DNA-Encoded Bidirectional Regulation of the Peroxidase Activity of Pt Nanozymes for Bioanalysis.

Rational regulation of nanozyme activity can promote biochemical sensing by expanding sensing strategies and improving sensing performance, but the design of effective regulatory strategies remains a challenge. Herein, a rapid DNA-encoded strategy was developed for the efficient regulation of Pt nanozyme activity. Interestingly, we found that the catalytic activity of Pt nanozymes was sequence-dependent, and its peroxidase activity was significantly enhanced only in the presence of T-rich sequences. Thus, different DNA sequences realized bidirectional regulation of Pt nanozyme peroxidase activity. Furthermore, the DNA-encoded strategy can effectively enhance the stability of Pt nanozymes at high temperatures, freezing, and long-term storage. Meanwhile, a series of studies demonstrated that the presence of DNA influenced the reduction degree of H2PtCl6 precursors, which in turn affected the peroxidase activity of Pt nanozymes. As a proof of application, the sensor array based on the Pt nanozyme system showed superior performance in the accurate discrimination of antioxidants. This study obtained the regulation rules of DNA on Pt nanozymes, which provided theoretical guidance for the development of new sensing platforms and new ideas for the regulation of other nanozyme activities.

"One suction and one extrusion" mode-based wash-free platform for determination of breast cancer cell-derived exosomes.

A simple and wash-free POCT platform based on microcapillary was developed, using breast cancer cell-derived exosomes as a model. This method adopted the "one suction and one extrusion" mode. The hybridized complex of epithelial cell adhesion molecule (EpCAM) aptamer and complementary DNA-horseradish peroxidase conjugate (CDNA-HRP) was pre-modified on the microcapillary's inner surface. "One suction" meant inhaling the sample into the functionalized microcapillary. The exosomes could specifically bind with the EpCAM aptamer on the microcapillary's inner wall, and then the CDNA-HRP complex was released. "One extrusion" referred to squeezing the shedding CDNA-HRP into the 3,3',5,5'-tetramethylbenzidine (TMB)/H2O2 solution, and then the enzyme-catalyzed reaction would occur to make the solution yellow using sulfuric acid as the terminator. Therefore, exosome detection could be realized. The limit of detection was 2.69 × 104 particles mL-1 and the signal value had excellent linearity in the concentration range from 2.75 × 104 to 2.75 × 108 particles⋅mL-1 exosomes. In addition, the wash-free POCT platform also displayed a favorable reproducibility (RSD = 2.9%) in exosome detection. This method could effectively differentiate breast cancer patients from healthy donors. This work provided an easy-to-operate method for detecting cancer-derived exosomes without complex cleaning steps, which is expected to be applied to breast cancer screening.

One-step multiplex analysis of breast cancer exosomes using an electrochemical strategy assisted by gold nanoparticles.

Exosomes, as a non-invasive biomarker, perform an important role in breast cancer screening and prognosis monitoring. However, establishing a simple, sensitive, and reliable exosome analysis technique remains challenging. Herein, a one-step multiplex analysis electrochemical aptasensor based on a multi-probe recognition strategy was constructed to analyze breast cancer exosomes. HER2-positive breast cancer cell (SK-BR-3) exosomes were selected as the model targets and three aptamers including CD63, HER2 and EpCAM aptamers were used as the capture units. Methylene blue (MB) functionalized HER2 aptamer and ferrocene (Fc) functionalized EpCAM aptamer, which were modified on gold nanoparticles (Au NPs), i.e. MB-HER2-Au NPs and Fc-EpCAM-Au NPs, were used as signal units. When the mixture of target exosomes, MB-HER2-Au NPs and Fc-EpCAM-Au NPs were added on the CD63 aptamer modified gold electrode, two Au NPs modified by MB and Fc could be specifically captured on the electrode by the recognition of three aptamers with target exosomes. Then one-step multiplex analysis of exosomes was achieved by detecting two independent electrochemical signals. This strategy can not only distinguish breast cancer exosomes from other exosomes (including normal exosomes and other tumor exosomes) but also HER2-positive breast cancer exosomes and HER2-negative breast cancer exosomes. Besides, it had high sensitivity and can detect SK-BR-3 exosomes with a concentration as low as 3.4 × 103 particles mL-1. Crucially, this method can be applicable to the examination of exosomes in complicated samples, which is anticipated to afford assistance for the screening and prognosis of breast cancer.

CIRC_0012535 CONTRIBUTES TO LIPOPOLYSACCHARIDE-INDUCED FETAL LUNG FIBROBLAST APOPTOSIS AND INFLAMMATION TO REGULATE INFANTILE PNEUMONIA DEVELOPMENT BY MODULATING THE MIR-338-3P/IL6R SIGNALING.

ABSTRACT: Background: Infantile pneumonia is a respiratory infection disease, seriously threatening the life of neonatal patients. Circular RNA (circRNA) dysregulation is reported to be involved in pneumonia pathogenesis. Circ_0012535 was previously displayed to be upregulated in blood samples of patients with community-acquired pneumonia. However, circ_0012535's role in this disorder remains unclear. We thus aim to unveil the functions of circ_0012535 in infantile pneumonia. Methods: Fetal lung fibroblasts (WI38) treated with LPS were used as pneumonia cell models. Expression analysis for circ_0012535, miR-338-3p and IL6R was performed using quantitative real-time polymerase chain reaction. Cell counting kit 88), 5-ethynyl-2'-deoxyuridine, and flow cytometry assays were implemented for cell function detection. The release of inflammatory factors, and superoxide dismutase activity and malonaldehyde content were ascertained using commercial kits. The putative binding between miR-338-3p and circ_0012535 or IL6R was validated by dual-luciferase analysis, RIP analysis, and pull-down analysis. Results: Circ_0012535 was highly expressed in LPS-treated WI38 cells. Knockdown of circ_0012535 recovered LPS-inhibited cell viability and proliferation and attenuated LPS-induced cell apoptosis, cell cycle arrest, inflammation, and oxidative stress. Circ_0012535 bound to miR-338-3p and negatively regulated miR-338-3p expression. Inhibition of miR-338-3p reversed the role of circ_0012535 knockdown, thereby recovering LPS-induced WI38 cell apoptosis and inflammation. MiR-338-3p bound to IL6R 3'UTR, and circ_0012535 shared miR-338-3p binding site with IL6R. IL6R overexpression reversed the role of miR-338-3p, thereby recovering LPS-induced WI38 cell apoptosis and inflammation. Conclusion: Circ_0012535 supported LPS-induced WI38 cell apoptosis and inflammation to promote the progression of infantile pneumonia, and circ_0012535 functioned partly by targeting the miR-338-3p/IL6R signaling.

DNA Tetrahedron-Based Valency Controlled Signal Probes for Tunable Protein Detection.

Combined detection of multiple markers related to the same disease could improve the accuracy of disease diagnosis. However, the abundance levels of multiple markers of the same disease varied widely in real samples, making it difficult for the traditional detection method to meet the requirements of a wide detection range. Herein, three kinds of cardiac biomarkers, cardiac troponin I (cTnI), myoglobin (Myo), and C-reaction protein (CRP), which were from the pM level to the µM level in real samples, were selected as model targets. Valency-controlled signal probes based on DNA tetrahedron nanostructures (DTNs) and platinum nanoparticles (PtNPs) were constructed for tunable cardiac biomarker detection. PtNPs with high horseradish peroxidase-like activity and stability served as signal molecules, and DTNs with unique spatial structure and sequence specificity were used for precisely controlling the number of connected PtNPs. By controlling the number of PtNPs connected to DTNs, monovalent, bivalent, and trivalent signal probes were obtained and were used for the detection of cardiac markers in different concentration ranges. The limit of detection of cTnI, Myo, and CRP was 3.0 pM, 0.4 nM, and 6.7 nM, respectively. Furthermore, it performed satisfactorily for the detection of cardiac markers in 10% human serum. It was anticipated that the design of valency-controlled signal probes based on DTNs and nanozymes could be extended to the construction of other multi-target detection platforms, thus providing a basis for the development of a new precision medical detection platform.

Polymer-assisted Au@PDA nanoparticles lyophilized powder with high stability and low adsorption and its application in colorimetric biosensing.

Gold nanoparticles (Au NPs) has been widely used to develop label-free colorimetric biosensors. Since the lyophilization process of Au NPs might cause various stresses and lead to irreversible aggregation, Au NPs were usually preserved in an aqueous suspension, which was inconvenienced for transportation and storage. In addition, the potential adsorption interaction between target and Au NPs was often ignored, which may lead to false-signal for Au NPs based colorimetric strategy. Herein, polydopamine-coated gold nanoparticles (Au@PDA NPs) freeze-dried powder was prepared with the assistance of polyvinylpyrrolidone (PVP) (i.e. Au@PDA-PVP NPs) or polyethylene glycol (PEG) (i.e. Au@PDA-PEG NPs). After freeze-dried powder of Au@PDA nanoparticles was redissolved, not only their spectral properties can still be maintained, but also the Au@PDA nanoparticles have nice monodispersity. Besides, the freeze-dried powder has long-term stability and could be stored for at least nine months. Since kanamycin, an aminoglycoside antibiotic, can be absorbed on the surface of Au NPs and induce easily the false signal, it was difficult to be detected using conventional Au NPs-based colorimetric method. Thus, kanamycin was chosen as the model target, a simple, sensitive and label-free colorimetric sensor was established. Given that the adsorption between kanamycin and Au@PDA-PVP NPs was effectively avoided, the possibility of false-positive signal was also reduced. The detection limit of kanamycin was 0.22 nM (S/N = 3), which was met the requirements for the detection of kanamycin residues in milk. This work not only provided an effective and facile way to prepare the nanomaterial lyophilized powder, but also expanded the application of the Au NPs based colorimetric method.

Microcapillary-based multicolor assay for quantitative and sensitive point-of-care testing of proteins.

A microcapillary-based multicolor assay was developed for proteins quantification in serum sample with the assistance of manual centrifugal platform. The proposed assay only required the operation of "one suction and one extrusion" to realize the target detection. Myoglobin (Myo), a biomarker in the early stage of acute myocardial infarction (AMI), was chosen as the model target. The microcapillary was first modified with polydopamine (PDA), then Myo aptamer was immobilized on the PDA modified microcapillary and hybridized with glucose oxidase (Gox) functionalized DNA probe (DNA-Gox). The step "one suction" referred to the inhalation of the sample into the functionalized microcapillary. Then the target Myo in the sample could bind to the Myo aptamer on the microcapillary so that DNA-Gox complexes were released from the microcapillary into solution. Through the step "one extrusion", the DNA-Gox complexes in the solution could catalyze glucose to generate hydrogen peroxide, and then the etching of gold nanorods (AuNRs) was initiated, causing a color change from brown to yellow. According to the color change based on the etching of AuNRs, as low as 0.1 nM Myo was detected with naked eyes. Combined with the manual centrifugal platform, even the Myo in the serum samples could be detected without power supply. It was expected to build a universal and adaptable sensing platform for different targets more quickly and efficiently.

The mechanisms of HSA@PDA/Fe nanocomposites with enhanced nanozyme activity and their application in intracellular H2O2 detection.

Nanozymes have drawn increasing attention with their broad applications but most nanozymes lack enzyme-like molecular structures, resulting in weak selectivity and low activity. Bioinspired molecular assembly provides an extremely promising strategy to mimic natural enzyme processes and develop function enhanced architectures. Herein, a new bioinspired molecular assembly strategy based on human serum albumin@polydopamine/Fe nanocomposites (HSA@PDA/Fe NCs) was proposed, in which Fe(iii)/Fe(ii) were anchored on HSA supported on PDA. HSA@PDA/Fe NCs with iron as the active center and HSA@PDA as the skeleton showed excellent peroxidase-like activity, which was nearly 1000 times higher than that of free Fe(iii). This may be attributed to the phenomenon that the cycle of quinones and the hydroxyl group on the nanocomposite surface greatly accelerate the conversion of Fe(iii)/Fe(ii) in acidic microenvironments. Systematic experimental studies illustrated that its activity was mainly affected by the metal active center, followed by the polymeric ligand, while the protein framework has little effect on its activity. Meanwhile, even after freeze-thaw and thermal cycle tests, it also showed excellent catalytic stability. Besides, a colorimetric assay based on HSA@PDA/Fe NCs was developed for detection of H2O2in vitro and in situ detection of H2O2 generated from live cells. This work will facilitate the developments on theoretical analysis, rational design and practical applications of nanozymes based on bioinspired molecular assemblies.

Photothermal and fluorescent dual-mode assay based on the formation of polydopamine nanoparticles for accurate determination of organophosphate pesticides.

A photothermal and fluorescent dual-mode assay for sensitive organophosphate pesticides (Ops) determination is reported based on alkaline phosphatase (ALP)-inhibition-induced formation of polydopamine (PDA) nanoparticles. In the presence of ALP, ascorbic acid 2-phosphate (AAP) can be catalyzed to produce ascorbic acid (AA). AA can reduce MnO2 nanosheets, further inhibiting the oxidation of dopamine (DA). Ops as an inhibitor for ALP activity prevents the formation of AA and the reduction of MnO2 nanosheets. Eventually, the formation of PDA nanoparticles is promoted. The inhibitory effect of Ops on ALP activity causes obvious changes of photothermal signals and fluorescence signal at 495 nm. The detection limit (LOD) of dimethoate is 0.1 µM. The method displays excellent sensing capability for the dimethoate assay in real water with good recoveries of 99.4-107.6%. Graphical abstract A photothermal and fluorescent dual-mode biosensor for sensitive Ops detection was reported based on alkaline phosphatase (ALP)-inhibition-induced formation of polydopamine (PDA) nanoparticles. The dual-mode method significantly improved the accuracy and reliability of the results.

DNA Hydrogelation-Enhanced Imaging Ellipsometry for Sensing Exosomal microRNAs with a Tunable Detection Range.

Conventional imaging ellipsometry-based biosensing faces the challenges of poor sensitivity and narrow dynamic range, especially for some small molecules such as microRNA. Given that detection of various exosomal miRNAs with tunable range could provide high-precision disease information and improve the accuracy of diagnosis, a sensitive imaging ellipsometry sensor was introduced to improve sensitivity with a tunable detection range by terminus-regulated DNA hydrogelation. Tetrahedron DNA probes with complementary sequence to the target miRNA were used as biorecognition elements to form DNA hydrogelation. This DNA hydrogelation was formed by template-independent and isothermal amplification on the Au film. Due to its high dielectric constant, DNA hydrogelation structure could be used for improving the sensitivity of imaging ellipsometry significantly. Importantly, by changing the cycle of the DNA hydrogelation amplification, this strategy showed a tunable detection range from fM to nM for miRNA with a limit of detection of 0.2 fM for let-7a, 10 fM for miR-375, and 40 pM for miR-21. Furthermore, it also performed satisfactorily for the miRNA sensing in 50% human serum and 50% human plasma. This DNA hydrogelation-enhanced imaging ellipsometry could broaden the applications of conventional imaging ellipsometry in biosensing and provide a sensitive method for sensing miRNAs at different abundances.