|
Lipopolysaccharide (LPS) an endotoxin which existed in the
outer membrane of cell wall of Gram negative bacteria G(-) could
induce some diseases such as sepsis, endotoxin shock, and so on.
Limulus amebocyte lysate (LAL) test is a conventional method, it
was performed by gel-clotting between LPS and ambeocyte.
However,this method defeats in high cost and semi-
quantification,Quartz crystal microbalance (QCM), was developed
to be a less cost and highly sensitive transducer. Adsorbing the
analyte on the quartz surface, it would cause a proportionally
decreasingon resonance frequency of oscillation. In this study,
we used an antibiotics, polymyxin B (PmB), as a sensing
elementwhich was coupled on the surface of QCM that
couldintroduce a new function for sensing LPS and show the shift
ofoscillation frequency. PmB immobilized on the surface of QCM
was via coupling method, immersing the gold-deposited crystal
into the ethanol solution containing 4,4''-dithiodi-(n-
butyricacid) (DTBA) for 2 hr at room temperature. After washing,
the treated crystal was activated with coupling agent,
1-ethyl-3-(3-dimethyl-amionpropyl) carbodiimine(EDC) and N-
hydrosuccinimide (NHS). Subsequently, the crystalwas immered
into aqueous solution containing 2 mg/ml of PmB. The resulting
sensor showed a linear range of LPS concentration between 2.5
ng/ml and 22.5 ng/ml and allow limit detection as 25 pg/ml.
However, we found such a PmB modified sensor would be interfered
by the adsorptionof bovine serum albumin (BSA). To extend a
method to the clinical application, an available deprotein
treatment was necessary. We discussed two deprotein agents,
ZnSO4 and trichloroacetic acid (TCA). As a result, the LPS
sample mixed with BSA was treatedZnSO4 that mainly precipitated
BSA showed a higher effect thanTCA method.Keyword: Quartz
crystal microbalance (QCM), Biosensor, Antibiotics,
Lipopolysaccharide, Deprotein
|