Analytica Chimica Acta最新文献

{"title":"Development of DNA aptamers towards detection of tuberculosis biomarker Ag85B in a fluorescence-based sensing platform","authors":"Satakshi Hazra ,&nbsp;Manish Gupta ,&nbsp;Rakesh Bhatnagar ,&nbsp;Prithwi Chayan Chatterjee ,&nbsp;Sanjukta Patra","doi":"10.1016/j.aca.2025.344029","DOIUrl":"10.1016/j.aca.2025.344029","url":null,"abstract":"<div><h3>Background</h3><div>Timely diagnosis of tuberculosis (TB) remains a critical challenge, highlighting the need for better screening tools. Traditional antibody-based detection methods for TB are often costly and cumbersome. To address this, we developed a streamlined centrifugal SELEX approach using a 69-nucleotide DNA library and the recombinant TB biomarker Ag85B, towards fabrication of an aptasensing platform offering a simpler and faster alternative.</div></div><div><h3>Results</h3><div>Two high affinity DNA aptamers were screened through 12 rounds of SELEX and verified with <em>in silico</em> docking, circular dichroism spectroscopy and electrophoretic shift assays for binding interactions with Ag85B. The aptamer with highest binding affinity (K_D values 76.36 ± 10.76 nM in binding buffer and 86.62 ± 6.20 nM in spiked serum) was used for fabrication of a fluorescence based aptasensing platform using graphene oxide as a quencher. The aptamer demonstrated specificity towards Ag85B without interference from two other recombinant TB proteins MPT64 and ESAT6. The aptasensing platform offered limits of detection of 5.83 nM in binding buffer and 6.51 nM in spiked serum.</div></div><div><h3>Significance</h3><div>This work developed a modified SELEX approach combining a centrifugal filter and streptavidin-biotin magnetic separation technique for isolation of DNA aptamers. We report for the first time, a DNA aptamer against Ag85B biomarker that holds high prospects for clinical applications in diagnosing TB.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1357 ","pages":"Article 344029"},"PeriodicalIF":5.7,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143819858","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":"Smartphone-assisted dual-sided capillary microfluidic device for multiplex detection of heavy metals and nutrients in drinking water","authors":"Pornchanok Punnoy ,&nbsp;Prakash Aryal ,&nbsp;Claire E. Hefner ,&nbsp;Eric Brack ,&nbsp;Nadnudda Rodthongkum ,&nbsp;Pranut Potiyaraj ,&nbsp;Charles S. Henry","doi":"10.1016/j.aca.2025.344031","DOIUrl":"10.1016/j.aca.2025.344031","url":null,"abstract":"<div><h3>Background</h3><div>Heavy metal and nutrient contamination are growing global issues necessitating monitoring water resources. While laboratory-based platforms for detecting these contaminants are sensitive and accurate, they require centralized facilities, trained personnel, and significant costs. Microfluidic paper-based analytical devices have emerged as a low-cost alternative for on-site detection of these water contaminants; however, these platforms struggle with slow assay times, loss of analyte, and the need for precise volumetric pipetting. Moreover, these platforms often focus on detecting only one subgroup of contaminants, limiting the potential for comprehensive measurements.</div></div><div><h3>Results</h3><div>We developed a capillary flow-driven, single-dip dual-sided detection system, enabling rapid, multiplex detection of heavy metals and nutrients in a single user step. The sensor enables both qualitative visual analysis and quantitative analysis via a smartphone app for real-time and on-site detection of Ni, Fe, Cu, NO₂⁻ and PO₄³⁻. The limits of detection (LoD) and quantification (LoQ) were calculated as 1.3 and 4.4 ppm for Ni, 0.3 and 0.9 ppm for Fe, 0.2 and 0.6 ppm for Cu, 0.4 and 1.2 ppm for NO₂⁻, and 0.5 and 1.6 ppm for PO₄³⁻. Selectivity was achieved through masking strategies in each detection zone. The sensors were stable for &gt;4 weeks under ambient conditions. Spike-recovery analysis was performed using river, tap, pond, and commercial drinking water, achieving recoveries between 86 and 112 % with accuracy and precision below 15 % RSD for all samples.</div></div><div><h3>Significance</h3><div>This multiplex sensor offers a solution to overcome the current limitations of paper-based devices, allowing for a more comprehensive analysis of multiple contaminant classes.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1356 ","pages":"Article 344031"},"PeriodicalIF":5.7,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143819875","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 fully automated paper-based smartphone-assisted microfluidic chemiluminescence sample to result immunoassay platform","authors":"Jihong Sun ,&nbsp;Sixuan Duan ,&nbsp;Weiqin Xu ,&nbsp;Wenjun He ,&nbsp;Tong Li ,&nbsp;Sanli Liu ,&nbsp;Kai Hoettges ,&nbsp;Junhui Zhu ,&nbsp;Mark Leach ,&nbsp;Pengfei Song","doi":"10.1016/j.aca.2025.344013","DOIUrl":"10.1016/j.aca.2025.344013","url":null,"abstract":"<div><h3>Background</h3><div>In recent years, adapting immunoassay technologies for point-of-care testing (POCT) to enable low-cost, on-site diagnostics has gained great attention. Among the various approaches to achieving this goal, microfluidic paper-based analytical devices (μPADs) have emerged as a promising platform. These devices are particularly appealing due to their low cost, simple procedures, and minimal sample consumption. Among the detection methods used in μPADs, chemiluminescence (CL) has attracted widespread attention for its high sensitivity. However, existing CL immunoassays often require expensive detection equipment and extensive manual operation, which limits their use in resource-poor areas or where trained personnel are lacking.</div></div><div><h3>Results</h3><div>In this study, we developed a newly designed μPAD for CL immunoassays, leveraging smartphone-based image capture, valve-regulated reaction process, and app-driven result analysis to enable fully automated detection. Each element of the device was highly integrated and designed for programmable operation, enhancing reliability and stability. An app was developed to manage the entire process, maximizing the convenience of smartphones. The device's effectiveness was calibrated using rabbit IgG as a model, achieving a limit of detection (LOD) of 62.4 pg/mL, which represents an improvement compared to the colorimetric ELISA method with a LOD of 3 ng/mL (20pM). We then applied the device to detect tau protein, a biomarker associated with Alzheimer's disease and achieved a detection limit of 26.1 pg/mL, which is lower than the clinical cut-off value.</div></div><div><h3>Significance</h3><div>Our device achieves complete automation and integrates the versatility of μPADs with the precision of CL immunoassays, offering an effective and accessible solution for POCT. By incorporating a user-friendly smartphone interface, it simplifies operation while maintain detection sensitivity, providing a practical and valuable improvement in the development of POCT technologies.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1358 ","pages":"Article 344013"},"PeriodicalIF":5.7,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143813911","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":"Real-time amplification and high resolution melt analysis on a rapid microfluidic instrument","authors":"Renna L. Nouwairi ,&nbsp;Killian C. O'Connell ,&nbsp;Rachelle A. Turiello ,&nbsp;Larissa L. Cunha ,&nbsp;Leah M. Gunnoe ,&nbsp;Allison C. Burton ,&nbsp;Ryan M. Gibiser ,&nbsp;Margaret E. Straub ,&nbsp;James P. Landers","doi":"10.1016/j.aca.2025.344046","DOIUrl":"10.1016/j.aca.2025.344046","url":null,"abstract":"<div><h3>Background</h3><div>Real-time nucleic acid amplification represents a robust, ubiquitous technology that often requires additional downstream analysis to identify sequence polymorphisms or differentiate specific from non-specific amplification (NSA). For example, common post-amplification analysis methods for the polymerase chain reaction (PCR) include sequencing and electrophoresis, both of which are labor- and time-intensive techniques that require expensive additional reagents and consumables. In contrast, high resolution melt (HRM) analysis presents a simpler alternative that can elucidate sequence differences and distinguish specific from non-specific amplification without requiring separate instrumentation or additional reagents beyond those used for real-time amplification.</div></div><div><h3>Results</h3><div>We have previously reported a microfluidic real-time amplification system that could complete PCR in 8 minutes with comparable sensitivity to conventional instruments that require 50+ minutes for the same assay. Here, we describe expanding the capability of the system to include post-amplification HRM analysis. Sequence differentiation was demonstrated using PCR to detect epigenetic targets with different methylation percentages. Moreover, isothermal amplification methods that commonly experience NSA due to excessive primer noise, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), were coupled with HRM to distinguish true positive results. All microfluidic experiments were completed in parallel on conventional benchtop instrumentation, the results of which were found to be comparable with the microfluidic system consuming a fraction of the total analysis time required on the conventional instrument.</div></div><div><h3>Significance</h3><div>With these modifications, the microfluidic platform, which has demonstrated 8 minute PCR, can perform HRM in under 4 minutes following amplification for differentiation of sequences containing mutations and elucidating NSA. We demonstrate enhanced applicability of this microfluidic instrument for point-of-need applications, including clinical diagnostics, where rapid and accurate genomic analysis is paramount.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1356 ","pages":"Article 344046"},"PeriodicalIF":5.7,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143819860","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":"Automated microfluidic-based polyurethane/lactate oxidase-chitosan/m-phenylenediamine biosensing platform for lactate monitoring in breast cancer cell culture","authors":"Hnin Thandar Lwin ,&nbsp;Kewarin Phonklam ,&nbsp;Thanaporn Khunpitak ,&nbsp;Pasarat Khongkow ,&nbsp;Tonghathai Phairatana","doi":"10.1016/j.aca.2025.344049","DOIUrl":"10.1016/j.aca.2025.344049","url":null,"abstract":"<div><h3>Background</h3><div>Lactate is a significant indicator of cancer characteristics. Monitoring lactate dynamics in cell metabolism provides crucial insights into cellular events, essential for developing novel therapeutic strategies, including drug screening. However, conventional methods for lactate detection in cell culture are typically time-consuming, labor-intensive, require large sample volumes, and offer only endpoint analysis.</div></div><div><h3>Results</h3><div>We introduce an automated microfluidic-based amperometric biosensor system for real-time lactate monitoring during a cell-culture experiment. An in-house combined needle microelectrode was modified with polyurethane/lactate oxidase-chitosan/m-phenylenediamine. The polyurethane layer was optimized to extend the sensor's linearity. Additionally, a computer-controlled microfluidic platform was developed to enable tracking of biosensor performance, ensuring precision in fluid delivery and improving the reliability of the monitoring process. The biosensor performances were monitored over time, demonstrating a sensitivity of 95.50 ± 6.30 nA/mM mm² with a detection limit of 82.60 ± 0.07 μM, and a wide linear range up to 6.0 mM. The lactate biosensor exhibited good selectivity for other potential molecules, while maintaining stability over 18 h during continuous monitoring. To assess its application in paclitaxel anti-cancer drug testing, we connected the analysis platform to a culture flask of MDA-MB-231 breast cancer cells and delivered the cell culture media samples to the analysis platform through a microdialysis probe.</div></div><div><h3>Significance</h3><div>The results revealed that the released extracellular lactate concentrations in the media were positively correlated with the CCK-8 (Cell Counting Kit-8) cell viability test results. Our lactate monitoring system shows significant potential as a practical tool for drug screening and for monitoring metabolic processes across various fields.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1356 ","pages":"Article 344049"},"PeriodicalIF":5.7,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143814034","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 Z-scheme photoelectrochemical biosensing platform based on Cu2O-sensitized hollow covalent organic frameworks for sensitive microcystin-LR detection","authors":"Yiyuan Yang,&nbsp;Hejie Zheng,&nbsp;Cuicui Du,&nbsp;Huan Wang,&nbsp;Guizhen Luo,&nbsp;Xiaohua Zhang,&nbsp;Jinhua Chen","doi":"10.1016/j.aca.2025.344050","DOIUrl":"10.1016/j.aca.2025.344050","url":null,"abstract":"<div><div>Photoactive materials play a crucial role in enhancing the sensitivity of photoelectrochemical (PEC) biosensors. In this work, we developed a highly sensitive Z-scheme PEC biosensing platform based on Cu₂O-sensitized hollow structured covalent organic frameworks (HCOF–OMe) microspheres for the detection of microcystin-LR (MC-LR). The HCOF–OMe photoelectrode with enhanced PEC response can be sensitized by Cu₂O nanocubes through the formation of a Z-scheme system, enabling the construction of a signal-on PEC biosensing platform. To amplify the detection signal, a homogeneous biosensing strategy was employed by integrating a DNA walker nanomachine-assisted CRISPR/Cas12a system. A DNA walker nanomachine was assembled on streptavidin-modified magnetic bead (MB) and initially locked by MC-LR aptamer. Upon MC- LR recognition, the walker DNA was allowed to hybridize with support DNA, exposing cleavage sites for the nicking endonuclease Nb.<em>Bbv</em>CI, which can release the activator strand to trigger the <em>trans</em>-cleavage ability of CRISPR/Cas12a. As a result, Cu₂O nanocubes were released from the MB-ssDNA-Cu₂O complex and subsequently loaded on the HCOF–OMe photoelectrode, significantly improving the PEC signals. This enabled the sensitive assay of MC-LR over a wide linear range of 1.0 × 10⁻⁵−100 μg/L with a low detection limit of 4.6 × 10⁻⁶ μg/L. The proposed biosensing platform is highly versatile and can be extended to detect other environmental pollutants or biological disease markers.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1356 ","pages":"Article 344050"},"PeriodicalIF":5.7,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143819854","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":"Functional data analysis, a comprehensive framework for processing non-quadrilinear and low-selective data provided by four-way liquid chromatography analysis","authors":"Mirta R. Alcaraz ,&nbsp;Milagros Montemurro ,&nbsp;Pablo L. Pisano ,&nbsp;Juan M. Lombardi ,&nbsp;Santiago A. Bortolato","doi":"10.1016/j.aca.2025.344044","DOIUrl":"10.1016/j.aca.2025.344044","url":null,"abstract":"<div><div>The chemometric treatment of higher-order chromatographic (LC) data often crashes due to two main effects: the chromatographic band shifts/warpings across samples and quasi-full overlaps between component signals, affecting their analytical selectivities. From a chemometric point of view, these phenomena have independent effects, although they can jointly contribute to the failure of the algorithms: 1) data multilinearity breaking, leading to the poor performance of multilinear decomposition algorithms, and 2) linear dependence between the analytes signals, causing the failure of folded models. Under this scenario, making chemometric processing feasible involves defining specific experimental conditions that minimize these effects or increasing the number of instrumental ways to deal with selectivity lost.</div><div>This work presents the Functional Aligned of Pure Vectors (FAPV) algorithm for restoring four-way chromatographic data multilinearity and bearing the spectral overlap trouble. Simulated and experimental four-way data were used to test the FAPV analytical efficiency, covering a wide range of chromatographic artifacts. Based on a multi-injection procedure, the experimental case implied the chromatographic determination of two analytes with uncalibrated interferents in aqueous samples. Both data systems were subjected to FAPV and then processed by PARAFAC. Therefore, a comprehensive comparison was made with the most widely used chemometric models for non-multilinear chromatographic data (MCR-ALS and PARAFAC2). Moreover, the performance of the FAPV approach was compared with commonly used alignment procedures, e.g., correlation-optimized warping. The results (c.a. REPs of 10 % in both analytes from the experimental case) show the efficiency of the FAPV algorithm in solving the troubles observed in chromatographic/spectral data.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1356 ","pages":"Article 344044"},"PeriodicalIF":5.7,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143806566","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":"Label-free urinary protein detection through machine learning analysis of single droplet evaporation patterns","authors":"Ruyue Yang ,&nbsp;Nannan Cao ,&nbsp;Yan Yang ,&nbsp;Shujuan Guan ,&nbsp;Chao Yang ,&nbsp;Bo Situ ,&nbsp;Yongyu Rui ,&nbsp;Hongwei Zhou ,&nbsp;Lei Zheng","doi":"10.1016/j.aca.2025.344034","DOIUrl":"10.1016/j.aca.2025.344034","url":null,"abstract":"<div><h3>Background</h3><div>Chronic kidney disease (CKD) is a major global public health issue, with a steadily increasing incidence. Urinary protein detection serves as a crucial indicator for the diagnosis, monitoring and management of CKD. However, current methods for urinary protein measurement, such as urine dipstick tests, colorimetric assays, and 24-h total urine protein analysis, have certain limitations that restrict their routine application in CKD screening and follow-up. Therefore, there is an urgent need for a simple, convenient, and rapid diagnostic approach for kidney function assessment.</div></div><div><h3>Results</h3><div>In this study, we have developed and validated a novel method for urine protein quantification based on dried droplet morphology analysis. Our approach demonstrates robust performance across a wide range of protein concentrations and is resilient to common interfering substances and variations in sample processing. It's worth noting that while our method shows excellent agreement with the colorimetric assay, it offers several potential advantages. These include reduced sample volume requirements, simplified sample preparation, and rapid analysis time. These factors could make our method particularly suitable for point-of-care testing or resource-limited settings where traditional laboratory infrastructure may be unavailable.</div></div><div><h3>Significance</h3><div>The novel method for protein quantification in urine based on the morphology of a dried droplet uses only one drop of urine specimen. Combined with machine learning models, by identifying protein content in urine droplet drying patterns without the need for staining or antibody binding, may provide a more convenient alternative to current techniques for the assessment of proteinuria. Simple, low-cost, and fast, the system can be used as a powerful tool for CKD surveillance at the point of care.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1356 ","pages":"Article 344034"},"PeriodicalIF":5.7,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143806564","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":"The catalytic hairpin assembly induces a conformational change of the RNA aptamer enabled in situ imaging of circRNAs in tumor cells","authors":"Yanheng Yao ,&nbsp;Feifan Yin ,&nbsp;Qiufeng Wang ,&nbsp;Murilege Chao ,&nbsp;Zhongyun Wang ,&nbsp;Yang Xiang","doi":"10.1016/j.aca.2025.344048","DOIUrl":"10.1016/j.aca.2025.344048","url":null,"abstract":"<div><div>Circular RNAs (circRNAs) are ubiquitously expressed across all cell types and tissues, playing a pivotal role in regulating diverse biological processes and being implicated in various human cancers. Their inherent stability renders them highly promising for practical applications in diagnosing and treating numerous human diseases, especially as biomarkers. In this study, we have successfully engineered a novel luminescent RNA sensor utilizing catalytic hairpin assembly (CHA) to mediate aptamer conformational changes for both extracellular and intracellular circRNA detection. The CHA process is specifically triggered by the unique back-splice junction (BSJ) present in circRNA, enabling precise discrimination between circular and homologous linear RNA. Through rational design and modification of the RNA aptamer sequence to enhance its affinity for the CHA product, the modified RNA aptamer undergoes specific conformational changes that facilitate binding with small molecule dyes, thereby generating a pronounced fluorescence signal. Our approach exhibits robust performance and stability in complex biological systems, making it suitable for visualizing circRNA in tumor cells and detecting extracellular circRNA. Additionally, this method demonstrates excellent biocompatibility and minimal cytotoxicity in live cell imaging, along with superior specificity and sensitivity for target molecules. This technique offers a valuable tool for elucidating intricate physiological processes involving circRNA in live cells.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1356 ","pages":"Article 344048"},"PeriodicalIF":5.7,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143806565","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":"Bead-free digital enzyme-linked immunoassay on PDMS microwell array","authors":"Han Wu ,&nbsp;Huaiheng Zhou ,&nbsp;Zhanqiang Sun ,&nbsp;Jiayao Fang ,&nbsp;Weiran An ,&nbsp;Xiaoli Yu ,&nbsp;Bo Zheng","doi":"10.1016/j.aca.2025.344045","DOIUrl":"10.1016/j.aca.2025.344045","url":null,"abstract":"<div><h3>Background</h3><div>Proteins which are important biomarkers for disease diagnosis, prognosis and treatment are often present at very low concentrations in biological samples. Measuring these low-level proteins is important yet challenging. Conventional enzyme-linked immunoassays (ELISAs) usually lack the sensitive required. While ultrasensitive detection methods, such as digital ELISA have recently significantly advanced in sensitivity, they usually face high-cost of using paramagnetic beads or require complicated substrate fabrication, limiting their accessibility for large-scale screening or application in resource-limited settings. Therefore, there is a pressing need to develop cost-effective detection methods with ultrahigh sensitivity to accurately detecting low-level protein biomarkers.</div></div><div><h3>Results</h3><div>In the study, we developed a cost-effective and easily prepared PDMS microwell array platform for bead-free digital enzyme-linked immunoassay (ELISA) to achieve ultrasensitive protein detection. Both the immunocomplexes formation and separation process, which are two key steps in digital ELISA occur within the PDMS microwells, eliminating the need for paramagnetic beads and complex bead-separation systems. This approach significantly reduces detection costs compared to the widely used single-molecule arrays (SiMoAs)-based digital ELISA, which relies on large quantities of paramagnetic beads for immunocomplexes immobilization and intricate microwell array chips for immunocomplexes separation. Here, the PDMS microwell array chip was modified to generate large-scale femtoliter droplet arrays for single-molecule reactions, with minimized droplet evaporation and no cross-contamination. The PDMS microwell array platform was further successfully applied to single-molecule enzyme detection and cytokine analysis, achieving detection limits of 1.57 fM for streptavidin-β-galactosidase (SβG) and 12.3 fg/ml for human interleukin-6.</div></div><div><h3>Significance</h3><div>A low-cost PDMS microwell array platform for digital ELISA was developed. The fabrication of PDMS microwell array chip was straightforward by soft lithography, enabling mass production with small batch-to-batch variation. With its cost-effective fabrication process and high detection sensitivity, the PDMS microwell array would be a promising platform for ultrasensitive protein detection, especially in the area of early disease screening.</div></div>","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"1356 ","pages":"Article 344045"},"PeriodicalIF":5.7,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143806569","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}