14 research outputs found
Non-Redox Modulated Fluorescence Strategy for Sensitive and Selective Ascorbic Acid Detection with Highly Photoluminescent Nitrogen-Doped Carbon Nanoparticles via Solid-State Synthesis
Highly photoluminescent nitrogen-doped
carbon nanoparticles (N-CNPs)
were prepared by a simple and green route employing sodium alginate
as a carbon source and tryptophan as both a nitrogen source and a
functional monomer. The as-synthesized N-CNPs exhibited excellent
water solubility and biocompatibility with a fluorescence quantum
yield of 47.9%. The fluorescence of the N-CNPs was intensively suppressed
by the addition of ascorbic acid (AA). The mechanism of the fluorescence
suppression of the N-CNPs was investigated, and the synergistic action
of the inner filter effect (IFE) and the static quenching effect (SQE)
contributed to the intensive fluorescence suppression, which was different
from those reported for the traditional redox-based fluorescent probes.
Owing to the spatial effect and hydrogen bond between the AA and the
groups on the N-CNP surface, excellent sensitivity and selectivity
for AA detecting was obtained in a wide linear relationship from 0.2
Ī¼M to 150 Ī¼M. The detection limit was as low as 50 nM
(signal-to-noise ratio of 3). The proposed sensing systems also represented
excellent sensitivity and selectivity for AA analysis in human biological
fluids, providing a valuable platform for AA sensing in clinic diagnostic
and drug screening
Eu,Sm,Mn-Doped CaS Nanoparticles with 59.3% Upconversion-Luminescence Quantum Yield: Enabling Ultrasensitive and Facile Smartphone-Based Sulfite Detection
Eu,Sm,Mn-doped
CaS (ESM-CaS) nanoparticles demonstrate a remarkable
upconversion luminescence (UCL) efficiency with a quantum yield of
nearly 60%, enabling many new applications and devices. We describe
an ESM-CaS nanoparticle-based paper test strip for one-shot quantitative
measurement of sulfite concentration using a smartphone-based reader.
The integrated UCL-based sulfite detection system features high sensitivity
and facile operation without the need for separation and pretreatment.
Moreover, the design principles are general in nature and so can be
tailored for the detection and quantification of a variety of other
analytes
Graphene OxideāPeptide Nanocomplex as a Versatile Fluorescence Probe of Protein Kinase Activity Based on Phosphorylation Protection against Carboxypeptidase Digestion
The
research on complicated kinomics and kinase-target drug discovery
requires the development of simple, cost-effective, and multiplex
kinase assays. Herein, we propose a novel and versatile biosensing
platform for the detection of protein kinase activity based on graphene
oxide (GO)āpeptide nanocomplex and phosphorylation-induced
suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed
phosphorylation protects the fluorophore-labeled peptide probe against
CPY digestion and induces the formation of a GO/peptide nanocomplex
resulting in fluorescence quenching, while the nonphosphopeptide is
degraded by CPY to release free fluorophore as well as restore fluorescence.
This GO-based nanosensor has been successfully applied to sensitively
detect two model kinases, casein kinase (CKII) and cAMPādependent
protein kinase (PKA) with low detection limits of 0.0833 mU/Ī¼L
and 0.134 mU/Ī¼L, respectively. The feasibility of this GO-based
sensor was further demonstrated by the assessment of kinase inhibition
by staurosporine and H-89, in vitro kinase assay in cell lysates,
and simultaneous detection of CKII and PKA activity. Moreover, the
GO-based fluorescence anisotropy (FA) kinase assay has been also developed
using GO as a FA signal amplifier. The proposed sensor is homogeneous,
facile, universal, label-free, and applicable for multiplexed kinase
assay, presenting a promising
method for kinase-related biochemical fundamental research and inhibitor
screening
Aptameric Peptide for One-Step Detection of Protein Kinase
Protein kinases are significant regulators in the cell
signal pathway, and it is difficult to achieve quick kinase detection
because traditional kinase assays normally rely on a time-consuming
kinase phosphorylation process. Herein, we present a novel one-step
strategy to detect protein kinase by using a kinase-specific aptameric
peptide-functionalized quartz crystal microbalance (QCM) electrode,
in which the detection can be finished in less than 10 min. A peptide
kinase inhibitor (IP<sub>20</sub>) was used as the aptameric peptide
because of its selective and strong interaction with the target protein
kinase (cyclic adenosine monophosphate-dependent protein kinase A,
PKA), high stability, and ease of inexpensive synthesis, presenting
a new direct recognition element for kinase. The aptameric peptide
was immobilized on the Au-coated quartz electrode through dual-thiol
anchoring and the binding of His-tagged peptide with a nitrilotriacetic
acid/NiĀ(II) complex, fabricating a highly specific and stable detection
platform. The interaction of aptameric peptide with kinase was monitored
with the QCM in real time, and the concentration of protein kinase
was sensitively measured by the frequency response of the QCM with
the low detection limit for PKA at 0.061 mU Ī¼L<sup>ā1</sup> and a linear range from 0.64 to 22.33 mU Ī¼L<sup>ā1</sup>. This method is rapid and reagentless and does not require a phosphorylation
process. The versatility of our aptameric peptide-based strategy has
also been demonstrated by the application in kinase assay using electrochemical
impedance spectroscopy. Moreover, this method was successfully applied
to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation
of PKA in cell lysate
Versatile Electrochemiluminescent Biosensor for ProteināNucleic Acid Interaction Based on the Unique Quenching Effect of Deoxyguanosine-5ā²-phosphate on Electrochemiluminescence of CdTe/ZnS Quantum Dots
In this paper, the efficient quenching
effect of deoxyguanosine-5ā²-phosphate
(dGMP) on anodic electrochemiluminescence (ECL) of the CdTe/ZnS quantum
dots (QDs) is reported for the first time. This ECL quenching was
found to be specific for free dGMP and not observed for dGMP residues
in different DNA structures. The unique dGMP-based QDs ECL quenching
was then utilized to develop a versatile biosensing strategy to determine
various proteināDNA interactions with the assistance of exonuclease,
Exo I, to hydrolyze DNA and liberate dGMP. Taking single-stranded
DNA binding protein (SSB) and thrombin as examples, two novel detection
modes have been developed based on dGMPāQDs ECL strategy. The
first method used hairpin probes and SSB-promoted probe cleavage by
Exo I for facile signal-off detection of SSB, with a wide linear range
of 1ā200 nM and a low detection limit of 0.1 nM. The second
method exploited aptamerāthrombin binding to protect probes
against Exo I degradation for sensitive signal-on detection of thrombin,
giving a linear response over a range of 1ā150 nM and a detection
limit as low as 0.1 nM. Both methods were homogeneous and label-free
without QDs or DNA modification. Therefore, this dGMP-specific QDs
ECL quenching presents a promising detection mechanism suitable for
probing various proteinānucleic acid interactions
Enzyme-Activated GāQuadruplex Synthesis for in Situ Label-Free Detection and Bioimaging of Cell Apoptosis
Fluorogenic
probes targeting G-quadruplex structures have emerged
as the promising toolkit for functional research of G-quadruplex and
biosensor development. However, their biosensing applications are
still largely limited in in-tube detection. Herein, we proposed a
fluorescent bioimaging method based on enzyme-generated G-quadruplexes
for detecting apoptotic cells at the cell and tissue level, namely,
terminal deoxynucleotidyl transferase (TdT)-activated de novo G-quadruplex
synthesis (TAGS) assay. The detection target is genomic DNA fragmentation,
a biochemical hallmark of apoptosis. The TAGS assay can efficiently
ātagā DNA fragments via using their DNA double-strand
breaks (DSBs) to initiate the de novo synthesis of G-quadruplexes
by TdT with an unmodified G-rich dNTP pool, followed by a rapid fluorescent
readout upon the binding of thioflavin T (ThT), a fluorogenic dye
highly specific for G-quadruplex. The feasibility of the TAGS assay
was proved by in situ sensitive detection of individual apoptotic
cells in both cultured cells and tissue sections. The TAGS assay has
notable advantages, including being label-free and having quick detection,
high sensitivity and contrast, mix-and-read operation without tedious
washing, and low cost. This method not only shows the feasibility
of G-quadruplex in tissue bioanalysis but also provides a promising
tool for basic research of apoptosis and drug evaluation for antitumor
therapy
Time-Resolved Luminescence Biosensor for Continuous Activity Detection of Protein Acetylation-Related Enzymes Based on DNA-Sensitized Terbium(III) Probes
Protein
acetylation of histone is an essential post-translational
modification (PTM) mechanism in epigenetic gene regulation, and its
status is reversibly controlled by histone acetyltransferases (HATs)
and histone deacetylases (HDACs). Herein, we have developed a sensitive
and label-free time-resolved luminescence (TRL) biosensor for continuous
detection of enzymatic activity of HATs and HDACs, respectively, based
on acetylation-mediated peptide/DNA interaction and Tb<sup>3+</sup>/DNA luminescent probes. Using guanine (G)-rich DNA-sensitized Tb<sup>3+</sup> luminescence as the output signal, the polycationic substrate
peptides interact with DNA with high affinity and subsequently replace
Tb<sup>3+</sup>, eliminating the luminescent signal. HAT-catalyzed
acetylation remarkably reduces the positive charge of the peptides
and diminishes the peptide/DNA interaction, resulting in the signal
on detection via recovery of DNA-sensitized Tb<sup>3+</sup> luminescence.
With this TRL sensor, HAT (p300) can be sensitively detected with
a wide linear range from 0.2 to 100 nM and a low detection limit of
0.05 nM. The proposed sensor was further used to continuously monitor
the HAT activity in real time. Additionally, the TRL biosensor was
successfully applied to evaluating HAT inhibition by two specific
inhibitors, anacardic acid and C464, and satisfactory <i>Z</i>ā²-factors above 0.73 were obtained. Moreover, this sensor
is feasible to continuously monitor the HDAC (Sirt1)-catalyzed deacetylation
with a linear range from 0.5 to 500 nM and a detection limit of 0.5
nM. The proposed sensor is a convenient, sensitive, and mix-and-read
assay, presenting a promising platform for protein acetylation-targeted
epigenetic research and drug discovery
Resurfaced Fluorescent Protein as a Sensing Platform for Label-Free Detection of Copper(II) Ion and Acetylcholinesterase Activity
Protein
engineering by resurfacing is an efficient approach to
provide new molecular toolkits for biotechnology and bioanalytical
chemistry. H<sub>39</sub>GFP is a new variant of green fluorescent
protein (GFP) containing 39 histidine residues in the primary sequence
that was developed by protein resurfacing. Herein, taking H<sub>39</sub>GFP as the signal reporter, a label-free fluorometric sensor for
Cu<sup>2+</sup> sensing was developed based on the unique multivalent
metal ion-binding property of H<sub>39</sub>GFP and fluorescence quenching
effect of Cu<sup>2+</sup> by electron transfer. The high affinity
of H<sub>39</sub>GFP with Cu<sup>2+</sup> (<i>K</i><sub>d</sub>, 16.2 nM) leads to rapid detection of Cu<sup>2+</sup> in
5 min with a low detection limit (50 nM). Using acetylthiocholine
(ATCh) as the substrate, this H<sub>39</sub>GFP/Cu<sup>2+</sup> complex-based
sensor was further applied for the turn-on fluorescence detection
of acetylcholinesterase (AChE) activity. The assay was based on the
reaction between Cu<sup>2+</sup> and thiocholine, the hydrolysis product
of ATCh by AChE. The proposed sensor is highly sensitive (limit of
detection (LOD) = 0.015 mU mL<sup>ā1</sup>) and is feasible
for screening inhibitors of AChE. Furthermore, the practicability
of this method was demonstrated by the detection of pesticide residue
(carbaryl) in real food samples. Hence, the successful applications
of H<sub>39</sub>GFP in the detection of metal ion and enzyme activity
present the prospect of resurfaced proteins as versatile biosensing
platforms
Phospholipid-Tailored Titanium Carbide Nanosheets as a Novel Fluorescent Nanoprobe for Activity Assay and Imaging of Phospholipase D
As
one of the emerging inorganic graphene analogues, two-dimensional
titanium carbide (Ti<sub>3</sub>C<sub>2</sub>) nanosheets have attracted
extensive attention in recent years because of their remarkable structural
and electronic properties. Herein, a sensitive and selective nanoprobe
to fluorescently probe phospholipase D activity was developed on the
basis of an ultrathin Ti<sub>3</sub>C<sub>2</sub> nanosheets-mediated
fluorescence quenching effect. Ultrathin Ti<sub>3</sub>C<sub>2</sub> nanosheets with ā¼1.3 nm in thickness were synthesized from
bulk Ti<sub>3</sub>AlC<sub>2</sub> powder by a two-step exfoliation
procedure and further modified by a natural phospholipid that is doped
with rhodamine B-labeled phospholipid (RhB-PL-Ti<sub>3</sub>C<sub>2</sub>). The close proximity between RhB and Ti<sub>3</sub>C<sub>2</sub> leads to efficient fluorescence quenching (>95%) of RhB
by
energy transfer. Phospholipase D-catalyzed lipolysis of the phosphodiester
bond in RhB-PL results in RhB moving away from the surface of Ti<sub>3</sub>C<sub>2</sub> nanosheets and subsequent fluorescence recovery
of RhB, providing a fluorescent āswitch-onā assay for
the phospholipase D activity. The proposed nanoprobe was successfully
applied to quantitatively determine phospholipase D activity with
a low limit of detection (0.10 U L<sup>ā1</sup>) and to measure
its inhibition. Moreover, in situ monitoring and imaging the activity
of phospholipase D in living cells were achieved using this biocompatible
nanoprobe. These results reveal that Ti<sub>3</sub>C<sub>2</sub> nanosheets-based
probes exhibit great potential in fluorometric assay and clinical
diagnostic applications
Self-Assembled DNA Hydrogel Based on Enzymatically Polymerized DNA for Protein Encapsulation and Enzyme/DNAzyme Hybrid Cascade Reaction
DNA hydrogel is a promising biomaterial
for biological and medical applications due to its native biocompatibility
and biodegradability. Herein, we provide a novel, versatile, and cost-effective
approach for self-assembly of DNA hydrogel using the enzymatically
polymerized DNA building blocks. The X-shaped DNA motif was elongated
by terminal deoxynucleotidyl transferase (TdT) to form the building
blocks, and hybridization between dual building blocks via their complementary
TdT-polymerized DNA tails led to gel formation. TdT polymerization
dramatically reduced the required amount of original DNA motifs, and
the hybridization-mediated cross-linking of building blocks endows
the gel with high mechanical strength. The DNA hydrogel can be applied
for encapsulation and controllable release of protein cargos (for
instance, green fluorescent protein) due to its enzymatic responsive
properties. Moreover, this versatile strategy was extended to construct
a functional DNAzyme hydrogel by integrating the peroxidase-mimicking
DNAzyme into DNA motifs. Furthermore, a hybrid cascade enzymatic reaction
system was constructed by coencapsulating glucose oxidase and Ī²-galactosidase
into DNAzyme hydrogel. This efficient cascade reaction provides not
only a potential method for glucose/lactose detection by naked eye
but also a promising modular platform for constructing a multiple
enzyme or enzyme/DNAzyme hybrid system