Cu-MOFs/GOx Bifunctional Probe-Based Synergistic Signal Amplification Strategy: Toward Highly Sensitive Closed Bipolar Electrochemiluminescence Immunoassay.

A closed bipolar electrochemiluminescence (BP-ECL) platform for sensitive prostate specific antigen (PSA) detection was proposed based on a novel synergistic signal amplification strategy. Specifically, glucose oxidase-loaded Cu-based metal-organic frameworks (Cu-MOFs/GOx) as bifunctional probes were bridged on the anodic interface with the target PSA as the intermediate unit. In virtue of the large loading capacity of Cu-MOFs, a large amount of a co-reactant, i.e., H2O2 in this L-012-based ECL system and gluconic acid were generated on the anodic pole in the presence of glucose. The generated gluconic acid could effectively degrade the Cu-MOFs to release Cu2+ which greatly accelerates the formation of highly active intermediates from co-reactant H2O2, boosting the ECL intensity. As for the cathodic pole, K3Fe(CN)6 with a lower reduction potential is used to reduce the driving voltage and speed up the reaction rate, further strengthening the ECL intensity. Thanks to the synergistic signal amplification effect at both two electrode poles of the BP-ECL system, highly sensitive detection of PSA was realized with a detection limit of 5.0 × 10-14 g/mL and a wide linear range of 1.0 × 10-13-1.0 × 10-7 g/mL. The strategy provides a novel way for signal amplification in the BP-ECL biosensing field.

Liposome-assisted enzyme catalysis: toward signal amplification for sensitive split-type electrochemiluminescence immunoassay.

Developing an efficient signal amplification strategy is very important to improve the sensitivity of bioanalysis. In this paper, a liposome-assisted enzyme catalysis signal amplification strategy was developed for electrochemiluminescence (ECL) immunoassay of prostate specific antigen (PSA) in a split-type mode. The sandwich immunoreaction occurred in a 96-well plate, and glucose oxidase (GOx) encapsulated and antibody-modified liposomes were used as labels. The ECL detection was carried out using a rGO-Au NP modified glassy carbon electrode (GCE). The large amount of generated H2O2, i.e. the coreactant of the luminol system, and the excellent catalytic behavior of rGO-Au NPs greatly boosted the ECL signal, resulting in the signal amplification. The developed ECL immunosensor for detecting PSA achieved a wider linear range from 1.0 × 10-13 to 1.0 × 10-8 g mL-1 and a detection limit of 1.7 × 10-14 g mL-1. The application of the proposed strategy was demonstrated by analyzing PSA in human serum samples with recoveries from 89.0% to 113.0%, and relative standard deviations (RSDs) were less than 6.6%. This work provides a new horizon to expand the application of liposomes for ECL bioanalysis.

A ternary CdS@Au-g-C3N4 heterojunction-based photoelectrochemical immunosensor for prostate specific antigen detection using graphene oxide-CuS as tags for signal amplification.

Photoactive materials with high photo-electron transfer efficiency and stable signal output hold a key role in constructing the photoelectrochemical (PEC) biosensing systems. In this study, the ternary CdS@Au-g-C3N4 heterojunction was first prepared and characterized, and its application in PEC bioanalysis was explored. The gold nanoparticles sandwiched between CdS and g-C3N4, acting as both plasmonic photosensitizer and electron relay, significantly boosted the light absorption and accelerated the charge transfer from g-C3N4 to CdS, both of which contributed to the enhancement of photoelectric conversion efficiency. Signal quenching with graphene oxide-CuS efficiently weakened the photocurrent from CdS@Au-g-C3N4. The combination of the excellent PEC properties of CdS@Au-g-C3N4 and the remarkable quenching effects of graphene oxide-CuS enabled construction of a sandwich-type PEC immunosensor for prostate specific antigen (PSA) detection. This immunosensor achieved sensitive PSA analysis by multiple signal amplification mechanisms, with a detection limit of 0.6 pg mL-1 and a wide linear range from 1.0 pg mL-1 to 10 ng mL-1. This work not only demonstrates the great potential of noble metal sandwiched ternary heterojunctions in the PEC field, but also lays a foundation for developing the general PEC immunoassays.

A novel nitrogen and sulfur co-doped carbon dots-H2O2 chemiluminescence system for carcinoembryonic antigen detection using functional HRP-Au@Ag for signal amplification.

A novel nitrogen and sulfur co-doped carbon dots (NS-CDs)-H2O2 chemiluminescence (CL) system was developed to detect carcinoembryonic antigen (CEA) by taking advantage of dual-signal amplification of functional Au@Ag nanoparticles (NPs) nanoprobes. Horseradish peroxidase (HRP) and the complementary DNA were co-immobilized onto Au@Ag NPs surface to shape the functional nanoprobes (HRP-Au@Ag-cDNA) for signal amplification. In this proposal, HRP-Au@Ag-cDNA was specifically hybridized with CEA aptamer-functionalized magnetic beads to form the double-strand hybridization nanocomposites (HRP-Au@Ag-dsDNA-MB). Upon the addition of CEA, the CEA aptamer preferred to bind with CEA instead of double-strand hybridization interaction, thus HRP-Au@Ag-dsDNA-MB was dehybridized and the HRP-Au@Ag-cDNA nanoprobe was released. The synergistic catalytic effects of HRP and Au @Ag NPs endow the nanoprobe producing a dual CL signal amplification in the NS-CDs-H2O2 CL system. The CL intensity of the developed strategy enhanced with CEA concentration increasing in the range of 0.3-80 ng mL-1. Benefiting from the synergistic effect, a detection limit as low as 94 pg mL-1 was obtained. Moreover, successful application of this CL sensing platform was achieved for the determination of CEA in human serum samples, demonstrating the promising prospect in the early tumor warning and therapeutic monitoring.

Spatial-Resolved Photoelectrochemical Biosensing Array Based on a CdS@g-C3N4 Heterojunction: A Universal Immunosensing Platform for Accurate Detection.

The detection of biomarkers with high sensitivity and accuracy in real biosamples remains challenging. Herein, a universal spatial-resolved photoelectrochemical (PEC) ratiometry for biodetection of prostate-specific antigen (PSA) as model biomarker was designed for the first time based on a dual-electrode array modified by CdS@g-C3N4 heterojunction coupled with CuS quantum dots (QDs) as signal amplification tags. Specifically, a new kind of photoactive material, the CdS@g-C3N4 p-n heterojunction with high photoelectric conversion efficiency and good chemical stability, was synthesized and immobilized on two spatial-resolved electrodes (WE1 and WE2). After immobilizing gold nanoparticles and capturing PSA antibodies on the electrodes, WE1 incubated with various concentrations of PSA was taken as a working electrode, whereas WE2 with a fixed concentration of PSA was used as an internal reference electrode. Next, signal antibodies of PSA-labeled CuS QDs as PEC signal quenchers were immobilized on the electrodes to form a sandwich-type immunocomplex. With the aid of a multiplexed disjunctor, the PEC responses of the dual electrodes were recorded, and the PSA was quantified via the ratio values of photocurrent intensities from WE1 and WE2. Combining the fine PEC performance of the CdS@g-C3N4 heterojunction with the superior quenching effect of CuS QDs in the spatial-resolved platform, the ratiometric system exhibits a linear range from 1.0 × 10-11 to 5.0 × 10-8 g mL-1 with a limit of detection of 4.0 pg mL-1. The results demonstrated herein may provide a new pattern for biomarker detection with high accuracy and good specificity as well as satisfactory applicability in real biosamples.

Graphene oxide@gold nanorods-based multiple-assisted electrochemiluminescence signal amplification strategy for sensitive detection of prostate specific antigen.

A novel and competitive electrochemiluminescence (ECL) aptasensor for prostate specific antigen (PSA) assay was constructed using gold nanorods functionalized graphene oxide (GO@AuNRs) multilabeled with glucose oxidase (GOD) and streptavidin (SA) toward luminol-based ECL system. A strong initial ECL signal was achieved by electrodeposited gold (DpAu) on the electrode because of gold nanoparticles (AuNPs) motivating the luminol ECL signal. The signal probes prepared by loading GOD and SA-biotin-DNA on GO@AuNRs were used for achieving multiple signal amplification. In the absence of PSA, the signal probes can be attached on the electrode by hybridization reaction between PSA aptamer and biotin-DNA. In this state, the GOD loaded on the probe could catalyze glucose to in situ produce H2O2 and then AuNRs catalyze H2O2 to generate abundant reactive oxygen species (ROSs) in luminol ECL reaction. Both the high-content GOD and AuNRs in the signal probe amplified the ECL signal in the ECL system. Moreover, the combination of SA with biotin-DNA further expands ECL intensity. The integration of such amplifying effects in this protocol endows the aptasensor with high sensitivity and good selectivity for PSA detection. This aptasensor exhibits a linear relation in the range of 0.5pgmL-1 to 5.0ngmL-1 with the detection limit of 0.17pgmL-1 (S/N = 3). Besides, the strategy was successfully applied in determination of human serum samples with recovery of 81.4-116.0%.

A novel immunosensing platform for highly sensitive prostate specific antigen detection based on dual-quenching of photocurrent from CdSe sensitized TiO2 electrode by gold nanoparticles decorated polydopamine nanospheres.

Herein, a novel photoelectrochemical (PEC) immunosensing platform for highly sensitive detection of prostate specific antigen (PSA) was constructed based on dual-quenching of photocurrent from CdSe sensitized TiO2 electrode by gold nanoparticles decorated dopamine-melanin nanospheres (AuNPs-Dpa-melanin CNSs). In this proposal, CdSe sensitized TiO2 was used as photoelectrochemical matrix and the functional AuNPs-Dpa-melanin CNSs were used as signal quenching element. The dual quenching of the gold nanoparticles decorated Dpa-melanin CNSs to the CdSe sensitized TiO2 was achieved as follows: (i) the strong energy transfer between the CdSe quantum dots (QDs) and Au NPs diminishes the photocurrent signal of CdSe QDs; (ii) the steric hindrance of AuNPs-Dpa-melanin CNSs partly obstructs the diffusion of the electron donor, i.e. ascorbic acid, to the surface of photoelectrode, which make the depleting efficiency of the photogenerated holes decrease, leading to a declined photocurrent intensity. On the basis of the dual quenching effect of AuNPs-Dpa-melanin CNSs, a competitive immunosensing platform for PSA was designed upon the specific binding of anti-PSA to PSA and PSA functionalized AuNPs-Dpa-melanin CNSs conjugates. This proposed immunosensor possesses wide linear range from 1.0×10-11gmL-1 to 1.0×10-7gmL-1 with the detection limit of 2.7pgmL-1. Moreover, the applicability of the present method was demonstrated in the determination of PSA in human serum. The strategy creates new paradigms for PSA and other tumor markers detection and demonstrates high sensitivity, good specificity, and satisfied applicability in complex biological samples.

[Inhibition of Paeoniflorin on TNF-α-induced TNF-α Receptor Type I /Nuclear Factor-κB Signal Transduction in Endothelial Cells].

OBJECTIVE: To study the inhibitory effect of paeoniflorin (PAE) on TNF-α-induced TNF receptor type I (TNFR1)-mediated signaling pathway in mouse renal arterial endothelial cells (AECs) and to explore its underlying molecular mechanisms. METHODS: Mouse AECs were cultured in vitro and then they were treated by different concentrations PAE or TNF-α for various time periods. Expression levels of intercellular cell adhesion molecule-1 (ICAM-1) were detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 6-h TNF-α 30 ng/mL), the low dose PAE group (cultured by 2-h PAE 0.8 µmo/L plus 6-h TNF-α 30 ng/mL), the middle dose PAE group (cultured by 2-h PAE 8 µmol/L plus 6-h TNF-α 30 ng/mL), the high dose PAE group (cultured by 2-h PAE 80 µmol/L plus 6-h TNF-α 30 ng/mL) with Western blot analysis. Nuclear translocation of transcription factor NF-κB (NE-κB) was detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 45-mm TNF-α 30 ng/mL), and the high dose PAE group (cultured by 2-h PAE 80 µmol/L plus 45-min TNF-α 30 ng/mL) by immunofluorescent staining. Expression levels of the phosphorylation of extracellular signal-regulated (protein) kinase (ph-ERK) and p38 (ph- p38) were detected in the normal group (cultured by serum-free culture media) and the high dose PAE group (2-h PAE 80 µmol/L culture) by Western blot. NF-κB inhibitor-α (IκBα) protein expressions were detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 30-min TNF-α 30 ng/mL), the high dose PAE group (cultured by 2-h PAE 80 µmol/L plus 30-min TNF-α 30 ng/mL), the p38 inhibitor group (SB group, pretreatment with SB238025 25 µmol/L for 30 min, then treated by PAE 80 µmol/L for 2 h, and finally treated by TNF-α 30 ng/mL for 30 min), the ERK inhibitor group (PD group, treated by PD98059 50 µmol/L for 30 min, then treated by PAE 80 µmol/L for 2 h, and finally treated by TNF-α 30 ng/mL for 30 min) by Western blot. RESULTS: Compared with the normal group, ICAM-1 protein expression levels obviously increased (P < 0.01). Compared with the TNFα group, ICAM-1 protein expression levels were obviously inhibited in the high dose PAE group (P < 0.05). Protein expression levels of ph-p38 and ph-ERK were obviously higher in the hIgh dose PAE group (P < 0.05). Compared with the normal group, IκBα protein expression levels obviously decreased in the TNF-α group (P < 0.01). Compared with the TNFα group, TNF-α-induced IκBα degradation could be significantly inhibited in the high dose PAE group (P < 0.01); the inhibition of PAE on IκBα degradation could be significantly inhibited in the SB group (P < 0.05). NF-κB/p65 signal was mainly located in cytoplasm in the normal group. NF-κB/p65 was translocated from cytoplasm to nucleus after stimulated by 45 min TNF-α in the TNF-α group, while it could be significantly inhibited in the high dose PAE group. CONCLUSIONS: PAE inhibited TNF-α-induced expression of lCAM-1. Its action might be associated with inhibiting TNFR1/NF-κB signaling pathway. p38 participated and mediated these actions.

A comparative study on inhibition of total astragalus saponins and astragaloside IV on TNFR1-mediated signaling pathways in arterial endothelial cells.

BACKGROUND: Both total astragalus saponins (AST) and it's main component astragaloside IV (ASIV) have been used in China as cardiovascular protective medicines. However, the anti-inflammatory activities that are beneficial for cardiovascular health have never been compared directly and the molecular mechanisms remain unresolved. This study was conducted to compare the inhibitory effects of these drugs on TNFα-induced cell responses, related signaling pathways, and the underlying mechanisms in mouse arterial endothelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Real-time qRT-PCR was performed to determine the expression of cell adhesion molecule (CAM) genes. Immunofluorescent staining was used to detect the nuclear translocation of transcription factor NF-κB-p65. Western Blot analysis was used to identify TNFα-induced NF-κB-p65 phosphorylation, IκBα degradation, and caspase-3 cleavage. Cell surface proteins were isolated and TNFα receptor-1(TNFR1) expression was determined. The results suggest that both AST and ASIV attenuate TNFα-induced up-regulation of CAMs mRNA and upstream nuclear translocation and phosphorylation of NF-κB-p65. However, TNFR1-mediated IκBα degradation, cleavage of caspase-3 and apoptosis were inhibited only by AST. These differences in the actions of AST and ASIV could be explained by the presence of other components in AST, such as ASII and ASIII, which also had an inhibitory effect on TNFR1-induced IκBα degradation. Moreover, AST, but not ASIV, was able to reduce TNFR1 protein level on the cell surface. Furthermore, mechanistic investigation demonstrated that TNFR1-mediated IκBα degradation was reversed by the use of TAPI-0, an inhibitor of TNFα converting enzyme (TACE), suggesting the involvement of TACE in the modulation of surface TNFR1 level by AST. CONCLUSION: ASIV was not a better inhibitor than AST, at least on the inhibition of TNFα-induced inflammatory responses and TNFR1-mediated signaling pathways in AECs. The inhibitory effect of AST was caused by the reduction of cell surface TNFR1 level, and TACE could be involved in this action.