Abstract
In recent years, food allergy has risen as a worldwide public concern as the rapid
development of economy and the improvement of living standards. Due to the rich nutrients
and savory flavor, fish is recognized as one of the popular food among consumers. However,
as one of the Big-eight allergenic food sources, fish has been considered to be related to 13%
of food allergy, 90% of which is caused by parvalbumin (PV). Moreover, it is difficult to
eliminate the allergenicity through common food processing and cooking process due to its
resistance to high temperature and enzymatic hydrolysis. For now, the major methods for
quantitative detection of PV are ELISA and PCR, which show limits in the complicated
operation procedure, high cost, and long detection time. Therefore, this study aims to develop
a dual-mode biosensor for rapid detection of PV. The main contents are as follows:
(1) The PV in cod was extracted and purified by extraction ammonium sulfate precipitation
and DEAE ion exchange chromatography. SDS-PAGE, ELISA and Western blot were used to
analyze the properties of the purified protein，and mass spectrometry was used to identify the
PV. The results showed that the purity of the extracted PV was over 99% with a molecular
weight being 11.5 kDa. The PV was found to possess strong allergenicity.
(2) The specific aptamer for PV was screened from random ssDNA library by GO-SELEX.
Six candidate sequences were obtained by high-throughput sequencing after 18 rounds of
selection. The obtained sequences were subjected to familial analysis, homology analysis and
secondary structure analysis by DNAMAN. The candidate sequences were also subject to
affinity and specificity analysis by BLI and GO-based fluorescence assay. Finally, a aptamer
with good affinity and specificity was selected to be used for further experiment.
(3) The thiolated aptamer DNA1 and its complementary short chain DNA2 were used to
modify the surface of AuNPs to prepare the AuNPs-DNA1 and AuNPs-DNA2 probes. Then,
the third short chain DNA3 that was fluorescently-labeled and complementary to the aptamer,
was used to complementary with the AuNPs-DNA1 and AuNPs-DNA2 probes to construct the
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