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

Adsorption of chitin derivatives onto liposomes: Optimization of adsorption conditions

A 2′ factorial design is used to optimize the adsorption conditions of the hydrophilic anionic polyelectrolytes, Carboxymethylchitin (CMC) and Carboxymethyl/Glycolchitin (CO) onto liposomes at physiological ionic strength (I) and pH using phosphate buffered saline (PBS, I= 154mM, pH = 7.4). Positive ([+]) or high surface affinity liposomes (DSPC:CHOL:DMTAP, 5:4:1), and Neutral ([N]) or low surface affinity liposomes (DSPC:CHOL, 1:1) were used as adsorption surfaces. Results of the calculations of the main effects indicate that polymer molecular weight (mwt), Surface Affinity (S), Number of Adsorption Shots (Sh), Temperature (T), and the combinations mwt ×S, mwt ×Sh are the most important process parameters. Results of a study conducted at T = 37 °C show that no loss occurs from the positive surface at the highest particle concentratiqn, Np = 4.043 × 1011. Finally, the extent of polymer-induced particle aggregation PTIS decreased when the diameter of the uncoated liposomes is doubled from 0.22 to 0.45m. These results are as expected, given the stiffness and the dimensions of the macromolecules

Purification and Quantitative Determination of Carboxymethylchitin Incorporation Into Submicron Bilayer-Lipid Membrane Artificial Cells (Liposomes) Encapsulating Hemoglobin

The separation of carboxymethylchitin-coated hemoglobin-loaded liposomes (CMC-LEHb)s from the non-adsorbed CMC has been achieved by gel chromatography. This purification takes place at the physiological pH of 7.4 favoring HbO2 preservation. A comparative study between experimental techniques for the quantitative determination of the adsorption of carboxymethylchitin (CMC) onto liposomes encapsulating hemoglobin (LEHb)s has been conducted. Results suggest that FT-IR spectroscopy gives a more accurate quantitative adsorption index while the chitinase-based enzymatic assay should be used as a qualitative detection tool. Quantitative bilayer and surface characterization show that the RBC membrane composition has been closely simulated by that of CMC-LEHbs in terms of total lipids and carbohydrates at 87.8% (phospholipids and cholesterol) and 12.2% CMC respectively

Kinetic Aspects of Polyelectrolyte Adsorption: Adsorption of Chitin Derivatives onto Liposomes as a Model System

Carboxymethylchitin (CMC) and Carboxymethyl/Glycolchitin (CO) have been adsorbed onto liposomes at physiological ionic strength (I) and pH using phosphate buffer saline (PBS, I= 154 mM, pH =7.4). Adsorption isotherms at different polymer weight average molecular weights (Mw), for Positive ([+]) or high surface affinity liposomes (DSPC:CHOL:DMTAP, 5:4:1), and Neutral ([N]) or low surface affinity liposomes (DSPC:CHOL, 1:1), have been obtained at T=25°C. For all CMCs, the adsorbed amount increases with polymer concentration ([P]o) and no true plateau is observed. The CMC Mw = 4.19 × 105 adsorbed on a positive surface fits Langmuir kinetics with maximal coverage γGMmax =325.4μg/mg (polymer/lipid), and inverse of equilibrium constant K* = 4.743 × 10−4 mg/ml. For all isotherms the predicted amount of polymer needed to coat the entire surface based on the Radius of Gyration (Rg?) is inferior to the amount adsorbed. This fact in conjunction with the dependence of γmax on the number of adsorption shots, suggests that the adsorption is not thermodynamically but kinetically controlled

Comparison of polymerically stabilized PEG-grafted liposomes and physically adsorbed carboxymethylchitin and carboxymethyl/glycolchitin liposomes for biological applications

The stabilities of two types of polymerically stabilized liposomes consisting of PEG-grafted (DSPC:CHOL:DSPE-PEG1900, 5:4:1) and physically adsorbed carboxymethylchitin (CMC) and carboxymethyl/glycolchitin (CO) are compared. The polyelectrolyte is adsorbed on positive (DSPC:CHOL:DMTAP, 5:4:1) and neutral (DSPC:CHOL, 1:1) liposomes at different molecular weights (Mw). In PBS buffer (=154 mm, pH=7.4) the theoretical stability ratios (W) calculated using the classical DLVO Theory, indicate that the CMC-coated vesicles and the negative liposomes (DSPC:CHOL:DMPG, 5:4:1) are highly stable (W≫1) compared to the PEG-grafted (W=0.9511) and CO-coated (W=0.9550) liposomes. Meanwhile, experimentally determined values of W, prove that the PEG-grafted is the most stable suspension (W=5.5). Computation of the theoretical values of W for liposome-red blood cell and liposome-macrophage indicates that the electrosterically stabilized suspensions and the negative liposomes are stable. Light scattering results show that the flocculation of liposomes in blood and plasma depends on polymer molecular weight, type of polyelectrolyte and surface charge of the uncoated liposome. Neutral liposomes coated with CMC of Mw=1.01×105 and negative liposomes provide a more effective barrier to plasma macromolecular protein adsorption than the grafted PEG groups and are easy to resuspend in blood

The Importance of Standardization of Carboxymethylchitin Concentration by the Dye-Binding Capacity of Alcian Blue Before Adsorption on Liposomes

A new method based on the measurement of the relative dye-binding capacity of Alcian Blue to carboxymethylchitin (CMC) at various molecular weights (MW) has been developed to facilitate the standardization of the initial polyelectrolyte concentration. In the absence of standardization, non-reproducible adsorption patterns are encountered during the adsorption of the MW CMC on neutral and positively charged liposomes. This method is sensitive down to a concentration of 5 μg/ml of polymer in water. Static Light Scattering (SLS) measurements are used to obtain the weight average molecular weight (Mw) and the size of the polyelectrolyte (Rg) and overlap concentrations (c*). The Mws are then used to determine the constants K and a of the Mark-Houwink equation which are 1.65 × 10−2dl/g and 0.4701, respectively, evaluated at K=0.154 M, pH= 7.4 and T= 25°C. The critical electrolyte concentration decreases with molecular weight for Mws ranging from 5.0×104-1.2×106. The dye-binding capacity changes with the molecular weight distribution of the polyelectrolyte demonstrating the sensitivity of this technique to polydispersity

Purification and Characterization of Liposomes Encapsulating Hemoglobin as Potential Blood Substitutes

In view of the desirability to increase the survival time of the liposome-based artificial red blood cells in vivo, the variables influencing optimum hemoglobin capture and preservation for the bovine hemoglobin-loaded liposomes (LEHb) are investigated. In order to predict the in vivo response, the necessary experiments for the in vitro system characterization have been carried out. The liposomes are prepared by the Reverse Phase Evaporation technique and then purified using a Sepharose 4B column. The purified LEHbs display a unimodal size distribution in the submicron range with a volume average diameter of 0.115 μm and a particle count of 1.25*1015 per ml of suspension. Analysis of the lipid/Hb content of the liposomes reveals that the variations in the ratio of Hb encapsulated to lipid entrapped (Hb/L)f as a function of the initial Hb concentration ([Hb]o) is insignificant compared to the net augmentation of (Hb/L)f as a function of the increasing initial lipid to Hb loading ([L]o). Meanwhile high [Hb]os are necessary for the preservation of oxyhemoglobin

Acoustic interrogation and optical visualization of ultrasound contrast agents within microcapsules

The effectiveness of localized drug delivery as a treatment for breast cancer requires sufficiently high therapeutic dose, as well as an ability to image the drug for proper spatial targeting. To balance treatment potential and imaging capabilities, we have begun to design a novel drug reservoir using microcapsules that are large in size (\u3e; 30 μm) but functionalized with microbubbles or ultrasound contrast agents (UCAs). We term these carriers as `Acoustically Sensitive Microcapsules\u27 (ASMs). In previous work, we have demonstrated preparation of ASM carriers and their structural changes under therapeutic ultrasound by imaging static changes. In this paper, we describe a combined optical-acoustic setup coupled with a microfluidic device to trap these carriers for imaging and sonication. Using the setup, continuous wave ultrasound (180 kPa, 2.25 MHz, 3 s) produced an average displacement of 3.5 μm in UCAs near the ASM boundary, and exhibited displacement as high as 90 μm near the center of the microcapsule. Longer exposure time and higher acoustic pressure increased UCA displacement within an ASM. These two parameters can be carefully optimized in the future to cause these UCAs to travel to the membrane boundary to help in the drug elution process