3a1742acb05a259e0be1f06150b2cf1d pmc_9496280.pdf 4f9c50831207cb561238db0b46dec96e27611032 pmc_9496280.pdf 9255171077eef17c720cdcb2255e6e0a3f2949d1df2c8ad58d18f4ce8bac1316 pmc_9496280.pdf Title: Magnetic Bead Handling Using a Paper-Based Device for Quantitative Point-of-Care Testing Subject: Microfluidic paper-based analytical devices (PADs) have been extensively proposed as ideal tools for point-of-care (POC) testing with minimal user training and technical requirements. However, most PADs use dried bioreagents, which complicate production, reduce device reproducibility and stability, and require transport and storage under temperature and humidity-controlled conditions. In this work, we propose a PAD produced using an affordable craft-cutter and stored at room temperature, which is used to partially automate a single-step colorimetric magneto-immunoassay. As a proof-of-concept, the PAD has been applied to the quantitative detection of Plasmodium falciparum lactate dehydrogenase (Pf-LDH), a biomarker of malaria infection. In this system, detection is based on a single-step magneto-immunoassay that consists of a single 5-min incubation of the lysed blood sample with immuno-modified magnetic beads (MB), detection antibody, and an enzymatic signal amplifier (Poly-HRP). This mixture is then transferred to a single-piece paper device where, after on-chip MB magnetic concentration and washing, signal generation is achieved by adding a chromogenic enzyme substrate. The colorimetric readout is achieved by the naked eye or using a smartphone camera and free software for image analysis. This PAD afforded quantitative Pf-LDH detection in <15 min, with a detection limit of 6.25 ng mL-1 when the result was interpreted by the naked eye and 1.4 ng mL-1 when analysed using the smartphone imaging system. Moreover, the study of a battery of clinical samples revealed concentrations of Pf-LDH that correlated with those provided by the reference ELISA and with better sensitivity than a commercial rapid diagnostic test (RDT). These results demonstrate that magneto-immunoassays can be partly automated by employing a PAD, achieving a level of handling that approaches the requirements of POC testing. Keywords: immuno-modified magnetic beads; paper-based diagnostic device; low-cost assay automation; smartphone colorimetric detection; point-of-care testing; malaria quantitative diagnosis Author: Kevin Arias-Alpízar, Ana Sánchez-Cano, Judit Prat-Trunas, Elena Sulleiro, Pau Bosch-Nicolau, Fernando Salvador, Inés Oliveira, Israel Molina, Adrián Sánchez-Montalvá and Eva Baldrich Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Aug 25 10:07:06 2022 CEST ModDate: Thu Aug 25 10:54:25 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2216774 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AEITLL+VnURWPalladioL Type 1 Custom yes yes no 10 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 15 0 OSPRJF+URWPalladioL-Bold Type 1 Custom yes yes no 19 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IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Material and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Reagents and Biocomponents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Device Fabrication Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Smartphone Imaging System Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Pf-LDH Magneto-Immunodetection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Magneto-Immunoassay in Tubes (Classical Approach) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Magneto-Immunoassay Using the PAD (POC Approach) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Pf-LDH Detection in Whole Blood Clinical Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Data Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Children: Item: Title: Production of the PAD Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Magneto-Immunoassay Handling and Detection Using the PAD Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Quantitative Detection of Pf-LDH Using a Smartphone Detection System Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Analysis of Clinical Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Info: Title: Magnetic Bead Handling Using a Paper-Based Device for Quantitative Point-of-Care Testing Author: Kevin Arias-Alpízar, Ana Sánchez-Cano, Judit Prat-Trunas, Elena Sulleiro, Pau Bosch-Nicolau, Fernando Salvador, Inés Oliveira, Israel Molina, Adrián Sánchez-Montalvá and Eva Baldrich Subject: Microfluidic paper-based analytical devices (PADs) have been extensively proposed as ideal tools for point-of-care (POC) testing with minimal user training and technical requirements. However, most PADs use dried bioreagents, which complicate production, reduce device reproducibility and stability, and require transport and storage under temperature and humidity-controlled conditions. In this work, we propose a PAD produced using an affordable craft-cutter and stored at room temperature, which is used to partially automate a single-step colorimetric magneto-immunoassay. As a proof-of-concept, the PAD has been applied to the quantitative detection of Plasmodium falciparum lactate dehydrogenase (Pf-LDH), a biomarker of malaria infection. In this system, detection is based on a single-step magneto-immunoassay that consists of a single 5-min incubation of the lysed blood sample with immuno-modified magnetic beads (MB), detection antibody, and an enzymatic signal amplifier (Poly-HRP). This mixture is then transferred to a single-piece paper device where, after on-chip MB magnetic concentration and washing, signal generation is achieved by adding a chromogenic enzyme substrate. The colorimetric readout is achieved by the naked eye or using a smartphone camera and free software for image analysis. This PAD afforded quantitative Pf-LDH detection in <15 min, with a detection limit of 6.25 ng mL-1 when the result was interpreted by the naked eye and 1.4 ng mL-1 when analysed using the smartphone imaging system. Moreover, the study of a battery of clinical samples revealed concentrations of Pf-LDH that correlated with those provided by the reference ELISA and with better sensitivity than a commercial rapid diagnostic test (RDT). These results demonstrate that magneto-immunoassays can be partly automated by employing a PAD, achieving a level of handling that approaches the requirements of POC testing. 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