Nanotechnology Enhanced Immunoassay for Preeclampsia
MS Thesis Defense – Syeda Fatima Zaidi
N-isopropylacrylamide (NIPAm)-based, bait-loaded hydrogel particles are successfully used to capture, sequester and protect from enzymatic degradation low abundance, low molecular weight labile proteins in body fluids.
In current protocols, proteins captured by the nanoparticles are eluted with chemical buffers and analyzed with immunoassays or mass spectrometry techniques. Here, a novel, in situ ELISA is presented in which captured proteins are probed with a labeling antibody directly inside the nanoparticles without the need of chemical elution.
Dually crosslinked (degradable N,N’-(1,2-Dihydroxyethylene)- bisacrylamide [DHEA] and nondegradable N,N′-methylenebis(acrylamide) BIS] crosslinkers) NIPAm particles functionalized with trypan blue were used. DHEA can be degraded by an oxidizing agent (e.g. NaIO4) thus increasing the pore size of the hydrogel nanoparticles and allowing the antibody to access the antigen previously trapped in the nanoparticle.
Interleukin 6 (IL6) was chosen as a model molecule to test the nanoparticle based ELISA. IL-6 is a protein associated with preeclampsia, an autoimmune, hypertensive pregnancy-related medical problem that can lead to serious complications that can be life threatening to mother and child.
Dually crosslinked NIPAm particles efficiently sequestered IL6 from human urine. Oxidative degradation of DHEA crosslinker by NaIO4, caused a partial erosion of the DHEA crosslinker resulting in loose network of hydrogel polymer and increased access of antibody to the captured IL6 protein. Captured IL6 was preserved from oxidative degradation by virtue of the interaction between the protein and trypan blue chemical bait.
The IL6 ELISA, based on these dually cross linked NIPAm particles, showed a limit of detection of 600 picograms and two orders of magnitude dynamic range. Coefficient of variations were constantly below 5%. Sensitivity of the test can be improved by increasing the volume of urine processed. It is anticipated that this technology can be successfully used to detect true positive preeclamptic patients from a group of gestational hypertensive population.
Dr. Alessandra Luchini
Dr. Lance Liotta
Dr. Robin Couch
Notes: The thesis is on reserve in the Johnson Center Library, Fairfax Campus. All members of the George Mason University community are invited to attend.