P. Julicher et al., IN-SITU ANTIGEN IMMOBILIZATION FOR STABLE ORGANIC-PHASE IMMUNOELECTRODES, Analytical chemistry (Washington), 70(16), 1998, pp. 3362-3367
A new method based on enzymatic single-step in situ synthesis of hapte
n-carrier conjugates on electrodes is described yielding stable, repro
ducible, and reusable organic-phase immunoelectrodes (OPIEs), The elec
trodes developed were tailored for analyte detection in organic solven
ts and allow for the analysis of soil extracts without further sample
processing and cleanup. Catalyzed by transglutaminase from a variant o
f Streptoverticillium mobaraense, the reaction proceeds in aqueous sol
ution with and without addition of organic media in only 1.5 hours. In
this study, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was
chosen as model compound and chemically amino functionalized prior to
its enzymatic immobilization, The high reproducibility of the immobili
zation procedure allowed for batch calibration of the immunoelectrodes
. Moreover, pure methanol or treatment with diluted sulfuric acid used
for regeneration studies did not disturb the hapten layer. The OPIE c
onsists of screen-printed carbon electrodes, monoclonal anti-2,4-D ant
ibodies, and the immunochemical recognition reaction and was optimized
with regard to a high stability in organic media. For electrochemical
detection, horseradish peroxidase was used as enzyme label together w
ith H2O2 as substrate and hexacyanoferrate(II)/(III) as mediator. The
OPIE showed high stability upon storage over 93 days. Response times o
f 17 s (t(95)) were found to be advantageous compared to those of othe
r biosensors, Including the immunochemical reactions, the complete ass
ay takes 30 min. A calibration curve for 2,4-D in 30% methanol/buffer
obtained with 70 electrodes within 4 weeks revealed a detection limit
of 9 mg/L, a sensitivity of 1.3 nA L mg(-1) cm(-2), and a repeatabilit
y of 6.8%. Although we calculated a lowered repeatability for reused e
lectrodes of 13.4% and a slightly decreased sensitivity of 0.9 nA L mg
(-1) cm(-2), multiple-used OPIEs could also be applied for calibration
.