Hypoxia is a common characteristic of solid tumors, which is caused by the imbalance of oxygen supply and consumption. As the expression level of nitroreductase (NTR) increases in hypoxic solid tumors, NTR is one of the common biomarkers of hypoxia and widely used to evaluate the degree of tumor hypoxia. In this study, we designed and synthesized a highly water-soluble chemiluminescent probe, CL-NTR, for the detection of NTR activity in hypoxic tumors. It was found that the probe could be used to detect NTR with high sensitivity, and the total light photons increased tremendously with 6000-fold after the probe was treated with NTR. The chemiluminescence total light photons emission was directly proportional to the concentration of nitroreductase in the range of 3−55 ng/mL, with a detection limit of 0.947 ng/mL. Finally, the probe was successfully used to evaluate NTR activity in living mice by chemiluminescent imaging. In general, this probe has a remarkable response to NTR, which provides a promising method for the determination of NTR activity in vivo.
A novel chemiluminescent probe for
detection of cysteine (Cys)
from other biothiols has been reported by utilizing the excellent
chemiluminescent Schaap’s adamantylidene-dioxetane scaffold.
After careful assessment, the probe CL-Cys could detect
Cys with high sensitivity and total light photons increased by 28-fold
after the probe was treated with Cys, with the detection limit of
7.5 × 10–8 M. Finally, CL-Cys was
further utilized for the chemiluminescent imaging of endogenous Cys
in a living mouse. In general, this probe has a remarkable ability
to detect Cys, which provides a valuable method for interrogation
of the Cys roles in more biological and pathological processes.
Multiplex
biomolecular analysis with inductively coupled plasma
mass spectrometry (ICP-MS) becomes increasingly important in clinical
diagnosis and single cell analysis. However, the sensitivity of ICP-MS-based
immunoassay is only comparable or lower than those of fluorescence
methods at the present stage. Therefore, designing elemental tags
with a large number of metal atoms is necessary to achieve high-sensitive
detection. In this work, we proposed a new strategy to build up elemental
tag loading with hundreds of rare earth ions by coupling alkyne-DNA
chains with rare earth element (REE)-DOTA complexes a click chemistry
reaction. There are about 2 orders of magnitude improvement in sensitivity
compared with single metal-ion tags. DNA chains with multialkynyl
groups were facilely prepared by PCR synthesis. Moreover, the DNA-based
elemental tags own excellent water-solubility and biocompatibility.
The tags would be potentially applied to mass cytometry and clinical
diagnosis.
The tool box of site-specific cleavage
for nucleic acid has been
an increasingly attractive subject. Especially, the recent emergence
of the orthogonally activatable DNA device is closely related to the
site-specific scission. However, most of these cleavage strategies
are based on exogenous assistance, such as laser irradiation. Endogenous
strategies are highly desirable for the orthogonally regulatable DNA
machine to explore the crucial intracellular biological process and
cell signal network. Here, we found that the accurate site-specific
cleavage reaction of phosphorothioate (PT) modified DNA by using myeloperoxidase
(MPO). A scissors-like mechanism by which MPO breaks PT modification
through chloride oxidation has been revealed. Furthermore, we have
successfully applied the scissors to activate PT-modified hairpin-DNA
machines to produce horseradish peroxidase (HRP)-mimicking DNAzyme
or initiate hybridization chain reaction (HCR) amplification. Since
MPO plays an important role in the pathway related to oxidative stress
in cells, through the HCR amplification activated by this tool box,
the oxidative stress in living cells has been robustly imaged. This
work proposes an accurate and endogenous site-specific cleavage tool
for the research of biostimuli and the construction of DNA molecular
devices.
Objectives
In this study, a new immunoassay for the simultaneous determination of pepsinogen I (PGI) and pepsinogen II (PGII) in serum based on element labeling strategy coupled with inductively coupled plasma mass spectrometry (ICP‐MS) detection was proposed.
Methods
The sandwich‐type immunoassay was used to simultaneously detect PGI and PGII in serum. PGI and PGII were captured by anti‐PGI and anti‐PGII antibody immobilized on the magnetic beads and then banded with Eu
3+
labeled anti‐PGI detection antibody and Sm
3+
labeled anti‐PGII detection antibody, followed by ICP‐MS detection.
Results
The linear correlation coefficient (
R
2
) of PGI and PGII standard curves was .9938 and .9911, with the dynamic range of 0‐200 ng/mL and 0‐60 ng/mL, respectively. The limit of detection for PGI and PGII was 1.8 ng/mL and 0.3 ng/mL, respectively. The average recovery was 101.41% ± 6.74% for PGI and 101.47% ± 4.20% for PGII. Good correlations were obtained between the proposed method and CLIA (
r
= .9588 for PGI,
r
= .9853 for PGII).
Conclusions
We established a mass spectrometry‐based immunoassay for the simultaneous detection of PGI and PGII in a single analysis. The element tagged immunoassay coupled with ICP‐MS detection has high sensitivity, accuracy, and specificity in clinical serum sample analysis.
AbstractIntroductionElement-tagged immunoassay coupled with inductively coupled plasma-mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis in clinical detection; however, a systematic evaluation with the standard guidelines of the assay is needed to ensure its performance meets the requirements of the clinical laboratory.MethodsCarcinoembryonic antigen (CEA) was chosen for analysis using the proposed method. A systematic evaluation of the proposed assay was carried out according to the Clinical and Laboratory Standards Institute (CLSI). The 469 clinical samples were analyzed using the new method and compared with the electrochemiluminescent immunoassay (ECLIA) method.ResultsThe measurement range of the assay was 1–900 ng/mL, with a detection limit of 0.83 ng/mL. The inter-assay and intra-assay imprecision were 4.67% and 5.38% with high concentration samples, and 9.27% and 17.64% with low concentration samples, respectively. The cross-reactivity (%) for different antigens was less than 0.05%, and the recovery was between 94% and 108%. Percentage deviation of all the dilutions was less than 12.5% during linearity estimation. The interference bias caused by different substances was less than 10%. The reference interval of the assay was 0–4.442 ng/mL. Comparison with the commercial ECLIA method for clinical sample detection, the proposed method showed a correlation of 0.9878 and no significant differences between the methods were observed (p = 0.6666).ConclusionsThe ICP-MS based immunoassay was successfully developed, and the analytical performance of the assay met the requirements of the CLSI, which fully proved the clinical transferability and application of the new method.
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