Bioactive papers: printing, activity and stability
thesisposted on 15.01.2017, 23:02 by Khan, Mohammad Mohidus Samad Khan
The fundamental and applied engineering knowledge required issues to develop stable and functional bioactive papers and paper fluidic devices for health and environmental diagnostics were investigated. Bioactive papers are designed to be wetted by a biofluid or some solution of interest; biomolecule retention and behaviour on paper must be maximized. Two enzymes, alkaline phosphatase (ALP) and horseradish peroxidase (HRP) were directly physisorbed on paper or retained on paper with a polymer. Three model polymers were investigated: a high molecular weight cationic polyacrylamide (CPAM), an anionic polyacryilc acid (PAA) or a high molecular weight polyethylene oxide (PEO). The reactivity and the thermal stability of enzymatic bioactive papers were quantified using an advanced colorimetric technique. The enzymes adsorbed on paper retained their functionality and selectivity. Adsorption on paper increased the enzyme thermal stability by 2 to 3 orders of magnitude compared to the same enzyme in solution. The thermal degradation of the adsorbed enzyme follows two sequential first order reactions, indication of a reaction system. The model polymers used as retention aids were efficient at increasing the enzyme concentration on paper (by 50%) and to prevent enzyme desorption/leaching upon the rewetting of the paper. The polymers affect the thermal stability and the aging of ALP enzyme on paper; the rapid initial deactivation becomes predominant, while it was negligible for the enzyme simply physisorbed on paper. As a result, the thermal stability significantly decreases. A mathematical model predicting the enzymatic paper’s half-life time was developed. The reaction kinetics of ALP enzymatic paper reacting with its substrate was measured and shown to follow a first order reaction with respect to the enzyme concentration. ALP immobilized on paper has a reaction rate 2 to 3 orders of magnitude lower than the free ALP in buffer solution. No increase in reaction rate was achieved by immobilizing ALP on paper with polymer as retention aids; this suggests that enzyme orientation was not significantly affected through preferentially linking with its anionic or cationic groups. Paper bioassays to identify antigens and antibodies in a biofluid, such as blood, were investigated. Two series of experiments were performed. In the first, blood samples were mixed with different amounts of antibodies and a droplet of the mixture was deposited onto a paper strip. Agglutinated blood phase separated, with the red blood cells forming a distinct spot upon contact with paper while the serum wicked; in contrast, stable blood wicked uniformly. In the second series, blood droplets were deposited onto the paper strips pretreated with solutions of antibodies. The wicking of blood droplets on paper strips was characterized. Blood agglutination by interaction with a specific antibody caused a chromatographic separation. The concept of blood typing using a paper diagnostic was demonstrated with a prototype. The feasibility of thermal ink jet printing was demonstrated for the precise deposition of biomolecules on paper and poly(ε-caprolactone) (PCL) with a protein (albumin-FITC) and an enzyme (HRP) as model biomolecules. Complex patterns of HRP and albumin-FITC were ink jet printed on paper. Microfluidic channels were also printed on paper to demonstrate the concept of paper based bioassays as diagnostic devices. Discreet and continuous concentration gradients of proteins on PCL scaffolds were achieved by ink jet printing; these protein concentration gradients can serve as potential guidance cues for cell growth generation in tissue engineering. Ink jet printing of biomolecules onto paper involves liquid-liquid interaction; acceptance of biomolecules on the porous surface is rather affected by the solid-liquid interaction. Both phenomena reduce print resolution. The liquid-liquid and solid-liquid interaction were quantified and modelled for sessile droplets. Satellite drops from the ink jet nozzle can form non-coalescence droplets (NCD), due to liquid-liquid interaction, and roll from their target. The Weber number (We) of falling liquid drops was used to quantify the NCD generation. To quantify the solid-liquid interaction, the liquid wicking of a droplet impacting an open V-groove at different velocities (groove angle, β= 60º, 90º, 120º) was studied. A new mathematical model was developed to calculate liquid wicking distance in V-groove. The dynamics of wetting of sessile liquid droplets impinging, at different impact velocities, a groove surface (120º) was investigated experimentally. The dynamics of V-groove wicking and wetting is important to optimize paper for ink jet printing and microfluidic devices.
Awards: Winner of the Mollie Holman Doctoral Medal for Excellence, Faculty of Engineering, 2010.