Journal: Lab on a chip

Article Title: Integrated device for plasma separation and nucleic acid extraction from whole blood toward point-of-care detection of bloodborne pathogens.

doi: 10.1039/d4lc00571f

Figure Lengend Snippet: Fig. 1 Design of PRECISE cartridge, which integrates plasma separation and RNA extraction for processing of whole blood at the point of care. Overview of workflow for blood sample preparation for HCV detection using the PRECISE cartridge. The PRECISE cartridge operates with as little as 50 μL of whole blood sample (which is compatible with fingerstick blood collection methods), drawn up by user with a tool that is then used to dispense the blood into the plasma separation component of the device. The dual-membrane filter separates out plasma from the sample, which is then withdrawn by user with a syringe from the filter to be dispensed into the extraction component of the device. Extraction is accomplished by using a magnetofluidic immiscible phase reagent separation approach to purify the RNA for PCR performed off-device. Overall, for the user, no pipetting steps are needed upstream of the cartridge (commonly used blood collection tools such as Minivette POCT (Sarstedt) or MICROSAFE tube (Drummond Scientific) fit right into our filter), the plasma separation step involves fluid handling with similar complexity to running LFA rapid tests, and the nucleic acid extraction requires the user to move two magnets either around a chamber to mix or along tracks to move the beads.

Article Snippet: Lab ChipThis journal is © The Royal Society of Chemistry 2024 reported methods for membrane-based plasma filtration typically utilize polycarbonate track-etched membranes2 or a commercially-available asymmetric polysulfone membrane, the Vivid plasma separation membrane (Cytiva).28–31 We explored using polycarbonate membranes (of 3 μm and 0.4 μm pore sizes) due to their more consistent pore sizes and densities, but observed a higher degree of hemolysis compared to the dual glass fiber design (ESI† Fig. S2a).

Techniques: Clinical Proteomics, RNA Extraction, Sample Prep, Membrane, Extraction

Journal: Lab on a chip

Article Title: Integrated device for plasma separation and nucleic acid extraction from whole blood toward point-of-care detection of bloodborne pathogens.

doi: 10.1039/d4lc00571f

Figure Lengend Snippet: Fig. 2 Design and characterization of dual-membrane size-exclusion filter for separating plasma from whole blood. (a) The filter allows for filtration of plasma from blood. The filter is constructed with an input and output piece, O-rings incorporated internally on each end, and a larger pore membrane (2.7 μm, glass fiber) followed by a smaller pore membrane (0.7 μm, glass fiber). The larger pore membrane functions to block larger blood components (such as white blood cells and some red blood cells), while the smaller pore membrane blocks the remaining cells and allows plasma and any present virus particles to pass through. (b) Workflow of plasma separation. The user dispenses the blood sample (e.g. collected from a Minivette, as demonstrated in this study) into the filter, flushes 450 μL of PBS (that was pre-loaded in a syringe) into the filter, and flips the filter upside down and draws the filtrate from the upward-facing outlet (see Fig. 4a for how the flipped filter looks in relation to the rest of the PRECISE cartridge.). (c) Plasma separation performance of the filter demonstrates its ability to separate the majority of cells from the blood sample with no significant difference from lab-based centrifugation. (d) Filtration causes minimal hemolysis of red blood cells, with no significant difference from centrifugation control. Hemolysis measured via hemoglobin absorbance peak at 414 nm peak and compared to fully lysed whole blood. (e) Images of filter after filtering blood. The red color at input (left) from blood sample on the membrane contrasts with the yellow color at output (middle) where only PBS-diluted plasma filtrate exits the filter, indicating successful retention of blood cells in the membranes. Images of the filtrate (right) are included, for three replicates after processing of 50 μL of whole blood. (f) Comparison of volume recovery of filter and centrifugation methods of plasma separation. The filter produces 201.8 ± 18.75 μL filtrate after input of 50 μL blood and 450 μL PBS, and centrifugation of 50 μL blood produces 24.6 ± 3.35 μL plasma. (g) Virus recovery from plasma separated from blood, spiked with whole inactivated HCV virus at 6.77 × 103 IU mL−1 final concentration in blood, using filter and traditional centrifugation methods, extracted via benchtop RNA extraction protocol and amplified via RT-PCR. Recovery was calculated by comparing Ct values to RNA standard curve and demonstrated no significant difference between filter and centrifugation separation methods. (h) Recovered volumes of filtrate across hematocrit levels (50%, 55%, and 60%) and blood sample input volumes (25, 50, and 75 μL). There was no significant difference in recovered volumes across hematocrit levels. There was an observed trend in higher recovered volume with higher blood input, with significantly higher filtrate volume for 75 μL blood vs. 25 μL irrespective of hematocrit (p = 0.0012). (i) Hemolysis level in filtrate across hematocrit levels and blood sample input volumes, measured at absorbance peak 414 nm for hemoglobin. Within blood sample input volumes, there was no significant difference in hemolysis between hematocrit levels. There was a significant difference between 25 μL and 75 μL (p = 0.0219) and 50 μL and 75 μL (p = 0.0237) irrespective of hematocrit level, but there was no significant difference in hemolysis between 25 μL and 50 μL groups. Error bars in all figures indicate mean ± standard deviation. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: Lab ChipThis journal is © The Royal Society of Chemistry 2024 reported methods for membrane-based plasma filtration typically utilize polycarbonate track-etched membranes2 or a commercially-available asymmetric polysulfone membrane, the Vivid plasma separation membrane (Cytiva).28–31 We explored using polycarbonate membranes (of 3 μm and 0.4 μm pore sizes) due to their more consistent pore sizes and densities, but observed a higher degree of hemolysis compared to the dual glass fiber design (ESI† Fig. S2a).

Techniques: Membrane, Clinical Proteomics, Filtration, Construct, Blocking Assay, Virus, Centrifugation, Control, Comparison, Concentration Assay, RNA Extraction, Amplification, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Lab on a chip

Article Title: Integrated device for plasma separation and nucleic acid extraction from whole blood toward point-of-care detection of bloodborne pathogens.

doi: 10.1039/d4lc00571f

Figure Lengend Snippet: Fig. 3 Design and characterization of nucleic acid extraction module. (a) Image (left) and schematic (right) of PRECISE device design. Device includes a magnet with a lower track for manually operated, power-free guided transfer of magnetic beads across the microfluidic extraction module. An upper magnet with a track facilitates mixing (see Fig. 3b). A holder for the filter sits to the left of the extraction module for movement of filter above and away from the first chamber of module for sample addition when whole blood starting samples are used, as in Fig. 2 and 4. (b) Design of magnetofluidic immiscible-phase extraction module (65 × 18 mm). The module includes an initial large chamber to accommodate appropriate lysis/binding buffer mix and 200 μL sample input, two wash chambers with wash buffer, and an elution chamber with elution buffer, all separated by chambers for mineral oil. Chambers are pre-loaded with reagents, and the extraction process functions as follows: 1) plasma sample is added (via dual-membrane filtration as in Fig. 2, or conventionally separated plasma as is used in this figure) to the first chamber containing lysis buffer, purification buffer, proteinase K, and magnetic beads functionalized for binding nucleic acids. Magnet-assisted mixing is performed by alternating presence of upper and lower magnets above and below the chamber, respectively, to draw beads through solution. Beads are then collected with the lower magnet and transferred through the oil chambers into the subsequent washes and finally into the elution chamber, where the magnet mixing is performed again before removal of eluate from the final chamber. (c) Use of the upper magnet for magnet- assisted mixing improves RNA extraction performance on the module, as demonstrated by the differences in RT-PCR amplification between no magnet-assisted mixing, mixing 3 times, and mixing 10 times in the lysis/binding and elution steps. (d) Representative RT-PCR curves for HCV RNA extraction on PRECISE cartridge and with benchtop magnetic bead extraction kit, from whole, inactivated HCV spiked into 25 μL of DNA-cleared plasma (estimated volume of plasma from 50 μL blood sample) and diluted by PBS to 200 μL to represent the estimated sample volumes determined from Fig. 2f, shown at 6770 IU mL−1 starting concentration in plasma sample. (e) RT-PCR amplification at varying concentrations of whole, inactivated HCV spiked into 25 μL of DNA-cleared plasma and diluted by PBS to 200 μL. All replicates amplified for both cartridge and benchtop comparison at 855 IU mL−1, with no significant differences between extraction methods for any concentration. Error bars in all figures indicate mean ± standard deviation. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: Lab ChipThis journal is © The Royal Society of Chemistry 2024 reported methods for membrane-based plasma filtration typically utilize polycarbonate track-etched membranes2 or a commercially-available asymmetric polysulfone membrane, the Vivid plasma separation membrane (Cytiva).28–31 We explored using polycarbonate membranes (of 3 μm and 0.4 μm pore sizes) due to their more consistent pore sizes and densities, but observed a higher degree of hemolysis compared to the dual glass fiber design (ESI† Fig. S2a).

Techniques: Extraction, Magnetic Beads, Lysis, Binding Assay, Clinical Proteomics, Membrane, Filtration, Purification, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Amplification, Concentration Assay, Comparison, Standard Deviation

Journal: Lab on a chip

Article Title: Integrated device for plasma separation and nucleic acid extraction from whole blood toward point-of-care detection of bloodborne pathogens.

doi: 10.1039/d4lc00571f

Figure Lengend Snippet: Fig. 4 Demonstration of PRECISE device for sample-to-answer HCV detection. (a) Integrated workflow for PRECISE device for preparing whole blood sample for HCV detection via RT-PCR involves a plasma separation step via plasma filter and an RNA extraction step via a magnetofluidic immiscible-phase cartridge manually operated with a guided magnet track. 50 μL of fingerstick blood collected with a blood collection tool is dispensed into the inlet of filter, followed by 450 μL PBS flushed with a syringe. The filter is flipped and the syringe is used to draw out filtrate from the outlet, then dispense into first chamber of extraction cartridge. The user performs on-cartridge extraction by guiding a magnet to move RNA- bound functionalized magnetic beads through chambers containing lysis/binding buffers, wash buffers, and finally elution buffer all in connected sequence and separated by mineral oil. The user then transfers the eluate from the final chamber to a PCR plate for RT-PCR on a QuantStudio 3 instrument. (b) Detection of varying concentrations of whole, inactivated HCV spiked into 50 μL blood when processed with lab-based workflow (centrifugation for plasma separation, benchtop magnetic bead protocol for RNA extraction) and PRECISE device (integrated blood filter for plasma separation and magnetofluidic immiscible-phase cartridge for RNA extraction). Extracted samples from both approaches were amplified for detection via RT-PCR on QuantStudio 3 system. All replicates (n = 3, each technical replicate run in triplicate on PCR plate) for PRECISE device and lab-based workflow amplified at 6770 IU mL−1 with no significant difference between workflows. (c) Real-time fluorescence curves of RT-PCR comparing lab-based workflow and PRECISE device for sample preparation from whole blood at 13 540 IU mL−1. Error bars in all figures indicate mean ± standard deviation. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: Lab ChipThis journal is © The Royal Society of Chemistry 2024 reported methods for membrane-based plasma filtration typically utilize polycarbonate track-etched membranes2 or a commercially-available asymmetric polysulfone membrane, the Vivid plasma separation membrane (Cytiva).28–31 We explored using polycarbonate membranes (of 3 μm and 0.4 μm pore sizes) due to their more consistent pore sizes and densities, but observed a higher degree of hemolysis compared to the dual glass fiber design (ESI† Fig. S2a).

Techniques: Reverse Transcription Polymerase Chain Reaction, Clinical Proteomics, RNA Extraction, Extraction, Magnetic Beads, Lysis, Binding Assay, Sequencing, Centrifugation, Amplification, Fluorescence, Sample Prep, Standard Deviation

Journal: Plant and Soil

Article Title: Myxospermous seed-mucilage quantity correlates with environmental gradients indicative of water-deficit stress: Plantago species as a model

doi: 10.1007/s11104-019-04335-z

Figure Lengend Snippet: Fig. 3 GLM models effect of pedoclimatic conditions on germination and seedlings development incubated at 15°C and 20°C FAMD’s second axis scores. a Maximum temperature at 15°C germination; Minimum precipitation at 15°C germination; c soil penetrometer resistance (−300 kPa) at 15°C germination; d Maximum temperature at 20°C germination; e Minimum precipitation at 20°C germination; f soil penetrometer resistance −300 kPa at 20°C germination; g Maximum temperature at 15°C seedlings; h Maximum temperature at 20°C seedlings; i Minimum precipitation at 20°C seedlings; j soil penetrometer resistance (−300 kPa) at 20°C seedlings

Article Snippet: Germinated seeds were carefully transferred to germination paper (WhatmanTM, grade 1, #1001-824), pre- moistened with 15mL of the same osmotic solution and held within transparent re-sealable plastic boxes (17 × 12 × 5 cm).

Techniques: Incubation

Journal: Plant and Soil

Article Title: Myxospermous seed-mucilage quantity correlates with environmental gradients indicative of water-deficit stress: Plantago species as a model

doi: 10.1007/s11104-019-04335-z

Figure Lengend Snippet: Fig. 5 GLM model regressions of mucilage quantity effect on water base (Ψb). a Mucilage quantity effect on germination water base (ΨbG) in 15°C (dashed line) and 20°C (solid line) and b mucilage quantity effect on seedling development water base (ΨbS) in 15°C (dashed line) and 20°C (solid line). GLM models were performed with the mean of three replicates for each accession for each species

Article Snippet: Germinated seeds were carefully transferred to germination paper (WhatmanTM, grade 1, #1001-824), pre- moistened with 15mL of the same osmotic solution and held within transparent re-sealable plastic boxes (17 × 12 × 5 cm).

Techniques: