyue Liu, Weixin You, Qianqian You, Boan Li, Huiming Ye, Rui Zhang","doi":"10.1016/j.aca.2025.344204","DOIUrl":"https://doi.org/10.1016/j.aca.2025.344204","url":null,"abstract":"<h3>Background</h3>The CRISPR/Cas12a system has emerged as a versatile molecular diagnostic tool due to its dual cis- and trans-cleavage activities. However, two key limitations hinder its broad application: high tolerance to single-base mismatches in DNA targets and strict reliance on DNA activators. To address these challenges, we hypothesized that structural reengineering of crRNA could enhance specificity and functional versatility. This study aimed to develop a modified Cas12a system capable of detecting DNA and RNA targets with improved single-base resolution, thereby expanding its utility in molecular diagnostics and clinical subclassification.<h3>Results</h3>We engineered split crRNAs by introducing a split site within the spacer region, creating a spacer-split crRNA-activated Cas12a system (SPCas12a). This system exhibited three key advantages: First, SPCas12a demonstrated significantly enhanced specificity in discriminating single-base mutations compared to conventional full-sized crRNA systems. Second, it bypassed the DNA activator requirement, enabling direct detection of miRNA targets without reverse transcription. In addition, AlphaFold Server predictive structural modeling analysis showed that the split site selected by SPCas12a gives the Cas12a complex an open structural domain, which is conducive to the stable function of Cas12a. Third, integration with isothermal amplification enabled constructing an \"AND\" logic gate detection platform that processes multiple inputs within 40 minutes. As a proof-of-concept, SPCas12a successfully distinguished triple-negative breast cancer (TNBC) subtype cell lines by analyzing miRNA-210 and miRNA-21 biomarkers in different cell lines.<h3>Significance</h3>SPCas12a overcomes fundamental limitations of current CRISPR diagnostics by unifying high-specificity DNA mutation detection and direct RNA sensing in a single platform. The split-crRNA design principle provides a universally adaptable strategy to enhance CRISPR-Cas systems, with immediate applications in precision oncology and infectious disease stratification where base-level discrimination and multi-target detection are critical.","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"30 1","pages":""},"PeriodicalIF":6.2,"publicationDate":"2025-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144067322","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":"SERS hydrogel designed using a microfluidic droplet platform for label-free therapeutic drug monitoring of doxorubicin in human serum","authors":"Ying Wang ,&nbsp;Jinru Cao ,&nbsp;Ziwei Xu ,&nbsp;Yaqin Zhan ,&nbsp;Ziyu Xu ,&nbsp;Hao Cheng ,&nbsp;Wenyi Huang ,&nbsp;Hongxing Kong ,&nbsp;Jun Feng","doi":"10.1016/j.aca.2025.344203","DOIUrl":"10.1016/j.aca.2025.344203","url":null,"abstract":"<div><h3>Background</h3><div>Doxorubicin (DOX) is an anthracycline anticancer drug that is commonly employed in the treatment of acute leukemia, malignant lymphoma, breast cancer and prostate cancer. Given the narrow safe dosage range and the strong toxic reaction associated with the drug, prolonged or excessive use can result in serious adverse reactions, which greatly limits its clinical application. It is therefore necessary to develop a rapid, sensitive and reproducible therapeutic drug monitoring (TDM) method for DOX in order to create an individualised dosing regimen based on the patient's specific situation, thus facilitating the precise treatment of the disease.</div></div><div><h3>Results</h3><div>We prepared hydrogel microspheres encapsulated with AgNPs as SERS substrates on a microfluidic droplet platform, utilising PEGDA as the monomer. The hydrogel microspheres have a 3D mesh structure with adjustable pore size, which facilitates the uniform dispersion of AgNPs in the hydrogel particles. This not only safeguards AgNPs from the biological matrix and enhances their stability and SERS signal reproducibility, but also facilitates the formation of denser SERS \"hotspots\" for highly sensitive SERS detection. Concurrently, the pore size of the hydrogel microspheres can preclude the interference of biomacromolecules in the biological matrix, thereby enabling the selective facilitation of the entry of small molecules. The selective, label-free SERS detection of DOX within human serum was successfully achieved by employing AgNPs@microgel SERS substrates. The linearity of DOX was demonstrated within the concentration range of 1.0–10⁵ ng mL⁻¹, with a detection limit of 1.0 ng mL⁻¹.</div></div><div><h3>Significance</h3><div>The method is characterised by its rapidity, sensitivity, reproducibility, the absence of any sample pretreatment requirements and minimal sample consumption. As a consequence, it provides a new avenue for the highly sensitive and reproducible label-free detection of DOX in human serum. Meanwhile, the new method has the potential to detect more small molecule drugs, which has a broad application potential in clinical TDM.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1363 ","pages":"Article 344203"},"PeriodicalIF":5.7,"publicationDate":"2025-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144067284","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}