10 research outputs found
Electrochemical Detection of Peanut Allergen Ara h 1 Using a Sensitive DNA Biosensor Based on Stem–Loop Probe
A novel electrochemical DNA sensor was developed by using
a stem–loop
probe for peanut allergen Ara h 1 detection. The probe was modified
with a thiol at its 5′ end and a biotin at its 3′ end.
The biotin-tagged “molecular beacon”-like probe was
attached to the surface of a gold electrode to form a stem–loop
structure by self-assembly through facile gold–thiol affinity.
6-Mercaptohexanol (MCH) was used to cover the remnant bare region.
The stem–-loop probe was “closed” when the target
was absent, and then the hybridization of the target induced the conformational
change to “open”, along with the biotin at its 3′
end moved away from the electrode surface. The probe conformational
change process was verified by circular dichroism (CD); meanwhile,
electron-transfer efficiency changes between probe and electrode were
proved by electrochemical impedance spectroscopy (EIS). The detection
limit of this method was 0.35 fM with the linear response ranging
from 10<sup>–15</sup> to 10<sup>–10</sup> M. Moreover,
a complementary target could be discriminated from one-base mismatch
and noncomplementarity. The proposed strategy has been successfully
applied to detect Ara h 1 in the peanut DNA extracts of peanut milk
beverage, and the concentration of it was 3.2 × 10<sup>–13</sup> mol/L
Allergenicity of alternative proteins: research hotspots, new findings, evaluation strategies, regulatory status, and future trends: a bibliometric analysis
As the world population rises, the demand for protein increases, leading to a widening gap in protein supply. There is an unprecedented interest in the development of alternative proteins, but their allergenicity has raised consumer concerns. This review aims to highlight and correlate the current research status of allergenicity studies on alternative proteins based on previously published studies. Current research keywords, hotspots and trends in alternative protein sensitization were analyzed using a mixed-method approach that combined bibliometric analysis and literature review. According to the bibliometric analysis, current research is primarily focused on food science, agriculture, and immunology. There are significant variations in the type and amount of allergens found in alternative proteins. A significant amount of research has been focused on studying plant-based proteins and the cross-reactivity of insect proteins. The allergenicity of alternative proteins has not been studied extensively or in depth. The allergenicity of other alternative proteins and the underlying mechanisms warrant further study. In addition, the lack of a standardized allergy assessment strategy calls for additional efforts by international organizations and collaborations among different countries. This review provides new research and regulatory perspectives for the safe utilization of alternative proteins in human food systems.</p
Development and Application of 3‑Chloro-1,2-propandiol Electrochemical Sensor Based on a Polyaminothiophenol Modified Molecularly Imprinted Film
In this work, a novel electrochemical
sensor for 3-chloro-1,2-propandiol
(3-MCPD) detection based on a gold nanoparticle-modified glassy carbon
electrode (AuNP/GCE) coated with a molecular imprinted polymer (MIP)
film was constructed. <i>p</i>-Aminothiophenol (<i>p</i>-ATP) and 3-MCPD were self-assembled on a AuNP/GCE surface,
and then a MIP film was formed by electropolymerization. The 3-MCPD
template combined with <i>p</i>-ATP during self-assembly
and electropolymerization, and the cavities matching 3-MCPD remained
after the removal of the template. The MIP sensor was characterized
by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and
scanning electron microscopy (SEM). Many factors that affected the
performance of the MIP membrane were discussed and optimized. Under
optimal conditions, the DPV current was linear with the log of the
3-MCPD concentration in the range from 1.0 × 10<sup>–17</sup> to 1.0 × 10<sup>–13</sup> mol L<sup>–1</sup> (<i>R</i><sup>2</sup> = 0.9939), and the detection limit was 3.8
× 10<sup>–18</sup> mol L<sup>–1</sup> (S/<i>N</i> = 3). The average recovery rate of 3-MCPD from spiked
soy sauce samples ranged from 95.0% to 106.4% (RSD < 3.49%). Practically,
the sensor showed high sensitivity, good selectivity, excellent reproducibility,
and stability during the quantitative determination of 3-MCPD
Mast-Cell-Based Fluorescence Biosensor for Rapid Detection of Major Fish Allergen Parvalbumin
In
this study, we developed a rat basophilic leukemia cell (RBL-2H3)
fluorescence sensor to detect and identify the major fish allergen
parvalbumin (PV). We constructed and transfected a CD63-enhanced green
fluorescent protein (EGFP) plasmid into RBL cells through a highly
efficient, lipid-mediated, DNA-transfection procedure. Stable transfectant
RBL cells were then obtained for a cell fluorescence assay with confocal
laser scanning microscopy. Results show that the cell surface expression
of CD63 reflects degranulation, indicating that a fluorescence assay
with these cells could efficiently measure the activation of antigen-stimulated
transfectant cells and detect antigens with a nanogram level. Therefore,
this cell-based fluorescence biosensor technique for detecting fish
PV exhibits promise for quantifying fish PV after anti-PV immunoglobulin
E (IgE) stimulation. Results show that fluorescence intensities increased
with purified PV concentrations from 1 to 100 ng/mL, with a detection
limit of 0.35 ng/mL [relative standard deviation (RSD) of 4.5%], confirmed
by β-hexosaminidase assays. These rat basophilic leukemia (RBL)
mast cells transfected with the CD63–EGFP gene and responded
to PV only when they were sensitized with the specific IgE antibody.
This demonstrates the utility of this highly sensitive biosensor for
food allergen detection and prediction
Effects of FB1 and HFB1 on Autonomous Exploratory and Spatial Memory and Learning Abilities in Mice
Fumonisin
B1 (FB1) is a representative form of fumonisin and is
widely present in food and feed. Hydrolyzed fumonisin B1 (HFB1) emerges
as a breakdown product of FB1, which is accompanied by FB1 alterations.
While previous studies have primarily focused on the liver or kidney
toxicity of FB1, with limited studies existing on its neurotoxicity
and even fewer on the toxicity of HFB1, this study focuses on the
neurotoxicity of FB1 and HFB1 exposure in mice investigated by the
open field test, Morris water maze test, histopathological analysis,
and nontargeted metabolomics. Further, the levels of oxidative stress-related
indices, neurotransmitters, and sphingolipids in the brain were measured
to analyze their correlation with behavioral outcomes. The results
showed that both FB1 (5 mg/kg) and HFB1 (2.8 mg/kg) reduced autonomous
exploratory behavior in mice, impaired spatial learning and memory,
and caused mild abnormalities in the brain structure. Quantitative
analysis further indicated that exposure to FB1 and HFB1 disrupted
neurotransmitter homeostasis, exacerbated oxidative stress, and significantly
increased the sphinganine/sphingosine (Sa/So) ratio. Moreover, HFB1
exhibited neurotoxic effects similar to those of FB1, emphasizing
the need to pay attention to the neurotoxicity effect of HFB1. These
findings underscore the importance of understanding the risks and
potential neurological damage associated with FB1 and HFB1 exposure,
highlighting the necessity for further research in this crucial field
Ultrasensitive Detection Strategy of Norovirus Based on a Dual Enhancement Strategy: CRISPR-Responsive Self-Assembled SNA and Isothermal Amplification
Spherical
nucleic acids (SNAs) have been used to construct various
nanobiosensors with gold nanoparticles (AuNPs) as nuclei. The SNAs
play a critical role in biosensing due to their various physical and
chemical properties, programmability, and specificity recognition
ability. In this study, CRISPR-responsive self-assembled spherical
nucleic acid (CRISPR-rsSNA) detection probes were constructed by conjugating
fluorescein-labeled probes to the surface of AuNPs to improve the
sensing performance. Also, the mechanism of ssDNA and the role of
different fluorescent groups in the self-assembly process of CRISPR-rsSNA
were explored. Then, CRISPR-rsSNA and reverse transcription-recombinase
polymerase amplification (RT-RPA) were combined to develop an ultrasensitive
fluorescence-detection strategy for norovirus. In the presence of
the virus, the target RNA sequence of the virus was transformed and
amplified by RT-RPA. The resulting dsDNA activated the trans-cleavage
activity of CRISPR cas12a, resulting in disintegrating the outer nucleic
acid structure of the CRISPR-rsSNA at a diffusible rate, which released
reporter molecules. Norovirus was quantitated by fluorescence detection.
This strategy facilitated the detection of the norovirus at the attomolar
level. An RT-RPA kit for norovirus detected would be developed based
on this method. The proposed method would be used for the detection
of different viruses just by changing the target RNA and crRNA of
the CRISPR cas12a system which provided a foundation for high-throughput
detection of various substances
Ultrasensitive “FRET-SEF” Probe for Sensing and Imaging MicroRNAs in Living Cells Based on Gold Nanoconjugates
MicroRNAs
(miRNAs), a kind of single-stranded small RNA molecule,
play significant roles in the physiological and pathological processes
of human beings. Currently, miRNAs have been demonstrated as important
biomarkers critically related to many diseases and life nature, including
several cancers and cell senescence. It is valuable to establish sensitive
assays for monitoring the levels of intracellular up-regulated/down-regulated
miRNA expression, which would contribute to the early prediction of
the tumor risk and cardiovascular disease. Here, an oriented gold
nanocross (AuNC)-decorated gold nanorod (AuNR) probe with “OFF-enhanced
ON” fluorescence switching was developed based on fluorescence
resonance energy transfer and surface enhanced fluorescence (FRET-SEF)
principle. The nanoprobe was used to specifically detect miRNA in
vitro, which gave two linear responses represented by the equation <i>F</i> = 1830.32 log <i>C</i> + 6349.27, <i>R</i><sup>2</sup> = 0.9901, and <i>F</i> = 244.41 log <i>C</i> + 1916.10, <i>R</i><sup>2</sup> = 0.9984, respectively,
along with a detection limit of 0.5 aM and 0.03 fM, respectively.
Furthermore, our nanoprobe was used to dynamically monitor the expression
of intracellular up-regulated miRNA-34a from the HepG2 and H9C2 cells
stimulated by AFB1 and TGF-β1, and the experimental results
showed that the new probe not only could be used to quantitively evaluate
miRNA oncogene in vitro, but also enabled tracking and imaging of
miRNAs in living cells
Cell Based-Green Fluorescent Biosensor Using Cytotoxic Pathway for Bacterial Lipopolysaccharide Recognition
Lipopolysaccharide (LPS), a characteristic
component of the outer
membrane of Gram-negative bacteria, can be used as an effective biomarker
to detect bacterial contamination. Here, we reported a 293/hTLR4A-MD2-CD14
cell-based fluorescent biosensor to detect and identify LPS, which
is carried out in a 96-well microplate which is nondestructive, user-friendly,
and highly efficient. The promoter sequence of the critical signaling
pathway gene <i>ZC3H12A</i> (encoding MCPIP1 protein) and
enhanced green fluorescence protein (EGFP) were combined to construct
a recombinant plasmid, which was transferred into 293/hTLR4A-MD2-CD14
cells through lipid-mediated, DNA-transfection way. LPS was able to
bind to TLR4 and coreceptors-induced signaling pathway could result
in green fluorescent protein expression. Results show that stable
transfected 293/hTLR4A-MD2-CD14 cells with LPS treatment could be
directly and continually observed under a high content screening imaging
system. The novel cell-based biosensor detects LPS at low concentration,
along with the detection limit of 0.075 ÎĽg/mL. The cell-based
biosensor was evaluated by differentiating Gram-negative and Gram-positive
bacteria and detecting LPS in fruit juices as well. This proposed
fluorescent biosensor has potential in sensing LPS optically in foodstuff
and biological products, as well as bacteria identification, contributing
to the control of foodborne diseases and ensurance of public food
safety with its high throughput detection way
Broad-Specificity Immunoassay for Simultaneous Detection of Ochratoxins A, B, and C in Millet and Maize
Ochratoxins
A, B, and C (OTA, OTB, and OTC) can be found in cereals
and feeds; the simultaneous detection of these ochratoxins holds a
great need in food safety. In this study, four antibodies raised from
two ochrotoxin haptens and two coating antigens were compared, and
then a sensitive and broad-specificity enzyme-linked immunosorbent
assay (ELISA) was established for the simultaneous determination of
three ochratoxins, where the detection limits were 0.005, 0.001, and
0.001 ng/mL for OTA, OTB, and OTC, respectively, and recoveries of
three ochratoxins were between 84.3% and 111.7%. This developed method
had been successfully applied to detect ochratoxins in both millet
and maize. Molecular modeling revealed that the broad-specificity
was related with the chlorine electronegativity on OTA and OTC and
the potential of the acetyl ester group on OTC. The proposed ELISA
can be used for simultaneous detection of three ochratoxins
Four Specific Hapten Conformations Dominating Antibody Specificity: Quantitative Structure–Activity Relationship Analysis for Quinolone Immunoassay
Antibody-based
immunoassay methods have been important tools for
monitoring drug residues in animal foods. However, because of limited
knowledge about the quantitative structure–activity relationships
between a hapten and its resultant antibody specificity, antibody
production with the desired specificity is still a huge challenge.
In this study, the three-dimensional quantitative structure–activity
relationship (3D QSAR) was analyzed in accordance with the cross-reactivity
of quinolone drugs reacting with the antibody raised by pipemidic
acid as the immunizing hapten and compared with the reported cross-reactivity
data and their hapten structures. It was found that the specificity
of a quinolone antibody was strongly related to the conformation of
the hapten used and that hapten conformations shaped like the letters
“I”, “P”, and “Φ”
were essential for the desired high specificity with low cross-reactivity,
but that the hapten conformation shaped like the letter “Y”
led to an antibody with broad specificity and high cross-reactivity.
Almost all of the antibodies against quinolones could result from
these four hapten conformations. It was first found that the concrete
conformations dominated the specificity of the antibody to quinolone,
which will be of significance for the accurate hapten design, predictable
antibody specificity, and better understanding the recognition mechanism
between haptens and the antibodies for immunoassays