Smart hydrogel particles: biomarker harvesting: one-step affinity purification, size exclusion, and protection against degradation

Abstract

Disease-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin. We have introduced an affinity bait molecule into N-isopropylacrylamide to produce a particle that will perform three independent functions within minutes, in one step, in solution: (a) molecular size sieving, (b) affinity capture of all solution-phase target molecules, and (c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis.

Figure 1

Chemical composition of particles. Structure of (A) N-isopropylacrylamide (NIPAm) and its polymer, (B) methylenebisacrylamide and (C) NIPAm and acrylic acid and their polymers.

Figure 2

Particle characterization. (A) Light scattering measurement of NIPAm particle size as a function of temperature (diameter decreases as temperature increases). (B) Plots of correlation of the size of NIPAm/AAc particles with temperature (diameter decreases as temperature increases) and pH (diameter decreases as pH decreases). AFM images of (C) NIPAm particles and (D) NIPAm/AAc particles on mica.

Figure 3

Schematic drawing of molecular sieving of particles in solution. Low molecular weight proteins are harvested; high molecular weight proteins are excluded.

Figure 4

Flow cytometry analyses of FITC-incubated particles. (A) Uptake is dose dependent. (B) Uptake rapidly reaches saturation with FITC concentration of 20 μM.

Figure 5

NIPAm particles incubated with FITC and FITC-labeled proteins: Flow cytometry measurements of (A) BSA and insulin, (B) myoglobin and free FITC. (C) SDS-PAGE of particles incubated with insulin: Lane 1) insulin solution (Control), 2) NIPAm supernatant (Out, substance excluded from the particles), 3) wash 1, 4) wash 2, 5) NIPAm particles (In, substance captured by the particles). (D) SDS-PAGE of NIPAm particles incubated with BSA and myoglobin: 1) BSA and myoglobin (Control), 2) NIPAm supernatant (Out), 3) wash 2, 4) NIPAm particles (In). BSA is totally excluded.

Figure 6

Schematic depiction of affinity-based sequestering.

Figure 7

Protein sequestering by NIPAm/AAc particles (+ bait ) versus NIPAm particles (– bait), SDS-PAGE analysis of (A) Myoglobin (aqueous solution, pH 5.5) sequestration by particles + and – bait. Lane 1) myoglobin, 2) NIPAm supernatant (Out), 3) NIPAm particle (In), 4) NIPAm/AAc supernatant (Out), 5) NIPAm/AAc particle (In), 6) NIPAm/AAc particles 1:64, 7) NIPAm/AAc particles 1:32, 8) NIPAm/AAc particles 1:128, and 9) NIPAm/AAc particles 1:256. (B) BSA and myoglobin sequestration by particles+bait (NIPAm/AAc) at two pH values. Lane 1) BSA and myoglobin, pH 5.5, 2) NIPAm/AAc supernatant (Out) pH 5.5, 3) wash 3 pH 5.5, 4) NIPAm/AAc particles (In) pH 5.5, 5) wash 2 pH 5.5, 6) wash 1 pH 5.5, 7) BSA and myoglobin, pH 8, 8) NIPAm/AAc supernatant (Out) pH 8, and 9) NIPAm/AAc particles (In) pH 8.

Figure 8

(A) SDS-PAGE analysis of particles- and +bait incubated with BSA and lysozyme: Lane 1) BSA and lysozyme solution prior to particle introduction, 2) NIPAm (− bait) supernatant (Out), 3) wash 3, 4) NIPAm (− bait) particles (In), 5) wash 2, 6) wash 1, 7) NIPAm/AAc (+ bait) supernatant (Out), 8) wash 3, and 9) NIPAm/AAc (+ bait) particles (In). (B) SDS-PAGE analysis of NIPAm/AAc particles (+ bait) incubated with molecular weight (MW) markers: 1) MW markers, 2) NIPAm/AAc supernatant (Out), and 3) NIPAm/AAc particles (In).

Figure 9

SDS PAGE analysis of particles + bait incubated with PDGF B and BSA. Lane 1) BSA and PDGF B, 2) NIPAm/AAc supernatant (Out), 3) NIPAm/AAc particles (In).

Figure 10

Flow cytometry analysis of (A) NIPAm and (B) NIPAm/AAc particles incubated with FITC-labeled insulin aqueous solution and FITC-labeled insulin spiked in serum, respectively.

Figure 11

Flow cytometry time course studies of (A) NIPAm and (B) NIPAm/AAc particles incubated with FITC-labeled insulin.

Figure 12

Uptake time course study (A) Mean values of the percentage, relative to the initial amount, of lysozyme in solution incubated with two quantities of NIPAm/AAc particles as measured by RPPAs (three replicate analyses and standard deviation shown). (B) SDS-PAGE analysis of a lysozyme and BSA solution incubated with NIPAm/AAc particles. Lane 1) BSA and lysozyme solution. 2-11) alternating supernatant (Out) and particles (In) for each of 5, 10, 20, 30, and 60 minutes incubation times. Lysozyme uptake is rapid and complete, while BSA exclusion is total.

Figure 13

Schematic drawing illustrating the ability of particles to protect proteins from enzymatic degradation.

Figure 14

NIPAm/AAc particles (+ bait) protect bound proteins from degradation by enzymes that may be present. (A) NIPAm/AAc particles incubated with a solution containing lysozyme and trypsin for 1 hr: Lane 1) lysozyme, 2) lysozyme incubated with trypsin, 3) NIPAm/AAc supernatant (Out), 4) NIPAm/AAc particles (In), 5) BSA and lysozyme, 6) BSA and lysozyme + protease, 7) NIPAm/AAc particles supernatant (Out), and 8) NIPAm/AAc particles (In). (B) NIPAm/AAc particles incubated overnight with BSA, lysozyme, and trypsin: Lane 1) lysozyme, 2) trypsin, 3) lysozyme + trypsin, 4) NIPAm/AAc supernatant (Out), 5) NIPAm/AAC particles (In), 6) lysozyme and BSA, 7) BSA and lysozyme + protease, 8) NIPAm/AAc supernatant (Out), and 9) NIPAm/AAc particles (In).

Figure 15

SDS PAGE analysis of reduced and alkylated lysozyme exposed to tryptic digestion and incubated with particles − bait, + bait, and core shell. Lane 1) reduced and alkylated lysozyme, 2) lysozyme + trypsin 1:50, 3) lysozyme + trypsin 1:100, 4) lysozyme with NIPAm particles (In), 5) lysozyme + trypsin + NIPAm particles supernatant (Out), 6) lysozyme + trypsin + NIPAm particles (In), 7) lysozyme + NIPAm/AAc particles (In), 8) lysozyme + trypsin + NIPAm/AAc particles supernatant (Out), 9) lysozyme + trypsin + NIPAm/AAc particles (In), 10) lysozyme + core shell (In), 11) lysozyme + trypsin + core shell particles supernatant (Out), 12) lysozyme + trypsin + core shell particles (In).

Figure 16

SDS PAGE analysis of trypsin incubated with NIPAm, NIPAm/AAc and core shell particles. Trypsin incubated with Lane 1) − bait particles, supernatant (Out), 2) − bait particles (In), 3) + bait particles, supernatant (Out), 4) + bait particles (In), 5) core shell particles, supernatant (Out), 6) core shell particles (In).