Rapid Determination Of Endotoxin Based On Molecularly Imprinted Polymer

Endotoxin exists widely in air, soil, water and other environmental media. Endotoxin contaminated water could enter the body and small doses of endotoxin (1-5ng/kg body weight) can increase body temperature, lead to endotoxin shock and disseminated intravascular coagulation. As a serious human health threat, sensitive and accurate methods to detect trace endotoxin in environmental water samples are in great need. Artificial antibodies (also known as molecularly imprinted polymer, MIP) could specificly recognize the target molecules and are often used as substitute for natural antibodies. MIP used with enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR) to establish new methods combining specific sample pre-treatment and rapid detction in one step. These new methods may have great applications in real-time monitoring of endotoxins in environmental water samples.Part I Detection of Trace Endotoxin using Artificial Antibody-Enzyme-Linked Immunosorbent AssayObjective To establish artificial antibody-enzyme-linked immunosorbent assay (MIP-ELISA) which combined specific sample pre-treatment and ELISA detction of trace endotoxin simultaneously.Methods MIP was prepared in microtiter plate using dopamine as functional monomer and endotoxin as template. The MIP was used as coated antibody for specific extraction of endotoxin. Then polyclonal rabbit anti-endotoxin antibody was added as the first antibody to form antigen-antibody conjugate with endotoxin bound to MIP. Enzyme-labeled second antibody was added to lable the antigen-antibody conjugate. Finally enhanced chemiluminescence (ECL) was used for determination of endotoxin. The sensitivity, linear range, stability and specificity of MIP-ELISA were evaluated. MIP-ELISA was further used to detect endotoxin in tap water, river water and hospital sewage.Result When100μL samples were loaded for MIP-ELISA, good linearity was obtained for endotoxin in concentrations ranged from10ng/mL to100μg/mL with LOD at10ng/mL Recoveries of MIP-ELISA for10-100ng/mL endotoxin spiked river water was83.6-101.7%, with inter-day and intra-day RSDs less than5%(n=6). The endotoxin concentrations of gram-negative bacteria-E. coli, Photobacterium phosphoreum and Pseudomonas aeruginosa (105cfu/mL) were99.15±2.79ng/mL,112.63±3.68ng/mL and113.74±3.58ng/mL respectively, while gram-positive bacteria Staphylococcus aureus which did not excrete endotoxin could not be detected by MIP-ELISA. The endotoxin levels in tap water, river water and hospital sewage were18.2±2.6ng/mL,52.6±2.9ng/mL,99.3±1.8ng/mL respectively determined by MIP-ELISA. Results of MIP-ELISA were not significantly different to standard method (Limulus amoebocyte lysate assays, LAL)(p>0.05).Conclusion MIP-ELISA can detect trace endotoxin in environmental water samples with high accurate and reliability. Part II Rapid Detection of Endotoxin using Artificial Antibody-SPRObjective To detect endotoxin rapidly using MIP combined with SPR (MIP-SPR).Methods MIP was prepared on the gold film of SPR sensor chip using dopamine as functional monomer and endotoxin as template. Then MIP-SPR chip was used on the SPR instrument for rapid detection of endotoxin. The sensitivity, linear range, stability and specificity of MIP-SPR were evaluated. Finally MIP-SPR was used to detect endotoxin levels in tap water, river water and hospital sewage.Result Good linearity was obtained for endotoxin in concentrations ranged from0.01-100ng/mL with LOD at0.01ng/mL by MIP-SPR. Recoveries of MIP-SPR for10-40ng/mL endotoxin spiked tap water was86.9-91.6%, with inter-day and intra-day RSDs less than5%(n=6). The endotoxin concentrations of gram-negative bacteria-E. coli, Photobacterium phosphoreum and Pseudomonas aeruginosa (103cfu/mL) were0.86±0.18ng/mL,0.73±0.13ng/mL,1.22±0.08ng/mL respectively, while gram-positive bacteria Staphylococcus aureus which did not excrete endotoxin could not be detected by MIP-SPR. The endotoxin levels in tap water, river water and hospital sewage were14.8±1.9ng/mL,50.1±2.2ng/mL,96.6±2.8ng/mL respectively determined by MIP-SPR. There was no significant difference between the results determined by MIP-SPR and standard method (LAL)(p>0.05).Conclusion MIP-SPR realized accurate, reliable and rapid detection of trace endotoxin in environmental water samples.