Dataset Information

Characterization and Optimization of Isotachophoresis Parameters for Pacific Blue Succinimidyl Ester Dye on a PDMS Microfluidic Chip.

ABSTRACT: Isotachophoresis (ITP) for Pacific Blue (PB) dye using a polydimethylsiloxane (PDMS) microfluidic chip is developed and characterized by determining the types and concentrations of electrolytes, the ITP duration, and the electric field density. Among candidate buffers for the trailing electrolyte (TE) and leading electrolyte (LE), 40 mM borate buffer (pH 9) and 200 mM trisaminomethane hydrochloride (Tris-HCl) (pH 8) were selected to obtain the maximum preconcentration and resolution of the PB bands, respectively. With the selected TE and LE buffers, further optimization was performed to determine the electric field (EF) density and the ITP duration. These ITP parameters showed a 20-170,000 preconcentration ratio from initial PB concentrations of 10 nM-100 fM. Further demonstration was implemented to preconcentrate PB-conjugated lactate dehydrogenase (LDH) using the PDMS microfluidic chip. By utilizing the quenching nature of PB-LDH conjugation, we were able to identify concentrations of LDH as low as 10 ng/mL. This simple PDMS microfluidic chip-based ITP for PB preconcentration enables highly sensitive biological and chemical analyses by coupling with various downstream detection systems.

SUBMITTER: Somaweera H

PROVIDER: S-EPMC7690402 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Characterization and Optimization of Isotachophoresis Parameters for Pacific Blue Succinimidyl Ester Dye on a PDMS Microfluidic Chip.

Somaweera Himali H Estlack Zachary Z Devadhasan Jasmine Pramila JP Kim Jungtae J Kim Jungkyu J

Micromachines 20201022 11

Isotachophoresis (ITP) for Pacific Blue (PB) dye using a polydimethylsiloxane (PDMS) microfluidic chip is developed and characterized by determining the types and concentrations of electrolytes, the ITP duration, and the electric field density. Among candidate buffers for the trailing electrolyte (TE) and leading electrolyte (LE), 40 mM borate buffer (pH 9) and 200 mM trisaminomethane hydrochloride (Tris-HCl) (pH 8) were selected to obtain the maximum preconcentration and resolution of the PB ba ...[more]

PMID: 33105673

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Project description:Nucleic acid amplification tests (NAATs) are a crucial diagnostic and monitoring tool for infectious diseases. A key procedural step for NAATs is sample preparation: separating and purifying target nucleic acids from crude biological samples prior to nucleic acid amplification and detection. Traditionally, sample preparation has been performed with liquid- or solid-phase extraction, both of which require multiple trained user steps and significant laboratory equipment. The challenges associated with sample preparation have limited the dissemination of NAAT point-of-care diagnostics in low resource environments, including low- and middle-income countries. We report on a paper-based device for purification of nucleic acids from whole blood using isotachophoresis (ITP) for point-of-care NAATs. We show successful extraction and purification of target nucleic acids from large volumes (33 µL) of whole human blood samples with no moving parts and few user steps. Our device utilizes paper-based buffer reservoirs to fully contain the liquid ITP buffers and does not require complex filling procedures, instead relying on the natural wicking of integrated paper membranes. We perform on-device blood fractionation via filtration to remove leukocytes and erythrocytes from our sample, followed by integrated on-paper proteolytic digestion of endogenous plasma proteins to allow for successful isotachophoretic extraction. Paper-based isotachophoresis purifies and concentrates target nucleic acids that are added directly to recombinase polymerase amplification (RPA) reactions. We show consistent amplification of input copy concentrations of as low as 3 × 103 copies nucleic acid per mL input blood with extraction and purification taking only 30 min. By employing a paper architecture, we are able to incorporate these processes in a single, robust, low-cost design, enabling the direct processing of large volumes of blood, with the only intermediate user steps being the removal and addition of tape. Our device represents a step towards a simple, fully integrated sample preparation system for nucleic acid amplification tests at the point-of-care.

Dataset Information

Characterization and Optimization of Isotachophoresis Parameters for Pacific Blue Succinimidyl Ester Dye on a PDMS Microfluidic Chip.

Publications

Characterization and Optimization of Isotachophoresis Parameters for Pacific Blue Succinimidyl Ester Dye on a PDMS Microfluidic Chip.

Similar Datasets

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