Silk-based blood stabilization for diagnostics

Effect of silk molecular mass and film thickness on solubility and recovery. (A) Schematic of silk and blood solution casting into thick and thin films. (B) Resolubility of silk films after simulated aging conditions. Films were formed from solutions of varying molecular mass by control of fibroin extraction time. Dotted line represents maximum concentration of silk based on mass of coupon. (C) Recovery of 100 µL of plasma from silk matrices of varying silk extraction time and film thickness; % recovery is the silk film assay value normalized to the control plasma value on day 0; 100% indicated by the dotted line. Solid lines indicate significance at the P < 0.05 level.

Stability profiles of plasma in silk films (plasma coupons), blood in silk films (blood coupon), and liquid plasma from three donors after storage at 37 and 22 °C for 30 d. Here, % recovery is the plasma or silk film assay value obtained from 100 µL of encapsulated plasma normalized to control plasma value on day 0. The three donor percentage recovery values were averaged and the error bars represent SD. Dashed line represents liquid plasma levels stored at −20 °C for 30 d.

Total IgE recovery and stability in silk compared with DBS. (A) Total IgE was assayed across three donor serum samples (A–C) via a commercial ELISA kit and compared with air-dried serum and silk solution, an air-dried whole blood and silk solution, and dried blood spots on Whatman 903 paper. Data represent of n = 4 replicates for each donor. (B–D) Liquid serum aliquots “neat serum,” dried blood spots “DBS”, and 50 µL of blood encapsulated in silk films “blood film” from the same three donors were subject to 22 or 45 °C storage over 84 d. Data are a pooled average ± SD of n = 4 replicates across three donors. Data were normalized to 22 °C recovery, the control condition.

Eliminating assay interferences and uncovering silk stabilization mechanisms. (A) Examples of recovery and stability of biomarkers not requiring any changes to silk solution formulation. (B) Recovery of osteopontin from films dissolved in water with and without lithium bromide (Left) and stability of the films, control plasma, and DPS groups over time (Right). Recovery of cancer antigen 15–3 (CA15-3) from films generated from 4 wt/vol% silk solution and reconstituted in DiH₂O compared with silk films generated from 1 wt/vol% silk solution and reconstituted with lithium bromide (Left) and stability of the films, control plasma, and DPS groups over time (Right). For all graphs the donor recovery values were averaged and the error bars represent SD. Lines indicate significance between groups at the P < 0.05 level. Asterisks indicate groups that were significantly different from their respective day 0 readings at the P < 0.05 level. (C) Recovery and stability of biomarkers stored in different matrices for two weeks at −80 °C (liquid plasma only), 22 and 45 °C. Groups are color coded to indicate agreement with day 0 liquid plasma: green indicates no significant differences, yellow indicates levels significantly lower (but within 20% of day 0 levels), and red indicates both significant differences and levels >20% lower than day 0. In most cases stability issues led to decreased analyte levels; however, where applicable, the white arrows indicate levels significantly higher than day 0.

Recovery of biomarkers is not dependent on patient health status. The x axis represents plasma levels as measured after storage at −80 °C, whereas the y axis represents plasma levels as measured after encapsulation in air-dried silk films. Blue data points represent readings from a healthy patient although red data points represent readings from a patient diagnosed with pancreatic cancer. Blue and red lines represent best fit lines (equations Inset) from linear regression. See Table S5 for raw data.

Silk powder for use as a stabilizing agent in the field. (A) Schematic showing the regeneration of silk powder with serum to incorporate stabilizing agent. The formulation is cast and the resulting air-dried matrix placed in an Eppendorf tube. (B, Left) Plasma NGAL can be recovered after addition of 16 mg of silk powder to 50 μL of plasma diluted 8× in water (“+silk”), after air-drying the powder/plasma solution (“film”), and also after lyophilizing said the powder/plasma solution (“foam”). Data are average ± SD of n = 4 replicate samples from a single donor. (B, Right) Luminex data demonstrating recovery of seven biomarkers from a silk powder-generated film. Black dots indicate average ± SD of n = 4 replicate samples from a single donor. Gray line indicates the best-fit line (equation Inset) from linear regression.