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1.
Biosensors (Basel) ; 12(12)2022 Nov 22.
Article in English | MEDLINE | ID: mdl-36551031

ABSTRACT

The fabrication, characterization and application of a nanoporous Silicon Rugate Filter (pSiRF) loaded with an enzymatically degradable polymer is reported as a bare eye detection optical sensor for enzymes of pathogenic bacteria, which is devoid of any dyes. The nanopores of pSiRF were filled with poly(lactic acid) (PLA), which, upon enzymatic degradation, resulted in a change in the effective refractive index of the pSiRF film, leading to a readily discernible color change of the sensor. The shifts in the characteristic fringe patterns before and after the enzymatic reaction were analyzed quantitatively by Reflectometric Interference Spectroscopy (RIfS) to estimate the apparent kinetics and its dependence on enzyme concentration. A clear color change from green to blue was observed by the bare eye after PLA degradation by proteinase K. Moreover, the color change was further confirmed in measurements in bacterial suspensions of the pathogen Pseudomonas aeruginosa (PAO1) as well as in situ in the corresponding bacterial supernatants. This study highlights the potential of the approach in point of care bacteria detection.


Subject(s)
Nanopores , Polymers , Polymers/chemistry , Silicon/chemistry , Pseudomonas aeruginosa , Polyesters/chemistry
2.
ACS Appl Mater Interfaces ; 13(11): 12928-12940, 2021 Mar 24.
Article in English | MEDLINE | ID: mdl-33709691

ABSTRACT

The fabrication of covalently cross-linked high-surface-area biopolymeric nanogel fibers by nanopore extrusion is reported for the first time. The biopolymer pullulan was functionalized with tert-butyl acetoacetate via a transesterification reaction to synthesize the water-soluble ketone-rich precursor pullulan acetoacetate (PUAA). PUAA and carbonic dihydrazide (CDH) as cross-linker were extruded through anodic aluminum oxide (AAO) nanoporous membranes, which possessed an average pore diameter of 61 ± 2 nm. By changing the concentration of PUAA, the flow rate, and extrusion time, the step polymerization cross-linking reaction was controlled so that the polymer can be extruded gradually during cross-linking through the membrane, avoiding the formation of macroscopic bulk hydrogels and rupture of the AAO membrane. Fibers with diameters on the order of 250 nm were obtained. This approach was also expanded to functionalized PUAA derivatives together with the fluorogenic substrate 4-methylumbelliferyl-ß-d-glucuronide MUGlcU in (PUAA-MUGlcU), which exhibited a mean equilibrium swelling ratio of 5.7 and 9.0 in Milli-Q water and in phosphate-buffered saline, respectively. ß-Glucuronidase was sensitively detected via fluorescence of 4-methylumbelliferone, which was liberated in the enzymatic hydrolysis reaction of PUAA-MUGlcU. Compared to hydrogel slabs, the rate of the hydrolysis was >20% higher in the nanogel fibers, facilitating the rapid detection of ß-glucuronidase-producing Escherichia coli (E. coli Mach1-T1). Nanopore extruded nanogel fibers are therefore considered a viable approach to enhance the functionality of hydrogels in surface-dominated processes.


Subject(s)
Escherichia coli/enzymology , Fluorescent Dyes/chemistry , Glucans/chemistry , Glucuronidase/analysis , Nanogels/chemistry , Acetoacetates/chemistry , Enzyme Assays/methods
3.
Langmuir ; 36(8): 1907-1915, 2020 03 03.
Article in English | MEDLINE | ID: mdl-32009415

ABSTRACT

The modification of cylindrical anodic aluminum oxide (AAO) nanopores by alternating layer-by-layer (LBL) deposition of poly(sodium-4-styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) was studied in situ by reflectometric interference spectroscopy (RIfS). In particular, the kinetics of polyelectrolyte deposition inside the pores with a diameter of 37 ± 3 nm and a length of 3.7 ± 0.3 µm were unraveled, and potential differences in the LBL multilayer growth compared to flat silicon substrates as well as the effect of different ionic strengths and different types of ions were investigated. RIfS measures the effective optical thicknesses, which is-for a constant pore length-proportional to the effective refractive index of the AAO sample, from which, in turn, the deposited mass of the polymer or the corresponding layer thickness can be estimated. Compared to the multilayer growth by the LBL deposition on the flat aminosilane-primed silicon wafers, which was assessed by spectroscopic ellipsometry, the thickness increment per deposited bilayer, as well as the dependence of this increment on the ionic strength (0.01-0.15) and the counterion type (Na+ vs Ca2+) inside the aminosilane-primed nanopores, was for the first bilayers to within the experimental error identical. For thicker multilayers, the pore diameter became smaller, which led to reduced thickness increments and eventually virtually completely filled the pores. The observed kinetics is consistent with the mass-transport-limited adsorption of the polyelectrolyte to the charged surface according to a Langmuir isotherm with a negligible desorption rate. In addition to fundamental insights into the buildup of polyelectrolyte multilayers inside the AAO nanopores, our results highlight the sensitivity of RIfS and its use as an analytical tool for probing processes inside the nanopores and for the development of biosensors.

4.
ACS Appl Bio Mater ; 3(4): 2419-2427, 2020 Apr 20.
Article in English | MEDLINE | ID: mdl-35025291

ABSTRACT

The effect of systematically varied mechanical properties and nano- and microscale surface topography on the adhesion and proliferation of human pancreatic cancer cells on fibronectin-functionalized poly(vinyl alcohol) (PVA) hydrogels was studied to understand the impact of these properties of the cell microenvironment on cell attachment and spreading. The mechanical properties of PVA, as assessed by atomic force microscopy (AFM) nanoindentation, were varied by the number of freezing-thawing cycles in the physical cross-linking process used for the generation of the hydrogels. Nano- and micropatterned hydrogel surfaces exposing nanosized PVA pillars and cuboids were fabricated by replicating ordered cylindrical nanopores of anodic aluminum oxide (AAO) and poly(dimethylsiloxane) (PDMS) templates, respectively. Softer PVA hydrogels, functionalized covalently with fibronectin, showed enhanced cell adhesion and proliferation of PaTu 8988t cells in comparison to stiffer hydrogels. In addition, PaTu 8988t cells favored the nanopatterned surfaces over micropatterned and flat hydrogels.

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