Fig 1: Exploring the plasma proteome and detecting tissue leakage proteins in plasma.(a) Individual protein expression levels (log2(relative ratios)) of the three out of eight proteins of the CancerSEEK test (Cohen et al., 2018) that were detected in a cohort of 30 blood donors. (b) Distribution of protein abundances based on precursor areas. Cancer-related proteins and FDA-approved drug target proteins (HPA classification) highlighted in green and blue, respectively. Classical plasma proteins (Anderson and Anderson, 2002) highlighted in red, proteins included in the CancerSEEK test in yellow. Among these proteins, some are specifically indicated by gene symbol. Spiked PSA (4 ng/mL) is marked with *. (c) Detection of tissue leakage proteins in plasma assessed by mRNA expression levels classified as enriched on particular tissue types. The anatomy image in panel c was adapted from https://commons.wikimedia.org/wiki/File:Female_shadow_anatomy_without_labels.png, which is available in the Public Domain.
Fig 2: The principle of the proposed microfluidic immunoassay based on vertically sheathing laminar flows of pAb and QD-sAb using Device C. When pAb and QD-sAb are flowing in the upper and lower laminar flows, respectively, pAb diffuses more rapidly to the reaction site where the immunoreactions between PSA, pAb, and QD-sAb instantaneously occur.
Fig 3: The concentration profiles across the T-shaped microfluidic channel at each longitudinal position were predicted by COMSOL®. When pAb and QD-sAb were infused into the device at a flow rate ratio of 1 : 1, we obtained the concentration profiles of (a) anti-PSA pAb and (b) QD-sAb. The concentration profiles were also predicted when (c) anti-PSA pAb and (d) QD-sAb were injected at a ratio of 4 : 1. The interface between two reagent solutions was shifted toward the right side of the channel due to the asymmetric input flow rates.
Fig 4: In vivo results of different measurements using the microneedle array. (a) Stabilization curves of PSA association at 10 pM (red curve) and 100 pM (blue curve) spiked-PBS solutions. The results indicate that the sensor requires approximately 60 s to achieve a differentiable signal. (b) One cycle close-up view taken once dissociation stabilization is achieved for PSA-spiked PBS buffer. (c) One cycle close-up view taken once dissociation stabilization is achieved for PSA-spiked serum. (d) Normalized response linear curves derived from (a) and (b). The dissociation phase was conducted in 5% EG in 100 µM phosphate buffer solution. Normalized reaction is in comparison to nonspiked buffer or serum, respectively. Pink data relates to nonspecific normalized response to 22 ng/mL cTnI and 21 ng/mL GFP. (e) In vivo intradermal capillary PSA concentrations were measured in four subjects using the microneedle array (green bars) compared to ELISA measurements of PSA concentration in venous blood (blue dots). (f) Multiplex experiment results of normalized response to PSA-spiked buffer from a device modified with PSA-specific antibody (aPSA, black curve) and a device modified with cTnI-specific antibody (acTnI, red curve). (g) Multiplex experiment results of normalized response to cTnI-spiked buffer from a device modified with PSA-specific antibody (aPSA, black curve) and a device modified with cTnI-specific antibody (acTnI, red curve). (i) Top: Deviation measurements performed on a single device via multiple entries to (100 pM spiked serum solution). Bottom: Variance measurements were performed between different devices via normalized response against a 100 pM spiked serum.
Fig 5: Detection of target cancer markers in coexisting conditions together with other markers. (a) PSA detection under a mixture with CEA of 5 ng/mL, presented on a linear scale. FL intensity coming from the PSA is shown using orange circles with error bars. Dashed line: a fitted line. Arrow: the medical criterion value of PSA, 4 ng/mL. (b) CEA detection under a mixture with PSA of 4 ng/mL. FL intensity coming from the CEA is shown using orange closed circles with error bars on a semi-log scale. Dashed curve: a fitted curve by the Hill equation (Equation (1)). A black horizontal bar represents the zero level, which is y0 in the Hill equation. Arrow: the medical criterion value of CEA, 5 ng/mL.
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