Research Progress

Activities

1.1 Immunoaggregation assay demonstration.

To prove the concept of immunoaggregation, goat IgG was used as a model biomarker and FITC-labeled rAb was conjugated to MP as a capture probe. The qualitative aggregation phenomenon of rAb-MP was studied using the fluorescent microscope though a GFP filter. Fluorescent microscope images (Fig. 1) show the dispersion state of particles in the rAb-MP solution of 53.4 μg/mL and the rAb-MP solution mixed with goat IgG with a final concentration was 36 ng/mL. In the absence of goat IgG, most rAb-MPs uniformly distributed and mostly separated from each other. After mixed with goat IgG, a large amount of rAb-MP aggregates formed.

Fig 1. Fluorescence Microscope images: (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model biomarker.

1.2 Antifouling materials development.

Protein fouling on particle and sensor surfaces can dramatically reduce the detection sensitivity. Using antifouling materials can improve the detection sensitivity. In this work we developed a versatile and high performance zwitterionic polysaccharide platform and studied the structure-property-function relationships of zwitterionic polysaccharides, so that this platform can be readily adapted to the biomarker detection. As shown in Scheme 1, CB-Dex was synthesized via one pot reaction (see Scheme 1). The molecular weight and the degree of substitution were characterized by GPC and 1H NMR spectroscopy respectively. Zwitterionic CB side chains were introduced onto dextran backbone using the rational design approach. Three methacrylate (MA) modified dextran derivatives, with different degree of CB substituent, from 0 % (Dex-MA), 35 % (CB-L-Dex-MA), to 158 % (CB-H-Dex-MA) were employed in this study. All samples were kept at a similar MA ratio around 25 % (one MA unit per four glucose units). We used NMR to confirm the structure of the polymer. The antifouling properties of the materials were evaluated in the form of hydrogel.

Scheme 1. Synthetic route of Zwitterionic CB-Dex-MA

Findings

Quantitative detection of biomarker is needed for many applications.We found that at the given particle concentration, the number or volume ratio of aggregates to total particles is proportional to the antigen concentration. The concentration of rAb-MP was kept constant at 53.4 μg/mL and mixed with different concentrations of goat IgG ranging from 0.1 ng/mL to 320 ng/mL. The number fraction of rAb-MP aggregates for each goat IgG concentration was calculated by subtracting the nonspecific aggregates volume fraction value (6.8%) and had a clear relationship with goat IgG concentration as shown in Fig. 2. At least 3 samples were prepared and counted for each goat IgG concentration. The max number fraction (fn = 65.7%) of rAb-MP aggregates were achieved at 40 ng/mL of goat IgG. Within the range from 0.1 ng/mL to 40 ng/mL, the volume fraction of aggregates went up with the increase of goat IgG concentration, while it went down with the increase of goat IgG concentration above the turning point (40 ng/mL). This is because goat IgG at high concentrations saturated rAb on MPs; therefore the number of unreacted rAb on MP was too low to aggregate. All the study showed that innovative microparticle-based immunoaggregation can be formed and biomarker concentration can be related to the volume fraction of aggregates.

Fig 2. The number fraction of rAb-MP aggregates to all particles as a function of goat IgG concentration in PBS containing 0.1% BSA. The standard deviation was calculated from three replicates.