Biology Reference
In-Depth Information
screen-printed electrodes as electrochemical transducers; among
the developed assay formats, a direct and an indirect competitive
assay was reported. The experimental work was also supported by
the use of the surface plasmon resonance (SPR) device Biacore X
TM
,
throughwhichdifferentinformationonthetestedassayformatswas
obtained. For the direct competitive assay the biotinylated 15-mer
aptamer was immobilized on streptavidin-coated magnetic beads,
whereas in the case of the indirect competitive assay, thrombin and
biotinylated thrombin was immobilized on the tosyl-activated and
streptavidinmagneticbeads,respectively.Usingadirectcompetitive
format, a detection limit of 430 nM of thrombin was achieved and a
goodspecificityoftheassaywasfoundusinghumanserumalbumin
as an interfering molecule.
Impedance spectroscopy, in this case faradic impedance spec-
troscopy (FIS), was used as transduction technique for a competi-
tive aptamer-based assay for the detection of neomycin B [19]. The
interestingfeatureofthisworkisthepossibilityofeasilydetectinga
small molecule like neomycin B with an electrochemical aptamer-
based assay. Actually, the detection of small molecules, such as
aminoglycoside antibiotics, is particularly challenging since only
time-consuming label-based immunoassays or HPLC methods have
been developed. On the contrary, in the present work an aptamer
specificforneomycinB[20]wasusedinacompetitive/displacement
assay format. In particular, neomycin B was immobilized onto gold
electrodes andthis modifiedsurface was saturated withthe specific
aptamer by a
nity binding. By exposing the modified system to dif-
ferent concentrations of neomycin B, a displacement of the bound
aptamer was observed resulting in a drop of the electron-transfer
resistanceconsistentwiththereductionofthenegativechargeofthe
electrode surface. The competitive assay resulted very fast (equilib-
rium in 5 minutes) with a linear range covering two orders of mag-
nitude (0.75-500
μ
M) and a submicromolar limitofdetection.
Very high specificity toward neomycin B was observed with
respect to other very similar antibiotics. Application of the method
to the analysis of real samples was also demonstrated by testing
neomycin spiked whole milk with a recovery of 102% and 109%,
respectively, for twodifferent neomycin concentrations.
