Scale Optimization of Milkguard Biosensor for Detecting E. coli in Human Breast Milk

Milkguard is an alginate-based biosensor developed to detect E. coli in human breast milk via the metabolism of X-gal (5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside) by β-galactosidase. In order to deconvolute metabolic reproducibility from scaling laws, the commercial enzyme β-galactosidase was used to mimic the biological function of the bacterial lac operon. Downscaling was explored as an optimization of the biosensor design based on numerical solutions to Fickian-based diffusion models. The characterization of large capsules (d ≅ 3 mm) and atomized microcapsules (d ≅ 300 ± 60 μm) yielded size-specific Michaelis-Menten constants. Small capsules (Km = 3.6 x 10-4 M; Vmax ’’ = 1.2 x 10-3) produced a significantly faster response time versus large capsules when loaded at a substrate concentration of 5 mg/mL (p = 7.7x 10-3 at = 0.01) and 2.5 mg/mL (p = 1.5 x 10-4 at \u3c 0.001). Comparisons of effectiveness factors between small (η = 0.58) and large (η = 0.43) capsules indicates a lesser degree of diffusion limitations in small capsules. Large bootstrapping errors produced by nonlinear regression of Michaelis-Menten models for the capsules suggests that additional mechanisms to diffusion are involved in producing sensor response. A new sensor mechanism combining Fickian diffusion and experimental results is proposed and modeled numerically

Modifying the size of nanopores of alginate microcapsules

Alginate hydrogels provide desirable biocompatibility and material properties for various biomedical applications, but are limited by the polymer\u27s natural pore size. With the rise of nanotechnology, the desired crosslinked pore size range of 30 nm to 100 nm has not yet been achieved. This project aimed to develop a method to increase the pore size of alginate-based hydrogels in a reproducible manner without compromising their structural integrity. Experimental methods included altering alginate composition using carboxymethyl cellulose or gelatin and inducing conformational changes via Mach-1TM mechanical compression. Fluorescence microscopy was used to visualize the diffusion of FITC-dextran weight markers and fluorescent polystyrene nanoparticles into the microcapsules (d = 300 μm) and macrocapsules (d = 3 mm) for all experimental conditions. Based on pilot experiments, altered alginate composition did not significantly increase the pore size of alginate capsules for the modeled diffusivity range D = 1 x 10-14 m2/s to D = 1 x 10-15 m2/s. Mechanical compressions did not significantly affect the porosity or diffusivity of alginate macrocapsules (p \u3e 0.05) under all conditions for Young’s moduli ranging from E = 76 kPa to E = 200 kPa. Based on image analysis results, it could be hypothesized that molecular weight cutoff cutoff may be increased to 500 kDa following 10 successive compressions. Additional work to optimize fluorescent microscopy methods and pore size manipulation methods is required for expanded use of alginate capsules with emerging nanotechnologies

Sustainable, Alginate-Based Sensor for Detection of Escherichia coli in Human Breast Milk

There are no existing affordable diagnostics for sensitive, rapid, and on-site detection of pathogens in milk. To this end, an on-site colorimetric-based sustainable assay has been developed and optimized using an L16 (54) Taguchi design to obtain results in hours without PCR amplification. To determine the level of Escherichia coli (E. coli) contamination, after induction with 150 µL of breast milk, the B-Per bacterial protein extraction kit was added to a solution containing an alginate-based microcapsule assay. Within this 3 mm spherical novel sensor design, X-Gal (5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside) was entrapped at a concentration of 2 mg/mL. The outward diffusing X-Gal was cleaved by β-galactosidase from E. coli and dimerized in the solution to yield a blue color after incubation at 40 °C. Color intensity was correlated with the level of E. coli contamination using a categorical scale. After an 8 h incubation period, a continuous imaging scale based on intensity normalization was used to determine a binary lower limit of detection (LOD), which corresponded to 102 colony forming unit per mL (CFU/mL) and above. The cost of the overall assay was estimated to be $0.81 per sample, well under the$3 benchmark for state-of-the-art immune-based test kits for pathogen detection in biofluids. Considering the reported binary LOD cutoff of 102 CFU/mL and above, this proposed hydrogel-based assay is suited to meet global requirements for screening breast milk or milk for pathogenic organisms of 104 CFU/mL, with a percentage of false positives to be determined in future efforts

Sustainable, Alginate-Based Sensor for Detection of Escherichia coli in Human Breast Milk

There are no existing affordable diagnostics for sensitive, rapid, and on-site detection of pathogens in milk. To this end, an on-site colorimetric-based sustainable assay has been developed and optimized using an L16 (54) Taguchi design to obtain results in hours without PCR amplification. To determine the level of Escherichia coli (E. coli) contamination, after induction with 150 µL of breast milk, the B-Per bacterial protein extraction kit was added to a solution containing an alginate-based microcapsule assay. Within this 3 mm spherical novel sensor design, X-Gal (5-Bromo-4-Chloro-3-Indolyl β-d-Galactopyranoside) was entrapped at a concentration of 2 mg/mL. The outward diffusing X-Gal was cleaved by β-galactosidase from E. coli and dimerized in the solution to yield a blue color after incubation at 40 °C. Color intensity was correlated with the level of E. coli contamination using a categorical scale. After an 8 h incubation period, a continuous imaging scale based on intensity normalization was used to determine a binary lower limit of detection (LOD), which corresponded to 102 colony forming unit per mL (CFU/mL) and above. The cost of the overall assay was estimated to be $0.81 per sample, well under the$3 benchmark for state-of-the-art immune-based test kits for pathogen detection in biofluids. Considering the reported binary LOD cutoff of 102 CFU/mL and above, this proposed hydrogel-based assay is suited to meet global requirements for screening breast milk or milk for pathogenic organisms of 104 CFU/mL, with a percentage of false positives to be determined in future efforts