Reduction of Non-Specific Protein Adsorption Using Poly(ethylene) Glycol (PEG) Modified Polyacrylate Hydrogels In Immunoassays for Staphylococcal Enterotoxin B Detection.

Three PEG molecules (PEG-methacrylate, -diacrylate and -dimethacrylate) were incorporated into galactose-based polyacrylate hydrogels and their relative abilities to reduce non-specific protein adsorption in immunoassays were determined. Highly crosslinked hydrogels containing amine-terminated functionalities were formed and used to covalently attach antibodies specific for staphylococcal enterotoxin B (SEB). Patterned arrays of immobilized antibodies in the PEG-modified hydrogels were created with a PDMS template containing micro-channels for use in sandwich immunoassays to detect SEB. Different concentrations of the toxin were applied to the hydrogel arrays, followed with a Cy3-labeled tracer antibody specific for the two toxins. Fluorescence laser scanning confocal microscopy of the tracer molecules provided both qualitative and quantitative measurements on the detection sensitivity and the reduction in non-specific binding as a result of PEG incorporation. Results showed the PEG-modified hydrogel significantly reduced non-specific protein binding with a detection limit for SEB of 1 ng/mL. Fluorescence signals showed a 10-fold decrease in the non-specific binding and a 6-fold increase in specific binding of SEB.

Optical solid-state detection of organophosphates using organophosphorus hydrolase.

We have developed a sensor surface for optical detection of organophosphates based on reversible inhibition of organophosphorus hydrolase (OPH) by copper complexed meso-tri(4-sulfonato phenyl) mono(4-carboxy phenyl) porphyrin (CuC1TPP). OPH immobilized onto glass microscope slides retains catalytic activity for more than 232 days. CuC1TPP is a reversible, competitive inhibitor of OPH, binding at the active site of the immobilized enzyme. The absorbance spectrum of the porphyrin-enzyme complex is measured via planar waveguide evanescent wave absorbance spectroscopy using a blue LED as a light source and an Ocean Optics USB2000 as the spectrophotometer. The characteristics of the absorbance spectrum of CuC1TPP are specific and different when the porphyrin is bound to the enzyme or is bound non-specifically to the surface of the slide. Addition of a substrate of OPH such as one of the organophosphates paraoxon, coumaphos, diazinon, or malathion displaces the porphyrin from the enzyme resulting in reduced absorbance intensity at 412 nm. Absorbance changes at 412 nm show log-linear dependence on substrate concentration. Paraoxon concentrations between 7 parts per trillion (ppt) and 14 parts per million (ppm) were investigated and a 3:1 S/N detection limit of 7 ppt was determined. Concentrations of 700 ppt to 40 ppm were investigated for diazinon, malathion, and coumaphos with detection limits of 800 ppt, 1 part per billion, and 250 ppt, respectively. This optical technique does not require the addition of reagents or solutions other than the sample and absorbance spectra can be collected in less than 6 s.

Extended lifetime of reagentless detector for multiple inhibitors of acetylcholinesterase.

Competitive inhibitors of acetylcholinesterase (AChE) are detected using an evanescent wave technique to monitor changes in the absorbance spectrum of an AChE-monosulfonate tetraphenyl porphyrin (TPPS(1)) complex immobilized on the surface of a glass slide. In this technique, porphyrin is displaced from the AChE active site by the inhibitor. The loss in absorbance intensity of the characteristic absorbance peak for the AChE-TPPS(1) complex at 446 nm is linearly dependent on the log of the inhibitor concentration. This technique yields detection limits at 3:1 S/N of 37 ppt for eserine, 50 ppt for galanthamine, 100 ppt for scopolamine, 250 ppt for tetracaine, 45 ppt for diazinon, and 83 ppb for Triton X-100. When stored under vacuum, the enzymatic lifetime of the immobilized AChE surface is greater than 73 days while the responsive lifetime of the immobilized AChE-TPPS(1) surface is currently 49 days.

Novel optical solid-state glucose sensor using immobilized glucose oxidase.

Meso-tetra(4-carboxyphenyl)porphine (CTPP(4)) binds reversibly to immobilized glucose oxidase (GOD), resulting in an absorbance peak for the CTPP(4)-GOD complex at 427nm. The absorbance intensity of the 427nm peak is reduced upon exposure to glucose, which causes the dissociation of CTPP(4) from GOD. The change in absorbance at 427nm shows linear dependence on glucose concentration from 20 to 200mg/dL (1.1-11.1mM).

Interaction of monosulfonate tetraphenyl porphyrin, a competitive inhibitor, with acetylcholinesterase.

Monosulfonate tetraphenyl porphyrin (TPPS(1)) forms a 1:1 complex with electric eel acetylcholinesterase (AChE) inducing a loss in TPPS(1) absorbance at 402 nm and the appearance of a new absorbance centered at 442 nm. In the presence of AChE, the fluorescence of TPPS(1) at 652 nm is slightly narrowed, with the maximal 652 nm fluorescence shifted from 407 to 412 nm excitation wavelength. The fluorescence peak of TPPS(1) at 712 nm shifts to 716 nm in the presence of AChE. TPPS(1) is a competitive inhibitor of AChE. The addition of acetylcholine iodide (AChI) or the competitive inhibitor tetracaine to the preformed AChE-TPPS(1) complex results in a loss of the 442 nm absorbance band as the porphyrin is displaced from AChE. The absorbance peak does not decrease in the presence of procaine, a non-competitive inhibitor.

Reagent-less detection of a competitive inhibitor of immobilized acetylcholinesterase.

The interaction of monosulfonate tetraphenyl porphine (TPPS(1)) with immobilized acetylcholinesterase (AChE) yields a characteristic absorbance peak at 446 nm. Addition of acetylcholine iodide or the competitive inhibitor tetracaine to the immobilized TPPS(1)-AChE complex results in a decrease in absorbance intensity at 446 nm due to displacement of the porphyrin from the active site. The loss in intensity at 446 nm is linearly dependent on tetracaine concentration at levels below 100 ppb. Tetracaine concentrations as low as 300 ppt have been detected.